Browsing by Subject "Virulence Factors"
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Item Characterization of Host and Microbiota Derived Signals that Regulate the Locus of Enterocyte Effacement of EHEC(2020-05-01T05:00:00.000Z) Jimenez Lopez, Angel Giovanni; Hooper, Lora V.; Winter, Sebastian E.; Greenberg, David; Sperandio, VanessaHumans are populated by an extensive community of microorganisms, primarily in organs such as the skin, mucosal membranes in the mouth, reproductive organs, and the gut. This complex community, termed the microbiota, is in part comprised of bacteria, many of which have intimate associations with their hosts to promote physiological homeostasis. These organisms, commonly termed commensal bacteria, have a rich and long history with their human hosts and accomplish important functions such as providing the host with nutrients, developing the immune system, and preventing colonization by pathogenic organisms in a process known as colonization resistance. These functions are especially apparent within the gastrointestinal (GI) tract, which contains the richest and most densely populated community of microbes in the body. Healthy gut function relies on the proper structure and balance of this microbial community. Disruption of the community, termed dysbiosis, has been associated with a plethora of diseases such as increased susceptibility to GI infections, neurological disorders, intestinal inflammation, and cancer progression. Dysbiosis is most commonly caused by pharmacological interventions with antibiotics or infection with a GI pathogen. The microbiota is regarded as a barrier against intestinal pathogens, partly due to intense competition for a limited supply of nutrients and space. This suggests that GI pathogens have evolved mechanisms to overcome colonization resistance and outcompete the resident microbiota for resources within the GI tract. Microbiota and host-derived metabolites have a significant impact on the abilities of GI pathogens to successfully establish intestinal infection and the subsequent development of disease. However, the precise mechanism by which microbiota or host metabolites affect the pathogenesis of GI pathogens is not well understood. Many of these nutrients, whether host-, diet-, or microbiota-derived, serve as chemical cues for incoming pathogens. These signals are used by pathogens to gauge resource availability, microbiota composition, host physiology, and location within the intestines to properly deploy virulence strategies that allow for colonization. Microbiota-derived small molecules include toxins, antimicrobials, oligopeptides, hormones, and products of microbial metabolism of host-derived and dietary molecules. Pathogens can directly sense many of these host- and microbiota-derived small molecules, which in turn can regulate their virulence mechanisms. Taken together, developing therapeutics that target the signaling pathways that control virulence-associated functions in pathogens represent an attractive alternative or secondary strategy to tackle bacterial infections. In a previous study, our group conducted a candidate-based screen of 372 independent mutants to look for novel regulators of the T3SS [1]. The candidates of this screen consisted primarily of transcription factors, two-component regulatory systems, anti-terminators and anti-toxins. This work generated a great number of hits that potentially regulate the T3SS of EHEC. Our work sought to characterize novel signaling pathways that directly affect the virulence of enterohemorrhagic Escherichia coli (EHEC) through characterization of some of the hits of said screen in particular the transcriptional regulators ExuR and FadR. Understanding of these signaling pathway could lead us to develop novel strategies to drive down the virulence of enteric pathogens and improve colonization resistance as an alternative approach to control bacterial infections. Here, we found that EHEC senses and utilizes galacturonic-acid (GalA) as a nutrient during infection and moonlights as a signal to downregulate the expression of virulence associated genes. Furthermore, we demonstrated that a pectin-rich diet, which is a source of GalA, increased mice tolerance towards a Citrobacter rodentium infection, a surrogate mice model for EHEC infection. AE pathogens like EHEC and C. rodentium thrive in an inflamed environment. During the onset phase of inflammation, the host-derived polyunsaturated omega-6 long-chain fatty acid (LCFA), arachidonic acid (AA) becomes elevated to produce endogenous lipid signaling molecules like prostaglandins and leukotrienes that act as inducers of inflammation. EHEC can sense long-chain fatty acids through the FadR response regulator. We found that AA is processed by EHEC using canonical LCFA signaling pathways involving the FadL LCFA transporter, the FadD acyl-CoA synthase and the FadR transcriptional regulator. In conclusion, we characterized the signaling pathways that mediate the sensing of galacturonic-acid and arachidonic-acid. We demonstrated that a diet high in pectin can effectively be used to control an infection by C. rodentium by effectively modulating the levels of GalA and affecting virulence in an ExuR dependent manner. We also showed that EHEC is capable of sensing a host produced long chain fatty acid like arachidonic acid to regulate its virulence. These studies highlight the complexity that underlies regulation of the locus of enterocyte effacement and perhaps will serve as a starting point for the development of new strategies to control enteric infections.Item Characterization of Mycobacterium tuberculosis Cor, a Protein Essential for Carbon Monoxide Resistance and Pathogenesis(2015-01-28) Zacharia, Vineetha Mariam; Hendrixson, David R.; Alto, Neal; McDonald, Jeffrey; Shiloh, MichaelTuberculosis, caused by Mycobacterium tuberculosis, is global health burden as it remains one of the most devastating human infectious diseases causing two million deaths annually and latently infecting a third of the world's population. We previously demonstrated that M. tuberculosis induces an enzyme, heme oxygenase (HO1), that produces carbon monoxide (CO) gas and that M. tuberculosis adapts its transcriptome during CO exposure. We now demonstrate that M. tuberculosis carries a novel resistance gene to combat CO toxicity. We screened an M. tuberculosis transposon library for CO-susceptible mutants and found that disruption of Rv1829 (carbon monoxide resistance, cor) leads to marked CO sensitivity. Heterologous expression of Cor and Cor homologue from Thermotoga maritima (TM0160) in Escherichia coli rescued it from CO toxicity, suggesting a conserved function across diverse microbial species. Importantly, the virulence of the cor mutant is attenuated in a mouse model of tuberculosis. Thus, Cor is necessary and sufficient to protect bacteria from host-derived CO. Evolutionary modeling suggested that Cor forms an active site, thus we predicted that Cor has enzymatic activity. To determine potential Cor enzymatic activity, we profiled the mycobacterial metabolome using liquid-chromatography mass spectrometry and, in vitro, monitored metabolite fluctuations in the presence and absence of recombinant Cor. Our activity-based metabolomic profiling data showed the accumulation of phosphatidic acid and multiple phospholipids in the presence of Cor, indicating its potential role in catalyzing a reaction involved in phospholipid biosynthesis. Our in vivo metabolomic analysis of a Cor mutant strain showed that in the presence of CO, levels of dihydroxyacetone phosphate is increased, whereas levels of glycerol-3-phosphate is reduced, which is required for phospholipid biosynthesis. Furthermore, we identified key metabolic enzymes in Mtb that physically interact with Cor using bioinformatics and immunoprecipitation techniques. Specifically, Cor interacted with glycerol-3-phosphate dehydrogenase 2, an enzyme that catalyzes the interconversion of glycerol-3-phosphate to dihydroxyacetone phosphate. Taken together, this represents the first report of a role for HO1-derived CO in controlling infection of an intracellular pathogen and the first identification of a CO resistance gene in a pathogenic organism, which may have a critical role in phospholipid biosynthesis.Item Identification and Characterization of Flagellar Co-expressed Determinants (Feds) of Campylobacter jejuni(2013-10-23) Barrero-Tobon, Angelica M.; Hansen, Eric J.; Hendrixson, David R.; Alto, Neal; Shiloh, MichaelCampylobacter jejuni is the leading cause of bacterial gastroenteritis in humans throughout the world. In contrast to infection of humans, C. jejuni is a commensal organism of the intestinal tracts of wild and agriculturally-significant animals and avian species. Flagellar motility is the only virulence and colonization factor proven to be required for infection of human volunteers to promote disease and infection of poultry for commensalism. Expression of many flagellar genes is dependent on two alternative sigma factors, σ54 and σ28. Μany rod and hook genes are dependent on σ54 for expression, whereas σ28 is involved in the expression of the major flagellin and other filament genes. We investigated the σ28 regulon and identified five genes that are dependent on σ28 and flagellar components for maximal expression, but are not required for motility. One gene, ciaI, has previously been shown to function in intracellular survival after invasion of human intestinal epithelial cells. The four remaining genes, which we annotated as fedA-fedD (for flagellar co-expressed determinants), encode proteins that have not been characterized. Mutants lacking any one of these feds or ciaI demonstrated a reduced commensal colonization capacity in a natural chick model of colonization. Similar to the σ28-dependent gene product FspA1, a subset of these Feds is secreted by the bacterium in a flagellar-dependent manner. To further investigate the secretion requirements of these σ28-dependent proteins (FedB, CiaI and FspA1), we examined putative flagellar chaperones, flagellar components and other aspects of flagellar biosynthesis such as flagellar protein glycosylation for a role in secretion of Feds. We discovered that, like in other motile organisms, the FliJ chaperone is required for secretion of flagellar components in general, and that FliS is likely the chaperone for the major flagellin, FlaA. However, FliS and other putative flagellar chaperones are not required for secretion of the Feds. We also discovered that secretion of the Feds occurs during or just after hook biosynthesis, suggesting that construction of a hook is required for maximal secretion of these proteins via the flagellum. In addition, in the absence of the flagellar cap or flagellin glycosylation, we observed an increase in secretion of FedB, CiaI and FspA1, suggesting a possible inverse correlation between the amount of Fed proteins secreted via the flagella and length of the flagellar filament. Furthermore, we have identified N- and C-terminal intramolecular determinants within FedB and CiaI that are required for maximal secretion. Based on how other flagellar proteins are secreted, these findings indicate that a flagellar Type III secretion system (T3SS)-specific signal sequence is likely found at the N-terminus, and that an unidentified chaperone may bind to the C-terminus. Both of these factors appear to be required for maximal flagellar-dependent secretion of the Feds. We also examined the importance of secretion of Feds during commensal colonization and invasion of human colonic epithelial cells in vitro. Gentamicin-protection assays revealed that secretion of CiaI is not required for invasion of T84 cells. Furthermore, preliminary studies using a chick model of commensal colonization showed that secretion of FedB is important for colonization of the chick intestinal tract. However, whereas CiaI is required for colonization, secretion of CiaI was not important for colonization of the chick cecum. In summary, our work provides evidence that the flagellar system is a global regulatory system that coordinates production of flagella with colonization and virulence determinants, some of which are secreted in a flagellar-dependent manner, to promote maximal fitness during colonization and virulence.Item Mycobacterium Tuberculosis Virulence Factor Mpt64 Targets the Endoplasmic Reticulum(2019-04-10) Stamm, Chelsea Elizabeth; Sperandio, Vanessa; Shiloh, Michael; Alto, Neal; Winter, Sebastian E.Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that M. tuberculosis can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, M. tuberculosis is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secret virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. I hypothesized that M. tuberculosis secreted proteins that target host membranes are vital for M. tuberculosis to adapt to and manipulate the host environment for survival. Thus, I characterized nearly 200 secreted proteins from M. tuberculosis for their ability to associate with eukaryotic membranes using a live-dead, temperature sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. I identified five M. tuberculosis secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during M. tuberculosis infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in M. tuberculosis pathogenesis and provide a basis for further investigation into their molecular mechanisms.Item Novel Activities of Kinase-Fold Enzymes from Legionella pneumophila(2020-08-01T05:00:00.000Z) Black, Miles; Cobb, Melanie H.; Tagliabracci, Vincent S.; Mendell, Joshua T.; Olson, Eric N.Protein kinases are fundamental mediators of cell signaling that transfer phosphate from ATP to their substrates. The protein kinase superfamily encompasses a vast and diverse trove of enzymes from all domains of life, including remote members that are barely recognizable by their primary amino acid sequence. SidJ (Substrate of Icm/Dot J) is a distant protein kinase homolog from the human pathogen Legionella pneumophila. Contamination of water supplies with Legionella bacteria is a frequent cause of deadly pneumonia outbreaks (Legionnaire's disease). SidJ is a secreted Legionella virulence factor required for bacterial intracellular replication, but it is unknown how SidJ contributes to pathogenesis of Legionnaire's disease, or if SidJ has maintained the kinase fold or catalytic activity. In this work, I determine that SidJ is a calmodulin-binding protein which adopts a protein kinase fold. However, instead of phosphorylation, it catalyzes protein polyglutamylation. SidJ utilizes ATP to form an isopeptide bond between the amino group of free glutamate and the 𝛾-carboxyl group of a glutamate of its substrate. During infection, SidJ polyglutamylates and inactivates a family of Legionella "all-in-one" ubiquitin ligases. Polyglutamylation is crucial step in the intracellular lifecycle of the bacterium and is required for full Legionella virulence in a eukaryotic host. SidJ reveals the unexpected catalytic versatility of the protein kinase fold, and highlights a unique strategy that pathogenic bacteria use to thrive within host cells. Interestingly, SidJ lacks key catalytic residues believed to be required for kinase activity. The discovery that SidJ is a polyglutamylating enzyme suggests that catalytically incompetent or 'pseudo' enzymes may lack activity only when assayed for the wrong reaction.Item A Systems Biology Approach to Study Type III and Type IV Bacterial Effector Properties(2014-07-23) Weigele, Bethany Auten; Shiloh, Michael; Alto, Neal; Yan, Nan; Goodman, Joel M.The interaction between Type III and Type IV bacterial effector proteins and host signal transduction enzymes is a critical interface that, in many cases, determines the outcome of infectious disease. While many pathogenic strategies, such as evasion of phagolysosomal fusion, have been identified as necessary for microbial survival within the host, the effectors responsible are still largely unknown. Since most Gram-negative bacterial pathogens secrete between 5 to 300 effector proteins, a "systems biology" approach offers an enormous discovery potential. To approach the problem of effector protein biology from a global perspective, I first developed a comprehensive library of Type III and Type IV effector proteins (from six diverse pathogens) and assayed this library of effectors for their ability to associate with eukaryotic membranes. Unexpectedly, 30% of the virulence factor repertoire exhibited transmembrane-spanning domains, fatty-acid acceptor sites, peripheral membrane-binding properties, and/or cryptic phospholipid-targeting motifs. From a global analysis of phospholipid-binding mechanisms and from specific studies on the Shigella flexneri invasion program, a membrane-dependent autocatalytic feedback loop that regulates bacterial effector protein functions in space and time was identified. Additionally, new tools to further understand the potential role(s) of bacteria effector molecules in usurping the tightly regulated endocytic trafficking pathway were developed. These tools were then used to identify and characterize the location at which three bacterial effectors EspG, VirA, and IpaJ disrupt the global secretory pathway. Lastly, the effector library was utilized in a bioinformatics approach to identify bacterial effectors from a newly sequenced pathogen found to encode a Type III secretion system. Exploiting previous knowledge of homologous characterized effectors within the library, the first bacterial effectors from the pathogen, Providencia alcalifaciens were identified. Taken together, these findings suggest that the evolution of bacterial membrane binding motifs promote higher-order signaling functions in host cells and provide a resource for further interrogation of these virulence properties across a broad range of bacterial pathogens.Item Trimming Fat Upon Infection: Proteolytic Demyristoylation as a Novel Bacterial Pathogenic Strategy(2014-08-28) Burnaevskiy, Nikolay; Seemann, Joachim; Chook, Yuh Min; Orth, Kim; Alto, NealHost-pathogen interaction is a complex process that involves an array of molecular tools on both sides of the conflict. In the light of growing antibiotics resistance, understanding of virulence factors utilized by pathogenic bacteria is crucial for developing better treatment for infectious diseases. Shigella spp are food-borne bacterial pathogens and are one of the leading causes of bacterial dysentery worldwide. Similarly to other gram-negative pathogens, Shigella injects effector proteins into an infected cell to control its responses. Dedicated type III secretion system delivers more than twenty effectors which highjack normal cell signaling by modulating activity of host proteins. By interfering with immune signaling, cytoskeleton, and other pathways, effector proteins promote survival of the bacterial pathogen. Inability to secrete effectors severely attenuates Shigella spp, pointing to the crucial role of these proteins in pathogenesis. Therefore, gaining an insight into the mechanism of action of secreted effectors may hold a key to better understanding and treatment of infectious diseases. Here I characterized the Shigella flexneri secreted virulence factor IpaJ. I found that IpaJ is a cysteine protease with novel specificity. IpaJ cleaves a myristoylated amino-terminal glycine of host proteins and therefore irreversibly delipidates them. Although IpaJ can target multiple host proteins, it strongly favors members of ADP-ribosylation factors family (ARF) and related ARF-like (ARL) proteins. By inactivating ARF and ARL proteins, IpaJ disrupts the Golgi structure and prevents secretion through general secretory pathway. I also found that by blocking protein export from the endoplasmic reticulum, IpaJ suppresses the activation of immune signaling pathways. In summary, I characterized a novel virulence factor of Shigella spp, IpaJ. I have shown that IpaJ promotes bacterial pathogenesis and therefore may be of potential interest for therapeutic interventions. The biochemical analysis of the enzyme provides the first look at the mechanism of substrate recognition and highlights the potential use of IpaJ as a research tool to study protein N-myristoylation and the mechanisms of membrane trafficking.Item [UT Southwestern Medical Center News](2006-05-25) McKenzie, Aline