Umeå University's logo

umu.sePublications
Change search
Refine search result
1 - 38 of 38
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1.
    Bueno, Emilio
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Pinedo, Victor
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Shinde, Dhananjay D.
    Center for Staphylococcal Research, Department of Pathology and Microbiology, University of Nebraska Medical Centergrid.266813.8, NE, Omaha, United States.
    Mateus, André
    Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Typas, Athanasios
    Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Savitski, Mikhail M.
    Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
    Thomas, Vinai C.
    Center for Staphylococcal Research, Department of Pathology and Microbiology, University of Nebraska Medical Centergrid.266813.8, NE, Omaha, United States.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Transient glycolytic complexation of arsenate enhances resistance in the enteropathogen Vibrio cholerae2022In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 13, no 5, article id e0165422Article in journal (Refereed)
    Abstract [en]

    The ubiquitous presence of toxic arsenate (AsV) in the environment has raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress-related systems to counter reactive oxygen species (ROS) production caused by intracellular AsV. Characterization of the var mutants suggested that these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions. IMPORTANCE Even though resistance to arsenate has been extensively investigated in environmental bacteria, how enteric pathogens tolerate this toxic compound remains unknown. Here, we found that the cholera pathogen V. cholerae exhibits increased resistance to arsenate compared to closely related enteric pathogens. Such resistance is promoted not by ArsC-dependent reduction of arsenate to arsenite but by an operon encoding an arsenate transporter (ArsJ), an alternative glyceraldehyde 3-phosphate dehydrogenase (VarG), and a putative, uncharacterized phosphatase (VarH). Mechanistically, we demonstrate that V. cholerae detoxifies arsenate by complexing it with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). 1As3PG is not transported outside the cell; instead, it is subsequently dissociated by VarH to enable extrusion of free arsenate through ArsJ. Collectively, this study proposes a novel mechanism for arsenate detoxification, entirely independent of arsenate reduction and arsenite extrusion, that enhances V. cholerae resistance to this metalloid compared to other enteric pathogens.

    Download full text (pdf)
    fulltext
  • 2.
    Campbell, Christopher
    et al.
    Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Fingleton, Claire
    Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Zeden, Merve S.
    Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Bueno, Emilio
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gallagher, Laura A.
    Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Shinde, Dhananjay
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Ahn, Jongsam
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Olson, Heather M.
    Biological Sciences Division, Pacific Northwest National Laboratory, WA, Richland, United States.
    Fillmore, Thomas L.
    Biological Sciences Division, Pacific Northwest National Laboratory, WA, Richland, United States.
    Adkins, Joshua N.
    Biological Sciences Division, Pacific Northwest National Laboratory, WA, Richland, United States.
    Razvi, Fareha
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Bayles, Kenneth W.
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Fey, Paul D.
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Thomas, Vinai C.
    Department of Pathology and Microbiology, University of Nebraska Medical Center, NE, Omaha, United States.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Clair, Geremy C.
    Biological Sciences Division, Pacific Northwest National Laboratory, WA, Richland, United States.
    O’gara, James P.
    Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
    Accumulation of succinyl coenzyme a perturbs the methicillin-resistant staphylococcus aureus (Mrsa) succinylome and is associated with increased susceptibility to beta-lactam antibiotics2021In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 12, no 3, article id e00530-21Article in journal (Refereed)
    Abstract [en]

    Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics.

    Download full text (pdf)
    fulltext
  • 3. Castanheira, Sonia
    et al.
    Cestero, Juan J.
    Rico-Perez, Gadea
    Garcia, Pablo
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ayala, Juan A.
    Graciela Pucciarelli, M.
    Garcia-del Portillo, Francisco
    A Specialized Peptidoglycan Synthase Promotes Salmonella Cell Division inside Host Cells2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 6, article id e01685-17Article in journal (Refereed)
    Abstract [en]

    Bacterial cell division has been studied extensively under laboratory conditions. Despite being a key event in the bacterial cell cycle, cell division has not been explored in vivo in bacterial pathogens interacting with their hosts. We discovered in Salmonella enterica serovar Typhimurium a gene absent in nonpathogenic bacteria and encoding a peptidoglycan synthase with 63% identity to penicillin-binding protein 3 (PBP3). PBP3 is an essential cell division-specific peptidoglycan synthase that builds the septum required to separate daughter cells. Since S. Typhimurium carries genes that encode a PBP3 paralog-which we named PBP3(SAL)-and PBP3, we hypothesized that there are different cell division events in host and nonhost environments. To test this, we generated S. Typhimurium isogenic mutants lacking PBP3(SAL) or the hitherto considered essential PBP3. While PBP3 alone promotes cell division under all conditions tested, the mutant producing only PBP3(SAL) proliferates under acidic conditions (pH <= 5.8) but does not divide at neutral pH. PBP3(SAL) production is tightly regulated with increased levels as bacteria grow in media acidified up to pH 4.0 and in intracellular bacteria infecting eukaryotic cells. PBP3(SAL) activity is also strictly dependent on acidic pH, as shown by beta-lactam antibiotic binding assays. Live-cell imaging microscopy revealed that PBP3(SAL) alone is sufficient for S. Typhimurium to divide within phagosomes of the eukaryotic cell. Additionally, we detected much larger amounts of PBP3(SAL) than those of PBP3 in vivo in bacteria colonizing mouse target organs. Therefore, PBP3(SAL) evolved in S. Typhimurium as a specialized peptidoglycan synthase promoting cell division in the acidic intraphagosomal environment. IMPORTANCE During bacterial cell division, daughter cells separate by a transversal structure known as the division septum. The septum is a continuum of the cell wall and therefore is composed of membrane(s) and a peptidoglycan layer. To date, actively growing bacteria were reported to have only a "cell division-specific" peptidoglycan synthase required for the last steps of septum formation and consequently, essential for bacterial life. Here, we discovered that Salmonella enterica has two peptidoglycan synthases capable of synthesizing the division septum. One of these enzymes, PBP3(SAL), is present only in bacterial pathogens and evolved in Salmonella to function exclusively in acidic environments. PBP3(SAL) is used preferentially by Salmonella to promote cell division in vivo in mouse target organs and inside acidified phagosomes. Our data challenge the concept of only one essential cell division-specific peptidoglycan synthase and demonstrate that pathogens can divide in defined host locations using alternative mechanisms.

    Download full text (pdf)
    fulltext
  • 4.
    Chan, Helena
    et al.
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Taib, Najwa
    Department of Microbiology, Unit Evolutionary Biology of the Microbial Cell, Paris, France; Hub Bioinformatics and Biostatistics, Department of Computational Biology, USR 3756 CNRS, Paris, France.
    Gilmore, Michael C.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Mohamed, Ahmed M T
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Hanna, Kieran
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Luhur, Johana
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Nguyen, Hieu
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Hafiz, Elham
    Australian Institute for Microbiology and Infection, University of Technology Sydneygrid.117476.2 (UTS), Sydney, Australia.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gribaldo, Simonetta
    Department of Microbiology, Unit Evolutionary Biology of the Microbial Cell, Paris, France; Hub Bioinformatics and Biostatistics, Department of Computational Biology, USR 3756 CNRS, Paris, France.
    Rudner, David
    Department of Microbiology, MA, Boston, United States.
    Rodrigues, Christopher D A
    School of Life Sciences, University of Warwickgrid.7372.1, Coventry, United Kingdom.
    Genetic screens identify additional genes implicated in envelope remodeling during the engulfment stage of bacillus subtilis sporulation2022In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 13, no 5, article id e0173222Article in journal (Refereed)
    Abstract [en]

    During bacterial endospore formation, the developing spore is internalized into the mother cell through a phagocytic-like process called engulfment, which involves synthesis and hydrolysis of peptidoglycan. Engulfment peptidoglycan hydrolysis requires the widely conserved and well-characterized DMP complex, composed of SpoIID, SpoIIM, and SpoIIP. In contrast, although peptidoglycan synthesis has been implicated in engulfment, the protein players involved are less well defined. The widely conserved SpoIIIAH-SpoIIQ interaction is also required for engulfment efficiency, functioning like a ratchet to promote membrane migration around the forespore. Here, we screened for additional factors required for engulfment using transposon sequencing in Bacillus subtilis mutants with mild engulfment defects. We discovered that YrvJ, a peptidoglycan hydrolase, and the MurA paralog MurAB, involved in peptidoglycan precursor synthesis, are required for efficient engulfment. Cytological analyses suggest that both factors are important for engulfment when the DMP complex is compromised and that MurAB is additionally required when the SpoIIIAH-SpoIIQ ratchet is abolished. Interestingly, despite the importance of MurAB for sporulation in B. subtilis, phylogenetic analyses of MurA paralogs indicate that there is no correlation between sporulation and the number of MurA paralogs and further reveal the existence of a third MurA paralog, MurAC, within the Firmicutes. Collectively, our studies identify two new factors that are required for efficient envelop remodeling during sporulation and highlight the importance of peptidoglycan precursor synthesis for efficient engulfment in B. subtilis and likely other endospore-forming bacteria. IMPORTANCE In bacteria, cell envelope remodeling is critical for cell growth and division. This is also the case during the development of bacteria into highly resistant endospores (spores), known as sporulation. During sporulation, the developing spore becomes internalized inside the mother cell through a phagocytic-like process called engulfment, which is essential to form the cell envelope of the spore. Engulfment involves both the synthesis and hydrolysis of peptidoglycan and the stabilization of migrating membranes around the developing spore. Importantly, although peptidoglycan synthesis has been implicated during engulfment, the specific genes that contribute to this molecular element of engulfment have remained unclear. Our study identifies two new factors that are required for efficient envelope remodeling during engulfment and emphasizes the importance of peptidoglycan precursor synthesis for efficient engulfment in the model organism Bacillus subtilis and likely other endospore-forming bacteria. Finally, our work highlights the power of synthetic screens to reveal additional genes that contribute to essential processes during sporulation.

    Download full text (pdf)
    fulltext
  • 5. Codemo, Mario
    et al.
    Muschiol, Sandra
    Iovino, Federico
    Nannapaneni, Priyanka
    Plant, Laura
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Henriques-Normark, Birgitta
    Immunomodulatory Effects of Pneumococcal Extracellular Vesicles on Cellular and Humoral Host Defenses2018In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 2, article id e00559-18Article in journal (Refereed)
    Abstract [en]

    Gram-positive bacteria, including the major respiratory pathogen Streptococcus pneumoniae, were recently shown to produce extracellular vesicles (EVs) that likely originate from the plasma membrane and are released into the extracellular environment. EVs may function as cargo for many bacterial proteins, however, their involvement in cellular processes and their interactions with the innate immune system are poorly understood. Here, EVs from pneumococci were characterized and their immunomodulatory effects investigated. Pneumococcal EVs were protruding from the bacterial surface and released into the medium as 25 to 250 nm lipid stained vesicles containing a large number of cytosolic, membrane, and surface-associated proteins. The cytosolic pore-forming toxin pneumolysin was significantly enriched in EVs compared to a total bacterial lysate but was not required for EV formation. Pneumococcal EVs were internalized into A549 lung epithelial cells and human monocyte-derived dendritic cells and induced proinflammatory cytokine responses irrespective of pneumolysin content. EVs from encapsulated pneumococci were recognized by serum proteins, resulting in C3b deposition and formation of C5b-9 membrane attack complexes as well as factor H recruitment, depending on the presence of the choline binding protein PspC. Addition of EVs to human serum decreased opsonophagocytic killing of encapsulated pneumococci. Our data suggest that EVs may act in an immunomodulatory manner by allowing delivery of vesicle-associated proteins and other macromolecules into host cells. In addition, EVs expose targets for complement factors in serum, promoting pneumococcal evasion of humoral host defense.

    Importance: Streptococcus pneumoniae is a major contributor to morbidity and mortality worldwide, being the major cause of milder respiratory tract infections such as otitis and sinusitis and of severe infections such as community-acquired pneumonia, with or without septicemia, and meningitis. More knowledge is needed on how pneumococci interact with the host, deliver virulence factors, and activate immune defenses. Here we show that pneumococci form extracellular vesicles that emanate from the plasma membrane and contain virulence properties, including enrichment of pneumolysin. We found that pneumococcal vesicles can be internalized into epithelial and dendritic cells and bind complement proteins, thereby promoting pneumococcal evasion of complement-mediated opsonophagocytosis. They also induce pneumolysin-independent proinflammatory responses. We suggest that these vesicles can function as a mechanism for delivery of pneumococcal proteins and other immunomodulatory components into host cells and help pneumococci to avoid complement deposition and phagocytosis-mediated killing, thereby possibly contributing to the symptoms found in pneumococcal infections.

    Download full text (pdf)
    fulltext
  • 6.
    Dai, Yunfei
    et al.
    Department of Biology, Northeastern University, MA, Boston, United States.
    Pinedo, Victor
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Tang, Amy Y.
    Department of Biology, Northeastern University, MA, Boston, United States.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Geisinger, Edward
    Department of Biology, Northeastern University, MA, Boston, United States.
    A New Class of Cell Wall-Recycling L,D-Carboxypeptidase Determines β-Lactam Susceptibility and Morphogenesis in Acinetobacter baumannii2021In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 12, no 6, article id e0278621Article in journal (Refereed)
    Abstract [en]

    The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for maintaining a characteristic short rod shape and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell wall-modifying L,D-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic L,D-carboxypeptidase (LDC) that provides a critical step in cell wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, morphology, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including L,D-cross-link formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on envelope physiology. We thus reveal a novel class of cell wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria.

    Download full text (pdf)
    fulltext
  • 7. De Pascalis, Roberto
    et al.
    Chou, Alicia Y.
    Ryden, Patrik
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Kennett, Nikki J.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Elkins, Karen L.
    Models Derived from In Vitro Analyses of Spleen, Liver, and Lung Leukocyte Functions Predict Vaccine Efficacy against the Francisella tularensis Live Vaccine Strain (LVS)2014In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 5, no 2, article id e00936Article in journal (Refereed)
    Abstract [en]

    Currently, there are no licensed vaccines and no correlates of protection against Francisella tularensis, which causes tularemia. We recently demonstrated that measuring in vitro control of intramacrophage bacterial growth by murine F. tularensis-immune splenocytes, as well as transcriptional analyses, discriminated Francisella vaccines of different efficacies. Further, we identified potential correlates of protection against systemic challenge. Here, we extended this approach by studying leukocytes derived from lungs and livers of mice immunized by parenteral and respiratory routes with F. tularensis vaccines. Liver and lung leukocytes derived from intradermally and intranasally vaccinated mice controlled in vitro Francisella Live Vaccine Strain (LVS) intramacrophage replication in patterns similar to those of splenocytes. Gene expression analyses of potential correlates also revealed similar patterns in liver cells and splenocytes. In some cases (e. g., tumor necrosis factor alpha [TNF-alpha], interleukin 22 [IL-22], and granulocyte-macrophage colony-stimulating factor [GM-CSF]), liver cells exhibited even higher relative gene expression, whereas fewer genes exhibited differential expression in lung cells. In contrast with their strong ability to control LVS replication, splenocytes from intranasally vaccinated mice expressed few genes with a hierarchy of expression similar to that of splenocytes from intradermally vaccinated mice. Thus, the relative levels of gene expression vary between cell types from different organs and by vaccination route. Most importantly, because studies comparing cell sources and routes of vaccination supported the predictive validity of this coculture and gene quantification approach, we combined in vitro LVS replication with gene expression data to develop analytical models that discriminated between vaccine groups and successfully predicted the degree of vaccine efficacy. Thus, this strategy remains a promising means of identifying and quantifying correlative T cell responses.

    IMPORTANCE

    Identifying and quantifying correlates of protection is especially challenging for intracellular bacteria, including Francisella tularensis. F. tularensis is classified as a category A bioterrorism agent, and no vaccines have been licensed in the United States, but tularemia is a rare disease. Therefore, clinical trials to test promising vaccines are impractical. In this report, we further evaluated a novel approach to developing correlates by assessing T cell immune responses in lungs and livers of differentially vaccinated mice; these nonprofessional immune tissues are colonized by Francisella. The relative degree of vaccine efficacy against systemic challenge was reflected by the ability of immune T cells, particularly liver T cells, to control the intramacrophage replication of bacteria in vitro and by relative gene expression of several immunological mediators. We therefore developed analytical models that combined bacterial replication data and gene expression data. Several resulting models provided excellent discrimination between vaccines of different efficacies.

    Download full text (pdf)
    fulltext
  • 8.
    Engström, Patrik
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Krishnan, K. Syam
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ngyuen, Bidong D.
    Chorell, Erik
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Normark, Johan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Silver, Jim
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bastidas, Robert J.
    Welch, Matthew D.
    Hultgren, Scott J.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Valdivia, Raphael H.
    Almqvist, Fredrik
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    A 2-Pyridone-Amide Inhibitor Targets the Glucose Metabolism Pathway of Chlamydia trachomatis2015In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 6, no 1, article id e02304-14Article in journal (Refereed)
    Abstract [en]

    In a screen for compounds that inhibit infectivity of the obligate intracellular pathogen Chlamydia trachomatis, we identified the 2-pyridone amide KSK120. A fluorescent KSK120 analogue was synthesized and observed to be associated with the C. trachomatis surface, suggesting that its target is bacterial. We isolated KSK120-resistant strains and determined that several resistance mutations are in genes that affect the uptake and use of glucose-6-phosphate (G-6P). Consistent with an effect on G-6P metabolism, treatment with KSK120 blocked glycogen accumulation. Interestingly, KSK120 did not affect Escherichia coli or the host cell. Thus, 2-pyridone amides may represent a class of drugs that can specifically inhibit C. trachomatis infection. IMPORTANCE Chlamydia trachomatis is a bacterial pathogen of humans that causes a common sexually transmitted disease as well as eye infections. It grows only inside cells of its host organism, within a parasitophorous vacuole termed the inclusion. Little is known, however, about what bacterial components and processes are important for C. trachomatis cellular infectivity. Here, by using a visual screen for compounds that affect bacterial distribution within the chlamydial inclusion, we identified the inhibitor KSK120. As hypothesized, the altered bacterial distribution induced by KSK120 correlated with a block in C. trachomatis infectivity. Our data suggest that the compound targets the glucose-6-phosphate (G-6P) metabolism pathway of C. trachomatis, supporting previous indications that G-6P metabolism is critical for C. trachomatis infectivity. Thus, KSK120 may be a useful tool to study chlamydial glucose metabolism and has the potential to be used in the treatment of C. trachomatis infections.

    Download full text (pdf)
    fulltext
  • 9.
    Fei, Keke
    et al.
    CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China.
    Yan, Huan
    CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China.
    Zeng, Xiaoyan
    CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China.
    Huang, Shaojia
    CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China.
    Tang, Wei
    CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China.
    Francis, Matthew S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chen, Shiyun
    CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China.
    Hu, Yangbo
    State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China.
    LcrQ coordinates with the YopD-LcrH complex to repress lcrF expression and control type III secretion by Yersinia pseudotuberculosis2021In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 12, no 3, article id e0145721Article in journal (Refereed)
    Abstract [en]

    Human-pathogenic Yersinia species employ a plasmid-encoded type III secretion system (T3SS) to negate immune cell function during infection. A critical element in this process is the coordinated regulation of T3SS gene expression, which involves both transcriptional and posttranscriptional mechanisms. LcrQ is one of the earliest identified negative regulators of Yersinia T3SS, but its regulatory mechanism is still unclear. In a previous study, we showed that LcrQ antagonizes the activation role played by the master transcriptional regulator LcrF. In this study, we confirm that LcrQ directly interacts with LcrH, the chaperone of YopD, to facilitate the negative regulatory role of the YopD-LcrH complex in repressing lcrF expression at the posttranscriptional level. Negative regulation is strictly dependent on the YopD-LcrH complex, more so than on LcrQ. The YopD-LcrH complex helps to retain cytoplasmic levels of LcrQ to facilitate the negative regulatory effect. Interestingly, RNase E and its associated protein RhlB participate in this negative regulatory loop through a direct interaction with LcrH and LcrQ. Hence, we present a negative regulatory loop that physically connects LcrQ to the posttranscriptional regulation of LcrF, and this mechanism incorporates RNase E involved in mRNA decay.

    Download full text (pdf)
    fulltext
  • 10. Felgner, S
    et al.
    Frahm, M
    Kocijancic, D
    Rohde, M
    Eckweiler, D
    Bielecka, A
    Bueno, Emilio
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Cava, Felipe
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Abraham, WR
    Curtiss, R
    Häussler, S
    Erhardt, M
    Weiss, S
    aroA-Deficient Salmonella enterica Serovar Typhimurium Is More Than a Metabolically Attenuated Mutant2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 5, article id e01220-16Article in journal (Refereed)
    Abstract [en]

    Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that Delta aroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB. Finally, Delta aroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of Delta aroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine. 

    IMPORTANCE Recombinant attenuated bacterial vector systems based on genetically engineered Salmonella have been developed as highly potent vaccines. Due to the pathogenic properties of Salmonella, efficient attenuation is required for clinical applications. Since the hallmark study by Hoiseth and Stocker in 1981 (S. K. Hoiseth and B. A. D. Stocker, Nature 291:238-239, 1981, http://dx.doi.org/10.1038/291238a0), the auxotrophic Delta aroA mutation has been generally considered safe and universally used to attenuate bacterial strains. Here, we are presenting the remarkable finding that a deletion of aroA leads to pronounced alterations of gene expression, metabolism, and cellular physiology, which resulted in increased immunogenicity, virulence, and adjuvant potential of Salmonella. These results suggest that the enhanced immunogenicity of aroA-deficient Salmonella strains might be advantageous for optimizing bacterial vaccine carriers and immunotherapy. Accordingly, we demonstrate a superior performance of Delta aroA Salmonella in bacterium-mediated tumor therapy. In addition, the present study highlights the importance of a functional shikimate pathway to sustain bacterial physiology and metabolism.

    Download full text (pdf)
    fulltext
  • 11. Fleurie, Aurore
    et al.
    Zoued, Abdelrahim
    Alvarez, Laura
    Hines, Kelly M.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Xu, Libin
    Davis, Brigid M.
    Waldor, Matthew K.
    A Vibrio cholerae BolA-Like Protein Is Required for Proper Cell Shape and Cell Envelope Integrity2019In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 4, article id e00790-19Article in journal (Refereed)
    Abstract [en]

    BolA family proteins are conserved in Gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well described. Here, we investigated the roles of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen's morphology. Notably, the bizarre-shaped, multipolar, elongated, and wide cells that predominated in exponential-phase Delta ibaG V. cholerae cultures were not observed in stationary-phase cultures. The V. cholerae Delta ibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall-acting antibiotics and bile, and was defective in intestinal colonization. Delta ibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant's morphological defects and sensitivity to envelope stressors. Transposon insertion sequencing analysis of ibaG's genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, copurification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins. IMPORTANCE BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae. The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster-containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving ironsulfur-containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

  • 12. Goormaghtigh, Frederic
    et al.
    Fraikin, Nathan
    Putrins, Marta
    Hallaert, Thibaut
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Institute of Technology, University of Tartu, Tartu, Estonia.
    Garcia-Pino, Abel
    Sjödin, Andreas
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Division of CBRN Security and Defence, FOI–Swedish Defence Research Agency, Umeå, Sweden.
    Kasvandik, Sergo
    Udekwu, Klas
    Tenson, Tanel
    Kaldalu, Niilo
    Van Melderen, Laurence
    Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells2018In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 3, article id e00640-18Article in journal (Refereed)
    Abstract [en]

    Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicillin. Moreover, microfluidic experiments performed with a strain reporting the induction of the yefM-yoeB TA system allowed the observation of a small number of type II persister cells that resume growth after removal of ampicillin. However, we were unable to establish a correlation between high fluorescence and persistence, since the fluorescence of persister cells was comparable to that of the bulk of the population and none of the cells showing high fluorescence were able to resume growth upon removal of the antibiotic. Altogether, these data show that there is no direct link between induction of TA systems and persistence to antibiotics. IMPORTANCE Within a growing bacterial population, a small subpopulation of cells is able to survive antibiotic treatment by entering a transient state of dormancy referred to as persistence. Persistence is thought to be the cause of relapsing bacterial infections and is a major public health concern. Type II toxin-antitoxin systems are small modules composed of a toxic protein and an antitoxin protein counteracting the toxin activity. These systems were thought to be pivotal players in persistence until recent developments in the field. Our results demonstrate that previous influential reports had technical flaws and that there is no direct link between induction of TA systems and persistence to antibiotics.

    Download full text (pdf)
    fulltext
  • 13. Goormaghtigh, Frederic
    et al.
    Fraikin, Nathan
    Putrins, Marta
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Institute of Technology, University of Tartu, Tartu, Estonia.
    Garcia-Pino, Abel
    Udekwu, Klas
    Tenson, Tanel
    Kaldalu, Niilo
    Van Melderen, Laurence
    Reply to Holden and Errington, "Type II Toxin-Antitoxin Systems and Persister Cells"2018In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 5, article id e01838-18Article in journal (Refereed)
  • 14. Greene, Sarah E.
    et al.
    Pinkner, Jerome S.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dodson, Karen W.
    Shaffer, Carrie L.
    Conover, Matt S.
    Livny, Jonathan
    Hadjifrangiskou, Maria
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hultgren, Scott J.
    Pilicide ec240 Disrupts Virulence Circuits in Uropathogenic Escherichia coli2014In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 5, no 6, p. UNSP e02038-Article in journal (Refereed)
    Abstract [en]

    Chaperone-usher pathway (CUP) pili are extracellular organelles produced by Gram-negative bacteria that mediate bacterial pathogenesis. Small-molecule inhibitors of CUP pili, termed pilicides, were rationally designed and shown to inhibit type 1 or P piliation. Here, we show that pilicide ec240 decreased the levels of type 1, P, and S piliation. Transcriptomic and proteomic analyses using the cystitis isolate UTI89 revealed that ec240 dysregulated CUP pili and decreased motility. Paradoxically, the transcript levels of P and S pilus genes were increased during growth in ec240, even though the level of P and S piliation decreased. In contrast, the most downregulated transcripts after growth in ec240 were from the type 1 pilus genes. Type 1 pilus expression is controlled by inversion of the fimS promoter element, which can oscillate between phase on and phase off orientations. ec240 induced the fimS phase off orientation, and this effect was necessary for the majority of ec240's inhibition of type 1 piliation. ec240 increased levels of the transcriptional regulators SfaB and PapB, which were shown to induce the fimS promoter phase off orientation. Furthermore, the effect of ec240 on motility was abolished in the absence of the SfaB, PapB, SfaX, and PapX regulators. In contrast to the effects of ec240, deletion of the type 1 pilus operon led to increased S and P piliation and motility. Thus, ec240 dysregulated several uropathogenic Escherichia coli (UPEC) virulence factors through different mechanisms and independent of its effects on type 1 pilus biogenesis and may have potential as an antivirulence compound. IMPORTANCE CUP pili and flagella play active roles in the pathogenesis of a variety of Gram-negative bacterial infections, including urinary tract infections mediated by UPEC. These are extremely common infections that are often recurrent and increasingly caused by antibiotic-resistant organisms. Preventing piliation and motility through altered regulation and assembly of these important virulence factors could aid in the development of novel therapeutics. This study increases our understanding of the regulation of these virulence factors, providing new avenues by which to target their expression.

    Download full text (pdf)
    fulltext
  • 15.
    Harrison, Gregory A.
    et al.
    Department of Molecular Microbiology, Center for Women’s Infectious Disease Research, Washington University School of Medicine, MO, St. Louis, United States.
    Wang, Erin R.
    Department of Molecular Microbiology, Center for Women’s Infectious Disease Research, Washington University School of Medicine, MO, St. Louis, United States.
    Cho, Kevin
    Department of Chemistry, Washington University in St. Louis, MO, St. Louis, United States; Department of Medicine, Washington University School of Medicine, MO, St. Louis, United States; Center for Metabolomics and Isotope Tracing, Washington University in St. Louis, MO, St. Louis, United States.
    Mreyoud, Yassin
    Department of Molecular Microbiology, Center for Women’s Infectious Disease Research, Washington University School of Medicine, MO, St. Louis, United States.
    Sarkar, Souvik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Patti, Gary J.
    Department of Chemistry, Washington University in St. Louis, MO, St. Louis, United States; Department of Medicine, Washington University School of Medicine, MO, St. Louis, United States; Center for Metabolomics and Isotope Tracing, Washington University in St. Louis, MO, St. Louis, United States.
    Stallings, Christina L.
    Department of Molecular Microbiology, Center for Women’s Infectious Disease Research, Washington University School of Medicine, MO, St. Louis, United States.
    Inducing vulnerability to InhA inhibition restores isoniazid susceptibility in drug-resistant Mycobacterium tuberculosis2024In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 15, no 3Article in journal (Refereed)
    Abstract [en]

    Of the approximately 10 million cases of Mycobacterium tuberculosis (Mtb) infections each year, over 10% are resistant to the frontline antibiotic isoniazid (INH). INH resistance is predominantly caused by mutations that decrease the activity of the bacterial enzyme KatG, which mediates the conversion of the pro-drug INH to its active form INH-NAD. We previously discovered an inhibitor of Mtb respiration, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a collection of INH-resistant mutants to INH through an unknown mechanism. To investigate the mechanism of action of C10, we exploited the toxicity of high concentrations of C10 to select for resistant mutants. We discovered two mutations that confer resistance to the disruption of energy metabolism and allow for the growth of Mtb in high C10 concentrations, indicating that growth inhibition by C10 is associated with inhibition of respiration. Using these mutants as well as direct inhibitors of the Mtb electron transport chain, we provide evidence that inhibition of energy metabolism by C10 is neither sufficient nor necessary to potentiate killing by INH. Instead, we find that C10 acts downstream of INH-NAD synthesis, causing Mtb to become particularly sensitive to inhibition of the INH-NAD target, InhA, without changing the concentration of INH-NAD or the activity of InhA, the two predominant mechanisms of potentiating INH. Our studies revealed that there exists a vulnerability in Mtb that can be exploited to render Mtb sensitive to otherwise subinhibitory concentrations of InhA inhibitor. IMPORTANCE Isoniazid (INH) is a critical frontline antibiotic to treat Mycobacterium tuberculosis (Mtb) infections. INH efficacy is limited by its suboptimal penetration of the Mtb-containing lesion and by the prevalence of clinical INH resistance. We previously discovered a compound, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a set of INH-resistant mutants to INH. Resistance is typically mediated by katG mutations that decrease the activation of INH, which is required for INH to inhibit the essential enzyme InhA. Our current work demonstrates that C10 re-sensitizes INH-resistant katG-hypomorphs without enhancing the activation of INH. We furthermore show that C10 causes Mtb to become particularly vulnerable to InhA inhibition without compromising InhA activity on its own. Therefore, C10 represents a novel strategy to curtail the development of INH resistance and to sensitize Mtb to sub-lethal doses of INH, such as those achieved at the infection site.

    Download full text (pdf)
    fulltext
  • 16. Herrador, Antonio
    et al.
    Fedeli, Chiara
    Radulovic, Emilia
    Campbell, Kevin P.
    Moreno, Hector
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology. TWINCORE - Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany.
    Kunz, Stefan
    Dynamic Dystroglycan Complexes Mediate Cell Entry of Lassa Virus2019In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 2, article id e02869-18Article in journal (Refereed)
    Abstract [en]

    Recognition of functional receptors by viruses is a key determinant for their host range, tissue tropism, and disease potential. The highly pathogenic Lassa virus (LASV) currently represents one of the most important emerging pathogens. The major cellular receptor for LASV in human cells is the ubiquitously expressed and evolutionary highly conserved extracellular matrix receptor dystroglycan (DG). In the host, DG interacts with many cellular proteins in a tissue-specific manner. The resulting distinct supramolecular complexes likely represent the functional units for viral entry, and preexisting protein-protein interactions may critically influence DG's function in productive viral entry. Using an unbiased shotgun proteomic approach, we define the largely unknown molecular composition of DG complexes present in highly susceptible epithelial cells that represent important targets for LASV during viral transmission. We further show that the specific composition of cellular DG complexes can affect DG's function in receptor-mediated endocytosis of the virus. Under steady-state conditions, epithelial DG complexes underwent rapid turnover via an endocytic pathway that shared some characteristics with DG-mediated LASV entry. However, compared to steady-state uptake of DG, LASV entry via DG occurred faster and critically depended on additional signaling by receptor tyrosine kinases and the downstream effector p21-activating kinase. In sum, we show that the specific molecular composition of DG complexes in susceptible cells is a determinant for productive virus entry and that the pathogen can manipulate the existing DG-linked endocytic pathway. This highlights another level of complexity of virus-receptor interaction and provides possible cellular targets for therapeutic antiviral intervention.

    Importance: Recognition of cellular receptors allows emerging viruses to break species barriers and is an important determinant for their disease potential. Many virus receptors have complex tissue-specific interactomes, and preexisting protein-protein interactions may influence their function. Combining shotgun proteomics with a biochemical approach, we characterize the molecular composition of the functional receptor complexes used by the highly pathogenic Lassa virus (LASV) to invade susceptible human cells. We show that the specific composition of the receptor complexes affects productive entry of the virus, providing proof-of-concept. In uninfected cells, these functional receptor complexes undergo dynamic turnover involving an endocytic pathway that shares some characteristics with viral entry. However, steady-state receptor uptake and virus endocytosis critically differ in kinetics and underlying signaling, indicating that the pathogen can manipulate the receptor complex according to its needs. Our study highlights a remarkable complexity of LASV-receptor interaction and identifies possible targets for therapeutic antiviral intervention.

    Download full text (pdf)
    fulltext
  • 17. Jacquier, Nicolas
    et al.
    Yadav, Akhilesh K.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Pillonel, Trestan
    Viollier, Patrick H.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Greub, Gilbert
    A SpoIID Homolog Cleaves Glycan Strands at the Chlamydial Division Septum2019In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 4, article id e01128-19Article in journal (Refereed)
    Abstract [en]

    Chlamydiales species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal peptidoglycan biosynthesis seems to be regulated by MreB actin and its membrane anchor RodZ rather than FtsZ tubulin in Chlamydiales, the mechanism of peptidoglycan remodeling is poorly understood. An amidase conserved in Chlamydiales is able to cleave peptide stems in peptidoglycan, but it is not clear how peptidoglycan glycan strands are cleaved since no classical lytic transglycosylase is encoded in chlamydial genomes. However, a protein containing a SpoIID domain, known to possess transglycosylase activity in Bacillus subtilis, is conserved in Chiamydiales. We show here that the SpoIID homologue of the Chlamydia-related pathogen Waddlia chondrophila is a septal peptidoglycan-binding protein. Moreover, we demonstrate that SpoIID acts as a lytic transglycosylase on peptidoglycan and as a muramidase on denuded glycan strands in vitro. As SpoIID-like proteins are widespread in nonsporulating bacteria, SpoIID might commonly be a septal peptidoglycan remodeling protein in bacteria, including obligate intracellular pathogens, and thus might represent a promising drug target. IMPORTANCE Chlamydiales species are obligate intracellular bacteria and important human pathogens that have a minimal division machinery lacking the proteins that are essential for bacterial division in other species, such as FtsZ. Chlamydial division requires synthesis of peptidoglycan, which forms a ring at the division septum and is rapidly turned over. However, little is known of peptidoglycan degradation, because many peptidoglycan-degrading enzymes are not encoded by chlamydial genomes. Here we show that an homologue of SpoIID, a peptidoglycan-degrading enzyme involved in sporulation of bacteria such as Bacillus subtilis, is expressed in Chlamydiales, localizes at the division septum, and degrades peptidoglycan in vitro, indicating that SpoIID is not only involved in sporulation but also likely implicated in division of some bacteria.

  • 18. Kaldalu, Niilo
    et al.
    Maiväli, Ülo
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Institute of Technology, University of Tartu, Tartu, Estonia.
    Tenson, Tanel
    Reanalysis of Proteomics Results Fails To Detect MazF-Mediated Stress Proteins2019In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 3, article id e00949-19Article in journal (Refereed)
  • 19.
    Lasswitz, Lisa
    et al.
    Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany; Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany.
    Zapatero-Belinchón, Francisco J.
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany; Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany.
    Moeller, Rebecca
    Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany; Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany.
    Hülskötter, Kirsten
    Department of Pathology, University of Veterinary Medicine Hanover, Hanover, Germany.
    Laurent, Timothée
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Carlson, Lars-Anders
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Goffinet, Christine
    Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Virology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany.
    Simmons, Graham
    Vitalant Research Institute, University of California, CA, San Francisco, United States; Department of Laboratory Medicine, University of California, CA, San Francisco, United States.
    Baumgärtner, Wolfgang
    Department of Pathology, University of Veterinary Medicine Hanover, Hanover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany; Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany.
    The tetraspanin CD81 is a host factor for Chikungunya virus replication2022In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 13, no 3, article id e0073122Article in journal (Refereed)
    Abstract [en]

    Chikungunya virus (CHIKV) is an arthritogenic reemerging virus replicating in plasma membrane-derived compartments termed "spherules." Here, we identify the human transmembrane protein CD81 as host factor required for CHIKV replication. Ablation of CD81 results in decreased CHIKV permissiveness, while overexpression enhances infection. CD81 is dispensable for virus uptake but critically required for viral genome replication. Likewise, murine CD81 is crucial for CHIKV permissiveness and is expressed in target cells such as dermal fibroblasts, muscle and liver cells. Whereas related alphaviruses, including Ross River virus (RRV), Semliki Forest virus (SFV), Sindbis virus (SINV) and Venezuelan equine encephalitis virus (VEEV), also depend on CD81 for infection, RNA viruses from other families, such as coronaviruses, replicate independently of CD81. Strikingly, the replication-enhancing function of CD81 is linked to cholesterol binding. These results define a mechanism exploited by alphaviruses to hijack the membrane microdomain-modeling protein CD81 for virus replication through interaction with cholesterol.

    IMPORTANCE: In this study, we discover the tetraspanin CD81 as a host factor for the globally emerging chikungunya virus and related alphaviruses. We show that CD81 promotes replication of viral genomes in human and mouse cells, while virus entry into cells is independent of CD81. This provides novel insights into how alphaviruses hijack host proteins to complete their life cycle. Alphaviruses replicate at distinct sites of the plasma membrane, which are enriched in cholesterol. We found that the cholesterol-binding ability of CD81 is important for its function as an alphavirus host factor. This discovery thus broadens our understanding of the alphavirus replication process and the use of host factors to reprogram cells into virus replication factories.

    Download full text (pdf)
    fulltext
  • 20.
    Lopes, Jose Pedro
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Stylianou, Marios
    Backman, Emelie
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Holmberg, Sandra
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Jass, Jana
    Claesson, Rolf
    Umeå University, Faculty of Medicine, Department of Odontology.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Evasion of Immune Surveillance in Low Oxygen Environments Enhances Candida albicans Virulence.2018In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 6, article id e02120-18Article in journal (Refereed)
    Abstract [en]

    Microbial colonizers of humans have evolved to adapt to environmental cues and to sense nutrient availability. Oxygen is a constantly changing environmental parameter in different host tissues and in different types of infection. We describe how Candida albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of polymorphonuclear leukocytes (PMNs) to the site of infection contributes to a low oxygen milieu in a murine subdermal abscess. A persistent hypoxic environment did not affect viability or metabolism of PMNs. Under oxygen deprivation, however, infection with C. albicans disturbed specific PMN responses. PMNs were not able to efficiently phagocytose, produce ROS, or release extracellular DNA traps. Failure to launch an adequate response was caused by C. albicans cell wall masking of β-glucan upon exposure to low oxygen levels which hindered PAMP sensing by Dectin-1 on the surfaces of PMNs. This in turn contributed to immune evasion and enhanced fungal survival. The cell wall masking effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen conditions. Finally, adaptation to oxygen deprivation increased virulence of C. albicans which we demonstrated using a Caenorhabditis elegans infection model.IMPORTANCE Successful human colonizers have evolved mechanisms to bypass immune surveillance. Infiltration of PMNs to the site of infection led to the generation of a low oxygen niche. Exposure to low oxygen levels induced fungal cell wall masking, which in turn hindered pathogen sensing and antifungal responses by PMNs. The cell wall masking effect was prolonged by increasing lactate amounts produced by neutrophil metabolism under oxygen deprivation. In an invertebrate infection model, C. albicans was able to kill infected C. elegans nematodes within 2 days under low oxygen conditions, whereas the majority of uninfected controls and infected worms under normoxic conditions survived. These results suggest that C. albicans benefited from low oxygen niches to increase virulence. The interplay of C. albicans with innate immune cells under these conditions contributed to the overall outcome of infection. Adaption to low oxygen levels was in addition beneficial for C. albicans by reducing susceptibility to selected antifungal drugs. Hence, immunomodulation of host cells under low oxygen conditions could provide a valuable approach to improve current antifungal therapies.

    Download full text (pdf)
    fulltext
  • 21.
    Meier, Karsten
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Jachmann, Lana H.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Türköz, Gözde
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Babu Sait, Mohammed Rizwan
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Pérez, Lucía
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Kepp, Oliver
    Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, Villejuif, France; Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France.
    Valdivia, Raphael H.
    Department of Molecular Genetics and Microbiology, Duke University School of Medicine, NC, Durham, United States.
    Kroemer, Guido
    Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, Villejuif, France; Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Department of Biology, Hôpital Européen Georges-Pompidou, AP-HP, Paris, France.
    Sixt, Barbara Susanne
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    The Chlamydia effector CpoS modulates the inclusion microenvironment and restricts the interferon response by acting on Rab352023In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 14, no 4, article id e0319022Article in journal (Refereed)
    Abstract [en]

    The obligate intracellular bacterium Chlamydia trachomatis inserts a family of inclusion membrane (Inc) proteins into the membrane of its vacuole (the inclusion). The Inc CpoS is a critical suppressor of host cellular immune surveillance, but the underlying mechanism remained elusive. By complementing a cpoS mutant with various natural orthologs and variants of CpoS, we linked distinct molecular interactions of CpoS to distinct functions. Unexpectedly, we found CpoS to be essential for the formation of inclusion membrane microdomains that control the spatial organization of multiple Incs involved in signaling and modulation of the host cellular cytoskeleton. While the function of CpoS in microdomains was uncoupled from its role in the suppression of host cellular defenses, we found the ability of CpoS to interact with Rab GTPases to be required not only for the manipulation of membrane trafficking, such as to mediate transport of ceramide-derived lipids (sphingolipids) to the inclusion, but also for the inhibition of Stimulator of interferon genes (STING)-dependent type I interferon responses. Indeed, depletion of Rab35 phenocopied the exacerbated interferon responses observed during infection with CpoS-deficient mutants. Overall, our findings highlight the role of Inc-Inc interactions in shaping the inclusion microenvironment and the modulation of membrane trafficking as a pathogenic immune evasion strategy.

    IMPORTANCE: Chlamydia trachomatis is a prevalent bacterial pathogen that causes blinding ocular scarring and urogenital infections that can lead to infertility and pregnancy complications. Because Chlamydia can only grow within its host cell, boosting the intrinsic defenses of human cells may represent a novel strategy to fight pathogen replication and survival. Hence, CpoS, a Chlamydia protein known to block host cellular defenses, or processes regulated by CpoS, could provide new opportunities for therapeutic intervention. By revealing CpoS as a multifunctional virulence factor and by linking its ability to block host cellular immune signaling to the modulation of membrane trafficking, the present work may provide a foundation for such rationale targeting and advances our understanding of how intracellular bacteria can shape and protect their growth niche.

    Download full text (pdf)
    fulltext
  • 22.
    Midonet, Caroline
    et al.
    Department of Microbiology, Harvard Medical School, Blavatnik Institute, Boston, Massachusetts, USA.
    Bisset, Sean
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Shlosman, Irina
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Blavatnik Institute, Boston, Massachusetts, USA.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Rudner, David Z.
    Department of Microbiology, Harvard Medical School, Blavatnik Institute, Boston, Massachusetts, USA.
    Bernhardt, Thomas G.
    Department of Microbiology, Harvard Medical School, Blavatnik Institute, Boston, Massachusetts, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
    MacP bypass variants of Streptococcus pneumoniae PBP2a suggest a conserved mechanism for the activation of bifunctional cell wall synthases2023In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 14, no 6, article id e0239023Article in journal (Refereed)
    Abstract [en]

    The peptidoglycan (PG) layer protects bacteria from osmotic lysis and defines their shape. The class A penicillin-binding proteins (aPBPs) are PG synthases that possess both glycan polymerization and crosslinking activities needed for PG biogenesis. In Gram-negative bacteria, aPBPs require activation by outer membrane lipoproteins, which are thought to stimulate their cognate synthase by inducing conformational changes that promote polymerase function. How aPBPs are controlled in Gram-positive bacteria is less clear. One of the few known regulators is MacP in Streptococcus pneumoniae (Sp). MacP is required for the activity of Sp PBP2a, but its mode of action has been obscure. We therefore selected for PBP2a variants capable of functioning in the absence of MacP. Amino acid substitutions that bypassed the MacP requirement for PBP2a function in vivo also activated its polymerase activity in vitro. Many of these changes mapped to the interface between the transmembrane (TM) helix and polymerase domain in a model PBP2a structure. This region is conformationally flexible in the experimentally determined structures of aPBPs and undergoes a structural transition upon binding the substrate-mimicking drug moenomycin. Our findings suggest that MacP promotes PG polymerization by altering the TM-polymerase domain interface in PBP2a and that this mechanism for aPBP activation may be broadly conserved. Furthermore, Sp cells expressing an activated PBP2a variant displayed heterogeneous shapes, highlighting the importance of proper aPBP regulation in cell morphogenesis.

    Importance: Class A penicillin-binding proteins (aPBPs) play critical roles in bacterial cell wall biogenesis. As the targets of penicillin, they are among the most important drug targets in history. Although the biochemical activities of these enzymes have been well studied, little is known about how they are regulated in cells to control when and where peptidoglycan is made. In this report, we isolate variants of the Streptococcus pneumoniae enzyme PBP2a that function in cells without MacP, a partner normally required for its activity. The amino acid substitutions activate the cell wall synthase activity of PBP2a, and their location in a model structure suggests an activation mechanism for this enzyme that is shared with aPBPs from distantly related organisms with distinct activators.

    Download full text (pdf)
    fulltext
  • 23. Murphy, Shannon G.
    et al.
    Alvarez, Laura
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Adams, Myfanwy C.
    Liu, Shuning
    Chappie, Joshua S.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Dörr, Tobias
    Endopeptidase Regulation as a Novel Function of the Zur-Dependent Zinc Starvation Response2019In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 1, article id e02620-18Article in journal (Refereed)
    Abstract [en]

    The cell wall is a strong, yet flexible, meshwork of peptidoglycan (PG) that gives a bacterium structural integrity. To accommodate a growing cell, the wall is remodeled by both PG synthesis and degradation. Vibrio cholerae encodes a group of three nearly identical zinc-dependent endopeptidases (EPs) that are predicted to hydrolyze PG to facilitate cell growth. Two of these (ShyA and ShyC) are conditionally essential housekeeping EPs, while the third (ShyB) is not expressed under standard laboratory conditions. To investigate the role of ShyB, we conducted a transposon screen to identify mutations that activate shyB transcription. We found that shyB is induced as part of the Zur-mediated zinc starvation response, a mode of regulation not previously reported for cell wall lytic enzymes. In vivo, ShyB alone was sufficient to sustain cell growth in low-zinc environments. In vitro, ShyB retained its D, D-endopeptidase activity against purified sacculi in the presence of the metal chelator EDTA at concentrations that inhibit ShyA and ShyC. This insensitivity to metal chelation is likely what enables ShyB to substitute for other EPs during zinc starvation. Our survey of transcriptomic data from diverse bacteria identified other candidate Zur-regulated EPs, suggesting that this adaptation to zinc starvation is employed by other Gram-negative bacteria. IMPORTANCE Bacteria encode a variety of adaptations that enable them to survive during zinc starvation, a condition which is encountered both in natural environments and inside the human host. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, we have identified a novel member of this zinc starvation response, a cell wall hydrolase that retains function and is conditionally essential for cell growth in low-zinc environments. Other Gram-negative bacteria contain homologs that appear to be under similar regulatory control. These findings are significant because they represent, to our knowledge, the first evidence that zinc homeostasis influences cell wall turnover. Anti-infective therapies commonly target the bacterial cell wall; therefore, an improved understanding of how the cell wall adapts to host-induced zinc starvation could lead to new antibiotic development. Such therapeutic interventions are required to combat the rising threat of drug-resistant infections.

    Download full text (pdf)
    fulltext
  • 24.
    Murphy, Shannon G.
    et al.
    Weill Institute for Cell and Molecular Biology, Cornell University, NY, Ithaca, United States; Department of Microbiology, Cornell University, NY, Ithaca, United States.
    Murtha, Andrew N.
    Weill Institute for Cell and Molecular Biology, Cornell University, NY, Ithaca, United States; Department of Microbiology, Cornell University, NY, Ithaca, United States.
    Zhao, Ziyi
    Weill Institute for Cell and Molecular Biology, Cornell University, NY, Ithaca, United States.
    Alvarez, Laura
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Diebold, Peter
    Department of Microbiology, Cornell University, NY, Ithaca, United States.
    Shin, Jung-Ho
    Weill Institute for Cell and Molecular Biology, Cornell University, NY, Ithaca, United States.
    Vannieuwenhze, Michael S.
    Department of Molecular and Cellular Biochemistry and Department of Biology, Indiana University, IN, Bloomington, United States.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Dörr, Tobias
    Weill Institute for Cell and Molecular Biology, Cornell University, NY, Ithaca, United States; Department of Microbiology, Cornell University, NY, Ithaca, United States; Cornell Institute of Host-Microbe Interactions and Disease, Cornell University, NY, Ithaca, United States.
    Class a penicillin-binding protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency in vibrio cholerae2021In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 12, no 2, article id e03596-20Article in journal (Refereed)
    Abstract [en]

    The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth, and structural integrity. PG is synthesized by class A/B penicillin-binding proteins (a/bPBPs) and shape, elongation, di-vision, and sporulation (SEDS) proteins like RodA (as part of the Rod system cell elongation machinery) and degraded by "autolytic" enzymes to accommodate growth processes. It is thought that autolysins (particularly endopeptidases [EPs]) are required for PG synthesis and incorporation by creating gaps that are patched and paved by PG syn-thases, but the exact relationship between autolysins and PG synthesis remains incom-pletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae. We found that EP depletion resulted in severe morphological and division defects, but these cells continued to increase in mass and aberrantly incorporated new cell wall material. Mass increase proceeded in the presence of Rod system inhibitors, but cells lysed upon inhibition of aPBPs, suggesting that aPBPs are required for structural integrity under these conditions. The Rod system, although not essential for the observed mass increase, remained functional even after prolonged EP depletion. Last, heterologous expression of an EP from Neisseria gonorrhoeae fully complemented growth and morphology of an EP-insufficient V. cholerae, highlighting the possibility that the PG synthases may not necessarily function via direct interaction with EPs. Overall, our findings suggest that during EP insufficiency in V. cholerae, aPBPs become essential for structural integrity while the Rod system is unable to promote proper cell expansion.

    IMPORTANCE: Synthesis and turnover of the bacterial cell wall must be tightly coordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines, the aPBPs and the Rod system. Our results suggest that in Vibrio cholerae, one class of turnover enzymes, the endopeptidases, are necessary for proper cell elongation and division. aPBPs become essential for maintaining structural integrity during EP insufficiency, while the Rod system remains active but contributes little to cell expansion under these conditions. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the coordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes.

    Download full text (pdf)
    fulltext
  • 25. Nielsen, Stine Vang
    et al.
    Turnbull, Kathryn Jane
    Centre for Bacterial Stress Response and Persistence, Section for Functional Genomics, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
    Roghanian, Mohammad
    Centre for Bacterial Stress Response and Persistence, Section for Functional Genomics, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
    Baerentsen, Rene
    Semanjski, Maja
    Brodersen, Ditlev E.
    Macek, Boris
    Gerdes, Kenn
    Serine-Threonine Kinases Encoded by Split hipA Homologs Inhibit Tryptophanyl-tRNA Synthetase2019In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 3, article id e01138-19Article in journal (Refereed)
    Abstract [en]

    Type II toxin-antitoxin (TA) modules encode a stable toxin that inhibits cell growth and an unstable protein antitoxin that neutralizes the toxin by direct protein-protein contact. hipBA of Escherichia coli strain K-12 codes for HipA, a serinethreonine kinase that phosphorylates and inhibits glutamyl-tRNA synthetase. Induction of hipA inhibits charging of glutamyl-tRNA that, in turn, inhibits translation and induces RelA-dependent (p) ppGpp synthesis and multidrug tolerance. Here, we describe the discovery of a three-component TA gene family that encodes toxin HipT, which exhibits sequence similarity with the C-terminal part of HipA. A genetic screening revealed that trpS in high copy numbers suppresses HipT-mediated growth inhibition. We show that HipT of E. coli O127 is a kinase that phosphorylates tryptophanyl-tRNA synthetase in vitro at a conserved serine residue. Consistently, induction of hipT inhibits cell growth and stimulates production of (p) ppGpp. The gene immediately upstream from hipT, called hipS, encodes a small protein that exhibits sequence similarity with the N terminus of HipA. HipT kinase was neutralized by cognate HipS in vivo, whereas the third component, HipB, encoded by the first gene of the operon, did not counteract HipT kinase activity. However, HipB augmented the ability of HipS to neutralize HipT. Analysis of two additional hipBSThomologous modules showed that, indeed, HipS functions as an antitoxin in these cases also. Thus, hipBST constitutes a novel family of tricomponent TA modules where hipA has been split into two genes, hipS and hipT, that function as a novel type of TA pair.

    IMPORTANCE: Bacterial toxin-antitoxin (TA) modules confer multidrug tolerance (persistence) that may contribute to the recalcitrance of chronic and recurrent infections. The first high-persister gene identified was hipA of Escherichia coli strain K-12, which encodes a kinase that inhibits glutamyl-tRNA synthetase. The hipA gene encodes the toxin of the hipBA TA module, while hipB encodes an antitoxin that counteracts HipA. Here, we describe a novel, widespread TA gene family, hipBST, that encodes HipT, which exhibits sequence similarity with the C terminus of HipA. HipT is a kinase that phosphorylates tryptophanyl-tRNA synthetase and thereby inhibits translation and induces the stringent response. Thus, this new TA gene family may contribute to the survival and spread of bacterial pathogens.

    Download full text (pdf)
    fulltext
  • 26.
    Okumura, Cheryl
    et al.
    University of California.
    Anderson, Ericka L
    University of California.
    Döhrmann, Simon
    Tran, Dan N
    University of California.
    Olson, Joshua
    University of California.
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Nizet, Victor
    University of California.
    IgG protease Mac/IdeS is not essential for phagocyte resistance or mouse virulence of M1T1 group A Streptococcus2013In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 4, no 4, p. e00499-e00513Article in journal (Refereed)
    Abstract [en]

    The Mac/IdeS protein of group A Streptococcus (GAS) is a secreted cysteine protease with cleavage specificity for IgG and is highly expressed in the GAS serotype M1T1 clone, which is the serotype most frequently isolated from patients with life-threatening invasive infections. While studies of Mac/IdeS with recombinant protein have shown that the protein can potentially prevent opsonophagocytosis of GAS by neutrophils, the role of the protein in immune evasion as physiologically produced by the living organism has not been studied. Here we examined the contribution of Mac/IdeS to invasive GAS disease by generating a mutant lacking Mac/IdeS in the hyperinvasive M1T1 background. While Mac/IdeS was highly expressed and proteolytically active in the hyperinvasive strain, elimination of the bacterial protease did not significantly influence GAS phagocytic uptake, oxidative-burst induction, cathelicidin sensitivity, resistance to neutrophil or macrophage killing, or pathogenicity in pre- or postimmune mouse infectious challenges. We conclude that in the highly virulent M1T1 background, Mac/IdeS is not essential for either phagocyte resistance or virulence. Given the conservation of Mac/IdeS and homologues across GAS strains, it is possible that Mac/IdeS serves another important function in GAS ecology or contributes to virulence in other strain backgrounds.

    IMPORTANCE Group A Streptococcus (GAS) causes human infections ranging from strep throat to life-threatening conditions such as flesh-eating disease and toxic shock syndrome. Common disease-associated clones of GAS can cause both mild and severe infections because of a characteristic mutation and subsequent change in the expression of several genes that develops under host immune selection. One of these genes encodes Mac/IdeS, a protease that has been shown to cleave antibodies important to the immune defense system. In this study, we found that while Mac/IdeS is highly expressed in hypervirulent GAS, it does not significantly contribute to the ability of the bacteria to survive white blood cell killing or produce invasive infection in the mouse. These data underscore the importance of correlating studies on virulence factor function with physiologic expression levels and the complexity of streptococcal pathogenesis and contribute to our overall understanding of how GAS causes disease.

    Download full text (pdf)
    fulltext
  • 27.
    Olofsson, Annelie
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nygård Skalman, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Uptake of Helicobacter pylori vesicles is facilitated by clathrin-dependent and clathrin-independent endocytic pathways2014In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 5, no 3, p. e00979-14-Article in journal (Refereed)
    Abstract [en]

    UNLABELLED: Bacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world's population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake.

    IMPORTANCE: Bacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake of Helicobacter pylori vesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs the H. pylori vesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

    Download full text (pdf)
    fulltext
  • 28. Reithuber, Elisabeth
    et al.
    Nannapaneni, Priyanka
    Rzhepishevska, Olena I
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindgren, Anders E. G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ilchenko, Oleksandr
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Microbiology, Virology and Biotechnology, Odessa II Mechnikov National University, Odessa, Ukraine.
    Normark, Staffan
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Henriques-Normark, Birgitta
    Mellroth, Peter
    The Bactericidal Fatty Acid Mimetic 2CCA-1 Selectively Targets Pneumococcal Extracellular Polyunsaturated Fatty Acid Metabolism2020In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 11, no 6, article id e03027-20Article in journal (Refereed)
    Abstract [en]

    Streptococcus pneumoniae, a major cause of pneumonia, sepsis, and meningitis worldwide, has the nasopharynges of small children as its main ecological niche. Depletion of pneumococci from this niche would reduce the disease burden and could be achieved using small molecules with narrow-spectrum antibacterial activity. We identified the alkylated dicyclohexyl carboxylic acid 2CCA-1 as a potent inducer of autolysin-mediated lysis of S. pneumoniae, while having low activity against Staphylococcus aureus. 2CCA-1-resistant strains were found to have inactivating mutations in fakB3, known to be required for uptake of host polyunsaturated fatty acids, as well as through inactivation of the transcriptional regulator gene fabT, vital for endogenous, de novo fatty acid synthesis regulation. Structure activity relationship exploration revealed that, besides the central dicyclohexyl group, the fatty acid-like structural features of 2CCA-1 were essential for its activity. The lysis-inducing activity of 2CCA-1 was considerably more potent than that of free fatty acids and required growing bacteria, suggesting that 2CCA-1 needs to be metabolized to exert its antimicrobial activity. Total lipid analysis of 2CCA-1 treated bacteria identified unique masses that were modeled to 2CCA-1 containing lysophosphatidic and phosphatidic acid in wild-type but not in fakB3 mutant bacteria. This suggests that 2CCA-1 is metabolized as a fatty acid via FakB3 and utilized as a phospholipid building block, leading to accumulation of toxic phospholipid species. Analysis of FabT-mediated fakB3 expression elucidates how the pneumococcus could ensure membrane homeostasis and concurrent economic use of host-derived fatty acids.IMPORTANCE Fatty acid biosynthesis is an attractive antibiotic target, as it affects the supply of membrane phospholipid building blocks. In Streptococcus pneumoniae, it is not sufficient to target only the endogenous fatty acid synthesis machinery, as uptake of host fatty acids may bypass this inhibition. Here, we describe a small-molecule compound, 2CCA-1, with potent bactericidal activity that upon interactions with the fatty acid binding protein FakB3, which is present in a limited number of Gram-positive species, becomes metabolized and incorporated as a toxic phospholipid species. Resistance to 2CCA-1 developed specifically in fakB3 and the regulatory gene fabT. These mutants reveal a regulatory connection between the extracellular polyunsaturated fatty acid metabolism and endogenous fatty acid synthesis in S. pneumoniae, which could ensure balance between efficient scavenging of host polyunsaturated fatty acids and membrane homeostasis. The data might be useful in the identification of narrow-spectrum treatment strategies to selectively target members of the Lactobacillales such as S. pneumoniae.

    Download full text (pdf)
    fulltext
  • 29.
    Resch, Ulrike
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Vascular Biology and Thrombosis Research, Medical University Vienna, Vienna, Austria.
    Tsatsaronis, James Anthony
    Le Rhun, Anais
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Stuebiger, Gerald
    Rohde, Manfred
    Kasvandik, Sergo
    Holzmeister, Susanne
    Tinnefeld, Philip
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Hannover Medical School, Hannover, Germany.
    A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 6, article id e00207-16Article in journal (Refereed)
    Abstract [en]

    Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to "anchorless surface proteins." Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response. IMPORTANCE Group A streptococcus (GAS) is a Gram-positive bacterial pathogen responsible for more than 500,000 deaths annually. Establishment of GAS infection is dependent on a suite of proteins exported via the general secretory pathway. Here, we show that GAS naturally produces extracellular vesicles with a unique lipid composition that are laden with proteins and RNAs. Interestingly, both virulence-associated proteins and RNA species were found to be differentially abundant in vesicles relative to the bacteria. Furthermore, we show that genetic disruption of the virulence-associated two-component regulator CovRS leads to an increase in vesicle production. This study comprehensively describes the protein, RNA, and lipid composition of GAS-secreted MVs and alludes to a regulatory system impacting this process.

    Download full text (pdf)
    fulltext
  • 30.
    Rosendal, Ebba
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Mihai, Ionut Sebastian
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). National Clinical Research School in Chronic Inflammatory Diseases (NCRSCID), Karolinska Institutet, Solna, Sweden.
    Becker, Miriam
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between The Medical School Hannover, The Helmholtz Centre for Infection Research, Hannover, Germany; Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Das, Debojyoti
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Frängsmyr, Lars
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Persson, B. David
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Swedish National Veterinary Institute (SVA), Uppsala, Sweden.
    Rankin, Gregory
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Section of Medicine. Swedish Defence Research Agency, CBRN Defence and Security, Umeå, Sweden.
    Gröning, Remigius
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Sartorius Corporate Research, Umeå, Sweden.
    Forsell, Mattias
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Ankarklev, Johan
    Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden; Microbial Single Cell Genomics Facility, SciLifeLab, Biomedical Center (BMC) Uppsala University, Uppsala, Sweden.
    Blomberg, Anders
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Section of Medicine.
    Henriksson, Johan
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Överby, Anna K.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lenman, Annasara
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Serine protease inhibitors restrict host susceptibility to SARS-CoV-2 infections2022In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 13, no 3, article id e00892-22Article in journal (Refereed)
    Abstract [en]

    The coronavirus disease 2019, COVID-19, is a complex disease with a wide range of symptoms from asymptomatic infections to severe acute respiratory syndrome with lethal outcome. Individual factors such as age, sex, and comorbidities increase the risk for severe infections, but other aspects, such as genetic variations, are also likely to affect the susceptibility to SARS-CoV-2 infection and disease severity. Here, we used a human 3D lung cell model based on primary cells derived from multiple donors to identity host factors that regulate SARS-CoV-2 infection. With a transcriptomics-based approach, we found that less susceptible donors show a higher expression level of serine protease inhibitors SERPINA1, SERPINE1, and SERPINE2, identifying variation in cellular serpin levels as restricting host factors for SARS-CoV-2 infection. We pinpoint their antiviral mechanism of action to inhibition of the cellular serine protease, TMPRSS2, thereby preventing cleavage of the viral spike protein and TMPRSS2-mediated entry into the target cells. By means of single-cell RNA sequencing, we further locate the expression of the individual serpins to basal, ciliated, club, and goblet cells. Our results add to the importance of genetic variations as determinants for SARS-CoV-2 susceptibility and suggest that genetic deficiencies of cellular serpins might represent risk factors for severe COVID-19. Our study further highlights TMPRSS2 as a promising target for antiviral intervention and opens the door for the usage of locally administered serpins as a treatment against COVID-19.

    Download full text (pdf)
    fulltext
  • 31. Shaffer, Carrie L.
    et al.
    Good, James A. D.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Kumar, Santosh
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Krishnan, K. Syam
    Gaddy, Jennifer A.
    Loh, John T.
    Chappell, Joseph
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Cover, Timothy L.
    Hadjifrangiskou, Maria
    Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 2, article id e00221-16Article in journal (Refereed)
    Abstract [en]

    Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. IMPORTANCE Many human and plant pathogens utilize complex nanomachines called type IV secretion systems (T4SSs) to transport proteins and DNA to target cells. In addition to delivery of harmful effector proteins into target cells, T4SSs can disseminate genetic determinants that confer antibiotic resistance among bacterial populations. In this study, we sought to identify compounds that disrupt T4SS-mediated processes. Using the human gastric pathogen H. pylori as a model system, we identified and characterized two small molecules that prevent transfer of an oncogenic effector protein to host cells. We discovered that these small molecules also prevented the spread of antibiotic resistance plasmids in E. coli populations and diminished the transfer of tumor-inducing DNA from the plant pathogen A. tumefaciens to target cells. Thus, these compounds are versatile molecular tools that can be used to study and disarm these important bacterial machines.

    Download full text (pdf)
    fulltext
  • 32.
    Simpson, Brent W.
    et al.
    Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, GA, Athens, United States.
    Nieckarz, Marta
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Pinedo, Victor
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    McLean, Amanda B.
    Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, GA, Athens, United States.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Trent, M. Stephen
    Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, GA, Athens, United States; Department of Microbiology, College of Art and Sciences, University of Georgia, GA, Athens, United States.
    Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis2021In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 12, no 6, article id e03099-21Article in journal (Refereed)
    Abstract [en]

    Gram-negative bacteria resist external stresses due to cell envelope rigidity, which is provided by two membranes and a peptidoglycan layer. The outer membrane (OM) surface contains lipopolysaccharide (LPS; contains O-antigen) or lipooligosaccharide (LOS). LPS/LOS are essential in most Gram-negative bacteria and may contribute to cellular rigidity. Acinetobacter baumannii is a useful tool for testing these hypotheses as it can survive without LOS. Previously, our group found that strains with naturally high levels of penicillin binding protein 1A (PBP1A) could not become LOS deficient unless the gene encoding it was deleted, highlighting the relevance of peptidoglycan biosynthesis and suggesting that high PBP1A levels were toxic during LOS deficiency. Transposon sequencing and follow-up analysis found that axial peptidoglycan synthesis by the elongasome and a peptidoglycan recycling enzyme, ElsL, were vital in LOS-deficient cells. The toxicity of high PBP1A levels during LOS deficiency was clarified to be due to a negative impact on elongasome function. Our data suggest that during LOS deficiency, the strength of the peptidoglycan specifically imparted by elongasome synthesis becomes essential, supporting that the OM and peptidoglycan contribute to cell rigidity. IMPORTANCE Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength. Recently, the most outer membrane of the cell was also proposed to contribute to rigidity due to properties of a unique molecule called lipopolysaccharide (LPS). LPS is located on the cell surface in the outer membrane and is typically required for growth. By using Acinetobacter baumannii, a Gram-negative bacterium that can grow without LPS, we found that key features of the peptidoglycan structure also become essential. This finding supports that both the outer membrane and peptidoglycan contribute to cell rigidity.

    Download full text (pdf)
    fulltext
  • 33.
    Springer Engdahl, Cecilia
    et al.
    W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.
    Caragata, Eric P.
    W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA; Florida Medical Entomology Laboratory, Department of Entomology & Nematology, Institute of Food & Agricultural Sciences, University of Florida, Vero Beach, Florida, USA.
    Tavadia, Mihra
    W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.
    Dimopoulos, George
    W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.
    Chromobacterium biopesticide exposure does not select for resistance in Aedes mosquitoes2023In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 14, no 2, article id e00480-23Article in journal (Refereed)
    Abstract [en]

    Developing effective tools to control mosquito populations is essential for reducing the incidence of diseases like malaria and dengue. Biopesticides of microbial origin are a rich, underexplored source of mosquitocidal compounds. We previously developed a biopesticide from the bacterium Chromobacterium sp. Panama that rapidly kills vector mosquito larvae, including Aedes aegypti and Anopheles gambiae. Here, we demonstrate that two independent Ae. aegypti colonies exposed to a sublethal dose of that biopesticide over consecutive generations persistently exhibited high mortality and developmental delays, indicating that resistance did not develop during the study period. Critically, the descendants of biopesticide-exposed mosquitoes experienced decreased longevity and did not display increased susceptibility to dengue virus or decreased susceptibility to common chemical insecticides. Through RNA sequencing, we observed no link between biopesticide exposure and the increased activity of xenobiotic metabolism and detoxification genes typically associated with insecticide resistance. These findings indicate that the Chromobacterium biopesticide is an exciting, emerging mosquito control tool.

    IMPORTANCE: Vector control is an essential part of mitigating diseases caused by pathogens that mosquitoes spread. Modern vector control is highly reliant on using synthetic insecticides to eliminate mosquito populations before they can cause disease. However, many of these populations have become resistant to commonly used insecticides. There is a strong need to explore alternative vector control strategies that aim to mitigate disease burden. Biopesticides, insecticides of biological origin, can have unique mosquitocidal activities, meaning they can effectively kill mosquitoes that are already resistant to other insecticides. We previously developed a highly effective mosquito biopesticide from the bacterium Chromobacterium sp. Csp_P. Here, we investigate whether exposure to a sublethal dose of this Csp_P biopesticide over 9 to 10 generations causes resistance to arise in Aedes aegypti mosquitoes. We find no evidence of resistance at the physiological or molecular levels, confirming that the Csp_P biopesticide is a highly promising new tool for controlling mosquito populations.

    Download full text (pdf)
    fulltext
  • 34.
    Tükenmez, Hasan
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). QureTech Bio, Umeå, Sweden.
    Singh, Pardeep
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sarkar, Souvik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Çakır, Melike
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Oliveira, Ana H.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lindgren, Cecilia
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vaitkevicius, Karolis
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bonde, Mari
    QureTech Bio, Umeå, Sweden.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    A highly substituted ring-fused 2-pyridone compound targeting PrfA and the efflux regulator BrtA in listeria monocytogenes2023In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 14, no 3, article id e0044923Article in journal (Refereed)
    Abstract [en]

    Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents.

    IMPORTANCE: Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed. In this work, we show that a new generation of substituted ring-fused 2-pyridones not only inhibit Listeria monocytogenes virulence gene expression, presumably by inactivating the PrfA virulence regulator, but also potentiate the bactericidal effects of cholic acid and different osmolytes. We identified a multidrug repressor as a second target of 2-pyridones. The repressor–2-pyridone interaction displaces the repressor from DNA, thus increasing the expression of a multidrug transporter. In addition, our data suggest that the new class of ring-fused 2-pyridones are efficient efflux inhibitors, possibly explaining why the simultaneous addition of 2-pyridones together with cholic acid or osmolytes is detrimental for the bacterium. This work proves conclusively that 2-pyridones constitute a promising scaffold to build on for future antibacterial drug design.

    Download full text (pdf)
    fulltext
  • 35. von Schaewen, Markus
    et al.
    Dorner, Marcus
    Hueging, Kathrin
    Foquet, Lander
    Gerges, Sherif
    Hrebikova, Gabriela
    Heller, Brigitte
    Bitzegeio, Julia
    Doerrbecker, Juliane
    Horwitz, Joshua A
    Gerold, Gisa
    Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany.
    Suerbaum, Sebastian
    Rice, Charles M
    Meuleman, Philip
    Pietschmann, Thomas
    Ploss, Alexander
    Expanding the Host Range of Hepatitis C Virus through Viral Adaptation2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 6, article id e01915-16Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) species tropism is incompletely understood. We have previously shown that at the level of entry, human CD81 and occludin (OCLN) comprise the minimal set of human factors needed for viral uptake into murine cells. As an alternative approach to genetic humanization, species barriers can be overcome by adapting HCV to use the murine orthologues of these entry factors. We previously generated a murine tropic HCV (mtHCV or Jc1/mCD81) strain harboring three mutations within the viral envelope proteins that allowed productive entry into mouse cell lines. In this study, we aimed to characterize the ability of mtHCV to enter and infect mouse hepatocytes in vivo and in vitro Using a highly sensitive, Cre-activatable reporter, we demonstrate that mtHCV can enter mouse hepatocytes in vivo in the absence of any human cofactors. Viral entry still relied on expression of mouse CD81 and SCARB1 and was more efficient when mouse CD81 and OCLN were overexpressed. HCV entry could be significantly reduced in the presence of anti-HCV E2 specific antibodies, suggesting that uptake of mtHCV is dependent on viral glycoproteins. Despite mtHCV's ability to enter murine hepatocytes in vivo, we did not observe persistent infection, even in animals with severely blunted type I and III interferon signaling and impaired adaptive immune responses. Altogether, these results establish proof of concept that the barriers limiting HCV species tropism can be overcome by viral adaptation. However, additional viral adaptations will likely be needed to increase the robustness of a murine model system for hepatitis C.

    IMPORTANCE: At least 150 million individuals are chronically infected with HCV and are at risk of developing serious liver disease. Despite the advent of effective antiviral therapy, the frequency of chronic carriers has only marginally decreased. A major roadblock in developing a vaccine that would prevent transmission is the scarcity of animal models that are susceptible to HCV infection. It is poorly understood why HCV infects only humans and chimpanzees. To develop an animal model for hepatitis C, previous efforts focused on modifying the host environment of mice, for example, to render them more susceptible to HCV infection. Here, we attempted a complementary approach in which a laboratory-derived HCV variant was tested for its ability to infect mice. We demonstrate that this engineered HCV strain can enter mouse liver cells but does not replicate efficiently. Thus, additional adaptations are likely needed to construct a robust animal model for HCV.

  • 36.
    Warsi, Omar M.
    et al.
    Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
    Upterworth, Lina M.
    Department of Evolutionary Ecology and Genetics, Zoological Institute, Kiel University, Kiel, Germany.
    Breidenstein, Annika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lustig, Ulrika
    Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
    Mikkelsen, Kasper
    Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Denmark.
    Nagy, Tamás
    Department of Laboratory Medicine, Medical School, University of Pécs, Pécs, Hungary.
    Szatmari, Dávid
    Department of Biophysics, Medical School, University of Pécs, Pécs, Hungary.
    Ingmer, Hanne
    Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Denmark.
    Andersson, Dan I.
    Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
    Staphylococcus aureus mutants resistant to the feed-additive monensin show increased virulence and altered purine metabolism2024In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 15, no 2, article id e0315523Article in journal (Refereed)
    Abstract [en]

    Ionophores are antibacterial compounds that affect bacterial growth by changing intracellular concentrations of the essential cations, sodium and potassium. They are extensively used in animal husbandry to increase productivity and reduce infectious diseases, but our understanding of the potential for and effects of resistance development to ionophores is poorly known. Thus, given their widespread global usage, it is important to determine the potential negative consequences of ionophore use on human and animal health. In this study, we demonstrate that exposure to the ionophore monensin can select for resistant mutants in the human and animal pathogen Staphylococcus aureus, with a majority of the resistant mutants showing increased growth rates in vitro and/or in mice. Whole-genome sequencing and proteomic analysis of the resistant mutants show that the resistance phenotype is associated with de-repression of de novo purine synthesis, which could be achieved through mutations in different transcriptional regulators including mutations in the gene purR, the repressor of the purine de novo synthesis pathway. This study shows that mutants with reduced susceptibility to the ionophore monensin can be readily selected and highlights an unexplored link between ionophore resistance, purine metabolism, and fitness in pathogenic bacteria.IMPORTANCEThis study demonstrates a novel link between ionophore resistance, purine metabolism, and virulence/fitness in the key human and animal pathogen Staphylococcus aureus. The results show that mutants with reduced susceptibility to the commonly used ionophore monensin can be readily selected and that the reduced susceptibility observed is associated with an increased expression of the de novo purine synthesis pathway. This study increases our understanding of the impact of the use of animal feed additives on both human and veterinary medicine.

    Download full text (pdf)
    fulltext
  • 37.
    Williams, Michelle A.
    et al.
    Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA.
    Aliashkevich, Alena
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Krol, Elizaveta
    Center for Synthetic Microbiology (SYNMIKRO), Philipps University Marburg, Marburg, Germany; Department of Biology, Philipps University Marburg, Marburg, Germany.
    Kuru, Erkin
    Department of Chemistry, Indiana University, IN, Bloomington, United States.
    Bouchier, Jacob M.
    Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA.
    Rittichier, Jonathan
    Department of Chemistry, Indiana University, IN, Bloomington, United States.
    Brun, Yves V.
    Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine ,Université de Montréal, QC, Montreal, Canada.
    VanNieuwenhze, Michael S.
    Department of Chemistry, Indiana University, IN, Bloomington, United States.
    Becker, Anke
    Center for Synthetic Microbiology (SYNMIKRO), Philipps University Marburg, Marburg, Germany; Department of Biology, Philipps University Marburg, Marburg, Germany.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Brown, Pamela J. B.
    Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA.
    Unipolar Peptidoglycan Synthesis in the Rhizobiales Requires an Essential Class A Penicillin-Binding Protein2021In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 12, no 5, article id e0234621Article in journal (Refereed)
    Abstract [en]

    Members of the Rhizobiales are polarly growing bacteria that lack homologs of the canonical Rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant pathogen Agrobacterium tumefaciens. The development of fluorescent d-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an additional mode of cell wall synthesis that requires ld-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation.

    IMPORTANCE

    While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation. Yet the vast majority of our understanding regarding bacterial elongation is derived from model organisms exhibiting lateral elongation. Here, we explore the role of penicillin-binding proteins in unipolar elongation of Agrobacterium tumefaciens and related bacteria within the Rhizobiales. Our findings suggest that penicillin-binding protein 1a, along with a subset of ld-transpeptidases, drives unipolar growth. Thus, these enzymes may serve as attractive targets for biocontrol of pathogenic Rhizobiales.

    Download full text (pdf)
    fulltext
  • 38.
    Zwack, Erin
    et al.
    University of Pennsylvania, USA.
    Snyder, Annelise
    University of Pennsylvania, USA.
    Wynosky-Dolfi, Meghan
    University of Pennsylvania, USA.
    Ruthel, Gordon
    University of Pennsylvania, USA.
    Philip, Naomi
    University of Pennsylvania, USA.
    Marketon, Melanie
    Indiana University, USA.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bliska, James
    SUNY Stony Brook, USA.
    Brodsky, Igor
    University of Pennsylvania, USA.
    Inflammasome activation in response to the Yersinia type III secretion system requires hyperinjection of translocon proteins YopB and YopD2015In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 6, no 1, article id e02095-14Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SS) translocate effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. However, recognition of T3SS activity by cytosolic pattern recognition receptors (PRRs) of the nucleotide-binding domain leucine rich repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Macrophages infected with Yersinia pseudotuberculosis strains lacking all known effectors or lacking the translocation regulator YopK induce rapid activation of both the canonical NLRP3 and noncanonical caspase-11 inflammasomes. While this inflammasome activation requires a functional T3SS, the precise signal that triggers inflammasome activation in response to Yersinia T3SS activity remains unclear. Effectorless strains of Yersinia as well as ΔyopK strains translocate elevated levels of T3SS substrates into infected cells. To dissect the contribution of pore formation and translocation to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions of the T3SS. Notably, YopD variants that abrogated translocation but not pore-forming activity failed to induce inflammasome activation. Furthermore, analysis of individual infected cells revealed that inflammasome activation at the single-cell level correlated with translocated levels of YopB and YopD themselves. Intriguingly, LcrH mutants that are fully competent for effector translocation but produce and translocate lower levels of YopB and YopD also fail to trigger inflammasome activation. Our findings therefore suggest that hypertranslocation of YopD and YopB is linked to inflammasome activation in response to the Yersinia T3SS.

    Download full text (pdf)
    fulltext
1 - 38 of 38
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf