Browsing by Subject "Bacterial Proteins"
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Item Analysis of Bacterial-Host Interactions Between Campylobacter jejuni and the Avian Host During Commensalism(2009-06-15) Bingham-Ramos, Lacey Kathleen; Hendrixson, David R.Campylobacter jejuni is a leading cause of bacterial enteritis in humans throughout the world. In contrast to the disease seen in humans upon infection, C. jejuni promotes an asymptomatic, intestinal colonization of many animals, especially avian species, to result in commensalism. The primary route of transmission to humans is through the consumption or handling of undercooked poultry meats, making C. jejuni of particular importance to the agricultural industry. The direct interplay between C. jejuni and the natural avian host was examined to better understand the interactions that contribute to commensalism. We analyzed the colonization dynamics of C. jejuni over 28 days and identified a previously uncharacterized prolonged, robust colonization of the bursa of Fabricius, a major lymphoid organ. C. jejuni localized to the mucus layer lining the epithelium of the bursal lumen, with no invasion of or damage to host tissue apparent. However, C. jejuni was detected invading the cecal epithelium of chicks but only at day 1 post-infection, which may contribute to the observed transient, infection of the spleen and liver. Additionally, certain colonization factors of C. jejuni were shown to promote persistence in specific organs. Mutants lacking catalase and the cytolethal distending toxin demonstrated a reduction in levels in the bursa but not the ceca during prolonged colonization, whereas an unencapsulated mutant showed a global colonization defect of all organs. These findings suggest that persistent colonization of the bursa and the ceca, and the ability of the avian host to largely confine C. jejuni to mucosal surfaces may be specific for the development of commensalism. Separate analyses of additional colonization factors of C. jejuni revealed the importance of two putative cytochrome c peroxidases (CCP), DocA and Cjj0382, in promoting efficient cecal colonization. Further analysis of DocA and Cjj0382 revealed that both proteins have typical characteristics of CCPs, as they are periplasmic proteins with heme-dependent peroxidase activity. Our data suggest that although DocA and Cjj0382 have characteristics of CCPs, they likely perform different physiological functions for the bacterium during colonization. Overall, this study enhances our understanding of the interactions between C. jejuni and a natural host that contribute to the development of commensalism.Item Analysis of Vibrio parahaemolyticus Virulence Systems(2014-08-14) Calder, Thomas James; Sperandio, Vanessa; Orth, Kim; Fontoura, Beatriz; Gardner, Kevin H.Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis from the consumption of raw or undercooked seafood. The pathogenicity of V. parahaemolyticus is attributed to several virulence factors, including two hemolysins and two type III secretion systems (T3SS1 and T3SS2). Herein, we compare the virulence of V. parahaemolyticus POR strains, which harbor a mutation in the T3SS needle apparatus, to the V. parahaemolyticus CAB strains, which contain mutations in transcriptional regulators for the T3SSs. Additionally, we characterize a novel T3SS2 effector termed VPA1380. From this study, we demonstrate that each structural or regulatory mutant of T3SS1 or T3SS2 alters the pathogenicity of the bacterium in a different manner. POR and CAB strains exhibited differences in biofilm growth, but shared similar levels of swarming motility and effector production/secretion. Additionally, while the cytotoxicity of these strains was similar, the CAB2 (T3SS1 regulatory mutant) strain was strikingly more invasive than the comparable POR2 (T3SS1 structural mutant) strain. In summary, by creating structural or regulatory mutations in either T3SS1 or T3SS2, differential downstream effects on other virulence systems were observed. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6)-inducible cysteine protease domain of several large bacterial toxins. Mutations of conserved catalytic residues and of residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in yeast cells deficient for the production of IP6. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator. Additionally, VPA1380 appeared to disrupt trafficking of dextran and transferrin, which may be due to VPA1380’s potential interaction with important retrograde factors. Elucidating the host targets and cellular effects of VPA1380 is important for understanding the pathogenic nature of V. parahaemolyticus for diagnostic and treatment and purposes.Item Antisense Blockade of Efflux Systems in Gram-Negative Pathogens(2018-01-23) Subramanian, Naveen G.; Felder-Scott, Christina F.; Sturge, Carolyn R.; Greenberg, DavidAntibiotic resistant bacteria, aka "super bugs", are a critical threat to public health worldwide, as the medical community is running out of effective antibiotics against a growing number of bacteria. One of the ways that bacteria develop resistance to antibiotics is by utilizing efflux systems that are used to pump the antibiotic out. A strategy that is currently being investigated is to restore the susceptibility of these bacteria to antibiotics by using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to suppress genes within these bacteria that encode components of efflux pumps. This project studied the effectiveness of PPMOs that target the AcrAB-TolC efflux pump, which is a major component of the intrinsic antibiotic resistance mechanisms of E. coli and K. pneumoniae. Experiments tested for the effect of the PPMO targeting the acrA gene, specific sequences within the acrA gene, and the tolC gene. The effect of the PPMO was measured by a change in the minimum inhibitory concentration (MIC) of common antibiotics such as Piperacillin/Tazobactam (Pip/Tazo), Azithromycin, and Levofloxacin on strains of these two bacteria. The results show that PPMOs targeted to the acrA gene have a 4-8 fold effectiveness at lowering antibiotic MICs for the bacterial strains. PPMOs that targeted the tolC gene, on the other hand, have no synergistic effect in lowering antibiotic MICs for the bacterial strains. In addition, changing the sequence of the PPMOs targeting the acrA gene was shown to have an effect, albeit small, on susceptibility to antibiotics, which suggests that targeting specific regions of a gene of interest can induce more or less susceptibility in the bacteria to antibiotics.Item An Atypical Minimal Kinase Inactivates the Molecular Chaperone Hsp90(December 2021) Park, Brenden Chul; Lehrman, Mark A.; Goodman, Joel M.; Zhang, Xuewu; Tagliabracci, Vincent S.Bacteria utilize a wide variety of proteins, termed effectors, to achieve virulence. We have identified an atypical kinase, HopBF1, found primarily in the plant pathogen Pseudomonas syringae. HopBF1 specifically inactivates Hsp90 causing the degradation of numerous Hsp90 clients, ultimately culminating in cell death and tissue necrosis. We further propose a remarkable betrayal mechanism in which HopBF1 masquerades as a host Hsp90 client to achieve specificity, followed by inactivation of the molecular chaperone.Item A Bacterial Cholesterol Sensor to Assess Cholesterol Accessibility in Red Blood Cells(2016-01-19) Chakrabarti, Rima Shah; Radhakrishnan, Arun; Cohen, Jonathan C.; Hobbs, Helen H.Mammals are able to gain cholesterol from two sources: diet and endogenous synthesis. However, the only means of cholesterol removal is reverse cholesterol transport (RCT), in which cholesterol is transported to the liver and exported into bile. While high density lipoprotein (HDL) is considered to be the major conduit for RCT, studies with HDL-deficient animals reveal no defect in tissue cholesterol balance. We hypothesize that red blood cells (RBCs), which contain 50% of blood cholesterol, also play a role in RCT. To measure accessible cholesterol in RBCs, we developed an assay that utilizes the cholesterol binding properties of the toxin Anthrolysin-O (ALO). We purified and fluorescently labeled domain 4 of ALO (fALOD4). We then incubated fALOD4 with RBCs from 164 subjects and measured fluorescence intensity using flow cytometry. Both intra-assay and intra-individual variability of the assay were less than 10%. In the test population, fALOD4 binding varied 10-fold. fALOD4 binding did not correlate with total RBC cholesterol but did correlate with RBC phosphatidylcholine (PC) (-0.42, p=6e-7) and lyso-phosphatidylcholine (LPC) (0.40, p=6e-6). Increasing the LPC:PC ratio in RBCs with phospholipase A2 (PLA2) increased fALOD4 binding by 3-fold. fALOD4 binding also correlated with plasma HDL (0.30, p=6e-4) and triglycerides (-0.57, p=2e-12). These data suggest that RBC accessible cholesterol varies in a population, is driven by intrinsic RBC phospholipid composition and interacts with known cholesterol transporters in the blood. Future studies will determine if variability in fALOD4 binding is driven by non-lipid RBC membrane components, is genetically determined, or contributes to atherosclerosis.Item Biochemical Characterization of the Yersinia Effector Protein, YopJ(2007-05-22) Mukherjee, Sohini; Orth, KimYersinia species, the causal agent of plague and gastroenteritis, uses a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ disrupts the mitogen-activated protein kinase and the nuclear factor κ B signaling pathways used in innate immune response by preventing activation of the family of mitogen-activated protein kinase kinases. The catalytic domain of YopJ is similar to Clan CE of cysteine proteases, and mutating the putative catalytic cysteine disrupts YopJ's inhibitory activity. YopJ binds mitogenactivated protein kinase kinases, including MKK1 through MKK6, and the related kinase, IκB kinase beta, however, the mechanism by which this binding leads to inactivation of these kinases is unknown. An in vitro cell-free signaling system was developed to recapitulate the inhibition of eukaryotic signaling by YopJ. Mass spectrometric studies were undertaken to determine the biochemical nature of modification of the mitogen-activated protein kinase kinases in the presence of YopJ. Based on the observations, a simple, molecular mechanism utilized by YopJ to block the signaling pathways was discovered. YopJ acted as an acetyltransferase, using acetyl coenzyme A, to modify the critical serine and threonine residues in the activation loop of mitogen-activated protein kinase kinases and thereby blocking phosphorylation. The acetylation on the kinase directly competed with phosphorylation, preventing activation of the modified protein. An essential characteristic feature of bacterial effector proteins is that they usurp or mimic a eukaryotic activity and refine this activity to produce an extremely efficient mechanism to combat eukaryotic signaling. Therefore, modification of amino acids, other than lysine, by acetylation could be a commonly used eukaryotic mechanism that has been undetected previously. The acetylation of these amino acids may compete with various other types of posttranslational modifications, such as ubiquitination, SUMOylation and glycosylation. Several questions that still need to be addressed are: Is this modification reversible? What are the eukaryotic proteins that add and remove this type of posttranslational modification? How do bacterial effectors use this activity? The characterization of a bacterial effector as a serine or threonine acetyltransferase presents a previously unknown paradigm to be considered for other biological signaling pathways.Item Biophysical and Biochemical Characterization of a REC Domain: Unfolded to Folded Transition of EL_LovR(2014-08-18) Ocasio, Victor J.; Hendrixson, David R.; Gardner, Kevin H.; Sperandio, Vanessa; Rizo-Rey, JoséProkaryotes frequently use two component systems to couple environmental stimuli to adaptive responses. These pathways use histidine kinases to detect environmental cues, harnessing these to control phosphorylation of the receiver domain of the response regulator, which convert this signal into a physiological response. Knowledge of how phosphorylation shifts receiver domains between their inactive and active states is limited, chiefly assembled from several prototypical receiver domains that switch between two similar and well-folded structures. However, it remains unclear how general these observations apply to other receiver domains, particularly for full-length proteins. Here we present a blue light-regulated two-component system from the marine α-proteobacterium Erythrobacter litoralis HTCC2594. The sensor domain of the 3 histidine kinases found in E. litoralis contain a LOV (Light-Oxygen-Voltage) domain, part of the widely used PAS (Per-ARNT-Sim) family of environmental sensors. Interestingly, one of the histidine kinases (EL362) contains a naturally occurring glycine to arginine mutation in the LOV domain that prevents chromophore binding, resulting in a "blind" histidine kinase. Reverting the arginine to a glycine residue allows blue light to trigger the autophosphorylation of EL362 and subsequent phosphotransfer towards the cognate response regulator EL_LovR. This arrangement of RRs is reminiscent of similar systems used in other bacterial general stress responses, most of which have been characterized entirely with genetic methods. Notably, EL_LovR is a single domain response regulator proposed to play a critical role in shutting off such systems via a potent phosphatase activity. Size exclusion chromatography, light scattering and NMR experiments show that phosphorylation and Mg(II) transitions EL_LovR between unfolded and folded monomeric states. Parallel functional assays show that EL_LovR has a fast dephosphorylation rate, consistent with its proposed function as a phosphate sink. Taken together, our findings provide evidence that EL_LovR undergoes drastic conformational changes that have not been seen in other response regulators, likely with effects on its autophosphatase activity. In conclusion, our work expands the kinds of conformational changes and regulation used by receiver domains, critical components of bacterial signaling systems.Item Butyrate Sensing by Campylobacter jejuni Impacts Bacterial-Host Interactions(2020-08-01T05:00:00.000Z) Goodman, Kyle Nicholas; Winter, Sebastian E.; Hendrixson, David R.; Sperandio, Vanessa; Pfeiffer, Julie K.The intestinal microbial ecosystem aids the host in digestion and nutrition by breaking down goods and providing beneficial vitamins and metabolites, including short-chain fatty acids (SCFAs) and lactate. Due to the abundance and intestinal distribution of these metabolites, bacterial pathogens can use them as biogeographical cues to discriminate among different regions of the host intestines. Indeed, Campylobacter jejuni, a commensal bacterium of the lower intestinal tract of avian species and a leading cause of bacterial diarrheal disease in humans, recognizes intestinal niches that support growth by sensing molecular cues produced by the microbiota. How C. jejuni senses and responds to microbiota-generated SCFAs and organic acids is not understood. Herein, I identified and characterized the C. jejuni BumSR two-component signal transduction system (TCS) that specifically directs a response to butyrate. Deletion of either C. jejuni bumS or bumR abolishes butyrate-modulated transcriptional changes in gene expression. Analysis of ΔbumS and ΔbumR mutants in a chick model of commensalism indicated that bumR is important for early colonization. This contrasts with a human volunteer infection study that demonstrated bumR is essential for infection of humans. Mutational analyses of genes within the BumSR regulon in the natural avian host revealed additional colonization factors including peb3, a putative glycoprotein adhesin/substrate binding protein, and Cjj0580, a putative d- and tri-carboxylate transporter. Through multiple biochemical assays, I discovered that BumS lacks kinase activity in vitro but possesses specific phosphatase activity towards BumR. These activities are not directly influenced by butyrate, suggesting that other metabolites, perhaps resulting from butyrate catabolism, are the direct cues sensed by BumS to modulate butyrate-dependent responses. By site-directed mutagenesis, I identified residues in the conserved H box that are required for BumS phosphatase activity. Consistent with previous work, phospho-BumR exhibits enhanced binding of target promoters in electrophoretic mobility shift assays, indicating that phosphorylated BumR likely has higher affinity to bind DNA at target promoters in vivo to either enhance or repress gene expression. Overall, this highlights BumSR as a non-canonical and first-identified TCS that directs a response to butyrate to modulate colonization gene expression through a phosphatase-dependent mechanism.Item CcpA-Mediated Carbon Catabolite Repression of Virulence in the Group A Streptoccus(2008-09-18) Kinkel, Traci L.; McIver, Kevin S.The group A streptococcus (GAS) is a strict human pathogen, which causes a broad spectrum of diseases ranging from the self-limiting diseases such as pharyngitis and impetigo to the more severe invasive disease such as necrotizing fasciitis. The coordinate expression of a wide array of virulence factors in response to the changing host environment represents a key step in the ability of the GAS to mediate disease in the human host. The present study investigates the role of the primary mediator of sugar metabolism regulation, carbon catabolite control protein (CcpA), in the regulation of virulence of the GAS. A putative CcpA-binding site or catabolite response element (cre) was identified upstream of the promoter for the virulence gene regulator, Mga. CcpA was shown to specifically bind to this cre, and activate the transcription of mga. In addition, both transcription of mga and expression of Mga were reduced in a ccpA mutant strain; however, the expression of the Mga-regulated genes were not affected. Additional studies analyzing the role of CcpA in pathogenesis of the GAS, showed a "hypervirulent" phenotype in the absence of CcpA using two mouse infection models. Microarray analysis of the delta ccpA strain determined that CcpA significantly represses the expression of saga, the gene encoding the potent cytolysin, streptolysin S (SLS). Moreover, hemolytic activity due to SLS was increased in the delta ccpA strain, and expression from Psaga demonstrated strong catabolite repression during growth in glucose compared to sucrose. Furthermore, purified GAS CcpA was shown to bind directly to the cre present in Psaga. The role of SLS in the increased pathogenesis of the delta ccpA strain was investigated by the creation of a double mutant strain, which lacks the ability to secrete SLS. Importantly, systemic infection of mice with the delta ccpA sagB double mutant resulted in complete attenuation of virulence and determined that the increased SLS expression is responsible for the "hypervirulent" phenotype in the absence of CcpA. Overall, these results have demonstrated a strong link between sugar metabolism regulation and virulence gene expression in the GAS.Item Characterization of VPA0450, A Type III Secreted Effector Protein from Vibrio Parahaemolyticus(2011-08-10) Broberg, Christopher Allen; Orth, KimVibrio parahaemolyticus is a Gram-negative, halophilic bacterium first isolated over 60 years ago after a major outbreak of food poisoning in Japan. It is now recognized as a significant cause of gastroenteritis associated with the consumption of raw or undercooked seafood. The recent emergence of pandemic strains has made the study of V. parahaemolyticus a priority in the field of bacterial pathogenesis. Virulence caused by V. parahaemolyticus has traditionally been attributed to the presence of one or more thermostable direct hemolysins. Genome sequencing of V. parahaemolyticus identified two distinct Type III Secretion Systems (T3SS). T3SS1, on chromosome 1, was shown to translocate four effectors, VopQ, VopR, VopS, and VPA0450, resulting in cytotoxicity of cultured host cells. VopQ has been shown to rapidly induce autophagy upon translocation into a host cell. VopS AMPylates Rho-family guanosine triphosphatases leading to the collapse of the actin cytoskeleton and host cell rounding prior to lysis. Herein we show that VPA0450 is a phosphatidylinositol phosphatase with homology to the inositol polyphosphate 5-phosphatase catalytic domain of the eukaryotic enzyme synaptojanin. VPA0450 was sufficient to induce membrane blebbing and the delocalization actin-binding proteins from the plasma membrane. VPA0450 contributes to cytotoxicity as strains deleted for vpa0450 induced cell lysis less efficiently than wild-type strains. VPA0450 compromised membrane integrity by hydrolyzing the D5 phosphate from phosphotidylinositide (4,5) bisphosphate, thereby disrupting adaptor protein binding sites required for proper membrane and cytoskeleton dynamics, likely contributing to cell death by facilitating lysis. Preliminary studies have shown the C-terminus of VPA0450 is necessary for localization of this effector to the plasma membrane, possibly by binding membranes and phosphoinositides. An improved system was developed for making chromosomal gene deletions in V. parahemaolyticus. New parent strains were created in which the positive regulators of each T3SS were deleted. Additional strains demonstrated that the cytotoxicity seen during infection with T3SS1 positive strains is attributed solely to T3SS1 effectors. Infection with a strain deleted for vopQ, vopS and vpa0450 uncovered the phenotype for VopR. Bioinformatic analysis of VopR identified effector homologs in other pathogens, homologous eukaryotic enzymes, and a catalytic triad.Item The Functional Characterization of the Quorum Sensing E. Coli Regulators B and C in EHEC(2005-12-19) Clarke, Marcie B.; Sperandio, VanessaEnterohemorrhagic E. coli (EHEC) is the causative agent of hemorrhagic colitis. During an infection, EHEC can sense and respond to environmental cues, including the cell density of the intestinal normal flora (through the floral-derived AI-3 signal) and the epinephrine/norepinephrine produced naturally by the host. This cell-to-cell signaling may aid in colonization and disease by allowing EHEC to up-regulate its flagella and motility genes to swim closer to the intestinal epithelium. Previously, Sperandio et al. (2002) have shown that the quorum sensing E. coli regulators B and C (QseB&C), a two-component system in EHEC, are responsible for the regulation of the master regulator of flagella and motility genes, flhDC, in response to cell-to-cell signaling [1]. Here, we show that QseC, the membrane-bound sensor kinase, can autophosphorylate itself in response to AI-3 or epinephrine/norepinephrine and then transfer this phosphate to the response regulator, QseB. The autophosphorylation of QseC is not affected by the addition of autoinducer-2 or intestinal hormones, including gastrin, galanin, and secretin. Additionally, autophosphorylation can be antagonized upon the addition of phentolamine, an a-adrenergic receptor antagonist. Given that enterocytes harbor a-adrenergic receptors, it would be consistent for a microbial adrenergic sensor (QseC) to mostly resemble (in an orthologous and not a homologous fashion) an a- and not a ᭡drenergic receptor. Taken together, these results suggest that QseC may be a microbial adrenergic receptor conserved amongst different bacterial and fungal species. After QseC has autophosphorylated and transferred its phosphate to QseB, QseB acts as a transcription factor to activate the expression of flhDC, the master regulator of flagella and motility genes. Nested deletion analyses of the flhDC promoter suggest that QseB may bind to three promoter regions, to either repress or activate transcription. Further transcriptional studies suggest that phosphorylated QseB autoregulates its own transcription in a similar manner. These analyses have identified a QseB consensus binding sequence, which was utilized in an in silico search to identify novel potential targets of QseB. Through the use of both biochemistry and genetics, a comprehensive model of the QseB&C signaling cascade was generated.Item High-Throughput Identification of Regulators of the Locus of Enterocyte Effacement from Enterohemorrhagic E. coli(2017-06-12) Pifer, Reed Allen; Winter, Sebastian E.; Sperandio, Vanessa; Alto, Neal; Kliewer, Steven A.Identification of pathways involved in the regulation of the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) of Enterohemorrhagic E. coli (EHEC) has been underway for more than twenty years, but significant knowledge gaps remain. We have created a generalizable, high-throughput method of identifying E. coli core genome components that facilitate T3S. Using this tool, we have generated a dataset of regulatory pathways that control the LEE under anaerobic conditions, a largely neglected but essential consideration for this enteric pathogen. As proof of principle as to the efficacy of this method, we have characterized two transcription factors, cutR and fadR, as previously unknown LEE regulators in EHEC and Citrobacter rodentium. We have determined that CutR functions as a transcriptional activator of the LEE in the presence of L-cysteine, in EHEC and C. rodentium. We have observed that CutR is capable of directly binding to the LEE1 regulatory region, suggesting a direct mechanism of action, while simultaneously controlling a network of LEE-governing genes. We have also determined that FadR functions as a transcriptional repressor of the LEE in EHEC and C. rodentium, directly binding to LEE1 of both organisms. Finally, we explore the broader dataset to begin assessing the potential of yet uncharacterized LEE regulators.Item Interplay Between Tryptophan Metabolites and the Virulence Factors of Enteric Pathogens(2020-08-01T05:00:00.000Z) Kumar, Aman; Winter, Sebastian E.; Sperandio, Vanessa; Conrad, Nicholas; Tu, BenjaminThe human gut consists of a complex milieu of several small molecules that helps in shaping its overall chemistry and biogeography. Trillions of bacteria, collectively known as the gut microbiota, colonize this landscape and occupy a specific niche. Small molecules derived from diet, gut microbial metabolism, and host metabolic activity have an impact on dictating the underlying gut microbiome composition. Gut bacterial populations effectively sense these molecular signatures and modulate gene expression to colonize this niche. An invading intestinal pathogen, to effectively colonize the gut, must sense and respond to the molecular signatures in the gut, which leads to effective colonization and infection. Diet is the principal source of energy by the intake of three main nutrients: carbohydrates, proteins, and fats. Tryptophan is an essential amino acid taken from the diet and plays an important role in protein biosynthesis. Tryptophan is also metabolized to different small molecules and its metabolic products are known to be present in high concentrations in the body. Upon ingestion, tryptophan is readily available in the luminal environment of the gut. Abundant levels of tryptophan are also absorbed by intestinal epithelial cells and is made available in different cellular compartments for normal physiological processes. Tryptophan present in the gut lumen can be further metabolized to numerous small molecules by the action of the gut microbial metabolic activity. Indole is one of the most abundant tryptophan-derived metabolites present in the gut and is absorbed by the host epithelial cells. The host can also metabolize tryptophan to various small molecules including serotonin that is made available in the gut lumen upon release from the host cells. Bacteria in the gut sense these bacterial and host-derived small molecules to colonize and maintain their niche. Similarly, an invading food-borne pathogen such as Enterohemorrhagic Escherichia coli (EHEC), which causes gastroenteritis by primarily colonizing the human colon, sense these small molecules and respond in a way that is conducive for its colonization and virulence gene expression. The role of these highly abundant tryptophan-derived small molecules in dictating the infectivity of an enteric pathogen remains unknown. Because the concentrations of these small molecules naturally present in the body are in the range of the drug concentrations that are used to treat certain diseases, it is possible to repurpose the information gained from these studies to treat intestinal infections. In the present study, we focused on two highly abundant tryptophan-derived small molecules present in the gut. Indole is derived from bacterial metabolism while serotonin is present via the action of host metabolism of tryptophan. Indole and serotonin are structurally similar and therefore may have similar effects against a pathogen in vivo. Indeed, we identified that both indole and serotonin decrease the virulence of the human pathogen Enterohemorrhagic E. coli (EHEC) and the mouse pathogen Citrobacter rodentium. We used multiple strategies including genetic manipulation, pharmacological inhibitors, and knock-out murine models to show that both indole and serotonin are inhibitory signals for virulence gene expression in EHEC and C. rodentium. We further investigated the mechanism used by these pathogens to sense these signals. We identified the first bacterial receptor for both indole and serotonin, and showed that these signals are sensed by a bacterial membrane bound histidine kinase CpxA. Upon sensing indole or serotonin, CpxA dephosphorylates itself and the transcription factor CpxR. In its phosphorylated state, CpxR directly activates expression of the virulence genes, its dephosphorylation prevents its action, decreasing expression of these genes. Together, our studies highlight the importance of sensing small molecules and understanding the gut biogeography by invading pathogens to successfully colonize the gut.Item Ion Selectivity and Gating in the NAK Channel(2009-06-15) Alam, Amer; Jiang, YouxingIon selectivity and gating are two fundamental properties central to proper physiological functioning of ion channels. In this work, a thorough structural characterization of ion binding profiles is presented for the NaK channel, a non selective cation channel from Bacillus cereus, along with an analysis of channel opening and closing mechanisms. An introduction to the overall structure of the full length channel is presented along with initial characterization of its ion selectivity properties. The body of the thesis encompasses a detailed analysis of Ca2+ binding within NaK, followed by additional gating and selectivity studies using high resolution structures of a truncated form of the channel. Two Ca2+ binding sites are observed, both utilizing a unique Ca2+ chelation chemistry involving only backbone carbonyl groups as ligands, with Ca2+ selectivity at the extracellular site mediated by a through space interaction with a conserved acidic residue, Asp66, seen in other Ca2+ conducting channels. In the high resolution structure of the truncated NaK channel, we observe the intracellular gate in an open conformation much like that seen in MthK, making NaK the first channel for which both open and closed state structures are known. This is combined with a structural analysis of ion binding within the NaK selectivity filter, which reveals several interesting features that shed light on the possible underlying mechanisms of ion non-selectivity.Item Localization and Function of Bacterial Type III and IV Effector Proteins(2017-07-31) Jimenez, Alyssa; Winter, Sebastian E.; Alto, Neal; Sperandio, Vanessa; Schoggins, John W.Eukaryotic cell signal transduction networks are highly dynamic and complex systems largely composed of signaling enzymes with modular protein interaction domains and subcellular localization motifs. These sophisticated regulatory mechanisms are crucial to the fidelity and efficacy of information relay in both space and time. Bacterial effector proteins are virulence factors that are directly secreted from the bacteria into the host cytosol and function to rewire eukaryotic signaling networks to establish an environment for bacterial survival. While much effort has gone into substrate identification and biochemical characterization of bacterial effector proteins, it remains unclear how these bacterial enzymes are able to amplify their signaling events to efficiently usurp the robust signaling networks of eukaryotic cells. To this end, I utilized a yeast genetic screen to ask whether bacterial effectors proteins are able to interact with eukaryotic membranes, structures that serve as organizational platforms for the assembly of multi-protein complexes critical for eukaryotic signal transduction. By focusing on a family of bacterial guanine nucleotide exchange factors (GEFs) that activate Rho-family GTPases, and are indispensable for the characteristic accumulation of actin at the site of bacterial attachment and invasion, I have identified a membrane-localization domain in the Salmonella effectors SopE and SopE2 that regulates the ability of these effectors to activate Rho GTPases and invade eukaryotic cells. This membrane-localization domain may function to concentrate these effectors to the highly curved membranes found during Salmonella invasion. Additionally, a polybasic domain identified in the Shigella GEF IpgB1 was found to spatially and temporally regulate actin dynamics. Furthermore, the subcellular location of previously uncharacterized Legionella effectors were found to localize to the vacuoles in yeast and may play a role in regulating vacuolar fusion. Lastly, to gain a greater comprehension of the complex interplay of Salmonella SPI-2 effectors, I developed a collection of Salmonella mutant strains consisting of single SPI-2 effector deletions and a series of combinatorial mutants that are deficient in 2 to 29 effectors. This study expands our knowledge on fundamental processes critical to host-pathogen interactions and provides important mechanistic insights into the spatiotemporal regulation of bacterial effector proteins.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 Physical Studies of Actin Nucleation and Conformational Dynamics(2017-09-06) Zahm, Jacob Aaron; Tomchick, Diana R.; Rosen, Michael K.; Rice, Luke M.; Yu, HongtaoActin is a 42 kilodalton ATPase that exists ubiquitously in eukaryotic cells. Unlike other ATPases, however, actin, under suitable conditions, can polymerize, forming helical filaments. Cells, in orchestrating their myriad cellular processes, utilize actin's intrinsic capacity to polymerize, but do so in a tightly controlled fashion, such that new filaments only appear when and where the cell needs them to suit specific purposes. Such control exists at two different levels. Firstly, the stability of actin filaments is subject to "intrinsic" control arising from the state of bound nucleotide. ATP binding favors incorporation of actin monomers into filaments. This incorporation augments actin's ATP hydrolysis activity, and the conversion of ATP to ADP in the nucleotide binding cleft considerably destabilizes filaments, facilitating the return of filament subunits to free monomers. The structural mechanism through which nucleotide conveys information throughout the actin monomer to influence polymerization behavior remains poorly understood and represents a persistent fundamental biological question. In this work I, for the first time, apply modern muti-resonance NMR methods to begin to answer these questions. In addition to the aforementioned intrinsic control, cellular actin is subject to "extrinsic" control via the action of nucleation factors. In order to form a growing filament, actin must proceed through a nucleation step in which monomers must assemble into a thermodynamically and kinetically disfavored nucleus, which ultimately proceeds to a growing filament. Nucleation factors accelerate the rate of filament formation by binding to actin monomers and arranging them into the prerequisite nucleus. In this work, I reveal the crystal structure of actin monomers in complex with the bacterially derived nucleation factor, VopL. The structure represents the first high resolution snapshot of a filament-like nucleation intermediate, and reveals general principles underlying the action of nucleation factors.Item The Regulation of Flagellar Biosynthesis and Cell Division in Campylobacter jejuni(2016-04-05) Gulbronson, Connor James; Hansen, Eric J.; Hendrixson, David R.; Norgard, Michael V.; Alto, NealFlagellar biosynthesis is one of the rare processes known to be spatially and numerically regulated in polarly-flagellated bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuni FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. FlhG also influences spatial regulation of division, which is essential for viability and is typically regulated by the Min system in most bacteria. However, C. jejuni lacks the Min system, but appears to utilize FlhG and components of the flagellar MS and C ring to influence spatial regulation of division. We utilized a variety imaging techniques to quantify the in vivo effects of mutations in C. jejuni and used purified proteins to assay the in vitro enzymatic activity of FlhG and FlhF (a GTPase) to determine the influence these factors have on both regulation of flagellar biogenesis and spatial regulation of division. We found that unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni instead, other regions of C. jejuni FlhG were discovered to be involved in numerical regulation of flagellar biogenesis. Mutations in the α6 and α7 helices of FlhG were found to influence aspects of FlhG biology, spatial regulation of division, and numerical regulation of flagellar biogenesis. We also found that C. jejuni FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. In this work, we propose a model in which FlhF in a GTP-bound ('active') state promotes the formation of the MS and C rings at the aflagellated pole after a division event. We then hypothesize that MS and C ring proteins influence FlhG localization to stimulate FlhF GTPase activity and, by extension, numerical regulation of flagellar biogenesis and spatial regulation of division at poles. Although some aspects of this model have yet to be fully tested, our data could potentially be applied in other polar flagellates to gain a better understanding of numerical regulation of flagellar biogenesis and spatial regulation of division in these organisms.Item Regulatory RNAs at the Heart of Sugar Metabolism: New Mechanisms and Novel Discoveries(2011-02-01) Irnov; Winkler, Wade C.Bacteria are adept at using a variety of posttranscriptional strategies to regulate gene expression. Specifically, various RNA-mediated genetic control elements have been discovered in the past decade through a combination of genetics, bioinformatics, and transcriptomic approaches. Together, these RNA elements control the expression of many genes involved in diverse cellular processes such as energy metabolism, stress response, biofilm formation, and pathogenesis. In the Gram-positive bacterium Bacillus subtilis, several RNA elements have been shown to be required for the precise coordination of genes involved in various sugar utilization pathways. These genetic switches typically regulate gene expression by modulating the formation of a transcription termination element in a ligand-dependent manner. Interestingly, two unique elements, the glmS ribozyme and the eps-associated RNA (EAR), are missing the signature elements required for control of transcription termination or translation initiation. The latter mechanism is more commonly found in Gram-negative bacteria. Our objective is to study the mechanisms by which these two RNAs control gene expression. Additionally, we would like to identify other regulatory RNAs that are important for sugar metabolism in Bacillus subtilis. Both the glmS ribozyme and EAR are positioned at the center of the sugar metabolism pathways in B. subtilis. The glmS RNA is a glucosamine-6-phosphate responsive element that regulates the expression of the GlmS enzyme, which directs sugar precursors from glycolysis into the cell wall biosynthesis pathway. The EAR element resides within the 16-kb eps operon that is required for biofilm exopolysaccharide production. Our data demonstrates that both RNAs employ novel mechanisms: the glmS ribozyme utilizes a ligand-specific RNase-mediated degradation event, while EAR uses a processive antitermination mechanism for complete synthesis of the long operon. Furthermore, by using high-throughput sequencing approach we have successfully identified many new regulatory RNA candidates, including various long 5`-UTR, toxin-antitoxin systems, prophage-encoded RNAs, and several developmentally regulated small RNAs. Their functions are still under investigation. Collectively, our studies provide important insights into the different aspects of bacterial physiology, including RNA decay pathways, transcription of long operons and cellular differentiation. We argue that posttranscriptional regulation is of greater importance to Bacillus subtilis (and probably all bacteria) than previously realized.