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  • 151.
    Espaillat, Akbar
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Forsmo, Oskar
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    El Biari, Khouzaima
    Björk, Rafael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lemaitre, Bruno
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Canada, Francisco Javier
    de Pedro, Miguel A.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Chemometric Analysis of Bacterial Peptidoglycan Reveals Atypical Modifications That Empower the Cell Wall against Predatory Enzymes and Fly Innate Immunity2016In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 138, no 29, p. 9193-9204Article in journal (Refereed)
    Abstract [en]

    Peptidoglycan is a fundamental structure for most bacteria. It contributes to the cell morphology and provides cell wall integrity against environmental insults. While several studies have reported a significant degree of variability in the chemical composition and organization of peptidoglycan in the domain Bacteria, the real diversity of this polymer is far from fully explored. This work exploits rapid ultraperformance liquid chromatography and multivariate data analysis to uncover peptidoglycan chemical diversity in the Class Alphaproteobacteria, a group of Gram negative bacteria that are highly heterogeneous in terms of metabolism, morphology and life-styles. Indeed, chemometric analyses revealed novel peptidoglycan structures conserved in Acetobacteria: amidation at the alpha-(L)-carboxyl of meso-diaminopimelic acid and the presence of muropeptides cross-linked by (1-3) L-Ala-D-(meso)diaminopimelate cross-links. Both structures are growth-controlled modifications that influence sensitivity to Type VI secretion system peptidoglycan endopeptidases and recognition by the Drosophila innate immune system, suggesting relevant roles in the environmental adaptability of these bacteria. Collectively our findings demonstrate the discriminative power of chemometric tools on large cell wall-chromatographic data sets to discover novel peptidoglycan structural properties in bacteria.

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

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

  • 153. Floyd, Kyle A.
    et al.
    Moore, Jessica L.
    Eberly, Allison R.
    Good, James A. D.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Shaffer, Carrie L.
    Zaver, Himesh
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Skaar, Eric P.
    Caprioli, Richard M.
    Hadjifrangiskou, Maria
    Adhesive Fiber Stratification in Uropathogenic Escherichia coli Biofilms Unveils Oxygen-Mediated Control of Type 1 Pili2015In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 11, no 3, article id e1004697Article in journal (Refereed)
    Abstract [en]

    Bacterial biofilms account for a significant number of hospital-acquired infections and complicate treatment options, because bacteria within biofilms are generally more tolerant to antibiotic treatment. This resilience is attributed to transient bacterial subpopulations that arise in response to variations in the microenvironment surrounding the biofilm. Here, we probed the spatial proteome of surface-associated single-species biofilms formed by uropathogenic Escherichia coli (UPEC), the major causative agent of community-acquired and catheter-associated urinary tract infections. We used matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) to analyze the spatial proteome of intact biofilms in situ. MALDI-TOF IMS revealed protein species exhibiting distinct localizations within surface-associated UPEC biofilms, including two adhesive fibers critical for UPEC biofilm formation and virulence: type 1 pili (Fim) localized exclusively to the air-exposed region, while curli amyloid fibers localized to the air-liquid interface. Comparison of cells grown aerobically, fermentatively, or utilizing an alternative terminal electron acceptor showed that the phase-variable fim promoter switched to the "FF" orientation under oxygen-deplete conditions, leading to marked reduction of type 1 pili on the bacterial cell surface. Conversely, S pili whose expression is inversely related to fim expression were upregulated under anoxic conditions. Tethering the fim promoter in the "ON" orientation in anaerobically grown cells only restored type 1 pili production in the presence of an alternative terminal electron acceptor beyond oxygen. Together these data support the presence of at least two regulatory mechanisms controlling fim expression in response to oxygen availability and may contribute to the stratification of extracellular matrix components within the biofilm. MALDI IMS facilitated the discovery of these mechanisms, and we have demonstrated that this technology can be used to interrogate subpopulations within bacterial biofilms.

  • 154.
    Forsell, Joakim
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Genetic subtypes in unicellular intestinal parasites with special focus on Blastocystis2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The development of molecular tools for detection and typing of unicellular intestinal parasites has revealed genetic diversities in species that were previously considered as distinct entities. Of great importance is the genetic distinction found between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar, two morphologically indistinguishable species. Blastocystis sp. is a ubiquitous intestinal parasite with unsettled pathogenicity. Molecular studies of Blastocystis sp. have identified 17 genetic subtypes, named ST1-17. Genetically, these subtypes could be considered as different species, but it is largely unknown what phenotypic or pathogenic differences exist between them. This thesis explores molecular methods for detection and genetic subtyping of unicellular intestinal parasites, with special focus on Blastocystis.

    We found that PCR-based methods were highly sensitive for detection of unicellular intestinal parasites, but could be partially or completely inhibited by substances present in faeces. A sample transport medium containing guanidinium thiocyanate was shown to limit the occurrence of PCR inhibition.

    The prevalence of Blastocystis in Swedish university students was over 40%, which is markedly higher than what was previously estimated. Blastocystis ST3 and ST4 were the two most commonly found Blastocystis subtypes in Sweden, which is similar to results from other European countries.

    Blastocystis sp. and Giardia intestinalis were both commonly detected in Zanzibar, Tanzania, each with a prevalence exceeding 50%. Blastocystis ST1, ST2, and ST3 were common, but ST4 was absent. While G. intestinalis was most common in the ages 2-5 years, the prevalence of Blastocystis increased with increasing age, at least up to young adulthood. We found no statistical association between diarrhoea and Blastocystis sp., specific Blastocystis subtype or G. intestinalis.

    Metagenomic sequencing of faecal samples from Swedes revealed that Blastocystis was associated with high intestinal bacterial genus richness, possibly signifying gastrointestinal health. Blastocystis was also positively associated with the bacterial genera Sporolactobacillus and Candidatus Carsonella, and negatively associated with the genus Bacteroides.

    Blastocystis ST4 was shown to have limited intra-subtype genetic diversity and limited geographic spread. ST4 was also found to be the major driver behind the positive association between Blastocystis and bacterial genus richness and the negative association with Bacteroides.

  • 155.
    Forsell, Joakim
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bengtsson-Palme, Johan
    Department of Infectious Diseases, Institute of Biomedicine, The Sahlgrenska Academy, and Centre for Antibiotic Resistance Research (CARe), University of Gothenburg, Gothenburg, Sweden.
    Angelin, Martin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Evengård, Birgitta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Granlund, Margareta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    The relation between Blastocystis and the intestinal microbiota in Swedish travellers2017In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 17, article id 231Article in journal (Refereed)
    Abstract [en]

    Background: Blastocystis sp. is a unicellular eukaryote that is commonly found in the human intestine. Its ability to cause disease is debated and a subject for ongoing research. In this study, faecal samples from 35 Swedish university students were examined through shotgun metagenomics before and after travel to the Indian peninsula or Central Africa. We aimed at assessing the impact of travel on Blastocystis carriage and seek associations between Blastocystis and the bacterial microbiota.

    Results: We found a prevalence of Blastocystis of 16/35 (46%) before travel and 15/35 (43%) after travel. The two most commonly Blastocystis subtypes (STs) found were ST3 and ST4, accounting for 20 of the 31 samples positive for Blastocystis. No mixed subtype carriage was detected. All ten individuals with a typable ST before and after travel maintained their initial ST. The composition of the gut bacterial community was not significantly different between Blastocystis-carriers and non-carriers. Interestingly, the presence of Blastocystis was accompanied with higher abundances of the bacterial genera Sporolactobacillus and Candidatus Carsonella. Blastocystis carriage was positively associated with high bacterial genus richness, and negatively correlated to the Bacteroides-driven enterotype. These associations were both largely dependent on ST4 – a subtype commonly described from Europe – while the globally prevalent ST3 did not show such significant relationships.

    Conclusions: The high rate of Blastocystis subtype persistence found during travel indicates that long-term carriage of Blastocystis is common. The associations between Blastocystis and the bacterial microbiota found in this study could imply a link between Blastocystis and a healthy microbiota as well as with diets high in vegetables. Whether the associations between Blastocystis and the microbiota are resulting from the presence of Blastocystis, or are a prerequisite for colonization with Blastocystis, are interesting questions for further studies.

  • 156.
    Forsell, Joakim
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Granlund, Margareta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Samuelsson, Linn
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Koskiniemi, Satu
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Edebro, Helen
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Evengård, Birgitta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    High occurrence of Blastocystis sp subtypes 1-3 and Giardia intestinalis assemblage B among patients in Zanzibar, Tanzania2016In: Parasites & Vectors, ISSN 1756-3305, E-ISSN 1756-3305, Vol. 9, article id 370Article in journal (Refereed)
    Abstract [en]

    Background: Blastocystis is a common intestinal parasite with worldwide distribution but the distribution of Blastocystis and its subtypes in East Africa is largely unknown. In this study, we investigate the distribution of Blastocystis subtypes in Zanzibar, Tanzania and report the prevalence of intestinal parasites using both molecular methods and microscopy.

    Methods: Stool samples were collected from both diarrhoeic and non-diarrhoeic outpatients in Zanzibar. In addition to microscopy, real-time PCR for Blastocystis, Entamoeba histolytica and E. dispar, Giardia intestinalis, Cryptosporidium spp., and Dientamoeba fragilis was used. Blastocystis subtypes were determined by a conventional PCR followed by partial sequencing of the SSU-rRNA gene. Genetic assemblages of Giardia were determined by PCR with assemblage specific primers.

    Results: Intestinal parasites were detected in 85 % of the 174 participants, with two or more parasites present in 56 %. Blastocystis sp. and Giardia intestinalis were the most common parasites, identified by PCR in 61 and 53 % of the stool samples respectively, but no correlation between carriage of Blastocystis and Giardia was found. The Blastocystis subtype distribution was ST1 34.0 %, ST2 26.4 %, ST3 25.5 %, ST7 0.9 %, and 13.2 % were positive only by qPCR (non-typable). The Giardia genetic assemblages identified were A 6.5 %, B 85 %, A + B 4.3 %, and non-typable 4.3 %. The detection rate with microscopy was substantially lower than with PCR, 20 % for Blastocystis and 13.8 % for Giardia. The prevalence of Blastocystis increased significantly with age while Giardia was most prevalent in children two to five years old. No correlation between diarrhoea and the identification of Giardia, Blastocystis, or their respective genetic subtypes could be shown and, as a possible indication of parasite load, the mean cycle threshold values in the qPCR for Giardia were equal in diarrhoeic and non-diarrhoeic patients.

    Conclusions: Carriage of intestinal parasites was very common in the studied population in Zanzibar. The most commonly detected parasites, Blastocystis and Giardia, had different age distributions, possibly indicating differences in transmission routes, immunity, and/or other host factors for these two species. In the Blastocystis subtype analysis ST1-3 were common, but ST4, a subtype quite common in Europe, was completely absent, corroborating the geographical differences in subtype distributions previously reported.

  • 157.
    Forsell, Joakim
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Granlund, Margareta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Stensvold, C. R.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Clark, G. C.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Evengård, Birgitta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Subtype analysis of Blastocystis isolates in Swedish patients2012In: European Journal of Clinical Microbiology and Infectious Diseases, ISSN 0934-9723, E-ISSN 1435-4373, Vol. 31, no 7, p. 1689-1696Article in journal (Refereed)
    Abstract [en]

    Blastocystis is a genetically diverse and widespread intestinal parasite of animals and humans with controversial pathogenic potential. At least nine subtypes of Blastocystis have been found in humans. The genetic diversity of Blastocystis was examined in stool samples from 68 patients from the Stockholm area, Sweden. Blastocystis was identified by light microscopy, and subtyped by sequencing the 5'-end of the small subunit ribosomal RNA gene. Five Blastocystis subtypes were identified in the 63 patients whose samples were successfully subtyped: ST1 (15.9%), ST2 (14.3%), ST3 (47.6%), ST4 (20.6%), and ST7 (1.6%). ST3 was more common in males compared to females (P = 0.049). Comparative molecular analysis of Blastocystis sequences revealed intra-subtype variations within the identified subtypes with the exception of ST4. Among ST4 sequences in this study, as well as in the majority of human GenBank sequences, a limited genetic diversity was found compared to what was found among the other common subtypes (ST1, ST2 and ST3). The relative prevalence of ST4 in this study was comparable to the overall distribution of ST4 in European cohorts (16.5%). This contrasts with the sparse reports of ST4 in studies from other continents, which may indicate that the distribution of this subtype is geographically heterogeneous.

  • 158.
    Forsell, Joakim
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Koskiniemi, Satu
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Hedberg, Ida
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Edebro, Helen
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Evengård, Birgitta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Granlund, Margareta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples2015In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 64, p. 1053-1062Article in journal (Refereed)
    Abstract [en]

    Although PCR offers the potential for sensitive detection of parasites:there are several pitfalls for optimal performance, especially when DNA is extracted from a complex sample material such as stool. With the aid of a sensitive inhibitor control in a duplex real-time PCR (qPCR) for identification of Entamoeba histolytica and Entamoeba dispar we have evaluated factors that influenced the performance of the qPCR and have suggested a rationale to be used in the analysis of clinical samples. Pre-PCR processing was found to be of outmost importance for an optimal amplification since inhibitors caused false-negative results when higher amounts of sample were used. Stool sampling with a flocked swab (ESwab, Copan), yielding on average 173 mg, gave positive qPCR results in samples with cysts of E. dispar that were negative in serially diluted stool samples. The degree of inhibition found varied between samples and was not an on-off phenomenon. Even low-grade inhibition, shown as an increase of two cycles in the qPCR for the inhibitor control, could lead to false negativity in samples with low amounts of parasites. Lack of amplification in the qPCR due to inhibition could be overcome by dilution of the extracted DNA by 1/10-1/20. We also describe the use of guanidinium thiocyanate buffer for transport and storage of samples as well as a time-saving semi-automated DNA extraction method in an Arrow instrument (Nordiag) preceded by bead beating.

  • 159.
    Forslund, Anna-Lena
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Salomonsson, Emelie Näslund
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Golovliov, Igor
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kuoppa, Kerstin
    Michell, Stephen
    Titball, Richard
    Oyston, Petra
    Noppa, Laila
    FOI.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis2010In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 10, p. 227-Article in journal (Refereed)
    Abstract [en]

    This suggests that expression and surface localisation of PilA contribute to virulence in the highly virulent type A strain, while PilT was dispensable for virulence in the mouse infection model.

  • 160.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Secretion systems and metabolism in the pathogenic Yersiniae2011In: Stress response in pathogenic bacteria / [ed] Stephen P. Kidd, United Kingdom: CAB International , 2011, 1, p. 185-220Chapter in book (Refereed)
    Abstract [en]

    The genus Yersinia comprises 11 species, three of which have clear etiology for causing human disease (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica). The obligate pathogen Y. pestis is the most infamous of these being the causal agent of plague, a bivalent disease that when left untreated is invariably fatal. The lifecycle of Y. pestis is complex, being dependent on two diverse hosts – the invertebrate flea Xenopsylla cheopis and a mammalian host (usually wild rodents). Although capable of catastrophic consequences, plague in humans is accidental – a consequence of being infected with Y. pestis via the bite of an infected flea that has been forced from its normal rodent host. In brief, the initial stage of disease presents as swollen lymph nodes (buboes) and is termed bubonic plague, whereas the second stage is a more vigorous systemic infection that results in bacterial colonization of multiple tissue organs including the lung. This form of disease is termed pneumonic plague; a highly contagious disease that enables bacteria to rapidly and effectively spread to new hosts via aerosol droplets. In light of this, global health organizations routinely list Y. pestis as a category A biowarfare agent. On the other hand, Y. pseudotuberculosis and Y. enterocolitica are essentially environmental bacteria that are capable of causing spasmodic enteric disease (known as yersiniosis) outbreaks linked to the ingestion of contaminated food or fluids. While these diseases cause gastrointestinal discomfort, they are usually self-limiting and rarely associated with systemic disease. In certain susceptible individuals however, chronic reactive arthritic sequelae can be attributed to these bacteria.

    On account of their ability to cause human disease, a prolific amount of information is available that describes these three human pathogens with respect to their ecology, epidemiology and the pathogenesis of disease. In contrast, very little information is available concerning the additional Yersinia species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, Y. aleksiciae, Y. mexicana and Y. aldovae). However, they might still be clinically relevant given their propensity to harbour a moderate number of genes that encode for products known to be associated with pathogenicity by other non-Yersinia bacteria. It is at least well established that Y. ruckeri is the causative agent of yersiniosis in infected salmonid fish, although the pathogenic mechanisms are comparatively poorly understood.

    In the first part of this chapter, the presence of known and suggested protein secretion mechanisms in the Yersiniae are described. Where appropriate, these individual processes are briefly discussed in the context of their contribution to bacterial pathogenesis to help the reader gain an understanding of their physiological importance within the various unique environments of an infected host. As considerably more is known about the pathogenic mechanisms of human pathogenic Yersinia, these examples will dominate the discussion. Then, focus will turn to the consequences of adaptation of pathogenic Yersinia to their surrounding environment. Where possible, emphasis will be given to the crosstalk between metabolism and the temporal and spatial regulatory control of these important secretion systems. This connection ensures that Yersinia conserve their valuable energy reserves to maximize their survival in stressful environments and only synthesize energetically expensive virulence determinants, such as multi-component secretion systems, when they will have utmost benefit during host infections.

  • 161.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The pathogenic Yersiniae: advances in the understanding of physiology and virulence2013In: Frontiers in Cellular and Infection Microbiology, ISSN 2235-2988, Vol. 3, no 51, p. 2p. 1-2Article, review/survey (Refereed)
  • 162.
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Type III secretion chaperones: a molecular toolkit for all occasions2010In: Handbook of molecular chaperones: roles, structures and mechanisms / [ed] Piero Durante and Leandro Colucci, New York: Nova Science Publishers , 2010, 1, p. 79-148Chapter in book (Refereed)
    Abstract [en]

    Common to many bacteria is the ability to establish a symbiotic relationship or to evade innate immune responses of an animal, plant, fish or insect host. Most often this capacity is mediated by a type III secretion system (T3SS). The function of these complex molecular machines is likened to a syringe-needle injection device that is dedicated to the translocation of effector proteins directly into target eukaryotic cells. Each translocated effector tends to possess a distinct enzymatic activity that aids in subverting host cell signaling for the benefit of the bacterium. Their translocation requires another class of secreted protein – the translocator – which form pores in the target eukaryotic cell plasma membrane through which the effectors may transit to gain entry into the cell interior. Most often, each secreted substrate requires a dedicated small, non-secreted cytoplasmic chaperone for their efficient secretion. Unlike traditional molecular chaperones, these specialized type III chaperones do not assist in protein folding and are not energized by ATP. Controversy still surrounds their primary role; as bodyguards to prevent premature aggregation or as pilots to direct substrate secretion through the correct T3SS. The later is supported by recent evidence that these chaperones can dock directly to the cytoplasmic face of the T3S machinery, possibly serving as a recognition motif for substrate secretion. Added to this functional complexity is their important contribution to system regulation, which can ultimately confer temporal order to substrate secretion. Moreover, some chaperones display a bewildering propensity to interact with several additional T3S-associated proteins – the relevance of which remains uncertain. Structural data has now appeared for several important type III chaperones, either alone or in complex with their cognate substrate. This is proving a fillip in our attempts to understand the mercurial ways in which these versatile proteins operate in nature. It is hoped that this article will provide information on type III chaperone function, as well as highlighting key recent advances in the field. May it also be a testament to the value of continued intense effort in unravelling the mysteries of type III chaperone biology.

  • 163.
    Francis, Matthew S.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Aili, Margareta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wiklund, Magda-Lena
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD2000In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 38, no 1, p. 85-102Article in journal (Refereed)
    Abstract [en]

    The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic alpha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.

  • 164.
    Francis, Matthew S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    YopD of Yersinia pseudotuberculosis is translocated into the cytosol of HeLa epithelial cells: evidence of a structural domain necessary for translocation.1998In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 29, no 3, p. 799-813Article in journal (Refereed)
    Abstract [en]

    Yersinia pseudotuberculosis YopB and YopD proteins are essential for translocation of Yop effector proteins into the target cell cytosol. YopB is suggested to mediate pore formation in the target cell plasma membrane, allowing translocation of Yop effector proteins, although the function of YopD is unclear. To investigate the role in translocation for YopD, a mutant strain in Y. pseudotuberculosis was constructed containing an in frame deletion of essentially the entire yopD gene. As shown recently for the Y. pestis YopD protein, we found that the in vitro low calcium response controlling virulence gene expression was negatively regulated by YopD. This yopD null mutant (YPIII/pIB621) was also non-cytotoxic towards HeLa cell monolayers, supporting the role for YopD in the translocation process. Although other constituents of the Yersinia translocase apparatus (YopB, YopK and YopN) are not translocated into the host cell cytosol, fractionation of infected HeLa cells allowed us to identify the cytosolic localization of YopD by the wild-type strain (YPIII/pIB102), but not by strains defective in either YopD or YopB. YopD was also identified by immunofluorescence in the cytoplasm of HeLa cell monolayers infected with a multiple yop mutant strain (YPIII/pIB29MEKA). These results demonstrate a dual function for YopD in negative regulation of Yop production and Yop effector translocation, including the YopD protein itself. To investigate whether an amphipathic domain near the C-terminus of YopD is involved in the translocation process, a mutant strain (YPIII/pIB155deltaD278-292) was constructed that is devoid of this region. Phenotypically, this small in frame deltayopD278-292 deletion mutant was indistinguishable from the yopD null mutant. The truncated YopD protein and Yop effectors were not translocated into the cytosol of HeLa cell monolayers infected with this mutant. The comparable regulatory and translocation phenotypes displayed by the small in frame deltayopD278-292 deletion and deltayopD null mutants suggest that regulation of Yop synthesis and Yop translocation are intimately coupled. We present an intriguing scenario to the Yersinia infection process that highlights the need for polarized translocation of YopD to specifically establish translocation of Yop effectors. These observations are contrary to previous suggestions that members of the translocase apparatus were not translocated into the host cell cytosol.

  • 165.
    Frisan, Teresa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Cell and Molecular Biology Karolinska Institutet, Stockholm, Sweden.
    Nagy, Noemi
    Chioureas, Dimitrios
    Terol, Marie
    Grasso, Francesca
    Masucci, Maria G.
    A bacterial genotoxin causes virus reactivation and genomic instability in Epstein-Barr virus infected epithelial cells pointing to a role of co-infection in viral oncogenesis2019In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 144, no 1, p. 98-109Article in journal (Refereed)
    Abstract [en]

    We have addressed the role of bacterial co-infection in viral oncogenesis using as model Epstein-Barr virus (EBV), a human herpesvirus that causes lymphoid malignancies and epithelial cancers. Infection of EBV carrying epithelial cells with the common oral pathogenic Gram-negative bacterium Aggregatibacter actinomycetemcomitans (Aa) triggered reactivation of the productive virus cycle. Using isogenic Aa strains that differ in the production of the cytolethal distending toxin (CDT) and purified catalytically active or inactive toxin, we found that the CDT acts via induction of DNA double strand breaks and activation of the Ataxia Telangectasia Mutated (ATM) kinase. Exposure of EBV-negative epithelial cells to the virus in the presence of sub-lethal doses of CDT was accompanied by the accumulation of latently infected cells exhibiting multiple signs of genomic instability. These findings illustrate a scenario where co-infection with certain bacterial species may favor the establishment of a microenvironment conducive to the EBV-induced malignant transformation of epithelial cells.

  • 166. Fulton, Joel
    et al.
    Mazumder, Bismoy
    Whitchurch, Jonathan B.
    Monteiro, Cintia J.
    Collins, Hilary M.
    Chan, Chun M.
    Clemente, Maria P.
    Hernandez-Quiles, Miguel
    Stewart, Elizabeth A.
    Amoaku, Winfried M.
    Moran, Paula M.
    Mongan, Nigel P.
    Persson, Jenny L.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Division of Experimental Cancer Research, Department of Translational Medicine, Lund University, Clinical Research Centre, Malmö, Sweden.
    Ali, Simak
    Heery, David M.
    Heterodimers of photoreceptor-specific nuclear receptor (PNR/NR2E3) and peroxisome proliferator-activated receptor-gamma (PPAR gamma) are disrupted by retinal disease-associated mutations2017In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 8, article id e2677Article in journal (Refereed)
    Abstract [en]

    Photoreceptor-specific nuclear receptor (PNR/NR2E3) and Tailless homolog (TLX/NR2E1) are human orthologs of the NR2E group, a subgroup of phylogenetically related members of the nuclear receptor (NR) superfamily of transcription factors. We assessed the ability of these NRs to form heterodimers with other members of the human NRs representing all major subgroups. The TLX ligand-binding domain (LBD) did not appear to form homodimers or interact directly with any other NR tested. The PNR LBD was able to form homodimers, but also exhibited robust interactions with the LBDs of peroxisome proliferator-activated receptor-gamma (PPAR gamma)/NR1C3 and thyroid hormone receptor b (TRb) TR beta/NR1A2. The binding of PNR to PPAR. was specific for this paralog, as no interaction was observed with the LBDs of PPAR alpha/NR1C1 or PPAR delta/NR1C2. In support of these findings, PPAR. and PNR were found to be co-expressed in human retinal tissue extracts and could be co-immunoprecipitated as a native complex. Selected sequence variants in the PNR LBD associated with human retinopathies, or a mutation in the dimerization region of PPAR. LBD associated with familial partial lipodystrophy type 3, were found to disrupt PNR/PPAR gamma complex formation. Wild-type PNR, but not a PNR309G mutant, was able to repress PPAR gamma-mediated transcription in reporter assays. In summary, our results reveal novel heterodimer interactions in the NR superfamily, suggesting previously unknown functional interactions of PNR with PPAR. and TR beta that have potential importance in retinal development and disease.

  • 167. Fulton, Kelly M.
    et al.
    Zhao, Xigeng
    Petit, Mireille D.
    Kilmury, Sara L. N.
    Wolfraim, Lawrence A.
    House, Robert V.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Twine, Susan M.
    Immunoproteomic analysis of the human antibody response to natural tularemia infection with Type A or Type B strains or LVS vaccination2011In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 301, no 7, p. 591-601Article in journal (Refereed)
    Abstract [en]

    Francisella tularensis is pathogenic for many mammalian species including humans, causing a spectrum of diseases called tularemia. The highly virulent Type A strains have associated mortality rates of up to 60% if inhaled. An attenuated live vaccine strain (LVS) is the only vaccine to show efficacy in humans, but suffers several barriers to licensure, including the absence of a correlate of protection. An immunoproteomics approach was used to survey the repertoire of antibodies in sera from individuals who had contracted tularemia during two outbreaks and individuals from two geographical areas who had been vaccinated with NDBR Lot 11 or Lot 17 LVS. These data showed a large overlap in the antibodies generated in response to tularemia infection or LVS vaccination. A total of seven proteins were observed to be reactive with 60% or more sera from vaccinees and convalescents. A further four proteins were recognised by 30-60% of the sera screened. These proteins have the potential to serve as markers of vaccination or candidates for subunit vaccines. Crown Copyright (C) 2011 Published by Elsevier GmbH. All rights reserved.

  • 168.
    Fällman, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gustavsson, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Yersinia inhibition of phagocytosis2006In: Phagocytosis of bacteria and Bacterial Pathogenicity, Cambridge: Cambridge University Press, 2006, p. 181-218Chapter in book (Other academic)
  • 169.
    Gaca, Anthony O.
    et al.
    Rochester, New York, USA .
    Kudrin, Pavel
    University of Tartu, Institute of Technology, Tartu, Estonia.
    Colomer-Winter, Cristina
    Rochester, New York, USA .
    Beljantseva, Jelena
    University of Tartu, Institute of Technology, Tartu, Estonia.
    Liu, Kuanqing
    Madison, Wisconsin, USA .
    Anderson, Brent
    Madison, Wisconsin, USA .
    Wang, Jue D.
    Madison, Wisconsin, USA .
    Rejman, Dominik
    Prague, Czech Republic.
    Potrykus, Katarzyna
    Gdańsk, Poland; Bethesda, Maryland, USA.
    Cashel, Michael
    Bethesda, Maryland, USA.
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). University of Tartu, Institute of Technology, Tartu, Estonia.
    Lemos, Jose A.
    Rochester, New York, USA .
    From (p)ppGpp to (pp)pGpp: characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis2015In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 197, no 18, p. 2908-2919Article in journal (Refereed)
    Abstract [en]

    The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p) ppGpp. In Enterococcus faecalis, (p) ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (Rel(Ef)) and the small alarmone synthetase (SAS) RelQ(Ef). Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQ(Ef) synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p) ppGpp synthesis from GDP and GTP, RelQ(Ef) also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p) ppGpp. We found that pGpp, like (p) ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQ(Ef) was activated only by ppGpp. Furthermore, enzymatic activity of RelQ(Ef) is insensitive to relacin, a (p) ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p) ppGpp. IMPORTANCE Accumulation of the nucleotide second messengers (p) ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p) ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p) ppGpp synthetase RelQ of Enterococcus faecalis (RelQ(Ef)), we found that, in addition to (p) ppGpp, RelQ(Ef) is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p) ppGpp. These findings provide a new regulatory feature of RelQ(Ef) and suggest that pGpp may represent a new member of the (pp) pGpp family of alarmones.

  • 170.
    Garbom, Sara
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A strategy to identify novel antimicrobial compounds: a bioinformatics and HTS approach2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Bacterial infections are again becoming difficult to treat because the microbes are growing increasingly resistant to the antibiotics in use today. The need for novel antimicrobial compounds is urgent and to achieve this new targets are crucial. In this thesis we present a strategy for identification of such targets via a bioinformatics approach. In our first study we compared proteins with unknown and hypothetical function of the spirochete Treponema pallidum to five other pathogens also causing chronic or persistent infections in humans (Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi and Streptococcus pneumoniae). T. pallidum was used as a starting point for the comparisons since this organism has a condensed genome (1.1 Mb). As we aimed at identifying conserved proteins important for in vivo survival or virulence of the pathogens we reasoned that T. pallidum would have deleted genes not important in the human host. This comparison yielded 17 ORFs conserved in all six pathogens, these were deleted in our model organism, Yersinia pseudotuberculosis, and the virulence of these mutant strains was evaluated in a mouse model of infection. Five genes were found to be essential for virulence and thus constitute possible antimicrobial drug targets.

    We have studied one of these virulence associated genes (vags), vagH, in more detail. Functional and phenotypic analysis revealed that VagH is an S-adenosyl-methionine dependent methyltransferase targeting Release factor 1 and 2 (RF1 and RF2). The analysis also showed that very few genes and proteins were differentially expressed in the vagH mutant compared to wild-type Yersinia. One major finding was that expression of the Type III secretion system effectors, the Yops, were down regulated in a vagH mutant. We dissected this phenotype further and found that the down regulation was due to lowered amounts of the positive regulator LcrF. This can be suppressed either by a deletion of yopD or by over expression of the Ribosomal Recycling Factor (RRF). These results indicate that YopD in addition to its role in translational regulation of the Yops also plays a part in the regulation of LcrF translation. We suggest also that the translation of LcrF is particularly sensitive to the amount of translation competent ribosomes and that one effect of a vagH mutation in Y. pseudotuberculosis is that the number of free ribosomes is reduced; this in turn reduces the amount of LcrF produced thereby causing a down regulation of the T3SS. This down regulation is likely the cause of the attenuated virulence of the vagH mutant.

    Finally, we set up a high throughput screening assay to screen a library of small molecules for compounds with inhibiting the VagH methyltransferase activity. Five such compounds were identified and two were found to inhibit VagH also in bacterial culture. Furthermore, analogues to one of the compounds showed improved inhibitory properties and inhibited the T3SS-dependent cytotoxic response induced by Y. pseudotuberculosis on HeLa cells.

    We have successfully identified five novel targets for antimicrobial compounds and in addition we have discovered a new class of molecules with antimicrobial properties.

  • 171.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Helicobacter pylori: molecular insights into regulation of adhesion properties2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Helicobacter pylori infects the human stomach and triggers an inflammatory response that damages the gastric tissue. This host-pathogen interplay has dire consequences as up to 20 % of infected individuals develop peptic ulcer disease or gastric cancer. Given that half of the world’s population is infected, the number of afflicted humans is staggering and also tells that H. pylori is extremely efficient in spreading and maintaining infection. To enable persistent infection many factors play a role, but one important feature of H. pylori is its impressive ability to adhere to the slimy gastric mucus layer and the underlying epithelial cells. This occurs mainly via the BabA and SabA proteins that bind ABO/Leb- and sLex/sLea-antigens. I have in my thesis studied how these two proteins are utilized and regulated.

    H. pylori transcription is in part controlled by two-component systems (TCSs) that use a sensor protein and a DNA-binding response regulator. We have studied how these systems control sabA and to some extent babA and indeed found a better map of how sabA and babA is regulated at the transcriptional level. We also found that variations in a polynucleotide T-tract located in the sabA promotor could fine-tune SabA expression/ sLex-binding. Thus we have exposed how strict regulation by TCSs combined with stochastic processes together shapes attachment in the bacterial population.

    As the buffering mucus layer is constantly exfoliated, placing H. pylori in bactericidal acid, we hypothesized that low pH should abrogate adhesion. SabA expression was indeed repressed in low pH, however BabA expression remained unaffected. The BabA/ Leb-binding was instead directly reversibly hampered by low pH and the degree of pH sensitivity was strain dependent and encoded in the BabA sequence. We believe that the pH dependent loss of binding is one key factor H. pylori utilizes to maintain persistent infection.

    BabA is divided in generalists that bind ABO antigens and specialists that only bind blood group (bg) O. We co-crystalized BabA bound to these receptors and established the structural basis for generalist vs. specialist discrimination. We furthermore found a disulfide-clasped loop (CL2) in the center of the binding domain crucial for binding. Breaking CL2 with N-Acetylcysteine (NAC) disrupted binding and H. pylori infection mice experiments revealed inflammatory reduction upon NAC-treatment.

    In sum, I have in my thesis dissected how H. pylori controls its adhesive abilities and how intrinsic properties in binding can be exploited for therapeutic purposes.

  • 172.
    Gillenius, Erik
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The adhesive protein invasin of Yersinia pseudotuberculosis induces neutrophil extracellular traps via β1 integrins2015In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 17, no 5, p. 327-336Article in journal (Refereed)
    Abstract [en]

    Yersinia pseudotuberculosis adhesive protein invasin is crucial for the bacteria to cross the intestine epithelium by binding to β1 integrins on M-cells and gaining access to the underlying tissues. After the crossing invasin can bind to β1 integrins on other cell surfaces, however effector proteins delivered by the type III secretion system Y. pseudotuberculosis efficiently inhibit potential immune responses induced by this interaction. Here, we use mutant Y. pseudotuberculosis strains lacking the type III secretion system and additionally invasin-expressing Escherichia coli to analyze neutrophil responses towards invasin. Our data reveals that invasin induces production of reactive oxygen species and release of chromatin into the extracellular milieu, which we confirmed to be neutrophil extracellular traps by immunofluorescence microscopy. This was mediated through β1 integrins and was dependent on both the production of reactive oxygen species and signaling through phosphoinositide 3-kinase. We therefore have gained insight into a potential role of integrins in inflammation and infection clearance that has not previously been described, suggesting that targeting of β1 integrins could be utilized as an adjunctive therapy against yersiniosis.

  • 173. Gnann, John W, Jr
    et al.
    Sköldenberg, Birgit
    Hart, John
    Aurelius, Elisabeth
    Schliamser, Silvia
    Studahl, Marie
    Eriksson, Britt-Marie
    Hanley, Daniel
    Aoki, Fred
    Jackson, Alan C
    Griffiths, Paul
    Miedzinski, Lil
    Hanfelt-Goade, Diane
    Hinthorn, Daniel
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Aksamit, Allen
    Cruz-Flores, Salvador
    Dale, Ilet
    Cloud, Gretchen
    Jester, Penelope
    Whitley, Richard J
    Herpes Simplex Encephalitis: Lack of Clinical Benefit of Long-term Valacyclovir Therapy2015In: Clinical Infectious Diseases, ISSN 1058-4838, E-ISSN 1537-6591, Vol. 61, no 5, p. 683-691Article in journal (Refereed)
    Abstract [en]

    Background: Despite the proven efficacy of acyclovir (ACV) therapy, herpes simplex encephalitis (HSE) continues to cause substantial morbidity and mortality. Among patients with HSE treated with ACV, the mortality rate is approximately 14%-19%. Among survivors, 45%-60% have neuropsychological sequelae at 1 year. Thus, improving therapeutic approaches to HSE remains a high priority. Methods: Following completion of a standard course of intravenous ACV, 87 adult patients with HSE (confirmed by positive polymerase chain reaction [PCR] for herpes simplex virus DNA in cerebrospinal fluid) were randomized to receive either valacyclovir (VACV) 2 g thrice daily (n = 40) or placebo tablets (n = 47) for 90 days (12 tablets of study medication daily). The primary endpoint was survival with no or mild neuropsychological impairment at 12 months, as measured by the Mattis Dementia Rating Scale (MDRS). Logistic regression was utilized to assess factors related to the primary endpoint. Results: The demographic characteristics of the 2 randomization groups were statistically similar with no significant differences in age, sex, or race. At 12 months, there was no significant difference in the MDRS scoring for VACV-treated vs placebo recipients, with 85.7% and 90.2%, respectively, of patients demonstrating no or mild neuropsychological impairment (P = .72). No significant study-related adverse events were encountered in either treatment group. Conclusions: Following standard treatment with intravenous ACV for PCR-confirmed HSE, an additional 3-month course of oral VACV therapy did not provide added benefit as measured by neuropsychological testing 12 months later in a population of relatively high-functioning survivors.

  • 174.
    Golovlev, Igor
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Twine, Susan M.
    Shen, Hua
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Conlan, Wayne
    A Delta clpB Mutant of Francisella tularensis Subspecies holarctica Strain, FSC200, Is a More Effective Live Vaccine than F. tularensis LVS in a Mouse Respiratory Challenge Model of Tularemia2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, p. e78671-Article in journal (Refereed)
    Abstract [en]

    Francisella tularensis subsp. tularensis is a highly virulent pathogen for humans especially if inhaled. Consequently, it is considered to be a potential biothreat agent. An experimental vaccine, F. tularensis live vaccine strain, derived from the less virulent subsp. holarctica, was developed more than 50 years ago, but remains unlicensed. Previously, we developed a novel live vaccine strain, by deleting the chaperonin clpB gene from F. tularensis subsp. tularensis strain, SCHU S4. SCHU S4 Delta clpB was less virulent for mice than LVS and a more effective vaccine against respiratory challenge with wild type SCHU S4. In the current study, we were interested to determine whether a similar mutant on the less virulent subsp. holarctica background would also outperform LVS in terms of safety and efficacy. To this end, clpB was deleted from clinical holarctica strain, FSC200. FSC200 Delta clpB had a significantly higher intranasal LD50 than LVS for BALB/c mice, but replicated to higher numbers at foci of infection after dermal inoculation. Moreover, FSC200 Delta clpB killed SCID mice more rapidly than LVS. However, dermal vaccination of BALB/c mice with the former versus the latter induced greater protection against respiratory challenge with SCHU S4. This increased efficacy was associated with enhanced production of pulmonary IL-17 after SCHU S4 challenge.

  • 175.
    Golovliov, Igor
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Lindgren, Helena
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Eneslätt, Kjell
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Conlan, Wayne
    Mosnier, Amandine
    Henry, Thomas
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    An In Vitro Co-culture Mouse Model Demonstrates Efficient Vaccine-Mediated Control of Francisella tularensis SCHU S4 and Identifies Nitric Oxide as a Predictor of Efficacy2016In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 152Article in journal (Refereed)
    Abstract [en]

    Francisella tularensis is a highly virulent intracellular bacterium and cell-mediated immunity is critical for protection, but mechanisms of protection against highly virulent variants, such as the prototypic strain F. tularensis strain SCHU S4, are poorly understood. To this end, we established a co-culture system, based on splenocytes from naive, or immunized mice and in vitro infected bone marrow-derived macrophages that allowed assessment of mechanisms controlling infection with F. tularensis. We utilized the system to understand why the clpB gene deletion mutant, Delta clpB, of SCHU S4 shows superior efficacy as a vaccine in the mouse model as compared to the existing human vaccine, the live vaccine strain (LVS). Compared to naive splenocytes, Delta clpB-, or LVS-immune splenocytes conferred very significant control of a SCHU S4 infection and the Delta clpB-immune splenocytes were superior to the LVS-immune splenocytes. Cultures with the Delta clpB-immune splenocytes also contained higher levels of IFN-gamma, IL-17, and GM-CSF and nitric oxide, and T cells expressing combinations of IFN-gamma, TNF-alpha, and IL-17, than did cultures with LVS-immune splenocytes. There was strong inverse correlation between bacterial replication and levels of nitrite, an end product of nitric oxide, and essentially no control was observed when BMDM from iNOS(-/-) mice were infected. Collectively, the co-culture model identified a critical role of nitric oxide for protection against a highly virulent strain of F. tularensis.

  • 176.
    Good, James A. D.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Silver, Jim
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nunez-Otero, Carlos
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bahnan, Wael
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Krishnan, K. Syam
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Salin, Olli
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Engström, Patrik
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Svensson, Richard
    Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden; The Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Chemical Biology Consortium Sweden, Uppsala University, SE-751 23 Uppsala, Sweden.
    Artursson, Per
    Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden; The Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Chemical Biology Consortium Sweden, Uppsala University, SE-751 23 Uppsala, Sweden.
    Gylfe, Åsa
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bergström, Sven
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Thiazolino 2-Pyridone Amide Inhibitors of Chlamydia trachomatis Infectivity2016In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 59, no 5, p. 2094-2108Article in journal (Refereed)
    Abstract [en]

    The bacterial pathogen Chlamydia trachomatis is a global health burden currently treated with broad-spectrum antibiotics which disrupt commensal bacteria. We recently identified a compound through phenotypic screening that blocked infectivity of this intracellular pathogen without host cell toxicity (compound 1, KSK 120). Herein, we present the optimization of 1 to a class of thiazolino 2-pyridone amides that are highly efficacious (EC50 <= 100 nM) in attenuating infectivity across multiple serovars of C. trachomatis without host cell toxicity. The lead compound 21a exhibits reduced lipophilicity versus 1 and did not affect the growth or viability of representative commensal flora at 50 mu M. In microscopy studies, a highly active fluorescent analogue 37 localized inside the parasitiphorous inclusion, indicative of a specific targeting of bacterial components. In summary, we present a class of small molecules to enable the development of specific treatments for C. trachomatis.

  • 177. Greber, Urs F.
    et al.
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Wadell, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Benko, Maria
    Kremer, Eric J.
    Adenoviruses: from pathogens to therapeutics: a report on the 10th International Adenovirus Meeting2013In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, no 1, p. 16-23Article in journal (Refereed)
  • 178. Greene, Sarah E.
    et al.
    Pinkner, Jerome S.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dodson, Karen W.
    Shaffer, Carrie L.
    Conover, Matt S.
    Livny, Jonathan
    Hadjifrangiskou, Maria
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hultgren, Scott J.
    Pilicide ec240 Disrupts Virulence Circuits in Uropathogenic Escherichia coli2014In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 5, no 6, p. UNSP e02038-Article in journal (Refereed)
    Abstract [en]

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

  • 179.
    Gripenland, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Regulatory roles of two small RNAs in the human pathogen Listeria monocytogenes and the evaluation of an alternative infection model2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Listeriosis is a potentially lethal disease caused by the Gram-positive facultative intracellular pathogen Listeria monocytogenes (L.m.). L.m. is found ubiquitously in the environment and infects humans via ingestion of contaminated food. Contaminated products are usually derived from ruminants and involve dairy products and different kinds of processed meat. Listeriosis is a potential lifethreatening disease with a total mortality rate of 20-30 %. The development of listeriosis may lead to meningitis and septicemia or other invasive diseases. Pregnant women are of increased risk of developing listeriosis and a materno-fetal infection commonly lead to spontaneous abortion or still-birth.

    Regulation of gene expression, and specifically virulence gene expression, is essential for pathogenic bacteria to be equipped for handling counteractions from the host as well as thriving in the often hostile environment. In pathogenic Listeria, virulence gene expression is under the control of the global virulence gene regulator PrfA. The expression of prfA is highly regulated at the transcriptional, post-transcriptional and post- translational level. We have identified a novel type of post-transcriptional regulation of prfA-mRNA by a trans-acting riboswitch element (SreA). By binding to the leader region of prfA-mRNA, SreA negatively regulates the expression of prfA. To our knowledge, this is the first description of a cis-acting riboswitch capable of functioning as a small RNA in trans, regulating targets on distant sites.

    To date, there have been around 100 sRNAs identified in Listeria monocytogenes, but experimental data is still limited. We have characterized a blood induced sRNA, Rli38, which is important for full virulence during oral infection of mice. Our data suggest that Rli38 regulates the expression of at least two proteins; OppD (Oligopeptide transport protein) and IsdG (heme degrading monooxygenase). Both of these proteins have been implicated in the infectious cycle of L.m. We speculate that the virulence phenotype of an ∆rli38 mutant is possibly mediated through the effect of these proteins.

    L.m. is a complex pathogen, able to infect and replicate in a variety of organs and cause several distinctive forms of disease. These qualities of L.m. generate difficulties in simulating human listeriosis in animal models, as entailed by the multitude of models used in the field. In this work, we have evaluated the use of an alternative animal model in studying listeriosis. Our results describe the differentiated virulence potential of wildtype bacteria and a ∆prfA mutant strain in the chicken embryo by live/death screening and organ colonization. Large differences in mean time to death were found between wild-type and the ∆prfA strain and ∆prfA cells displayed a considerable defect in colonization of the embryonal liver. The results presented in this thesis show that the chicken embryo infection model is a valuable and convenient tool in studying end-outcome and organ colonization of Listeria monocytogenes.

    Taken together, this thesis describes the characterization of two previously unknown sRNAs in the human pathogen Listeria monocytogenes and the use of an alternative infection model for simulating listeriosis.

  • 180.
    Gripenland, Jonas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Dussurget, Olivier
    Institut Pasteur, Paris, France.
    Sesto, Nina
    Institut Pasteur, Paris, France..
    Byström, Jonas
    Queen Mary, University of London, London, Great Britain..
    Vaitkevicius, Karolis
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bécavin, Christoph
    Institut Pasteur, Paris, France..
    Cossart, Pascale
    Institut Pasteur, Paris, France..
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rli38, a novel stress induced small RNA required for Listeria monocytogenes virulenceManuscript (preprint) (Other academic)
  • 181.
    Gripenland, Jonas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Netterling, Sakura
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Loh, Edmund
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Tiensuu, Teresa
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Toledo-Arana, Alejandro
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    RNAs: regulators of bacterial virulence2010In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 8, no 12, p. 857-866Article in journal (Refereed)
    Abstract [en]

    RNA-based pathways that regulate protein expression are much more widespread than previously thought. Regulatory RNAs, including 5' and 3' untranslated regions next to the coding sequence, cis-acting antisense RNAs and trans-acting small non-coding RNAs, are effective regulatory molecules that can influence protein expression and function in response to external cues such as temperature, pH and levels of metabolites. This Review discusses the mechanisms by which these regulatory RNAs, together with accessory proteins such as RNases, control the fate of mRNAs and proteins and how this regulation influences virulence in pathogenic bacteria.

  • 182.
    Gustafsson, Dan J
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Andersson, Emma K
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Hu, Yan-Ling
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Marttila, Marko
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Lindman, Kristina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Strand, Mårten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Wang, Li
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Mei, Ya-Fang
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Adenovirus 11p downregulates CD46 early in infection2010In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 405, no 2, p. 474-482Article in journal (Refereed)
    Abstract [en]

    Adenovirus 11 prototype (Ad11p), belonging to species B, uses CD46 as an attachment receptor. CD46, a complement regulatory molecule, is expressed on all human nucleated cells. We show here that Ad11p virions downregulate CD46 on the surface of K562 cells as early as 5min p.i. Specific binding to CD46 by the Ad11p fiber knob was required to mediate downregulation. The complement regulatory factors CD55 and CD59 were also reduced to a significant extent as a consequence of Ad11p binding to K562 cells. In contrast, binding of Ad7p did not result in downregulation of CD46 early in infection. Thus, the presumed interaction between Ad7p and CD46 did not have the same consequences as the Ad11p-CD46 interaction, the latter virus (Ad11p) being a promising gene therapy vector candidate. These findings may lead to a better understanding of the pathogenesis of species B adenovirus infections.

  • 183.
    Gustavsson, Anna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Armulik, Annika
    Brakebusch, Cord
    Fässler, Reinhard
    Johansson, Staffan
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Role of the β1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia2002In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 115, no 13, p. 2669-2678Article in journal (Refereed)
    Abstract [en]

    Invasin of Yersinia pseudotuberculosis binds to beta1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the beta1-integrin-mediated internalization of Yersinia, a beta1-integrin-deficient cell line, GD25, transfected with wild-type beta1A, beta1B or different mutants of the beta1A subunit was used. Both beta1A and beta1B bound to invasin-expressing bacteria, but only beta1A was able to mediate internalization of the bacteria. The cytoplasmic region of beta1A, differing from beta1B, contains two NPXY motifs surrounding a double threonine site. Exchanging the tyrosines of the two NPXYs to phenylalanines did not inhibit the uptake, whereas a marked reduction was seen when the first tyrosine (Y783) was exchanged to alanine. A similar reduction was seen when the two nearby threonines (TT788-9) were exchanged with alanines. It was also noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex structures was unable to mediate uptake of invasin-expressing bacteria.

  • 184.
    Habayeb, Mazen
    Molecular Biology.
    Nora virus as a model to study persistent infection in Drosophila melanogaster2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Drosophila melanogaster has been widely used as a model organism to study the immune responses against bacteria, fungi, parasites and viruses. Here, I present a D. melanogaster virus as a model to study persistent virus infections. I have discovered and characterized the Nora virus, a small picorna-like RNA virus able to persistently infect D. melanogaster. The Nora virus genome encodes four open reading frames; a feature not present in other picorna-like viruses. The Nora virus is not closely related to any other virus family, but rather is the first virus in a new family of picorna-like viruses. The major replicative proteins of this virus are encoded in the second open reading frame and the capsid proteins are encoded in the fourth open reading frame. The sequence of the capsid proteins are not obviously related to any other previously described protein. By looking at expressed sequence tags (EST) projects, we identified an EST sequence from the parasitic wasp Nasonia which appears to encode proteins that have sequence similarity to the Nora virus capsid proteins. I have shown that the Nora virus persists in the fly intestine however I did not observe serious pathological effects in the infected flies. The virus is shed through feces and the transmission occurs horizontally via the ingestion of virus-contaminated food. Moreover, I observed variability in the viral titers among single flies of the same infected stock. Some flies are able to clear the Nora virus but not others and this phenomenon seems to be titer-dependent. Surprisingly, none of the known Drosophila antiviral responses play a role against the Nora virus. In conclusion, my work shows that studying the Nora virus interaction with the Drosophila immune system can lead to new findings on viral persistence mechanisms of RNA viruses and of Drosophila viral innate immunity.

  • 185.
    Haubek, Dorte
    et al.
    Århus universitet.
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Pathogenicity of the highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans and its geographic dissemination and role in aggressive periodontitis2014In: Journal of Oral Microbiology, ISSN 2000-2297, E-ISSN 2000-2297, Vol. 6, article id 23980Article in journal (Refereed)
    Abstract [en]

    For decades, Aggregatibacter actinomycetemcomitans has been associated with aggressive forms of periodontitis in adolescents. In the middle of the 1990s, a specific JP2 clone of A. actinomycetemcomitans, belonging to the cluster of serotype b strains of A. actinomycetemcomitans and having a number of other characteristics, was found to be strongly associated with aggressive forms of periodontitis, particularly in North Africa. Although several longitudinal studies still point to the bacterial species, A. actinomycetemcomitans as a risk factor of aggressive periodontitis, it is now also widely accepted that the highly leukotoxic JP2 clone of A. actinomycetemcomitans is implicated in rapidly progressing forms of aggressive periodontitis. The JP2 clone strains are highly prevalent in human populations living in Northern and Western parts of Africa. These strains are also prevalent in geographically widespread populations that have originated from the Northwest Africa. Only sporadic signs of a dissemination of the JP2 clone strains to non-African populations have been found despite Africans living geographically widespread for hundreds of years. It remains an unanswered question if a particular host tropism exists as a possible explanation for the frequent colonization of the Northwest African population with the JP2 clone. Two exotoxins of A. actinomycetemcomitans are known, leukotoxin (LtxA) and cytolethal distending toxin (Cdt). LtxA is able to kill human immune cells, and Cdt can block cell cycle progression in eukaryotic cells and thus induce cell cycle arrest. Whereas the leukotoxin production is enhanced in JP2 clone strains thus increasing the virulence potential of A. actinomycetemcomitans, it has not been possible so far to demonstrate such a role for Cdt. Lines of evidence have led to the understanding of the highly leukotoxic JP2 clone of A. actinomycetemcomitans as an aetiological factor of aggressive periodontitis. Patients, who are colonized with the JP2 clone, are likely to share this clone with several family members because the clone is transmitted through close contacts. This is a challenge to the clinicians. The patients need intense monitoring of their periodontal status as the risk for developing severely progressing periodontal lesions are relatively high. Furthermore, timely periodontal treatment, in some cases including periodontal surgery supplemented by the use of antibiotics, is warranted. Preferably, periodontal attachment loss should be prevented by early detection of the JP2 clone of A. actinomycetemcomitans by microbial diagnostic testing and/or by preventive means.

  • 186.
    Hauryliuk, Vasili
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). University of Tartu, Institute of Technology, Tartu, Estonia.
    Atkinson, Gemma C.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Small Alarmone Synthetases as novel bacterial RNA-binding proteins2017In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 14, no 12, p. 1695-1699Article, review/survey (Refereed)
    Abstract [en]

    The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, antibiotic tolerance and pathogenicity. We have recently shown that the Small Alarmone Synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis has an RNA-binding activity (Beljantseva et al. 2017). RelQ's activities as an enzyme and as an RNA-binding protein are mutually incompatible: binding of single-stranded RNA potently inhibits (p)ppGpp synthesis in a sequence-specific manner, and RelQ's enzymatic activity destabilizes the RNA: RelQ complex. RelQ's allosteric regulator, pppGpp, destabilizes RNA binding and activates RelQ's enzymatic activity. Since SAS enzymes are widely distributed in bacteria, and, as has been discovered recently, are also mobilized by phages (Dedrick et al. 2017), RNA binding to SASs could be a widespread mechanism. The initial discovery raises numerous questions regarding RNA-binding function of the SAS enzymes: What is the molecular mechanism underlying the incompatibility of RNA: SAS complex formation with pppGpp binding and (p)ppGpp synthesis? What are the RNA targets in living cells? What is the regulatory output of the system - (p)ppGpp synthesis, modulation of RNA structure and function, or both?

  • 187.
    Hedberg, Maria
    et al.
    Umeå University, Faculty of Medicine, Odontology, Oral Microbiology.
    Nord, Carl Erik
    Karoloinska Institute,.
    Anaerobic bacteria2008In: Antimicrobial therapy and vaccines: volume I: Microbes, New York: Apple Trees Production, LLC , 2008, 3rdChapter in book (Other academic)
  • 188.
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Helicobacter pylori: multitalented adaptation of binding properties2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Helicobacter pylori infects and persistently colonizes the stomach, which results in gastritis and in some individuals peptic ulcer disease or gastric cancer. Adherence of H. pylori to the epithelium is an important factor for development of disease. Attachment is mediated by the adhesins BabA and SabA that binds the ABO/Leb blood group antigens and sialylated glycoconjugates respectively.  High-affinity attachment could be anticipated to be of disadvantage for H. pylori because epithelial cells have a fast turnover rate and the dislocated and shed epithelial cells would carry attached bacteria to the acidic gastric juice in the lumen. However, here we describe that H. pylori manage to adapt to this innate clearance mechanism by unique acid regulatory binding properties of its adhesins. We propose that pH regulated binding properties enable bacteria to detachment from host cells for chemotactic guided motility and successful return to the more neutral epithelium for a fresh restart of the infectious cycle. By comparison of BabA from different stomach loci we identified amino acid key position for acid regulated binding activity.

    Previous studies found lower prevalence of Leb-binding among H. pylori isolates from southern Europe compared to Sweden. Here we tested if the reduced prevalence of Leb-binding could be explained by a novel binding mode; in among Spanish strains, we identified S812 that demonstrates preference for multivalent binding to ABO antigens in glycolipids; we found that 812 BabA had drifted in its preferred binding epitope away from the consensus a1,2fucosylation and towards the blood group A and B derivatives. Such epitope drift might in particular optimize binding to ABO antigens in densely packed lipid rafts.

    In parallel, we studied the influence of BabA for disease progression by an inventory of gastric biopsies. BabA correlated both with the oncoprotein CagA, the VacAs1 toxin and, in addition, to severe disease progression. We further correlate BabA expression with positive secretor phenotype and stronger adhesion of H. pylori in vitro.

    For functional adherence studies in vitro, we constructed a recombinant Leb-expressing cell lineage that supports BabA mediated H. pylori attachment.

  • 189.
    Henriksson, Sara
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mendez, Melissa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bugaytsova, Jeanna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nordén, Jenny
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Berg, Douglas E
    Blixt, Ola
    Teneberg, Susann
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Clinical isolates of Helicobacter pylori demonstrates alternative BabA-mediated adherence to human gastric mucosaManuscript (preprint) (Other academic)
    Abstract [en]

    Helicobacter pylori infection is life-long and can cause peptic ulcer disease and gastric cancer. The H. pylori BabA adhesin binds the ABO/Leb blood group (bg) antigens (Leb), which mediates attachment to the gastric epithelium. The prevalence of ABO binding is high worldwide and also in northern Europe. However, prevalence is reduced by 50% in Germany and is further reduced in Spain and Portugal. An inventory of strains from different European populations resulted in strains with high level of BabA expression but very little or no binding to Leb. The majority of such strains could not bind to human gastric mucosa in vitro. We further characterized a Spanish isolates, strain 812, that binds only weakly to soluble Leb-conjugate but still adheres firmly to gastric mucosa indicative of that it might bind to an alternative set of receptor. Receptor analysis by glycan arrays revealed higher binding of strain 812 to ALeb and Bleb glycans than to Leb, indicating that BabA from strain 812 has shifted its binding epitope somewhat away from the central Fuca1.2Gal bg domain and closer to the very terminal bg A and B determinants, i.e. GalNAca1.3Gal (bgA) or the Gala1.3Gal (bgB). By a colony screening approach we identified a subpopulation of 812 clones adapted for stronger Leb binding. Such affinity shifts comes from replacement of distinguishing amino acids by mechanisms of recombination with a BabA-related outer membrane protein.

  • 190. Hernandez, Jorge
    et al.
    Bonnedahl, Jonas
    Eliasson, Ingvar
    Wallensten, Anders
    Comstedt, Pär
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Granholm, Susanne
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Melhus, Asa
    Olsen, Björn
    Drobni, Mirva
    Globally disseminated human pathogenic Escherichia coli of O25b-ST131 clone, harbouring bla(CTX-M-15), found in Glaucous-winged gull at remote Commander Islands, Russia2010In: Environmental Microbiology Reports, ISSN 1758-2229, E-ISSN 1758-2229, Vol. 2, no 2, p. 329-332Article in journal (Refereed)
    Abstract [en]

    With focus on environmental dissemination of antibiotic resistance among clinically relevant bacteria, such as the rising ESBL type of resistance among Escherichia coli, we investigated antibiotic resistance levels in wild birds in the Commander Islands and Kamchatka, Russia. Despite overall low resistance levels in randomly selected E. coli (one from each sample), we found multi-resistant ESBL-producing E. coli harbouring bla(CTX-M-14) and bla(CTX-M-15) using selective screening. Among these multi-resistant ESBL-producing E. coli we found one bla(CTX-M-15) harbouring strain belonging to the O25b-ST131 clone, recognized for its clonal disseminated worldwide as a human pathogen. The potential in acquiring resistant bacteria of human origin, especially highly pathogenic clones, as well as downstream consequences of that, should not be underestimated but further investigated.

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

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

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

  • 192.
    Hessle, Pontus
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    IgdE homologues - a potential novel immunomodulating enzyme family of Streptococci: Proteases of high specificity linking Streptococcal species to their host organisms2016Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
  • 193.
    Hjalmarsson, Karin J.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli1983In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 258, no 2, p. 1343-1351Article in journal (Refereed)
    Abstract [en]

    The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme. The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is probably a single polypeptide chain of molecular weight 32,000. The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro. The enzyme has a pI of 5.2. The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine. S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM. Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively. Ammonium ion is not required and is inhibitory at concentrations above 0.15 M. Magnesium ion inhibited the activity at a concentration as low as 2 mM. Sodium and potassium ions were inhibitory at concentrations above 0.1 M. The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1. It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.

  • 194. Hmelo, Laura R.
    et al.
    Borlee, Bradley R.
    Almblad, Henrik
    Love, Michelle E.
    Randall, Trevor E.
    Tseng, Boo Shan
    Lin, Chuyang
    Irie, Yasuhiko
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Storek, Kelly M.
    Yang, Jaeun Jane
    Siehnel, Richard J.
    Howell, P. Lynne
    Singh, Pradeep K.
    Tolker-Nielsen, Tim
    Parsek, Matthew R.
    Schweizer, Herbert P.
    Harrison, Joe J.
    Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange2015In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 10, no 11, p. 1820-1841Article in journal (Refereed)
    Abstract [en]

    Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNANA. The entire procedure requires similar to 2 weeks.

  • 195.
    Holmberg, Sandra
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    The Role of Neutrophils in Anaerobic Environments: Periodontitis as Infectious Model2016Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
  • 196.
    Holmström, Anna
    et al.
    Swedish Defence Research Agency, Division of CBRN Defence and Security, SE-901 82 Umeå, Sweden.
    Olsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cherepanov, Peter
    Maier, Elke
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Pettersson, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Benz, Roland
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Forsberg, Åke
    LcrV is a channel size-determining component of the Yop effector translocon of Yersinia2001In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 39, no 3, p. 620-632Article in journal (Refereed)
    Abstract [en]

    Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact‐dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127–195 of LcrV and to amino acids 106–173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size‐determining structural component of the Yop translocon.

  • 197.
    Honn, Marie
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    The oxidative stress response of Francisella tularensis2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Francisella tularensis is capable of infecting numerous cell types, including professional phagocytes. Upon phagocytosis, F. tularensis resides within the phagosome before escaping into the cytosol to replicate. Phagocytes constitute a hostile environment rich in ROS, which are employed as a means of killing pathogens. ROS interact with and disrupt the function of vital molecules such as DNA, proteins and bacterial structures. Iron potentiates the danger of ROS through the Fenton reaction where ferrous iron reduces H2O2 causing the formation of highly reactive hydroxyl radicals and anions. Low levels of ROS are formed during normal aerobic metabolism and pathogens thus have a need for defense mechanisms to handle the ever present levels of ROS but even more so to combat the onslaught of ROS experienced within a host.

    This thesis was focused on the investigation of the iron status and oxidative stress response of F. tularensis; thereby identifying key players controlling the bacterial iron content, its adaptation to oxygen-rich environments and defense against ROS.

    We identified subspecies-specific differences in iron content, where F. tularensis subsp. tularensis was found to contain significantly less iron than strains of subsp. holarctica. The reduced iron content resulted in an increased tolerance to H2O2, despite simultaneously causing a decrease in the activity of catalase - the iron-dependent enzyme responsible for degrading H2O2 in F. tularensis. This strongly suggests that the restricted iron uptake and storage by subsp. tularensis strains is beneficial by rendering the bacteria less susceptible to H2O2, thereby evading the toxic effects of the iron-driven Fenton reaction. This evasion is likely to be an important part of the higher virulence displayed by subsp. tularensis as compared to subsp. holarctica.

    We further identified that the global regulator, MglA, is important for the adaptation of LVS to oxygen-rich environments. Deletion of mglA from LVS resulted in a mutant, ΔmglA, with impaired defense to oxidative stress, as manifested by an inability to grow to wild-type levels under aerobic conditions, an accumulation of proteins with oxidative damage, a suppressed expression of iron-uptake related genes, an increased catalase activity, and an increased tolerance to H2O2. This phenotype was reversed in a microaerobic environment. We therefore conclude that MglA is an important factor for the defense of LVS to oxidative damage under aerobic conditions and speculate that MglA is of greatest importance in oxygen-rich foci.

    We also studied the role of OxyR in LVS by creating a ΔoxyR mutant as well as a double mutant, ΔoxyR/ΔkatG. The in vitro response of these mutants, as well as of ΔkatG, to defined ROS was assessed using H2O2, the O2- generating agent paraquat, and the ONOO- generator SIN-1. ΔoxyR was more susceptible to all ROS than LVS as was ΔkatG, with the exception of O2- Strikingly, ΔoxyR/ΔkatG was significantly more susceptible to all ROS tested compared to either single deletion mutant. LVS, ΔoxyR and ΔkatG replicated efficiently in bone marrow-derived macrophages whereas ΔoxyRkatG showed no replication. In mice, the ΔoxyR mutant displayed impaired replication in liver, but intact replication vs. LVS in spleen. Collectively, our results demonstrate an important role of OxyR in the oxidative stress response and virulence of F. tularensis, and further reveal overlapping roles of OxyR and catalase in the defense against ROS. The results thus shed new light on the complexity of ROS defense in F. tularensis.

  • 198.
    Honn, Marie
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). MIMS.
    Lindgren, Helena
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). MIMS.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). MIMS.
    OxyR: an important regulator of the oxidative stress response of Francisella tularensis LVSManuscript (preprint) (Other academic)
    Abstract [en]

    An essential part of the oxidative stress response in Gram-negative bacteria is the H2O2-activated transcriptional regulator OxyR. Although it is much studied in common bacteria such as Escherichia coli, little is known about it about its role in the facultative intracellular bacterium Francisella tularensis. Here, we studied the role of OxyR in the strain F. tularensis LVS. We studied the effects of ROS on the LVS, ΔoxyR, ΔkatG and ΔoxyRkatG. The latter mutants lack expression of catalase, the function of which is important for degradation of reactive oxygen species, especially H2O2. The in vitro response of these mutants to defined ROS was assessed using H2O2, the O2- generating agent paraquat, and the ONOO- generator SIN-1. ΔoxyR was more susceptible to all ROS than LVS, as was ΔkatG, with the exception of O2-. Strikingly, ΔoxyR/ΔkatG was significantly more susceptible to all ROS tested compared to either single deletion mutant. Also the catalase activity was assessed and whereas LVS significantly upregulated the enzymatic activity in response to H2O2, this did not occur in the ΔoxyR mutant. Gene expression by ΔoxyR was compared to LVS and it was found that there was down-regulation of fur, katG, sodB, sodC, furA, and in particular of ahpC, in the mutant. LVS, ΔoxyR and ΔkatG replicated efficiently in bone marrow-derived macrophages, whereas ΔoxyRkatG showed no replication. In mice, the ΔoxyR mutant displayed impaired replication in liver but intact replication vs. LVS in spleen. Collectively, our results demonstrate an important role of OxyR in the oxidative stress response and virulence of F. tularensis. The combined mutation of ΔoxyRkatG led to severely impaired handling of oxidative stress.

  • 199.
    Horcajo, Pilar
    et al.
    Centro de Biología Molecular ‘‘Severo Ochoa,’’ Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain.
    de Pedro, Miguel A
    Centro de Biología Molecular ‘‘Severo Ochoa,’’ Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain.
    Cava, Felipe
    Centro de Biología Molecular ‘‘Severo Ochoa,’’ Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain.
    Peptidoglycan plasticity in bacteria: stress-induced peptidoglycan editing by noncanonical D-amino acids2012In: Microbial Drug Resistance, ISSN 1076-6294, E-ISSN 1931-8448, Vol. 18, no 3, p. 306-313Article in journal (Refereed)
    Abstract [en]

    It has been generally assumed that the role of D-amino acids in bacterial physiology is rather limited. However, recent new evidence demonstrated that millimolar concentrations of noncanonical D-amino acids are synthesized and released to the environment by bacteria from diverse phyla. These D-amino acids help bacteria adapt to environmental challenges by modulating the structure and composition of the peptidoglycan (PG). This regulation, which appears to be well conserved among bacterial species, occurs principally through the incorporation of the D-amino acids into the terminus of the peptide moiety of muropeptides. These findings revived interest in studies investigating D-amino acids as an exciting and trendy topic in current microbiology, which considers them as fundamental players in different aspects of bacterial physiology. In this article, we provide an overview of the origins of research on the effects of D-amino acids in the biology of bacterial cell walls, including their recent implication as key factors for stress-associated PG remodeling.

  • 200.
    Hosseinzadeh, Ava
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Modulation of neutrophil extracellular trap formation in health and disease2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The critical prompt innate immune response is highly built upon the influx of neutrophils from the blood stream to the site of infection. In the battlefield, neutrophils sense pathogen-associated molecular patterns (PAMPs) through their pattern-recognition receptors (PRRs) to launch a number of responses with the goal to defeat the invading pathogen. Neutrophils’ wide spectrum of responses ranges from reactive oxygen species production (ROS), phagocytosis, cytokine and chemokine secretion, and neutrophil extracellular trap (NET) formation. The NET scaffold is composed of nuclear chromatin which is armed with antimicrobial proteins. DNA traps are able to ensnare and kill microbes in the extracellular space and NET release concurs with cell death of the neutrophil. An increasing body of literature describes that NETs impose deleterious effects on the host itself in addition to their antimicrobial activity. These hazardous effects mainly stem from pro-inflammatory and tissue-destructive activity of NETs. These two diverse outcomes of NETs result in a series of effects on both host and pathogen. Therefore, it seems rational that NET formation is tightly regulated and not happening spontaneously. The opportunistic fungal pathogen Candida albicans captured and killed by NETs. This fungus has the remarkable ability to grow as budding yeast or as filamentous hyphae, and reversibly alternate between these morphotypes. Hyphae are the tissue-destructive, invasive and pro-inflammatory form of C. albicans, whereas yeast is the proliferative, non-invasive form. Hence, it is important to find out how neutrophils discriminate between distinct growth forms of C. albicans and how NET release is regulated in this regard.

    To assess neutrophils responses towards each growth form of C. albicans, the mere ratio of each fungal morphotypes is an insufficient measure to describe comparable amounts used in infection experiments; we therefore used dry mass of fungal cells to serve as a common denominator for amounts of fungal cells with different morphotypes. As assessment of dry mass is laborious, we developed a quick correlative method, which quantified fungal metabolic activity corresponding to the actual dry mass. We applied this method in consecutive studies investigating the neutrophil responses specific to different morphotypes of C. albicans.

    Positive and negative regulators of NET formation were investigated for this thesis in a mechanistic fashion. To identify how NET release is negatively regulated during C. albicans infection we focused on anti-inflammatory receptors on neutrophils. We observed that adenosine signals via adenosine receptor reduces the amount of NETs exclusively in response to C. albicans hyphae, the invasive, pro-inflammatory form. We identified adenosine receptor A3 as the responsible receptor suggesting that targeting of adenosine A3 would be a promising approach to control invasive fungal infection, since particularly during immune reconstitution invasive mycoses are frequently accompanied by hyperinflammation which additionally worsens the patient’s state.

    As unbalanced inflammation is harmful to the host, a situation reflected in autoimmune diseases, such as systemic lupus erythematosus, we aimed to find molecules, which are able to inhibit NET formation. Thus, we introduced the non-toxic agent tempol’’. During ROS-depended stimulation of NET formation via C. albicans and phorbol esters, the stable redox-cycling nitroxide tempol efficiently blocked NET induction. We therefore proposed tempol as a potential treatment during inflammatory disorders where NET formation is out of balance. In quest for positive regulators of NET formation we found the major addictive component of tobacco and electronic cigarettes, nicotine, as compelling direct inducer of NET release. Interestingly, nicotine is associated with exacerbated inflammatory diseases exerting its pro-inflammatory activity via acetylcholine receptor by targeting protein kinase B (known as Akt) activation with no effect on NADPH oxidase complex in a ROS independent fashion. In consideration of neutrophils role in smoking-related diseases we propose targeting Akt could lower the undesirable effect of NET. 

    In conclusion, this thesis identified new modulators of NET formation in response to fungal infection and more broadly to other NET-inducing stimuli, which might have implications in forthcoming therapies.

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