Browsing by Subject "Vibrio cholerae"
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Item Biochemistry and evolution of Feo, the major ferrous iron transport system of Vibrio cholerae(2022-04-04) Gómez Garzón, Camilo Andrés; Payne, Shelley M.; Barrick, Jeffrey E; Browning, Karen S; Contreras, Lydia M; Torres, Alfredo GIron is an essential element for life, and its acquisition entails a challenge for pathogens as they compete with their host and associated microbiota for this element. A major mechanism used by bacterial pathogens to obtain iron from their host is Feo, a widespread prokaryotic system dedicated to the transport of ferrous iron (Fe²⁺), which represents the prevalent form of this metal in the human gut. Despite of the importance of Feo for bacterial pathogens, the mechanism for Feo-mediated iron transport remains poorly understood. In this work, I used the human pathogen Vibrio cholerae as a model organism to study Feo. In V. cholerae, Feo is made up of three components: FeoB, a large transmembrane protein with NTPase activity; and FeoA and FeoC, two small cytoplasmic proteins with unknown function. Current evidence indicate that Feo works as a multimeric complex embedded in the inner membrane and the FeoB NTPase activity is critical for iron transport. In section 1 of Results, I evaluated how different factors affect the enzymatic activity of full-length FeoB in vitro. I found that FeoA, FeoC, Fe²⁺, or K⁺ do not modulate the activity of FeoB. Therefore, FeoB is independent of stimulatory factors, and the roles of FeoA and FeoC might be other than regulation. In section 2, I studied the functional significance specific residues and domains of the Feo proteins for complex formation and iron uptake. According to my results, both the cytoplasmic and transmembrane domains of FeoB are necessary for protein-protein interactions and assembly of the large complex. Also, it may be possible that FeoA and FeoC interact, but this interaction would require the presence of FeoB. Finally, in section 3, I conducted a comprehensive bioinformatic analysis of the Feo protein sequences in bacteria. Complementing the findings from this analysis with in vivo assessments in V. cholerae, I proposed an evolutionary model of the Feo system, focusing on how FeoC might have been lost in several lineages. In summary, this work contributes to the understanding of the Feo system by providing insight into the relationship between its structural organization of and its function.Item Characterization of the Vibrio cholerae ferrous iron transport system, feo(2015-11-23) Stevenson, Gladys Begoña; Payne, Shelley M.; Browning, Karen S; Davies, Bryan W; Hoffman, David W; Trent, Michael SFeo is the major ferrous iron transport system in prokaryotes and has only been partially characterized, as its assembly and mechanism of transport have not been determined. The feo operon in V. cholerae encodes three proteins, FeoA, FeoB, and FeoC, which are all required for function of the Feo system. FeoA and FeoC are both small cytoplasmic proteins and their function remains unclear. FeoB, thought to function as a ferrous iron permease, is a large integral membrane protein made up of an N-terminal GTPase domain and a C-terminal membrane-spanning region. To date, structural studies of FeoB have been carried out using a truncated form of the protein encompassing only the N-terminal GTPase region. However, in this study, a model of the topology of the C-terminal membrane-spanning region of FeoB, based on in vivo labeling experiments, is proposed. Further, through the use of scanning cysteine accessibility mutagenesis, it is determined that the N- and C- termini of FeoB are located in the cytoplasm of V. cholerae. Moreover, epitope-tagged FeoB and FeoC are used to show that these proteins form higher order complexes when cross-linked in vivo in V. cholerae. Further analysis reveals that FeoB simultaneously associates with both FeoA and FeoC to form a large inner membrane complex, an observation that has not been reported previously. It is found that FeoA is required for complex formation, while FeoC is required for wildtype protein levels of FeoB. It is also determined that certain amino acid residues in the GTPase region of FeoB are required for function of the Feo system and for complex formation.Item Creation of a viable csrA mutant in Vibrio cholerae(2013-08) Thomas, Martha Barnett; Payne, Shelley M.Vibrio cholerae, the causative agent of cholera, has been a lethal enteric pathogen to humans for most of recorded history. Even though it is well studied, it still kills many people every year due to rapid and severe dehydrations from diarrhea. Part of what makes V. cholerae such an effective pathogen is its ability to control virulence factors depending on its environment. ToxR is a major virulence protein that has upstream control of most of the virulence genes that are turned on when in a human host. Two of the most critical virulence factors, toxin coregulated pilus and cholera toxin are controlled by ToxR. CsrA is a protein that regulates many cellular functions in V. cholerae, including glycogen synthesis, motility, and biofilm production. Preliminary data suggests a link between CsrA and the regulation of ToxR. In order to study CsrA as it relates to ToxR regulation, a csrA mutant must be generated in V. cholerae. CsrA plays such an important role in glycogen metabolism that a csrA mutant is not viable due to excessive glycogen levels. In order to make a viable csrA mutant, glycogen synthesis has to be turned off. In this research, I attempt to make a viable V. cholerae csrA mutant by deleting csrA in a strain that is deficient for glycogen synthesis (glg). Normally without CsrA, glycogen in the cell would increase to a detrimental level. Since a glg⁻ csrA⁻ mutant lacks the ability to make glycogen, the levels never reach a lethal level, allowing the mutant to survive without functional CsrA. Such a glg- csrA- double mutant's ToxR regulation can be studied by growth in various media by measuring OmpU and OmpT expression. Using PCR, restriction enzymes, and DNA ligase, a suicide plasmid was created containing sequences that flank the csrA gene but instead of the csrA gene, a chloramphenicol resistance cassette was inserted. Through bacterial conjugation this plasmid was introduced into three V. cholerae glg- strains. Allelic exchange was carried out utilizing the homology between the DNA flanking wild type csrA and the csrA deletion with chloramphenicol cassette. This first crossover event was initiated with the requirement of the [pi] protein for the plasmid to replicate. Without the pir gene to create [pi] protein, selection for antibiotic resistance required that the plasmid integrate into the genome. This was selected based on the plasmid encoded ampicillin resistance. After the second crossover event, there were two possible outcomes of excision: reverting to wild type csrA or retention of the csrA mutation. The csrA mutant was selected based on its sucrose and chloramphenicol resistance and ampicillin sensitivity.Item Creation of Vibrio cholerae strains to test the induction of the Feo iron transport system using the recombination-based in vivo expression technology (RIVET) vector(2009) Maupin, Suzanna; Shelley PayneCholera is a diarrheal disease caused by the bacterium Vibrio cholerae. Iron is important for survival for this pathogen, both as a regulatory mechanism and as part of enzymes. Although the iron-transport mechanisms of V. cholerae have been studied in great detail, it is still unclear when these mechanisms are active during infection. But, there is now a method to test the expression of these mechanisms. This method is known as the recombination-based in vivo expression technology (RIVET) vector. This is a new technology with which one can look at the induction of transcription of different genes during infection of infant mice. I have inserted the feo gene promoter, which is a ferrous iron uptake mechanism, into this vector. I have then incorporated this vector into two cholera strains, so that we can better understand when this particular iron uptake mechanism is expressed in vivo. These strains will be able to successfully identify when the iron transport genes are expressed during infection.Item Effects of fatty acids in bile on the phospholipids of Vibrio cholerae(2010) Lynch, Stephanie; Stephen TrentEnteric bacteria, such as E. coli and V. cholerae, encounter the digestive secretion bile in the small intestine. Bile is composed of mainly bile acids, lecithin, bilirubin, bicarbonate ions and fatty acids. Since bile possesses detergent like activity, bacteria have evolved mechanisms to avoid its bactericidal effect. V. cholerae senses bile and activates virulence genes that mediate motility, chemotaxis and adherence in preparation for colonization. The present study identifies a difference in the phospholipid profile of V. cholerae, but not E. coli, when grown in the presence and absence of bile. Mass spectrometry analysis of crude bile showed a variety of fatty acids including several longchain and polyunsaturated fatty acids. To determine if the fatty acids in bile could be responsible for the differences in the phospholipids, E. coli and V. cholerae were grown in the presence of the individual fatty acids prior to phospholipid analysis. Growth of E. coli in fatty acids showed no significant changes in any phospholipids. However, growth of V. cholerae in fatty acids resulted in an upward shift of cardiolipin in longer chained fatty acids as well as the appearance of two species of phosphatidylethanolamine. There were also fatty acids that caused an appearance of lyso-phosphatidylethanolamine. These results demonstrate that V. cholerae can utilize a wider range of fatty acids than E. coli as reflected in membrane phospholipids. Specific long chain and polyunsaturated fatty acids are identified as being incorporated into V. cholerae phospholipids. V. cholerae is also associated with fatty acid rich marine environments. These environments include colonization of zooplankton and insect egg masses, as well as surviving in aquatic sediment. The survival advantage that this adaptation may provide is discussed.Item Elucidating factors affecting H-NS-mediated regulation of horizontally acquired genes in Vibrio cholerae(2018-05-01) Conrado, Aaron Ross; Davies, Bryan William; Marcotte, Edward; Barrick, Jeffrey; Upton, JasonHorizontal gene transfer amongst bacteria plays a critical role in the evolution, emergence, and virulence of both currently recognized and novel pathogens. Vibrio cholerae exemplifies this process, as benign environmental isolates emerge as pandemic pathogens through the acquisition and incorporation of genetic elements encoding virulence factors into their progenitor genomes. In V. cholerae, these genes are localized to distinct areas of the chromosome, known as horizontally acquired islands (HAIs), which are characterized by a lower GC-content than the progenitor genome. In cholera and many other Gram-negative enterics, AT-rich DNA is bound by the H-NS protein immediately upon entrance into the cell, silencing the expression of this potentially toxic DNA. In other enterics, some proteins can remove H-NS from the DNA, allowing the cell to probe these novel genes, while others can interact with H-NS to maintain regulatory control and ensure derepression only happens when appropriate. However, such interactions with H-NS have not been observed in V. cholerae. Here, we discover and characterization of the genetic relationships between H-NS and an H-NS antagonist (ToxR, an essential protein for virulence), as well as an H-NS enhancer (TsrA, a poorly understood protein) in V. cholerae. ToxR is known to be a master activator of virulence, but we demonstrate that ToxR’s critical role is not direct activation, but rather to antagonize H-NS binding at shared binding loci related to host colonization and biofilm formation. Furthermore, TsrA, previously shown to be involved in regulation of type-VI secretion, was detected through immunoprecipitation of H-NS, followed by LC-MS/MS. Subsequent genetic analyses revealed that TsrA enhances H-NS repression of virulence genes on HAIs. Interestingly, TsrA enhances H-NS enrichment at areas of low-GC content, similar to the H-NS interactors in other enterics that V. cholerae was thought to lack. TsrA deletion also affects uptake of a mobile genetic element, establishing the first Vibrio-specific modulator of H-NS function that influences the regulation and acquisition of virulence-defining genetic elements. Knowledge of these interactions sheds light on HNS’ role in defining the virulence potential of V. cholerae and reveals a novel H-NS interactor similar to those present in other Gram-negative bacteriaItem Endogenous MMTV Proviruses Induce Susceptibility to Both Viral and Bacterial Pathogens(Public Library of Science, 2006-12-01) Bhadra, Sanchita; Lozano, Mary M; Payne, Shelley M; Dudley, Jaquelin PMost inbred mice carry germline proviruses of the retrovirus, mouse mammary tumor virus (MMTV) (called Mtvs), which have multiple replication defects. A BALB/c congenic mouse strain lacking all endogenous Mtvs (Mtv-null) was resistant to MMTV oral and intraperitoneal infection and tumorigenesis compared to wild-type BALB/c mice. Infection of Mtv-null mice with an MMTV-related retrovirus, type B leukemogenic virus, also resulted in severely reduced viral loads and failure to induce T-cell lymphomas, indicating that resistance is not dependent on expression of a superantigen (Sag) encoded by exogenous MMTV. Resistance to MMTV in Mtv-null animals was not due to neutralizing antibodies. Further, Mtv-null mice were resistant to rapid mortality induced by intragastric inoculation of the Gram-negative bacterium, Vibrio cholerae, but susceptibility to Salmonella typhimurium was not significantly different from BALB/c mice. Susceptibility to both MMTV and V. cholerae was reconstituted by the presence of any one of three endogenous Mtvs located on different chromosomes and was associated with increased pathogen load. One of these endogenous proviruses is known to encode only Sag. Therefore, Mtv-encoded Sag appears to provide a unique genetic susceptibility to specific viruses and bacteria. Since human endogenous retroviruses also encode Sags, these studies have broad implications for pathogen-induced responses in mice and humans.Item feoA, feoB, and feoC encode essential components of the Vibrio cholerae ferrous iron transport system(2010-12) Helton, Emily Ann; Payne, Shelley M.; Whiteley, Marvin; Stevens, Scott W.; Trent, M. Stephen; Robertus, JonVibrio cholerae, the causative agent of the diarrheal disease cholera, must acquire iron to survive. Although iron is relatively abundant, it forms insoluble ferric complexes in the presence of oxygen. The more soluble ferrous iron is limited to anaerobic or reducing environments. To meet the nutritional needs of the cell, V. cholerae encodes many different ferric iron transport systems but only one characterized ferrous iron transporter, Feo. Feo is widely distributed in bacteria and archaea, but the mechanism for transport is not known. In this study, basic characterization of the V. cholerae feoABC operon was performed to gain further understanding about a critical iron transport system. Each gene in the operon, feoA, feoB, and feoC, was found to be required for ferrous iron uptake. FeoB, an inner membrane protein, is considered to be the ferrous permease but functions for FeoA and FeoC are not known. These studies show that neither FeoA nor FeoC is required for expression of feoB, suggesting that these proteins are required for Feo function. Analysis of the composition of the Feo transporter using a bacterial adenylate cyclase two-hybrid system indicated interactions between Feo proteins, specifically, between FeoC and the cytoplasmic portion of FeoB. This result indicates that feoC encodes a protein that interacts with FeoB and is necessary for ferrous iron transport.Item Heme utilization in Vibrio cholerae and analysis of domains involved in the specificity of TonB for TonB-dependent receptors(2002) Mey, Alexandra Rebecca; Payne, Shelley M.Vibrio cholerae has multiple iron transport systems, one of which involves heme uptake through the outer membrane receptor HutA. This study demonstrates that V. cholerae encodes two additional TonB-dependent heme receptors, HutR and HasR. HutR has significant homology to HutA and to other bacterial outer membrane heme receptors, and the role of HutR in hemin utilization and its localization in the outer membrane were confirmed. The hutR gene was co-transcribed with the upstream gene ptrB, and expression from the ptrB promoter was negatively regulated by iron. HasR is most similar to the hemophore-utilizing heme receptors from Pseudomonas aeruginosa and Serratia marcescens. A mutant defective in all three heme receptors was unable to utilize hemin as an iron source. HutA and HutR functioned with either V. cholerae TonB1 or TonB2. In contrast, hemin uptake through HasR was TonB2- dependent. Efficient utilization of hemoglobin as an iron source required HutA and TonB1. The triple heme receptor mutant exhibited no defect in its ability to compete with its Vib- parental strain in an infant mouse model of infection, indicating that additional iron sources are present in vivo. V. cholerae utilized heme derived from marine invertebrate hemoglobins, suggesting that heme may be available to V. cholerae growing in the marine environment. Although E. coli TonB and V. cholerae TonB1 exhibit different specificities for outer membrane receptors, these TonB proteins are similar enough that functional chimeras can be created between them. The activities of the chimeric TonB proteins demonstrated that the C-terminal one-third of TonB constitutes a functional domain responsible for receptor specificity. A Pro238Thr substitution in V. cholerae TonB1 resulted in the ability of TonB1 to recognize a wider range of receptors, indicating that very C-terminal end of TonB1 determines receptor specificity. Domain-switching experiments between E. coli ChuA and V. cholerae HutA showed that the TonB box heptapeptide at the Nterminus of these receptors does not contain specificity determinants. Instead, specificity was controlled by the residue immediately preceding the TonB box. Taken together, these data suggest that functional interactions take place between the C-terminus of TonB and the very N-terminal domain of TonB-dependent receptors.Item Medium alkalization due to carbon metabolism is largely responsible for inhibition of bacterial growth by Vibrio cholerae supernatants(2017-05) Becker, Miranda; Payne, ShelleyVibrio cholerae is the causative agent of the diarrheal disease cholera. Many Vibrio species secrete antimicrobial factors, though the identity of such a factor has not been determined for any V. cholerae strain. Such an antimicrobial factor could be relevant to pathogenesis of cholera, which disrupts the intestinal microbiome. In this study, we investigated the antimicrobial effects of supernatant from 72 hour old cultures of V. cholerae C6706 on Shigella flexneri CFS100. Inhibition of S. flexneri growth was found to be dependent on the alkaline pH of the supernatant. A 1:1 mixture of pH-adjusted supernatant and LB was found to inhibit S. flexneri growth at alkaline but not neutral pH, as was pH-adjusted LB alone. In minimal medium, elevation of supernatant pH by V. cholerae was dependent on nutritional factors, and this elevation of medium pH also correlated with increased S. flexneri growth inhibition. Though medium alkalization in LB is often attributed to amino acid catabolism and the consequent production of ammonia, supplementation of V. cholerae cultures in minimal medium with amino acids had a weaker effect on alkalization and inhibition than did supplementation with selected carbon sources. This suggests that some feature of carbon metabolism causes medium alkalization and the resultant antimicrobial activity. Several V. cholerae mutants in potentially relevant pathways were screened for alkalization and S. flexneri growth inhibition, but none had any effect.Complicating this picture is the finding that V. cholerae grown under microaerobic conditions produce a less alkaline supernatant with stronger S. flexneri growth inhibition. The significance of this is unknown.Item RpoS Controls the Vibrio cholerae Mucosal Escape Response(Public Library of Science, 2006-10-20) Nielsen, Alex Toftgaard; Dolganov, Nadia A; Otto, Glen; Miller, Michael C; Wu, Cheng Yen; Schoolnik, Gary KVibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the “mucosal escape response,” this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle.Item The role and regulation of CsrA in Vibrio cholerae pathogenesis(2019-05-06) Butz, Heidi Ann; Payne, Shelley M.; Davies , Bryan W; Xhemalce , Blerta; Contreras, Lydia M; Macdonald , Paul MVibrio cholerae is a natural inhabitant of the aquatic environment; however, if ingested, it can be a deadly human pathogen. A V. cholerae infection requires a rapid change in gene expression in response to host-specific environmental cues. This response enables the bacterium to overcome microbial deterrents found throughout the gastrointestinal tract while promoting colonization within the small intestine. This study shows that the post-transcriptional global regulator, CsrA, has an instrumental role in this process. In this study, I show that in response to amino acid supplementation, the levels of the CsrA-antagonistic Csr small RNAs (sRNAs) decrease. This decrease in the Csr sRNAs likely shifts the equilibrium from more sequestered CsrA to more available CsrA, enabling CsrA to regulate more direct RNA targets. This shift is reflected in the CsrA-dependent increase in ToxR protein levels in the presence, but not absence, of nutrient supplementation. This change in CsrA availability in response to nutrient supplementation is one example of V. cholerae altering gene expression in response to environmental cues. Additionally, I demonstrate that CsrA autoregulates its availability by controlling the expression of the Csr sRNAs through their primary regulator, VarA. Because the activity of CsrA must be tightly regulated, this intrinsic regulatory feedback loop prevents major fluctuations in CsrA availability, which would be deleterious to the cell. I also show through transcriptomic approaches that CsrA-mediated regulation controls the gene expression of nearly 25% of the V. cholerae transcriptome. From this analysis, I found that CsrA represses the expression of genes required for survival in the aquatic environment, including biofilm production, while simultaneously activating expression of genes required for host colonization, such as motility and virulence factors. Approximately 70% of all flagellar assembly genes were downregulated in the csrA mutant, and the csrA mutant had impaired motility compared to the wild-type, suggesting that CsrA positively regulates motility. Additionally, CsrA directly activates the protein production of the quorum sensing master regulator, AphA, which is required for toxin production. Taken together, these data indicate that CsrA regulation controls multiple pathways that are important both outside and inside the host