Download or read book Virulence Gene Regulation in Francisella Tularensis written by Anna Brotcke Zumsteg and published by . This book was released on 2008 with total page 250 pages. Available in PDF, EPUB and Kindle. Book excerpt: The MglA regulon was identified by comparing the transcriptional profiles of wild-type to mglA mutant bacteria. There are 102 MglA-regulated genes, the majority of which were positively regulated, including all of the FPI genes. Among these, we identified 5 novel MglA-regulated genes that contribute to replication in macrophages and virulence in mice. In addition, our gene expression data suggested that additional F. tularensis regulatory elements were involved in the expression of virulence genes.
Download or read book Transcription Regulation of Virulence Gene Expression in Francisella Tularensis written by James Carl Charity and published by . This book was released on 2010 with total page 346 pages. Available in PDF, EPUB and Kindle. Book excerpt: F. tularensis also contains two additional critical regulators of virulence gene expression: ppGpp (a small molecule that signals nutrient stress) and PigR (a putative DNA-binding protein). Genome-wide transcription analyses indicated that MglA, SspA, PigR, and ppGpp regulate the same set of genes. Bacterial two-hybrid assays showed that MglA, SspA, and PigR form a three-protein complex, and in vivo crosslinking experiments showed that ppGpp facilitates the interaction between PigR and the MglA-SspA complex. These findings provide a framework for understanding the mechanistic basis for virulence gene control in F. tularensis and uncover a novel mechanism by which ppGpp influences gene expression--by modulating the interaction between a transcription activator and the RNAP-associated MglA-SspA complex.
Download or read book Examining Mechanisms of Virulence Gene Regulation and the Early Host Interactions in Francisella Tularenisis written by Matthew Leon Faron and published by . This book was released on 2014 with total page 121 pages. Available in PDF, EPUB and Kindle. Book excerpt: Francisella tularensis is a facultative intracellular pathogen and is the etiological agent of tularemia. One key aspect to the success of Francisella as a pathogen is ability of the organism to establish infection with a low inoculum, as few as 10 colony forming units (cfu). Essential to this process is the Francisella pathogenicity island (FPI). Several studies have been performed to understand how the FPI is regulated; however, the working model is not complete, as the signals important for regulation are unknown. Additionally, the mechanisms of the proteins MigR, TrmE, and CphA, which are important for activation of the FPI, are unknown. I initiated the study of this regulatory system by measuring the ability of various cellular stresses to activate an iglA-lacZ reporter. I identified that amino acid starvation and growth in basic pH activated expression of the reporter in both LVS and Schu S4. By combining these two stresses I was able to induce iglA-lacZ reporter expression in an additive manner. As it was previously demonstrated that ppGpp is important for stabilization of the regulatory complex that transcribes FPI genes, I demonstrated by TLC that both amino acid starvation and basic pH effected iglA-lacZ expression by increasing ppGpp. Due to the importance of ppGpp in FPI expression and because MigR, TrmE, and CphA each appear to be involved in a metabolic process: fatty acid metabolism (migR) t-RNA modification (trmE) and amino acid storage (cphA), I had hypothesized that the effect on these mutations were due to decreased levels of the small alarmone ppGpp. I compared ppGpp accumulation of LVS mutants in migR, trmE, and cphA to the parent strain and observed that loss of these genes resulted in reduced ppGpp. To better understand the importance of ppGpp synthesis in F. tularensis pathogenesis, I compared the phenotypes of these strains in primary human macrophages and two immortalized epithelial cell lines. These experiments demonstrated that although each of these strains had reduced ppGpp, there were cell line specific growth phenotypes. Mice infected with these strains survived suggesting tight regulation of the FPI is required for virulence. When similar mutations were characterized in the Schu S4 background these mutations retained their regulatory role; however, mutation of migR did not significantly decrease virulence in mice. As my data demonstrated that there are different challenges that Francisella must overcome to successfully replicate within cells, I developed an in vitro model to study the interactions of F. tularensis with human alveolar type II cells (AT-II). Interestingly, Schu S4 internalizes and replicates in these recently immortalized human AT-II cells whereas, LVS internalizes, but replicates poorly within these cells. Finally, to better understand the role of AT-II cells in vivo, I performed Transmission Electron Microscopy (TEM) of infected mice. These data confirmed that Schu S4 infected both alveolar macrophages and AT-II cells. Together, this work contributes to the understanding of how Francisella adapts to various environments by modulating virulence gene expression and highlights differences between virulent Schu S4 and LVS, which may partially contribute to virulence differences observed between strains.
Download or read book Coordinate Control of Virulence Gene Expression in Francisella Tularensis written by Amy Elizabeth Rohlfing and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The small molecule ppGpp has previously been shown to promote the interaction between the MglA-SspA complex and PigR in F. tularensis. It is unknown if ppGpp directly or indirectly promotes this interaction. We determined that ppGpp is required to detect an interaction between the MglA-SspA complex and PigR in the E. coli bridge-hybrid assay, indicating that ppGpp is either directly involved in promoting this interaction or works through an indirect mechanism that is conserved between F. tularensis and E. coli. One potential conserved mechanism through which ppGpp may be influencing the interaction between the MglA-SspA complex and PigR is through regulation of the levels of the molecule polyphosphate. However, we determined that polyphosphate is not required in order for the MglA-SspA complex and PigR to detectably interact with one another in the E. coli bridge-hybrid assay. Furthermore, analysis of the role of polyphosphate in gene expression in F. tularensis revealed that polyphosphate is a negative regulator of virulence gene expression.
Download or read book Regulation of Bacterial Virulence written by Michael L. Vasil and published by American Society for Microbiology Press. This book was released on 2012-12-05 with total page 1189 pages. Available in PDF, EPUB and Kindle. Book excerpt: A comprehensive compendium of scholarly contributions relating to bacterial virulence gene regulation. • Provides insights into global control and the switch between distinct infectious states (e.g., acute vs. chronic). • Considers key issues about the mechanisms of gene regulation relating to: surface factors, exported toxins and export mechanisms. • Reflects on how the regulation of intracellular lifestyles and the response to stress can ultimately have an impact on the outcome of an infection. • Highlights and examines some emerging regulatory mechanisms of special significance. • Serves as an ideal compendium of valuable topics for students, researchers and faculty with interests in how the mechanisms of gene regulation ultimately affect the outcome of an array of bacterial infectious diseases.
Download or read book Examining the Regulation of Virulence Factors in Francisella Tularensis written by Blake Wade Buchan and published by . This book was released on 2009 with total page 302 pages. Available in PDF, EPUB and Kindle. Book excerpt: Comparison of LVS and Schu S4 in primary human airway epithelial cell infections revealed an inability of LVS to replicate within these cells, which is in contrast to the robust replication of LVS in cultured epithelial cell lines. Together, this work contributes to the understanding of regulatory mechanisms governing virulence gene expression in F. tularensis and highlights differences between LVS and Schu S4 strains.
Download or read book Dissecting the Interactions Between Francisella Tularensis and Its Murine Host written by Kaitian Peng and published by Stanford University. This book was released on 2011 with total page 163 pages. Available in PDF, EPUB and Kindle. Book excerpt: Francisella is a gram-negative bacterium that causes tularemia. It is capable of infecting a remarkably broad host range including humans, mammals, birds and fish via multiple different routes of infection, establishing a successful colonization event within the various organs. This facultative, intracellular pathogen is also capable of invading a broad range of host cell types ranging from macrophages to fibroblasts. This is an extremely fascinating facet of the bacterium. The ability of Francisella to infect such a wide range of hosts and cell types suggests that the bacterium either co-opts cellular mechanisms common to all hosts and cell types or has the requisite bacterial genes to adapt to many different intraorganismal environments, or both. We were interested in studying the diverse repertoire of interactions that may occur between the bacterium and its murine host. In this thesis, the transposon site hybridization (TraSH) negative selection strategy was applied in a range of in vivo and in vitro systems to identify novel host-pathogen interactions in Francisella. We subsequently demonstrated that Francisella require tryptophan for virulence specifically in the lungs due to lung-specific induction of a host innate immune molecule indoleamine 2,3-dioxygenase 1. Alveolar macrophages may also deplete intracellular trytophan via a novel mechanism and microbial lung-specific requirement of tryptophan for virulence may be widely applicable to all bacterial species. We also demonstrate that Francisella hypercytotoxic mutants, unlike previously suggested, induce macrophage hypercytotoxicity due to increased bacteriolysis in the intracellular milieu. Identification and characterization of bacterial mutants that are attenuated under different in vitro and in vivo conditions have led to further insights into the interactions that occur between Francisella and its murine host.
Download or read book Genetic Regulation of Virulence in Francisella Tularensis written by Stephen A. Rodriguez and published by . This book was released on 2010 with total page 312 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Francisella Tularensis Insertion Sequence Elements Contribute to Differential Gene Expression written by Amanda Marie Bartling and published by . This book was released on 2018 with total page 156 pages. Available in PDF, EPUB and Kindle. Book excerpt: Francisella tularensis is the causative agent of the disease tularemia and a select agent. The genome of this pathogen contains many insertion sequence (IS) elements with ISFtu1 being the most abundant. ISFtu1 expression and the contribution of this IS element to differential gene expression in the F. tularensis subpopulations was evaluated. Full-length and truncated ISFtu1 sense and antisense transcriptional expression was detected. The prototype A.I strain (Schu S4) had considerably higher expression levels of ISFtu1 and the adjacent genes than the wild type A.I strain (NE061598). The A.II strains (WY96-3418 and WY-00W4114) had similar expression levels for ISFtu1 and the adjacent genes. In the highly virulent A.I strains, a bicistronic transcript encoding the universal stress protein (Usp) and a downstream ISFtu1 was highly expressed during early and mid log growth phase when provisions were plentiful, and was induced 2-fold by nitric oxide and a polyamine. During late log growth and stationary phase, only monocistronic transcripts for usp and ISFtu1 were being moderately expressed. Intrinsic transcription termination sequences were not apparent between usp and ISFtu1. Secondary RNA structure models indicated that the bicistronic transcript will form more readily than the monocistronic usp transcript. The co-expression of ISFtu1 and the adjacent gene(s) may provide a fitness advantage by regulating expression and/or transcript stability of the co-transcribed gene(s). Additional study will provide a better understanding of the contribution of IS elements to the differential gene expression and virulence observed for the various F. tularensis clades.
Download or read book Regulation of Virulence Gene Transcripts by the Francisella Orphan Response Regulator PmrA written by Brian Len Bell and published by . This book was released on 2009 with total page 156 pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: Francisella tularensis subspecies tularensis is the etiologic agent of tularemia and has been designated a category A biothreat agent by the CDC. Tularemia is characterized by replication and dissemination within host phagocytes. Intramacrophage growth is dependent upon the regulation of Francisella Pathogenicity Island (FPI) virulence genes, which is poorly understood. Two-component regulatory systems (TCS) are widely employed by Gram negative bacteria to monitor and respond to environmental signals. Virulent strains of F. tularensis are devoid of classical, tandemnly arranged TCS, but orphaned members, such as the response regulator PmrA, have been identified. In the F. novicida model system, previous work has shown that a pmrA mutant is deficient for intramacrophage growth and is avirulent in the mouse model. Here we determine that phosphorylation aids PmrA binding to regulated promoters pmrA and the FPI encoded pdpD, and KdpD is the histidine kinase primarily responsible for phosphorylation of PmrA at the aspartic acid at position 51 (D51). A strain expressing PmrA D51A retains some DNA binding but exhibits reduced expression of the PmrA-regulon, is deficient for intramacrophage replication and is attenuated in the mouse model. PmrA co-precipitates with the FPI transcription factors MglA and SspA, which bind RNA polymerase. Together this data suggests a model of Francisella gene regulation that includes a TCS consisting of KdpD and PmrA. Once phosphorylated, PmrA binds to regulated gene promoters recruiting free or RNA polymerase bound MglA and SspA to initiate FPI gene transcription.
Download or read book Analysis of Francisella Tularensis Mutants Defective in Intracellular Survival written by Frances Valencia and published by . This book was released on 2007 with total page 50 pages. Available in PDF, EPUB and Kindle. Book excerpt: Francisella tularensis is a gram-negative bacterium categorized as a category A agent of biological warfare that causes the disease known as tularemia. To identify virulence factors of the F. tularensis type A strain Schu S4 a transposon-insertion library as generated and screened for mutant strains defective in intracellular survival. We focused in particular on two mutant strains, BJM1005 and BJM1026. BJM1005 has a transposon insertion in the dsbB gene, that encodes a protein that is required for disulfide bond formation. DsbB appear to be part of an operon with two genes FTT1016c and FTT105c, that potentially encode a multidrug efflux pump. We hypothesize that the dsbB null mutation prevented expression of the downstream FTT1016c and FTT105c genes. Therefore, we attempted to determine if dsbB and the downstream genes FTT0106c and FTT0105c are cotranscribed on a polycistronic mRNA. Oligonucleotide primer pairs for reverse-transcription PCR (RT-PCR) were designed to test this hypothesis. The preliminary results do not confirm cotranscription because we were unable to detect gene expression above background. BJM1026 has a transposon in FTT1236 (hypothetical protein) that is potentially cotranscribed with FTT1237, a putative glycosyl transferase. We hypothesized that the mutation of FTT1236 in Francisella tularensis has a polar effect on the putative glycosyl transferase, causing a defect in lipopolysaccharide (LPS) synthesis. To investigate this we performed a western blot with primary rabbit anti-LVS, antibodies that are able to detect F. tularensis LPS. Our results demonstrated a defect in the O-antigen of the LPS which both supports our hypothesis and suggests that LPS synthesis and its role in F. tularensis virulence needs to be investigated further.
Download or read book Bacterial Pathogens and Their Virulence Factors written by Douglas I. Johnson and published by Springer. This book was released on 2017-11-23 with total page 451 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bacterial Pathogens and their Virulence Factors contains a detailed description of 32 major bacterial pathogens that affect human health and their associated virulence determinants. Chapter 1 gives an overview of the different types and classes of general virulence factors involved in host cell adherence and invasion, dissemination within the host, host cell damage, and evasion of host defense systems, as well as mechanisms by which these virulence factors are regulated. Chapters 2 through 33 give concise descriptions of the disease states associated with the 32 bacterial genera and their major pathogenic species, along with an in-depth description of the individual virulence factors that have been found to be functionally involved in pathogenicity. A detailed bibliography derived from primary literature and review articles accompanies each of these chapters, allowing the reader to delve more deeply into individual pathogens and their virulence determinants. Chapter 34 discusses the exciting possibilities and initial successes of using detailed information on a pathogen’s virulence toolkit to design new therapeutics aimed at specific virulence traits.
Download or read book Francisella tularensis and tularemia written by Anders Sjöstedt and published by Frontiers E-books. This book was released on 2011-06-11 with total page 206 pages. Available in PDF, EPUB and Kindle. Book excerpt: The bacterium today known as Francisella tularensis was first identified 99 years ago and, since then, much research has been devoted to study it and the resulting disease, tularemia. F. tularensis became the focus of an intense research effort during the first half of the 20th century, in particularly in the United States and Soviet Union, since the disease was fairly common. Due to its high infectivity, ease of spread, and severity of the resulting disease, it was one of the agents given the highest priority in the biological weapon programs of the United States and Soviet Union. After termination of these programs in the 1960s, the interest in F. tularensis diminished significantly, but after several decades of little attention, the last decade has led to resurgence in the research on F. tularensis. In 2003, the Science magazine phrased it as follows: “an obscure weapon of the cold war edges into the limelight”. There were multiple reasons for this resurgence, one of which was an increase in the number of tularemia cases in several European countries and, moreover, the intense research effort on potential bioterrorist agents in the US post 9/11. Thereby, the number of annual publications on F. tularensis has tripled in 10 years, and many new research groups, in particular American, have entered the field. This has led to very rapid development of state-of-the-art research tools, and fast progress in the understanding of F. tularensis. A proof of the rapid progress was the publication of a comprehensive volume on Francisella and tularemia in 2007. Although only four years ago, the rapid pace of the research has led to many new discoveries since then and, to this end, we now present a collection of articles from leading scientists on the up-to-date knowledge regarding F. tularensis. The articles cover important areas such as molecular research tools, experimental models, genomics, virulence mechanisms, manipulation and subversion of host responses, host immune responses, and the ecology of F. tularensis.
Download or read book Identification of Two Novel in Vivo upregulated Francisella Tularensis Proteins Involved in Metal Acquisition and Virulence written by Xiaojun Wu and published by . This book was released on 2016 with total page 172 pages. Available in PDF, EPUB and Kindle. Book excerpt: Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal binding and uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. We have identified two new proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of those iron acquisition genes and demonstrated that both are upregulated during mouse infections. Based on sequence homology and in vivo upregulation, we hypothesized that FmvA and FmvB are OMPs involved in metal acquisition and virulence. Despite sequence similarity to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither fmvA nor fmvB mutants exhibited growth defects in iron limitation. However, among other metals examined in this study, magnesium limitation significantly induced fmvB expression, fmvB mutant was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on fmvB mutant in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of fmvB mutant revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in fmvB mutant, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in magnesium uptake, we demonstrated that FmvB was outer membrane-localized. Finally, both fmvA and fmvB mutants were found to be significantly attenuated in mice and cytokine analyses revealed that fmvB mutant-infected mice produced lower levels of pro-inflammatory cytokines, including GM-CSF, IL-3, and IL-10, compared with mice infected with wild-type F. tularensis. Taken together, these studies have characterized two previously-unstudied F. tularensis proteins, have shown that both play roles in F. tularensis virulence, and provide new insights into the importance of magnesium for intracellular pathogens.
Download or read book Localization of Francisella Pathogenicity Island encoded Secreted Proteins and Their Secretion System written by Rebekah Frances Hare and published by . This book was released on 2014 with total page 290 pages. Available in PDF, EPUB and Kindle. Book excerpt: Intracellular pathogens have evolved virulence genes that allow them to exploit host cells for their life cycles, and virulence genes are commonly located in pathogenicity islands, such as the Francisella pathogenicity island of Francisella tularensis. The Francisella pathogenicity island is linked to virulence, intracellular growth, and a type VI secretion system. Since the Francisella pathogenicity island encodes a secretion system, I hypothesize that Francisella pathogenicity island encoded proteins are secreted during infection of host cells. The molecular mechanisms involved in the pathogenesis of this bacterium are not well understood and there are no readily available tools for studying these mechanisms. Therefore, I developed expression plasmids of all Francisella pathogenicity island encoded proteins as C-terminal and N-terminal epitope FLAG-tagged proteins. The Francisella pathogenicity island encoded proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of their respective genes that were deficient for intramacrophage growth. Immuno-fluorescence microscopy experiments of cells infected with bacteria containing the expression plasmids showed some of the Francisella pathogenicity island encoded proteins were secreted. To test if protein localization is dependent on the type VI secretion system, localization observed in wild type was compared to the localization of Francisella pathogenicity island encoded proteins in a pdpB mutant, a gene that is homologous to a type VI secretion system structural inner membrane protein. The localization of FLAG-tagged proteins was significantly reduced when expressed in the pdpB mutant compared to expression in wild type. Two of the secreted proteins, pdpC and pdpE, were tested for their roles in pathogenicity. pdpC was required for virulence in vivo but not for growth within macrophages. Plasmid expression of PdpC-FLAG and FLAG-PdpC in the pdpC mutant restored the virulent phenotype to that of the wild type. PdpE was not required for intramacrophage growth or virulence in mice. These data further support the hypothesis that the Francisella pathogenicity island encodes a secretome that contributes to the virulence of Francisella.
Download or read book WHO Guidelines on Tularaemia written by World Health Organization and published by World Health Organization. This book was released on 2007-12-15 with total page 125 pages. Available in PDF, EPUB and Kindle. Book excerpt: Tularaemia is a bacterial zoonotic disease of the northern hemisphere. The bacterium (Francisella tularensis) is highly virulent for humans and a range of animals such as rodents hares and rabbits. Humans can infect themselves by direct contact with infected animals by arthropod bites by ingestion of contaminated water or food or by inhalation of infective aerosols. There is no human-to-human transmission. In addition to its natural occurrence F. tularensis evokes great concern as a potential bioterrorism agent. F. tularensis subspecies tularensis is one of the most infectious pathogens known in human medicine. In order to avoid laboratory-associated infection safety measures are needed and consequently clinical laboratories do not generally accept specimens for culture. However since clinical management of cases depends on early recognition there is an urgent need for diagnostic services. This first edition of WHO Guidelines on tularaemia provides background information on the disease describes the current best practices for its diagnosis and treatments in humans suggests measures to be taken in case of epidemics and provides guidance on how to handle F. tularensis in the laboratory. The target audience includes clinicians laboratory personnel public health workers veterinarians and any other person with an interest in zoonoses.
Download or read book The Prokaryotes written by Edward F. DeLong and published by Springer. This book was released on 2014-10-02 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Prokaryotes is a comprehensive, multi-authored, peer reviewed reference work on Bacteria and Achaea. This fourth edition of The Prokaryotes is organized to cover all taxonomic diversity, using the family level to delineate chapters. Different from other resources, this new Springer product includes not only taxonomy, but also prokaryotic biology and technology of taxa in a broad context. Technological aspects highlight the usefulness of prokaryotes in processes and products, including biocontrol agents and as genetics tools. The content of the expanded fourth edition is divided into two parts: Part 1 contains review chapters dealing with the most important general concepts in molecular, applied and general prokaryote biology; Part 2 describes the known properties of specific taxonomic groups. Two completely new sections have been added to Part 1: bacterial communities and human bacteriology. The bacterial communities section reflects the growing realization that studies on pure cultures of bacteria have led to an incomplete picture of the microbial world for two fundamental reasons: the vast majority of bacteria in soil, water and associated with biological tissues are currently not culturable, and that an understanding of microbial ecology requires knowledge on how different bacterial species interact with each other in their natural environment. The new section on human microbiology deals with bacteria associated with healthy humans and bacterial pathogenesis. Each of the major human diseases caused by bacteria is reviewed, from identifying the pathogens by classical clinical and non-culturing techniques to the biochemical mechanisms of the disease process. The 4th edition of The Prokaryotes is the most complete resource on the biology of prokaryotes. The following volumes are published consecutively within the 4th Edition: Prokaryotic Biology and Symbiotic Associations Prokaryotic Communities and Ecophysiology Prokaryotic Physiology and Biochemistry Applied Bacteriology and Biotechnology Human Microbiology Actinobacteria Firmicutes Alphaproteobacteria and Betaproteobacteria Gammaproteobacteria Deltaproteobacteria and Epsilonproteobacteria Other Major Lineages of Bacteria and the Archaea
