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  • 301.
    Loh, Edmund
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Memarpour, Faranak
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sondén, Berit
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    The first 20 codons of the prfA-mRNA are required for efficient translation in Listeria monocytogenesManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Expression of virulence factors in the human pathogen Listeria monocytogenes is almost exclusively regulated by the transcriptional activator PrfA. The translation of prfA is controlled by a thermosensor located in the 5´-untranslated RNA (UTR), which is high at 37°C and low at temperatures below 30°C. Also, translation of the prfA transcript is inhibited by a trans-acting riboswitch RNA, SreA, which binds to the 5´-end of the thermosensor. In order to develop a thermoregulated translational expression system in Mycobacterial species, the 5´-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the constructs retained their thermoregulation. However, the β-galactosidase expression was directly correlated to the length of the prfA-coding mRNA fused in front of lacZ. A similar regulation was also detected when gfp was used as a reporter gene. Transcriptional stability experiments indicated that the observed difference in expression was not due to a decreased stability of transcripts lacking more of the prfA-coding RNA. The gfp constructs behaved similarly in L. monocytogenes as in E. coli, emphasizing the requirement of the prfA-coding RNA for maximal expression, also in its natural genetic background. Moreover, the different PrfA-LacZ fusion proteins showed the same proteolytic stability, ruling out post-translational mechanisms. Instead, in vitro transcription/translation experiments suggest a role of the first 20 codons of the native prfA-mRNA for maximal expression. Our data indicated that the difference in expression was not due to rare codons, stretches of certain bases or a putative downstream box. We observed an inverse correlation between the stability of the RNA secondary structure and protein expression. The first 12 codons of prfA displayed a very weak RNA secondary structure. Similar weak stabilities were detected also for thermosensors in other species, indicating a common strategy of regulation. In summary, the present work determines the importance of an unstructured 5´-coding region of the prfA-RNA for efficient translation.

  • 302.
    Loh, Edmund
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Memarpour, Faranak
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Vaitkevicius, Karolis
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kallipolitis, Birgitte H.
    Univ So Denmark, Dept Biochem & Mol Biol, DK-5230 Odense M, Denmark.
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sonden, Berit
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    An unstructured 5'-coding region of the prfA mRNA is required for efficient translation2012Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 4, s. 1818-1827Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Expression of virulence factors in the human bacterial pathogen Listeria monocytogenes is almost exclusively regulated by the transcriptional activator PrfA. The translation of prfA is controlled by a thermosensor located in the 5'-untranslated RNA (UTR), and is high at 37 degrees C and low at temperatures < 30 degrees C. In order to develop a thermoregulated translational expression system, the 5'-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the beta-galactosidase expression was directly correlated to the length of the prfA-coding mRNA lying in front of lacZ. A similar effect was detected with gfp as a reporter gene in both L. monocytogenes and E. coli, emphasizing the requirement of the prfA-coding RNA for maximal expression. In vitro transcription/translation and mutational analysis suggests a role for the first 20 codons of the native prfA-mRNA for maximal expression. By toe-print and RNA-probing analysis, a flexible hairpin-loop located immediately downstream of the start-codon was shown to be important for ribosomal binding. The present work determines the importance of an unstructured part of the 5'-coding region of the prfA-mRNA for efficient translation.

  • 303.
    Lopes, Jose Pedro
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Evasion of immune surveillance under low oxygen enhances Candida albicans survivalManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Human colonizers have evolved to sense and adapt environmental cues and nutrient availability. Oxygen is a constantly changing environmental parameter within different host tissues upon different types of infection. We describe how C. albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of neutrophils to the site of infection contributes to a low oxygen milieu. A persistent low oxygen milieu did not affect viability nor metabolism of polymorphonuclear leukocytes (PMNs), however, disturbed anti-Candida responses. PMNs were not able to efficiently phagocytize, produce ROS or release extracellular DNA traps. This failure to respond was caused by C. albicans β-glucan cell wall masking upon exposure to low oxygen which hindered PAMP sensing by dectin-1. Overall, this contributed to immune evasion which in turn enhanced fungal survival. The effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen condition. Finally, low oxygen adaptation increased the pathogenicity and successful colonization of C. albicans in vivo which we confirmed using a Caenorhabditis elegans infection model. 

  • 304.
    Lopes, Jose Pedro
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Stylianou, Marios
    Backman, Emelie
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Holmberg, Sandra
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Jass, Jana
    Claesson, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Urban, Constantin F.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Evasion of Immune Surveillance in Low Oxygen Environments Enhances Candida albicans Virulence.2018Inngår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, nr 6, artikkel-id e02120-18Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 305.
    Lopes, Jose Pedro
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Stylianou, Marios
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Nilsson, Gunnar
    Stockholm, Sweden.
    Urban, Constantin F.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Opportunistic pathogen Candida albicans elicits a temporal response in primary human mast cells2015Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, artikkel-id 12287Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Immunosuppressed patients are frequently afflicted with severe mycoses caused by opportunistic fungal pathogens. Besides being a commensal, colonizing predominantly skin and mucosal surfaces, Candida albicans is the most common human fungal pathogen. Mast cells are present in tissues prone to fungal colonization being expectedly among the first immune cells to get into contact with C. albicans. However, mast cell-fungus interaction remains a neglected area of study. Here we show that human mast cells mounted specific responses towards C. albicans. Collectively, mast cell responses included the launch of initial, intermediate and late phase components determined by the secretion of granular proteins and cytokines. Initially mast cells reduced fungal viability and occasionally internalized yeasts. C. albicans could evade ingestion by intracellular growth leading to cellular death. Furthermore, secreted factors in the supernatants of infected cells recruited neutrophils, but not monocytes. Late stages were marked by the release of cytokines that are known to be anti-inflammatory suggesting a modulation of initial responses. C. albicans-infected mast cells formed extracellular DNA traps, which ensnared but did not kill the fungus. Our results suggest that mast cells serve as tissue sentinels modulating antifungal immune responses during C. albicans infection. Consequently, these findings open new doors for understanding fungal pathogenicity.

  • 306.
    Lopes, Jose Pedro
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Urban, Constantin F.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Visualizing Hypoxia in a Murine Model of Candida albicans Infection Using in vivo Biofluorencence2019Inngår i: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, nr 15, artikkel-id UNSP e3326Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Candida albicans is a leading human fungal pathogen that uses several metabolic adaptations to escape immune cells and causes systemic disease. Here, we describe a protocol for measuring one of these adaptations, the ability to thrive in hypoxic niches. Hypoxia was generated after successful subdermal infection with C. albicans in a murine infection model. Hypoxia was measured using a fluorescent dye for carbonic anhydrase 9, a host enzyme active under hypoxic conditions. Emitted fluorescence was subsequently quantified using an IVIS system. This protocol was optimized for the use in subdermal infection in mice but has the potential to be adapted to other models of fungal infection.

  • 307.
    Lopes, José Pedro
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Candida albicans adaption to host microenvironments drives immune evasion2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Immunosuppressed patients are frequently afflicted with severe mycoses caused by opportunistic fungal pathogens. Besides being a commensal colonizing predominantly skin and mucosal surfaces, Candida albicans is the most common human fungal pathogen. Mast cells are present in tissues prone to fungal colonization being expectedly among the first immune cells to get into contact with C. albicans. Here we describe how mast cells acted as tissue sentinels and modulated initial antifungal immune responses. Mast cells response was able to reduce fungal viability and signaled for neutrophil infiltration to the tissue. Upon chemokine sensing circulating neutrophils are rapid infiltrating to the mucosal to help fight infection. A high number of infiltrating cells coupled with the formation of multicellular structures such as biofilm comes with induction of hypoxic and anoxic micro niches. We found that a persistence anoxia hampered neutrophil responses by affecting fungi sensing and consequent antifungal due to cell wall masking. Adaption to low oxygen seems is important for a successful host infection. Hypoxic and anoxic environments do not allow neutrophils to efficiently produce ROS. Neutrophil oxidative burst is essential for antifungal activity and many fungal pathogens evolved antioxidative factors to mediate survival during infection. We reasoned that targeting of fungal redox balances could be a new therapy approach. We have tested tempol, a redox-cycling nitroxide Tempol as a new antifungal drug. Tempol proved an efficient compound in our testing. We found that Tempol affected fundamental pathways for fungal homeostases such as glycolysis and steroid biosynthesis. Additionally, Tempol helped curve fungal infectivity in a mouse model and leads for an enhanced immune system cytokine profile in human blood. The results obtained proposed tempol as a valid new antifungal compound and open new opportunities for the future development of therapies. Efficient antifungal therapies are still urgent since only 6 classes of antimycotics exist and all with few restricted fungal targets. Since primarily fungal infections affect patients with other immunosuppressive conditions, which are undergoing treatment, we reasoned that repurposing drugs could offer clinical benefits. We performed a screening of two US Food and Drug Administration (FDA)–approved compound libraries for compounds with anti-Candida activity. From 844 drugs, 26 agents showed activity against C. albicans. We identified 7 new off-target drugs all with potent anti- C. albicans activity. The use of these new drugs could be prophylactic or to treat both conditions simultaneously offering, therefore the intended benefit.

    Overall, in this thesis work, we have focused on the sensing clearing and management of fungal pathogens. These findings open new doors for understanding better fungal pathogenicity and purpose valid new antifungal compounds that pave the way for future development of therapies.

  • 308.
    Lopes, José Pedro
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Stylianou, Marios
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Backman, Emelie
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Holmberg, Sandra
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ekoff, Maria
    Nilsson, Gunnar
    Urban, Constantin F.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Cryptococcus neoformans Induces MCP-1 Release and Delays the Death of Human Mast Cells2019Inngår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 9, artikkel-id 289Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cryptococcosis, caused by the basidiomycete Cryptococcus neoformans, is a life-threatening disease affecting approximately one million people per year worldwide. Infection can occur when C. neoformans cells are inhaled by immunocompromised people. In order to establish infection, the yeast must bypass recognition and clearance by immune cells guarding the tissue. Using in vitro infections, we characterized the role of mast cells (MCs) in cryptococcosis. We found that MCs recognize C. neoformans and release inflammatory mediators such as tryptase and cytokines. From the latter group MCs released mainly CCL-2/MCP-1, a strong chemoattractant for monocytic cells. We demonstrated that supernatants of infected MCs recruit monocytes but not neutrophils. During infection with C. neoformans, MCs have a limited ability to kill the yeast depending on the serotype. C. neoformans, in turn, modulates the lifespan of MCs both, by presence of its polysaccharide capsule and by secreting soluble modulators. Taken together, MCs might have important contributions to fungal clearance during early stages of cryptocococis where these cells regulate recruitment of monocytes to mucosal tissues.

  • 309.
    Lundqvist, Jenny
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Larsson, Christer
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Nelson, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Andersson, Marie
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Persson, Cathrine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Concomitant Infection Decreases the Malaria Burden but Escalates Relapsing Fever Borreliosis2010Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 78, nr 5, s. 1924-1930Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    About 500 million cases of malaria occur annually. However, a substantial number of patients who actually have relapsing fever (RF) Borrelia can be misdiagnosed with malaria due to similar manifestations and geographic distribution of the two diseases. More alarmingly, high prevalence of concomitant infections with malaria and RF Borrelia has been reported. Therefore, we used a mouse model to study the effects of such mixed infection. We observed a 21-fold increase in spirochete titers, whereas the numbers of parasitized erythrocytes were reduced 15-fold. This may be explained by polarization of the host immune response towards the intracellular malaria parasite, resulting in unaffected extracellular spirochetes and hosts that succumb to sepsis. Mixed infection also resulted in severe malaria anemia with low hemoglobin levels, even though the parasite counts were low. Overall, co-infected animals had higher fatality rate and shorter time to death than both malaria and RF single infection. Furthermore, secondary malaria infection reactivated a quiescent RF brain infection, which is the first evidence of a clinically and biologically relevant cue for reactivation of RF Borrelia infection. Our study highlights the importance of investigating concomitant infections in vivo to elucidate the immune responses that are involved in the clinical outcome.

  • 310.
    Lärkeryd, Adrian
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Myrtennäs, Kerstin
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Division of CBRN Security and Defence, FOI, Swedish Defence Research Agency.
    Karlsson, Edvin
    Division of CBRN Security and Defence, FOI, Swedish Defence Research Agency.
    Dwibedi, Chinmay Kumar
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Division of CBRN Security and Defence, FOI, Swedish Defence Research Agency.
    Forsman, Mats
    Division of CBRN Security and Defence, FOI, Swedish Defence Research Agency.
    Larsson, Pär
    Division of CBRN Security and Defence, FOI, Swedish Defence Research Agency.
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sjödin, Andreas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    CanSNPer: a hierarchical genotype classifier of clonal pathogens2014Inngår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 30, nr 12, s. 1762-1764Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Advances in typing methodologies have recently reformed the field of molecular epidemiology of pathogens. The falling cost of sequencing technologies is creating a deluge of whole genome sequencing data that burdens bioinformatics resources and tool development. In particular, single nucleotide polymorphisms in core genomes of pathogens are recognized as the most important markers for inferring genetic relationships because they are evolutionarily stable and amenable to high-throughput detection methods. Sequence data will provide an excellent opportunity to extend our understanding of infectious disease when the challenge of extracting knowledge from available sequence resources is met. Here, we present an efficient and user-friendly genotype classification pipeline, CanSNPer, based on an easily expandable database of predefined canonical single nucleotide polymorphisms.

  • 311.
    Lécrivain, Anne-Laure
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Post-transcriptional regulation by RNases in Streptococcus pyogenes2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Ribonucleases (RNases) are proteins that adjust cellular RNA levels by processing RNA transcripts, leading to their stabilization or degradation. RNases are grouped based on their ability to cleave the transcript internally (endoRNases) or degrade the transcript starting from the ends (exoRNases). Specificities of RNA degradation vary among bacterial species, attributable to different sets of endo- and exoRNases. Most of the current knowledge gathered about the roles of RNases and their targets relies on the study of a few model bacteria, such as Escherichia coli and Bacillus subtilis. The aim of this thesis was to understand how Streptococcus pyogenes, a strict human pathogen, controls and adjusts gene expression by characterizing in vivo RNase activities.

    The transcriptome of S. pyogenes was inspected to identify cleavages in vivo performed by RNases of interest using RNA sequencing. For this purpose, we developed a method to compare transcript 5′ and 3′ ends in RNase deletion mutants with those in the parental strain. We first applied our method for the study of endoRNase III, which cleaves ds RNA, and endoRNase Y, which is specific for ss RNA. We accurately retrieved RNase III cleavage positions in structured regions, characterized by 2 nucleotide (nt) 3′ overhangs, and we showed RNase III nicking activity in vivo. We observed that RNase Y processed transcripts after a guanosine. The upstream and downstream fragments generated by a single cleavage event were never both identified, indicating that RNase Y processing always led to the degradation of one of the two fragments. To investigate further the degradation of the upstream fragment subsequent to RNase Y processing, we characterized the 3′-to-5′ exoRNases R, YhaM, and PNPase. RNase R did not have any detectable activity in standard laboratory conditions. YhaM is an intriguing enzyme that removed on average 3 nt of the majority of cellular transcripts. PNPase fully degraded fragments originating from endoRNase processing and is the main 3′-to-5′ exoRNase involved in RNA decay in S. pyogenes.

    To conclude, in this work, we developed a novel method to analyze RNA sequencing data. This method was successfully applied to the study of both endo- and exoRNases. Most importantly, we identified the targetomes of RNases III, Y, R, YhaM, and PNPase and we highlighted the distinctive features of these enzymes.

  • 312.
    Lécrivain, Anne-Laure
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Max Planck Unit for the Science of Pathogens, D-10117 Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, D-10117 Berlin, Germany..
    Broglia, Laura
    Renault, Thibaud
    Hahnke, Karin
    Ahmed-Begrich, Rina
    Le Rhun, Anaïs
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Charpentier, Emmanuelle
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Interplay between 3′-to-5′ exoRNases and RNase Y in Streptococcus pyogenes2018Manuskript (preprint) (Annet vitenskapelig)
  • 313.
    Lécrivain, Anne-Laure
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Le Rhun, Anaïs
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Renault, Thibaud T.
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; nstitute for Biology, Humboldt University, Berlin, Germany .
    Ahmed-Begrich, Rina
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Hahnke, Karin
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Charpentier, Emmanuelle
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; nstitute for Biology, Humboldt University, Berlin, Germany.
    In vivo 3′-to-5′ exoribonuclease targetomes of Streptococcus pyogenes2018Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, nr 46, s. 11814-11819Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    mRNA decay plays an essential role in the control of gene expression in bacteria. Exoribonucleases (exoRNases), which trim transcripts starting from the 5′ or 3′ end, are particularly important to fully degrade unwanted transcripts and renew the pool of nucleotides available in the cell. While recent techniques have allowed genome-wide identification of ribonuclease (RNase) targets in bacteria in vivo, none of the 3′-to-5′ exoRNase targetomes (i.e., global processing sites) have been studied so far. Here, we report the targetomes of YhaM, polynucleotide phosphorylase (PNPase), and RNase R of the human pathogen Streptococcus pyogenes. We determined that YhaM is an unspecific enzyme that trims a few nucleotides and targets the majority of transcript ends, generated either by transcription termination or by endonucleolytic activity. The molecular determinants for YhaM-limited processivity are yet to be deciphered. We showed that PNPase clears the cell from mRNA decay fragments produced by endoribonucleases (endoRNases) and is the major 3′-to-5′ exoRNase for RNA turnover in S. pyogenes. In particular, PNPase is responsible for the degradation of regulatory elements from 5′ untranslated regions. However, we observed little RNase R activity in standard culture conditions. Overall, our study sheds light on the very distinct features of S. pyogenes 3′-to-5′ exoRNases.

  • 314. Maciejewska, B
    et al.
    Roszniowski, B
    Espaillat, Akbar
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kęsik-Szeloch, A
    Majkowska-Skrobek, G
    Kropinski, AM
    Briers, Y
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lavigne, R
    Drulis-Kawa, Z
    Klebsiella phages representing a novel clade of viruses with an unknown DNA modification and biotechnologically interesting enzymes2017Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 101, nr 2, s. 673-684Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Lytic bacteriophages and phage-encoded endolysins (peptidoglycan hydrolases) provide a source for the development of novel antimicrobial strategies. In the present study, we focus on the closely related (96 % DNA sequence identity) environmental myoviruses vB_KpnM_KP15 (KP15) and vB_KpnM_KP27 (KP27) infecting multidrug-resistant Klebsiella pneumoniae and Klebsiella oxytoca strains. Their genome organisation and evolutionary relationship are compared to Enterobacter phage phiEap-3 and Klebsiella phages Matisse and Miro. Due to the shared and distinct evolutionary history of these phages, we propose to create a new phage genus BKp15virus^ within the Tevenvirinae subfamily. In silico genome analysis reveals two unique putative homing endonucleases of KP27 phage, probably involved in unrevealed mechanism of DNA modification and resistance to restriction digestion, resulting in a broader host spectrum. Additionally, we identified in KP15 and KP27 a complete set of lysis genes, containing holin, antiholin, spanin and endolysin. By turbidimetric assays on permeabilized Gram-negative strains, we verified the ability of the KP27 endolysin to destroy the bacterial peptidoglycan. We confirmed high stability, absence of toxicity on a human epithelial cell line and the enzymatic specificity of endolysin, which was found to possess endopeptidase activity, cleaving the peptide stem between L-alanine and D-glutamic acid.

  • 315. Maciejewska, Barbara
    et al.
    Zrubek, Karol
    Espaillat, Akbar
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wisniewska, Magdalena
    Rembacz, Krzysztof P.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Dubin, Grzegorz
    Drulis-Kawa, Zuzanna
    Modular endolysin of Burkholderia AP3 phage has the largest lysozyme-like catalytic subunit discovered to date and no catalytic aspartate residue2017Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikkel-id 14501Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Endolysins are peptidoglycan-degrading enzymes utilized by bacteriophages to release the progeny from bacterial cells. The lytic properties of phage endolysins make them potential antibacterial agents for medical and industrial applications. Here, we present a comprehensive characterization of phage AP3 modular endolysin (AP3gp15) containing cell wall binding domain and an enzymatic domain (DUF3380 by BLASTP), both widespread and conservative. Our structural analysis demonstrates the low similarity of an enzymatic domain to known lysozymes and an unusual catalytic centre characterized by only a single glutamic acid residue and no aspartic acid. Thus, our findings suggest distinguishing a novel class of muralytic enzymes having the activity and catalytic centre organization of DUF3380. The lack of amino acid sequence homology between AP3gp15 and other known muralytic enzymes may reflect the evolutionary convergence of analogous glycosidases. Moreover, the broad antibacterial spectrum, lack of cytotoxic effect on human cells and the stability characteristics of AP3 endolysin advocate for its future application development.

  • 316.
    Maicher, André
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gazy, Inbal
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Marjavaara, Lisette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Grinberg, Gilad
    Shemesh, Keren
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kupiec, Martin
    Rnr1, but not Rnr3, facilitates the sustained telomerase-dependent elongation of telomeres2017Inngår i: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 13, nr 10, artikkel-id e1007082Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Ribonucleotide reductase (RNR) provides the precursors for the generation of dNTPs, which are required for DNA synthesis and repair. Here, we investigated the function of the major RNR subunits Rnr1 and Rnr3 in telomere elongation in budding yeast. We show that Rnr1 is essential for the sustained elongation of short telomeres by telomerase. In the absence of Rnr1, cells harbor very short, but functional, telomeres, which cannot become elongated by increased telomerase activity or by tethering of telomerase to telomeres. Furthermore, we demonstrate that Rnr1 function is critical to prevent an early onset of replicative senescence and premature survivor formation in telomerase-negative cells but dispensable for telomere elongation by Homology-Directed-Repair. Our results suggest that telomerase has a "basal activity" mode that is sufficient to compensate for the "end-replication-problem" and does not require the presence of Rnr1 and a different "sustained activity" mode necessary for the elongation of short telomeres, which requires an upregulation of dNTP levels and dGTP ratios specifically through Rnr1 function. By analyzing telomere length and dNTP levels in different mutants showing changes in RNR complex composition and activity we provide evidence that the Mec1ATR checkpoint protein promotes telomere elongation by increasing both dNTP levels and dGTP ratios through Rnr1 upregulation in a mechanism that cannot be replaced by its homolog Rnr3.

  • 317. Makarova, Kira S
    et al.
    Haft, Daniel H
    Barrangou, Rodolphe
    Brouns, Stan J J
    Charpentier, Emmanuelle
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Horvath, Philippe
    Moineau, Sylvain
    Mojica, Francisco J M
    Wolf, Yuri I
    Yakunin, Alexander F
    van der Oost, John
    Koonin, Eugene V
    Evolution and classification of the CRISPR-Cas systems2011Inngår i: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 9, nr 6, s. 467-477Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR-Cas systems and Cas proteins. Three major types of CRISPR-Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR-Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a 'polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR-cas loci.

  • 318. Manav, Melek Cemre
    et al.
    Beljantseva, Jelena
    Bojer, Martin S.
    Tenson, Tanel
    Ingmer, Hanne
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia.
    Brodersen, Ditlev E.
    Structural basis for (p)ppGpp synthesis by the Staphylococcus aureus small alarmone synthetase RelP2018Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, nr 9, s. 3254-3264Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The stringent response is a global reprogramming of bacterial physiology that renders cells more tolerant to antibiotics and induces virulence gene expression in pathogens in response to stress. This process is driven by accumulation of the intracellular alarmone guanosine-5'-di(tri)phosphate-3'-diphosphate ((p)ppGpp), which is produced by enzymes of the RelA SpoT homologue (RSH) family. The Gram-positive Firmicute pathogen, Staphylococcus aureus, encodes three RSH enzymes: a multidomain RSH (Rel) that senses amino acid starvation on the ribosome and two small alarmone synthetase (SAS) enzymes, RelQ (SAS1) and RelP (SAS2). In Bacillus subtilis, RelQ (SAS1) was shown to form a tetramer that is activated by pppGpp and inhibited by single-stranded RNA, but the structural and functional regulation of RelP (SAS2) is unexplored. Here, we present crystal structures of S. aureus RelP in two major functional states, pre-catalytic (bound to GTP and the non-hydrolyzable ATP analogue, adenosine 5'-(alpha,beta-methylene) triphosphate (AMP-CPP)), and post-catalytic (bound to pppGpp). We observed that RelP also forms a tetramer, but unlike RelQ (SAS1), it is strongly inhibited by both pppGpp and ppGpp and is insensitive to inhibition by RNA. We also identified putative metal ion-binding sites at the subunit interfaces that were consistent with the observed activation of the enzyme by Zn2+ ions. The structures reported here reveal the details of the catalytic mechanism of SAS enzymes and provide a molecular basis for understanding differential regulation of SAS enzymes in Firmicute bacteria.

  • 319.
    Mansjö, Mikael
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    The Riboflavin analog roseoflavin targets an FMN-riboswitch and blocks Listeria monocytogenes growth, but also stimulates virulence gene-expression and infection2011Inngår i: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 8, nr 4, s. 674-680Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    During recent years, riboswitches have emerged as potential targets for novel antibacterial substances. In this study, we investigated how one flavin analog, roseoflavin, affected the gene-expression, growth and infectivity of the human bacterial pathogen Listeria monocytogenes to determine the potential of this analog to function as an antibacterial substance. The results indicate that roseoflavin has a profound inhibiting effect on the growth of L. monocytogenes at very low concentrations. Also, expression of the gene located downstream of the FMN riboswitch, a riboflavin transporter, was blocked by the addition of roseoflavin. Base-substitution mutations in the FMN riboswitch allowed the bacteria to grow in the presence of roseoflavin, showing that roseoflavin targeted the FMN riboswitch directly. Surprisingly, we found that roseoflavin stimulated L. monocytogenes virulence gene expression and infection abilities in a mechanism independent of the FMN riboswitch. Our results suggest that roseoflavin can block growth but also enhance Listeria virulence.

  • 320.
    Marcinowska, Renata
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Trygg, Johan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Mortiz, Thomas
    Surowiec, Izabella
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Optimization of a sample preparation method for the metabolomic analysis of clinically relevant bacteria2011Inngår i: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 87, nr 1, s. 24-31Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Metabolomics, or metabolite profiling, is an approach that is increasingly used to study the metabolism of diverse organisms, elucidate biological processes and/or find characteristic biomarkers of physiological states. Here, we describe the optimization of a method for global metabolomic analysis of bacterial cultures, with the following steps. Cells are grown to log-phase, starting from an overnight culture and bacterial concentrations are monitored by measuring the optical density of the cultures at 600nm. At an appropriate density they are harvested by centrifugation, washed three times with NaCl solution and metabolites are extracted using methanol and a bead-mill. Dried extracts are methoxymated and derivatized with methyltrimethylsilyltrifluoroacetamide (MSTFA) then analyzed using gas chromatography coupled to time-of-flight mass spectrometry (GC-MS/TOF). Finally, patterns in the acquired data are examined by multivariate data modeling. This method enabled us to obtain reproducible metabolite profiles of Yersinia pseudotuberculosis, with about 25% compound identification, based on comparison with entries in available GC-MS libraries. To assess the potential utility of the method for comparative analysis of other bacterial species we analyzed cultures of Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and methicillin-sensitive Staphylococcus aureus (MSSA). Multivariate analysis of the acquired data showed that it was possible to differentiate the species according to their metabolic profiles. Our results show that the presented procedure can be used for metabolomic analysis of a wide range of bacterial species of clinical interest.

  • 321. Marinho, Catarina M.
    et al.
    Dos Santos, Patricia T.
    Kallipolitis, Birgitte H.
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ignatov, Dmitriy
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Guerreiro, Duarte N.
    Piveteau, Pascal
    O'Byrne, Conor P.
    The σB-dependent regulatory sRNA Rli47 represses isoleucine biosynthesis in Listeria monocytogenes through a direct interaction with the ilvA transcript2019Inngår i: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 16, nr 10, s. 1424-1437Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The facultative intracellular pathogen Listeria monocytogenes can persist and grow in a diverse range of environmental conditions, both outside and within its mammalian host. The alternative sigma factor Sigma B (sigma(B)) plays an important role in this adaptability and is critical for the transition into the host. While some of the functions of the sigma(B) regulon in facilitating this transition are understood the role of sigma(B)-dependent small regulatory RNAs (sRNAs) remain poorly characterized. In this study, we focused on elucidating the function of Rli47, a sigma(B)-dependent sRNA that is highly induced in the intestine and in macrophages. Using a combination of in silico and in vivo approaches, a binding interaction was predicted with the Shine-Dalgarno region of the ilvA mRNA, which encodes threonine deaminase, an enzyme required for branched-chain amino acid biosynthesis. Both ilvA transcript levels and threonine deaminase activity were increased in a deletion mutant lacking the rli47 gene. The Delta rli47 mutant displayed a shorter growth lag in isoleucine-depleted growth media relative to the wild-type, and a similar phenotype was also observed in a mutant lacking sigma(B). The impact of the Delta rli47 on the global transcription profile of the cell was investigated using RNA-seq, and a significant role for Rli47 in modulating amino acid metabolism was uncovered. Taken together, the data point to a model where Rli47 is responsible for specifically repressing isoleucine biosynthesis as a way to restrict growth under harsh conditions, potentially contributing to the survival of L. monocytogenes in niches both outside and within the mammalian host.

  • 322.
    Marwaha, Sania
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Uvell, Hanna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Salin, Olli
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Lindgren, Anders E. G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Silver, Jim
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Gylfe, Åsa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    N-acylated derivatives of sulfamethoxazole and sulfafurazole inhibit intracellular growth of Chlamydia trachomatis2014Inngår i: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 58, nr 5, s. 2968-2971Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Antibacterial compounds with novel modes of action are needed for management of bacterial infections. Here we describe a high-content screen of 9,800 compounds identifying acylated sulfonamides as novel growth inhibitors of the sexually transmitted pathogen Chlamydia trachomatis. The effect was bactericidal and distinct from that of sulfonamide antibiotics, as para-aminobenzoic acid did not reduce efficacy. Chemical inhibitors play an important role in Chlamydia research as probes of potential targets and as drug development starting points.

  • 323.
    Massai, Francesco
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Saleeb, Michael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Doruk, Tugrul
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa2019Inngår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 9, artikkel-id 250Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Antibiotics are becoming less effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa. Antimicrobial therapies based on the inhibition of specific virulence-related traits, as opposed to growth inhibitors, constitute an innovative and appealing approach to tackle the threat of P. aeruginosa infections. The twin-arginine translocation (Tat) pathway plays an important role in the pathogenesis of P. aeruginosa, and constitutes a promising target for the development of anti-pseudomonal drugs. In this study we developed and optimized a whole-cell, one-well assay, based on native phospholipase C activity, to identify compounds active against the Tat system. Statistical robustness, sensitivity and consequently suitability for high-throughput screening (HTS) were confirmed by a dry run/pre-screening test scoring a Z' of 0.82 and a signal-to-noise ratio of 49. Using this assay, we evaluated ca. 40,000 molecules and identified 59 initial hits as possible Tat inhibitors. Since phospholipase C is exported into the periplasm by Tat, and subsequently translocated across the outer membrane by the type II secretion system (T2SS), our assay could also identify T2SS inhibitors. To validate our hits and discriminate between compounds that inhibited either Tat or T2SS, two separate counter assays were developed and optimized. Finally, three Tat inhibitors and one T2SS inhibitor were confirmed by means of dose-response analysis and additional counter and confirming assays. Although none of the identified inhibitors was suitable as a lead compound for drug development, this study validates our assay as a simple, efficient, and HTS compatible method for the identification of Tat and T2SS inhibitors.

  • 324. Matern, Andreas
    et al.
    Pedrolli, Danielle
    Großhennig, Stephanie
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Mack, Matthias
    Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes2016Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, nr 23, s. 3233-3243Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteria Streptomyces davawensis and Streptomyces cinnabarinus Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (vitamin B2), RoF, and AF in the human pathogen Listeria monocytogenes, a bacterium which is a riboflavin auxotroph. We show that the L. monocytogenes protein Lmo1945 is responsible for the uptake of riboflavin, RoF, and AF. Following import, these flavins are phosphorylated/adenylylated by the bifunctional flavokinase/flavin adenine dinucleotide (FAD) synthetase Lmo1329 and adenylylated by the unique FAD synthetase Lmo0728, the first monofunctional FAD synthetase to be described in bacteria. Lmo1329 generates the cofactors flavin mononucleotide (FMN) and FAD, whereas Lmo0728 produces FAD only. The combined activities of Lmo1329 and Lmo0728 are responsible for the intracellular formation of the toxic cofactor analogs roseoflavin mononucleotide (RoFMN), roseoflavin adenine dinucleotide (RoFAD), 8-demethyl-8-aminoriboflavin mononucleotide (AFMN), and 8-demethyl-8-aminoriboflavin adenine dinucleotide (AFAD). In vivo reporter gene assays and in vitro transcription/translation experiments show that the L. monocytogenes FMN riboswitch Rli96, which controls expression of the riboflavin transport gene lmo1945, is negatively affected by riboflavin/FMN and RoF/RoFMN but not by AF/AFMN. Treatment of L. monocytogenes with RoF or AF leads to drastically reduced FMN/FAD levels. We suggest that the reduced flavin cofactor levels in combination with concomitant synthesis of inactive cofactor analogs (RoFMN, RoFAD, AFMN, and AFAD) explain why RoF and AF contribute to antibiotic activity in L. monocytogenes IMPORTANCE: The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are small molecules which are produced by Streptomyces davawensis and Streptomyces cinnabarinus RoF and AF were reported to have antibacterial activity, and we studied how these compounds are metabolized by the human bacterial pathogen Listeria monocytogenes We found that the L. monocytogenes protein Lmo1945 mediates uptake of AF and RoF and that the combined activities of the enzymes Lmo1329 and Lmo0728 are responsible for the conversion of AF and RoF to toxic cofactor analogs. Comparative studies with RoF and AF (a weaker antibiotic) suggest that the reduction in FMN/FAD levels and the formation of inactive FMN/FAD analogs explain to a large extent the antibiotic activity of AF and RoF.

  • 325. Meinzer, Ulrich
    et al.
    Esmiol-Welterlin, Sophie
    Barreau, Frederick
    Berrebi, Dominique
    Dussaillant, Monique
    Bonacorsi, Stephane
    Chareyre, Fabrice
    Niwa-Kawakita, Michiko
    Alberti, Corinne
    Sterkers, Ghislaine
    Villard, Claude
    Lesuffleur, Thecla
    Peuchmaur, Michel
    Karin, Michael
    Eckmann, Lars
    Giovannini, Marco
    Ollendorff, Vincent
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinsk fakultet, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinsk fakultet, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hugot, Jean-Pierre
    Nod2 mediates susceptibility to Yersinia pseudotuberculosis in mice.2008Inngår i: PLoS ONE, ISSN 1932-6203, Vol. 3, nr 7, s. e2769-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs) in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs. We investigated the role of Nod2 during Y. pseudotuberculosis infection. Death was delayed in Nod2 deleted and Crohn's disease associated Nod2 mutated mice orogastrically inoculated with Y. pseudotuberculosis. In PPs, the local immune response was characterized by a higher KC level and a more intense infiltration by neutrophils and macrophages. The apoptotic and bacterial cell counts were decreased. Finally, Nod2 deleted mice had a lower systemic bacterial dissemination and less damage of the haematopoeitic organs. This resistance phenotype was lost in case of intraperitoneal infection. We concluded that Nod2 contributes to the susceptibility to Y. pseudotuberculosis in mice.

  • 326. Mellin, JR
    et al.
    Tiensuu, Teresa
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Becavin, Christophe
    Gouin, Edith
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Cossart, Pascale
    A riboswitch-regulated antisense RNA in Listeria monocytogenes2013Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, nr 32, s. 13132-13137Artikkel i tidsskrift (Fagfellevurdert)
  • 327.
    Mertz, Tony M
    et al.
    Omaha, Nebraska, USA.
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Shcherbakova, Polina V
    Omaha, Nebraska, USA.
    Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity2015Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, nr 19, s. E2467-E2476Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Defects in DNA polymerases δ (Polδ) and ε (Polε) cause hereditary colorectal cancer and have been implicated in the etiology of some sporadic colorectal and endometrial tumors. We previously reported that the yeast pol3-R696W allele mimicking a human cancer-associated variant, POLD1-R689W, causes a catastrophic increase in spontaneous mutagenesis. Here, we describe the mechanism of this extraordinary mutator effect. We found that the mutation rate increased synergistically when the R696W mutation was combined with defects in Polδ proofreading or mismatch repair, indicating that pathways correcting DNA replication errors are not compromised in pol3-R696W mutants. DNA synthesis by purified Polδ-R696W was error-prone, but not to the extent that could account for the unprecedented mutator phenotype of pol3-R696W strains. In a search for cellular factors that augment the mutagenic potential of Polδ-R696W, we discovered that pol3-R696W causes S-phase checkpoint-dependent elevation of dNTP pools. Abrogating this elevation by strategic mutations in dNTP metabolism genes eliminated the mutator effect of pol3-R696W, whereas restoration of high intracellular dNTP levels restored the mutator phenotype. Further, the use of dNTP concentrations present in pol3-R696W cells for in vitro DNA synthesis greatly decreased the fidelity of Polδ-R696W and produced a mutation spectrum strikingly similar to the spectrum observed in vivo. The results support a model in which (i) faulty synthesis by Polδ-R696W leads to a checkpoint-dependent increase in dNTP levels and (ii) this increase mediates the hypermutator effect of Polδ-R696W by facilitating the extension of mismatched primer termini it creates and by promoting further errors that continue to fuel the mutagenic pathway.

  • 328.
    Meyer, Lena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Bröms, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Alam, A.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    A mutagenesisbased approach to map functional domain(s) within the N- terminus of Francisella tularensis IglEManuskript (preprint) (Annet vitenskapelig)
  • 329.
    Meyer, Lena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Bröms, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Liu, Xijia
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Rottenberg, M.E.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Microinjection of Francisella tularensis and Listeria monocytogenes reveals the importance of bacterial and host factors for successful replication2015Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 83, nr 8, s. 3233-3242Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis, many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis; five selected FPI mutants thereof, i.e., Delta iglA, Delta iglC, Delta iglG, Delta iglI, and Delta pdpE strains; and Listeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC(-/-) BMDM, MyD88(-/-) BMDM, J774 cells, or HeLa cells, LVS, Delta pdpE and Delta iglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the Delta iglA and Delta iglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC(-/-) BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88(-/-) BMDM and LVS showed no replication in either BMDM or MyD88(-/-) BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection.

  • 330.
    Mistry, Nitesh
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Inoue, Hirotoshi
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Jamshidi, Fariba
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Storm, Rickard J.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Oberste, M. Steven
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Coxsackievirus A24 variant uses aialic acid-containing O-Linked glycoconjugates as cellular receptors on human ocular cells2011Inngår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 85, nr 21, s. 11283-11290Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Coxsackievirus A24 variant (CVA24v) is a main causative agent of acute hemorrhagic conjunctivitis (AHC), which is a highly contagious eye infection. Previously it has been suggested that CVA24v uses sialic acid-containing glycoconjugates as attachment receptors on corneal cells, but the nature of these receptors is poorly described. Here, we set out to characterize and identify the cellular components serving as receptors for CVA24v. Binding and infection experiments using corneal cells treated with deglycosylating enzymes or metabolic inhibitors of de novo glycosylation suggested that the receptor(s) used by CVA24v are constituted by sialylated O-linked glycans that are linked to one or more cell surface proteins but not to lipids. CVA24v bound better to mouse L929 cells overexpressing human P-selectin glycoprotein ligand-1 (PSGL-1) than to mock-transfected cells, suggesting that PSGL-1 is a candidate receptor for CVA24v. Finally, binding competition experiments using a library of mono- and oligosaccharides mimicking known PSGL-1 glycans suggested that CVA24v binds to Neu5Ac alpha 2,3Gal disaccharides (Neu5Ac is N-acetylneuraminic acid). These results provide further insights into the early steps of the CVA24v life cycle.

  • 331.
    Moell, Andrea
    et al.
    Boston, Massachusetts, USA .
    Doerr, Tobias
    Boston, Massachusetts, USA .
    Alvarez, Laura
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Chao, Michael C.
    Boston, Massachusetts, USA .
    Davis, Brigid M.
    Boston, Massachusetts, USA .
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Waldor, Matthew K.
    Boston, Massachusetts, USA .
    Cell Separation in Vibrio cholerae Is Mediated by a Single Amidase Whose Action Is Modulated by Two Nonredundant Activators2014Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 196, nr 22, s. 3937-3948Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Synthesis and hydrolysis of septal peptidoglycan (PG) are critical processes at the conclusion of cell division that enable separation of daughter cells. Cleavage of septal PG is mediated by PG amidases, hydrolytic enzymes that release peptide side chains from the glycan strand. Most gammaproteobacteria, including Escherichia coli, encode several functionally redundant periplasmic amidases. However, members of the Vibrio genus, including the enteric pathogen Vibrio cholerae, encode only a single PG amidase, AmiB. Here, we show that V. cholerae AmiB is crucial for cell division and growth. Genetic and biochemical analyses indicated that AmiB is regulated by two activators, EnvC and NlpD, at least one of which is required for AmiB's localization to the cell division site. Localization of the activators (and thus of AmiB) is dependent upon the cell division protein FtsN. These factors mediate septal PG cleavage in E. coli as well; however, their precise roles vary between the two organisms in a number of ways. Notably, even though V. cholerae EnvC and NlpD appear to be functionally redundant under most growth conditions tested, NlpD is specifically required for intestinal colonization in the infant mouse model of cholera and for V. cholerae resistance against bile salts, perhaps due to environmental regulation of AmiB or its activators. Collectively, our findings reveal that although the cellular components that enable cleavage of septal PG appear to be generally conserved between E. coli and V. cholerae, they can be combined into diverse functional regulatory networks.

  • 332.
    Mohan, Jagan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Morén, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Holst, Mikkel
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae2015Inngår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, nr 5, s. 979-991Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Caveolae are invaginations of the cell surface thought to regulate membrane tension, signalling, adhesion and lipid homeostasis due to their dynamic behaviour ranging from stable surface association to dynamic rounds of fission and fusion with the plasma membrane. The caveolae coat is generated by oligomerisation of the membrane protein caveolin and the family of cavin proteins. Here, we show that cavin3 is targeted to caveolae by cavin1 where it interacts with the scaffolding domain of caveolin1 and promote caveolae dynamics. We found that the N-terminal region of cavin3 binds a trimer of the cavin1 N-terminus in competition with a homologous cavin2 region, showing that the cavins form distinct subcomplexes via their N-terminal regions. Our data shows that cavin3 is enriched at deeply invaginated caveolae and that loss of cavin3 in cells results in an increase of stable caveolae and a decrease of caveolae with short duration time at the membrane. We propose that cavin3 is recruited to the caveolae coat by cavin1 to interact with caveolin1 and regulate the duration time of caveolae at the plasma membrane.

  • 333.
    Mojica, Sergio A.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Salin, Olli
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Bastidas, Robert J.
    Sunduru, Naresh
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Hedenström, Mattias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Andersson, C. David
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Núñez-Otero, Carlos
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Engström, Patrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Valdivia, Raphael H.
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Gylfe, Åsa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    N-acylated derivatives of sulfamethoxazole block Chlamydia fatty acid synthesis and interact with FabF2017Inngår i: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 61, nr 10, artikkel-id e00716-17Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The type II fatty acid synthesis (FASII) pathway is essential for bacterial lipid biosynthesis and continues to be a promising target for novel antibacterial compounds. Recently, it has been demonstrated that Chlamydia is capable of FASII and this pathway is indispensable for Chlamydia growth. Previously, a high-content screen with Chlamydia trachomatis-infected cells was performed, and acylated sulfonamides were identified to be potent growth inhibitors of the bacteria. C. trachomatis strains resistant to acylated sulfonamides were isolated by serial passage of a wild-type strain in the presence of low compound concentrations. Results from whole-genome sequencing of 10 isolates from two independent drug-resistant populations revealed that mutations that accumulated in fabF were predominant. Studies of the interaction between the FabF protein and small molecules showed that acylated sulfonamides directly bind to recombinant FabF in vitro and treatment of C. trachomatis-infected HeLa cells with the compounds leads to a decrease in the synthesis of Chlamydia fatty acids. This work demonstrates the importance of FASII for Chlamydia development and may lead to the development of new antimicrobials.

  • 334.
    Mojica, Sergio
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Eriksson, Anna U.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Davis, Rohan A.
    Bahnan, Wael
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Gylfe, Åsa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Red Fluorescent Chlamydia trachomatis Applied to Live Cell Imaging and Screening for Antibacterial Agents2018Inngår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, artikkel-id 3151Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In this study, we describe the application of a transformed Chlamydia trachomatis strain constitutively expressing the red fluorescent protein mCherry, to allow real-time monitoring of the infection cycle and screening for agents that block replication of C. trachomatis. The red fluorescent C. trachomatis strain was detected autonomously without antibody staining and was equally susceptible to doxycycline as the wild type strain. A high-throughput screening assay was developed using the transformed strain and automated fluorescence microscopy. The assay was used in a pilot screen of a 349 compound library containing natural products from Australian flora and fauna. Compounds with anti-chlamydial activity were tested for dose response and toxicity to host cells and two non-toxic compounds had 50% effective concentration (EC50) values in the low micromolar range. Natural products are valuable sources for drug discovery and the identified Chlamydia growth inhibition may be starting points for future drug development. Live cell imaging was used to visualize growth of the red fluorescent C. trachomatis strain over time. The screening assay reduced workload and reagents compared to an assay requiring immunostaining and could further be used to monitor the development of Chlamydia inclusions and anti-chlamydial effect in real time.

  • 335. Monteferrante, C. G.
    et al.
    Jirgensons, A.
    Varik, Vallo
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
    Hauryliuk, VVasili
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
    Goessens, W. H. F.
    Hays, J. P.
    Evaluation of the characteristics of leucyl-tRNA synthetase (LeuRS) inhibitor AN3365 in combination with different antibiotic classes2016Inngår i: European Journal of Clinical Microbiology and Infectious Diseases, ISSN 0934-9723, E-ISSN 1435-4373, Vol. 35, nr 11, s. 1857-1864Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aminoacyl tRNA synthetases are enzymes involved in the key process of coupling an amino acid to its cognate tRNA. AN3365 is a novel antibiotic that specifically targets leucyl-tRNA synthetase, whose development was halted after evaluation in phase II clinical trials owing to the rapid selection of resistance. In an attempt to bring AN3365 back into the developmental pipeline we have evaluated the efficacy of AN3365 in combination with different classes of antibiotic and characterized its mechanism of action. Although we detect no synergy or antagonism in combination with a range of antibiotic classes, a combination of AN3365 with colistin reduces the accumulation of AN3365-resistant and colistin resistance mutations. We also demonstrate that treatment with AN3365 results in the dramatic accumulation of the alarmone (p)ppGpp, the effector of the stringent response-a key player in antibiotic tolerance.

  • 336. Morell, M. L.
    et al.
    Pena Carcamo, J. R.
    Vazquez, C. A.
    Vatansever, S.
    Upadhyay, Arunkumar S.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Overby, A. K.
    Cordo, S. M.
    Garcia, C. C.
    Viperin exerts antiviral function against Junin mammarenavirus at different subcellular localizations2017Inngår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28Artikkel i tidsskrift (Annet vitenskapelig)
  • 337. Moreno-Guzman, Maria
    et al.
    Garcia-Carmona, Laura
    Molinero-Fernandez, Agueda
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Lopez Gil, Miguel Angel
    Escarpa, Alberto
    Bi-enzymatic biosensor for on-site, fast and reliable electrochemical detection of relevant D-amino acids in bacterial samples2017Inngår i: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 242, s. 95-101Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In this work, a bi-enzymatic biosensor allowed the total content of D-amino acids (DAAs) determination in highly relevant matrices involving bacteria. The strategy is based on the unique coimmobilization of D-amino acid oxidase (DAAO) and horseradish peroxidase (HRP) enzymes onto a multi-walled carbon nanotubes (MWCNTs) and gold nanoparticles (AuNPs) modified screen-printed electrode (SPCE). The greater amount of AuNPs deposited and hence the greater loading of both enzymes was observed when they were deposited after the activation of the carboxylated MWCNTs with EDC/Sulfo-NHS chemistry. These platforms provided a fast (300s) and selective quantification of DAAs with excellent precision (RSD < 5%) and accuracy (Recoveries 100-104%) in bacterial samples. Collectively, the electrochemical bi-enzymatic biosensor become an universal, fast, sensitive and easy-to-use approach to determine total content of DAAs in complex matrices.

  • 338.
    Mortezaei, Narges
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Epler, Chelsea
    Shao, Paul
    Shirdel, Mariam
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Yrkes- och miljömedicin.
    Bhupender, Singh
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    McVeigh, Annette
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Savarino, Stephen
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Bullitt, Esther
    Boston University School of Medicine.
    Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways2015Inngår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 95, nr 1, s. 116-126Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram-negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone-usher pathway and others by the alternate chaperone pathway. Here, we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared with colonization factor antigen I (CFA/I) fimbriae, which are two ETEC fimbriae assembled via different pathways, and with P-fimbriae from uropathogenic E.coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P-fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology.

  • 339.
    Mortezaei, Narges
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Epler, Chelsea
    Shao, Paul
    Shirdel, Mariam
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Singh, Bhupender
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    McVeigh, Annette
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Stephen, Savarino
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Bullitt, Esther
    Adhesion Pili from Enterotoxigenic Escherichia coli Share Similar Biophysical Properties Despite Their Different Assembly Pathways2015Inngår i: Microscopy and Microanalysis, ISSN 1431-9276, E-ISSN 1435-8115, Vol. 21, s. 915-916Artikkel i tidsskrift (Fagfellevurdert)
  • 340.
    Mortezaei, Narges
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Singh, Bhupender
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Bullitt, Esther
    Boston University School of Medicine.
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    P-fimbriae in the presence of anti-PapA antibodies: new insight of antibodies action against pathogens2013Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, artikkel-id 3393Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Uropathogenic strains of Escherichia coli establish urinary tract infections by attaching to host epithelial cells using adhesive organelles called fimbriae. Fimbriae are helix-like structures with a remarkable adaptability, offering safeguarding for bacteria exposed to changing fluid forces in the urinary tract. We challenged this property of P-fimbriae by cross-linking their subunits with shaft-specific antibodies and measuring the corresponding force response at a single organelle level. Our data show compromised extension and rewinding of P-fimbriae in the presence of antibodies and reduced fimbrial elasticity, which are important properties of fimbriae contributing to the ability of bacteria to cause urinary tract infections. The reduced elasticity found by cross-linking fimbrial subunits could thus be another assignment for antibodies; in addition to marking bacteria as foreign, antibodies physically compromise fimbrial function. We suggest that our assay and results will be a starting point for further investigations aimed at inhibiting sustained bacterial adhesion by antibodies.

  • 341.
    Mortezaei, Narges
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Singh, Bhupender
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Savarino, Stephen
    Bullitt, Esther
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Antibodies Change the Mechanics of Adhesion Fimbriae: a Case Study of CS20 Fimbriae Expressed by Enterotoxigenic Escherichia Coli2015Inngår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, s. 602-Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    Enterotoxigenic Escherichia coli (ETEC) express a variety of fimbriae that mediate adhesion to host epithelial cells. It has been shown that the ability of a fimbriated bacterial cell to attach and stay attached to host cells does not merely depend on the adhesin expressed distal of the fimbriae but also the biomechanical properties of the fimbriae are vital for sustained adhesion. Fimbriae can significantly extend under a constant force when exposed to an external force and therefore reduce the load on the adhesin, which is believed to help bacteria to withstand external forces applied by various body defense systems. Thus, it is thought that the fimbrial shaft and adhesin have co-evolved for optimal function when bacteria attach to host cells. To investigate if antibodies, normally found in the intestines, affects the biomechanical properties of fimbriae, we exposed CS20 fimbriae expressed by ETEC to anti-fimbrial antibodies and measured these properties using optical tweezers force spectroscopy. Our data show a change in the force required to extend the fimbriae and that the elasticity is significantly reduced by the presence of antibodies. The reduced elasticity, likely due to cross-linking of fimbrial subunits, could thus be another assignment for antibodies; in addition to their mission in marking bacteria as foreign, our data indicate that antibodies physically compromise fimbrial function. To further confirm interaction of antibodies to their specific target we performed western blot analysis, transmission electron microscopy and immunofluoresence microscopy. In the presence of antibodies, we suggest that our assay and results will be a starting point for further studies aimed at inhibiting bacterial adhesion by antibodies.

  • 342.
    Morén, Björn
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Shah, Claudio
    Howes, Mark T
    Schieber, Nicole L
    McMahon, Harvey T
    Parton, Robert G
    Daumke, Oliver
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization2012Inngår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 23, nr 7, s. 1316-1329Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Eps15 homology domain-containing 2 (EHD2) belongs to the EHD-containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes, resulting in stimulation of the intrinsic ATPase activity. In this paper, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1, and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolar integrity. While the EHD of EHD2 is dispensable for targeting, we identified a loop in the nucleotide-binding domain that, together with ATP binding, is required for caveolar localization. EHD2 was not essential for the formation or shaping of caveolae, but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2, resulting in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.

  • 343. Mostafavi, Ehsan
    et al.
    Ghasemi, Ahmad
    Rohani, Mahdi
    Molaeipoor, Leila
    Esmaeili, Saber
    Mohammadi, Zeinolabedin
    Mahmoudi, Ahmad
    Aliabadian, Mansour
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Molecular Survey of Tularemia and Plague in Small Mammals From Iran2018Inngår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 8, artikkel-id 215Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Introduction: Plague and tularemia are zoonoses and their causative bacteria are circulating in certain regions of Iran. This study was conducted to investigate potential disease reservoirs amongst small wildlife species in different regions of Iran.

    Methods: Rodents, insectivores and hares from 17 different provinces of the country were collected in 2014 and 2015. Samples were taken from the spleens of the animals and Real-time PCR was applied to detect nucleic acid sequences that are specific to Francisella tularensis and Yersinia pestis, respectively.

    Results: Among 140 collected rodents, 25 distinct species were identified out of which five were the most common: Microtus paradoxus (21% out of 140 rodents), Apodemus witherbyi (12%), Microtus irani (11%), Mus musculus (11%) and Microtus socialis (10%). Seventeen insectivores were collected and identified as Crocidura suaveolens (82%) and C. leucodon (18%). Fifty-one hares were collected and identified as Lepus europaeus (57%), Lepus tolai (14%) and Lepus sp. (29%). Three out of 140 explored rodents (1.91%) were positive for F. tularensis, an A. witherbyi, a Mus musculus domesticus, and a Chionomys nivalis collected from Golestan, Khuzestan and Razavi Khorasan provinces, respectively. Two hares (3.92%) were F. tularensis-positive, a L. europaeus from Khuzestan and a Lepus sp. from the Sistan and Baluchistan province. None of the tested animals were positive for Y. pestis.

    Conclusion: This is the first report of direct detection of F. tularensis in mammals of Iran and the first-time observation of the agent in a snow vole, C. nivalis worldwide. The results indicate that tularemia is more widespread in Iran than previously reported including the Northeast and Southwestern parts of the country. Future studies should address genetic characterization of F. tularensis positive DNA samples from Iran to achieve molecular subtyping and rule out assay cross-reactivity with near neighbor Francisella species.

  • 344.
    Murina, Victoriia
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kasari, Marje
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). University of Tartu, Institute of Technology, Tartu, Estonia.
    Atkinson, Gemma C.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest2018Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, nr 7, s. 3753-3763Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Several ATPases in the ATP-binding cassette F (ABCF) family confer resistance to macrolides, lincosamides and streptogramins (MLS) antibiotics. MLS are structurally distinct classes, but inhibit a common target: the peptidyl transferase (PTC) active site of the ribosome. Antibiotic resistance (ARE) ABCFs have recently been shown to operate through direct ribosomal protection, but the mechanistic details of this resistance mechanism are lacking. Using a reconstituted translational system, we dissect the molecular mechanism of Staphylococcus haemolyticus VgaA(LC) and Enterococcus faecalis LsaA on the ribosome. We demonstrate that VgaA(LC) is an NTPase that operates as a molecular machine strictly requiring NTP hydrolysis (not just NTP binding) for antibiotic protection. Moreover, when bound to the ribosome in the NTP-bound form, hydrolytically inactive EQ(2) ABCF ARE mutants inhibit peptidyl transferase activity, suggesting a direct interaction between the ABCF ARE and the PTC. The likely structural candidate responsible for antibiotic displacement by wild type ABCF AREs, and PTC inhibition by the EQ(2) mutant, is the extended inter-ABC domain linker region. Deletion of the linker region renders wild type VgaA(LC) inactive in antibiotic protection and the EQ(2) mutant inactive in PTC inhibition.

  • 345.
    Murina, Victoriia
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kasari, Marje
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Takada, Hiraku
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hinnu, Mariliis
    Kumar Saha, Chayan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grimshaw, James W.
    Seki, Takahiro
    Reith, Michael
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Putrins, Marta
    Tenson, Tanel
    Strahl, Henrik
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). University of Tartu, Institute of Technology, Tartu, Estonia.
    Atkinson, Gemma Catherine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    ABCF ATPases Involved in Protein Synthesis, Ribosome Assembly and Antibiotic Resistance: Structural and Functional Diversification across the Tree of Life2019Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 431, nr 18, s. 3568-3590Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterized VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase center and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance (ARE) factors from other ABCFs have not been explored. To address this, we analyzed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting that they were present in the last common ancestor of both. Surprisingly, currently known ARE ABCFs are not confined to a distinct lineage of the ABCF family tree, suggesting that ARE can readily evolve from other ABCF functions. Our data suggest that there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that the ribosome-binding resistance factor VmlR from Bacillus subtilis is localized to the cytoplasm, ruling out a role in antibiotic efflux.

  • 346. Murphy, Shannon G.
    et al.
    Alvarez, Laura
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Adams, Myfanwy C.
    Liu, Shuning
    Chappie, Joshua S.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Dorr, Tobias
    Endopeptidase Regulation as a Novel Function of the Zur-Dependent Zinc Starvation Response2019Inngår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, nr 1, artikkel-id e02620-18Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 347. Möll, Andrea
    et al.
    Dörr, Tobias
    Alvarez, Laura
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Davis, Brigid M
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Waldor, Matthew K
    A D, D-carboxypeptidase is required for Vibrio cholerae halotolerance2015Inngår i: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, nr 2, s. 527-540Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The biological roles of low molecular weight penicillin-binding proteins (LMW PBP) have been difficult to discern in Gram-negative organisms. In Escherichia coli, mutants lacking these proteins often have no phenotype, and cells lacking all seven LMW PBPs remain viable. In contrast, we report here that Vibrio cholerae lacking DacA-1, a PBP5 homologue, displays slow growth, aberrant morphology and altered peptidoglycan (PG) homeostasis in Luria-Bertani (LB) medium, as well as a profound plating defect. DacA-1 alone among V.cholerae'sLMW PBPs is critical for bacterial growth; mutants lacking the related protein DacA-2 and/or homologues of PBP4 or PBP7 displayed normal growth and morphology. Remarkably, the growth and morphology of the dacA-1 mutant were unimpaired in LB media containing reduced concentrations of NaCl (100mM or less), and also within suckling mice, a model host for the study of cholera pathogenesis. Peptidoglycan from the dacA-1 mutant contained elevated pentapeptide levels in standard and low salt media, and comparative analyses suggest that DacA-1 is V.cholerae's principal DD-carboxypeptidase. The basis for the dacA-1 mutant's halosensitivity is unknown; nonetheless, the mutant's survival in biochemically uncharacterized environments (such as the suckling mouse intestine) can be used as a reporter of low Na+ content.

  • 348.
    Müller, Claudia M
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Åberg, Anna
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Straseviçiene, Jurate
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Emody, Levente
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Balsalobre, Carlos
    Type 1 fimbriae, a colonization factor of uropathogenic Escherichia coli, are controlled by the metabolic sensor CRP-cAMP.2009Inngår i: PLoS pathogens, ISSN 1553-7374, Vol. 5, nr 2, s. e1000303-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues.

  • 349.
    Müller, Daniel C.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kauppi, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Edin, Alicia
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Gylfe, Åsa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sjöstedt, Anders B.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Phospholipid Levels in Blood during Community-Acquired Pneumonia2019Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, nr 5, artikkel-id e0216379Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Phospholipids, major constituents of bilayer cell membranes, are present in large amounts in pulmonary surfactant and play key roles in cell signaling. Here, we aim at finding clinically useful disease markers in community-acquired pneumonia (CAP) using comprehensive phospholipid profiling in blood and modeling of changes between sampling time points. Serum samples from 33 patients hospitalized with CAP were collected at admission, three hours after the start of intravenous antibiotics, Day 1 (at 12–24 h), Day 2 (at 36–48 h), and several weeks after recovery. A profile of 75 phospholipid species including quantification of the bioactive lysophosphatidylcholines (LPCs) was determined using liquid chromatography coupled to time-of-flight mass spectrometry. To control for possible enzymatic degradation of LPCs, serum autotaxin levels were examined. Twenty-two of the 33 patients with a clinical diagnosis of CAP received a laboratory-verified CAP diagnosis by microbial culture or microbial DNA detection by qPCR. All major phospholipid species, especially the LPCs, were pronouncedly decreased in the acute stage of illness. Total and individual LPC concentrations increased shortly after the initiation of antibiotic treatment, concentrations were at their lowest 3h after the initiation, and increased after Day 1. The total LPC concentration increased by a change ratio of 1.6–1.7 between acute illness and Day 2, and by a ratio of 3.7 between acute illness and full disease resolution. Autotaxin levels were low in acute illness and showed little changes over time, contradicting a hypothesis of enzymatic degradation causing the low levels of LPCs. In this sample of patients with CAP, the results demonstrate that LPC concentration changes in serum of patients with CAP closely mirrored the early transition from acute illness to recovery after the initiation of antibiotics. LPCs should be further explored as potential disease stage biomarkers in CAP and for their potential physiological role during recovery.

  • 350. Najdenski, H
    et al.
    Golkocheva-Markova, E
    Kussovski, V
    Vesselinova, A
    Garbom, Sara
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Attenuation and preserved immunogenic potential of Yersinia pseudotuberculosis mutant strains evidenced in oral pig model2009Inngår i: Zoonoses and Public Health, ISSN 1863-1959, E-ISSN 1863-2378, Vol. 56, nr 4, s. 157-168Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Experimental oral infection of pigs with a parental Yersinia pseudotuberculosis strain pIB102, serotype O:3 and two mutant isogenic strains - pIB155,DeltayopK and pIB44,DeltaypkA has been carried out. Clinical findings, microbiological and immunological parameters were examined in dynamics from day 7 to day 60 post-infection (p.i.). All types of infections ran asymptomatically, without hyperthermia, loss of appetite, etc. Experiments on the blood parameters demonstrated a transient leucocytosis with lymphocytosis and monocytosis better expressed after yopK infection. Even though pig is usually known as a reservoir of yersiniae, bacterial colonization was found in mesenterial lymph nodes and tonsils on day 7, respectively 14 p.i. with parental strain, and only in tonsils on day 14 p.i. with both mutant strains. The augmented sensitivity of mutants to the bactericidal effect of leukocytes and blood sera is the characteristic feature of attenuation in their pathogenicity, compared to the parental strain. Comparative in vitro experiments on the immune response and immunostimulating capacity of Y. pseudotuberculosis mutant strains verify their preserved immunogenic potential, predominantly in case of yopK. Hyperplasia and strong activation of the lymph tissue of Peyer's patches, mesenterial lymph nodes, tonsils and spleen of pigs challenged with both mutant strains were proved as immunomorphological rearrangements. The results obtained give the reason to claim that the genetically constructed yopK null mutant strain is significantly attenuated but is still immunogenic and has the potential for a live vaccine carrier strain.

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