scholarly journals Characterization of Ferric and Ferrous Iron Transport Systems in Vibrio cholerae

2006 ◽  
Vol 188 (18) ◽  
pp. 6515-6523 ◽  
Author(s):  
Elizabeth E. Wyckoff ◽  
Alexandra R. Mey ◽  
Andreas Leimbach ◽  
Carolyn F. Fisher ◽  
Shelley M. Payne
Keyword(s):  
Vibrio Cholerae ◽  
Transport System ◽  
Iron Transport ◽  
Iron Acquisition ◽  
Shigella Flexneri ◽  
Transport Systems ◽  
Infant Mouse ◽  
Mouse Intestine ◽  
Dependent Transport

ABSTRACT Vibrio cholerae has multiple iron acquisition systems, including TonB-dependent transport of heme and of the catechol siderophore vibriobactin. Strains defective in both of these systems grow well in laboratory media and in the infant mouse intestine, indicating the presence of additional iron acquisition systems. Previously uncharacterized potential iron transport systems, including a homologue of the ferrous transporter Feo and a periplasmic binding protein-dependent ATP binding cassette (ABC) transport system, termed Fbp, were identified in the V. cholerae genome sequence. Clones encoding either the Feo or the Fbp system exhibited characteristics of iron transporters: both repressed the expression of lacZ cloned under the control of a Fur-regulated promoter in Escherichia coli and also conferred growth on a Shigella flexneri mutant that has a severe defect in iron transport. Two other ABC transporters were also evaluated but were negative by these assays. Transport of radioactive iron by the Feo system into the S. flexneri iron transport mutant was stimulated by the reducing agent ascorbate, consistent with Feo functioning as a ferrous transporter. Conversely, ascorbate inhibited transport by the Fbp system, suggesting that it transports ferric iron. The growth of V. cholerae strains carrying mutations in one or more of the potential iron transport genes indicated that both Feo and Fbp contribute to iron acquisition. However, a mutant defective in the vibriobactin, Fbp, and Feo systems was not attenuated in a suckling mouse model, suggesting that at least one other iron transport system can be used in vivo.

scholarly journals Anaerobic Regulation of Shigella flexneri Virulence: ArcA Regulates fur and Iron Acquisition Genes

2007 ◽  
Vol 189 (19) ◽  
pp. 6957-6967 ◽  
Author(s):  
Megan L. Boulette ◽  
Shelley M. Payne
Keyword(s):  
Transcription Factors ◽  
Transport System ◽  
Iron Transport ◽  
Iron Acquisition ◽  
Shigella Flexneri ◽  
Plaque Assay ◽  
Plaque Formation ◽  
Transport Systems ◽  
Dependent Manner ◽  
Anaerobic Conditions

ABSTRACT Invasion and plaque formation in epithelial monolayers are routinely used to assess the virulence of Shigella flexneri, a causative agent of dysentery. A modified plaque assay was developed to identify factors contributing to the virulence of S. flexneri under the anaerobic conditions present in the colon. This assay demonstrated the importance of the ferrous iron transport system Feo, as well as the global transcription factors Fur, ArcA, and Fnr, for Shigella plaque formation in anoxic environments. Transcriptional analyses of S. flexneri iron transport genes indicated that anaerobic conditions activated feoABC while repressing genes encoding two other iron transport systems, the ABC transporter Sit and the Iuc/Iut aerobactin siderophore synthesis and transport system. The anaerobic transcription factors ArcA and Fnr activated expression of feoABC, while ArcA repressed iucABCD iutA. Transcription of fur, encoding the iron-responsive transcriptional repressor of bacterial iron acquisition, was also repressed anaerobically in an ArcA-dependent manner.

scholarly journals Iron and Fur Regulation in Vibrio cholerae and the Role of Fur in Virulence

2005 ◽  
Vol 73 (12) ◽  
pp. 8167-8178 ◽  
Author(s):  
Alexandra R. Mey ◽  
Elizabeth E. Wyckoff ◽  
Vanamala Kanukurthy ◽  
Carolyn R. Fisher ◽  
Shelley M. Payne
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Iron Acquisition ◽  
Bacterial Species ◽  
Regulation Of Gene Expression ◽  
Transport Systems ◽  
Infant Mouse ◽  
Genes Encoding ◽  
Regulatory Functions

ABSTRACT Regulation of iron uptake and utilization is critical for bacterial growth and for prevention of iron toxicity. In many bacterial species, this regulation depends on the iron-responsive master regulator Fur. In this study we report the effects of iron and Fur on gene expression in Vibrio cholerae. We show that Fur has both positive and negative regulatory functions, and we demonstrate Fur-independent regulation of gene expression by iron. Nearly all of the known iron acquisition genes were repressed by Fur under iron-replete conditions. In addition, genes for two newly identified iron transport systems, Feo and Fbp, were found to be negatively regulated by iron and Fur. Other genes identified in this study as being induced in low iron and in the fur mutant include those encoding superoxide dismutase (sodA), fumarate dehydratase (fumC), bacterioferritin (bfr), bacterioferritin-associated ferredoxin (bfd), and multiple genes of unknown function. Several genes encoding iron-containing proteins were repressed in low iron and in the fur mutant, possibly reflecting the need to reserve available iron for the most critical functions. Also repressed in the fur mutant, but independently of iron, were genes located in the V. cholerae pathogenicity island, encoding the toxin-coregulated pilus (TCP), and genes within the V. cholerae mega-integron. The fur mutant exhibited very weak autoagglutination, indicating a possible defect in expression or assembly of the TCP, a major virulence factor of V. cholerae. Consistent with this observation, the fur mutant competed poorly with its wild-type parental strain for colonization of the infant mouse gut.

scholarly journals Vibrio cholerae VciB Promotes Iron Uptake via Ferrous Iron Transporters

2008 ◽  
Vol 190 (17) ◽  
pp. 5953-5962 ◽  
Author(s):  
Alexandra R. Mey ◽  
Elizabeth E. Wyckoff ◽  
Lindsey A. Hoover ◽  
Carolyn R. Fisher ◽  
Shelley M. Payne
Keyword(s):  
Vibrio Cholerae ◽  
Transport System ◽  
Ferrous Iron ◽  
Iron Uptake ◽  
Iron Transport ◽  
Growth Enhancement ◽  
Outer Membrane Receptor ◽  
E Coli

ABSTRACT Vibrio cholerae uses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein in V. cholerae, VciB. The vciB gene and its linked gene, vciA, were isolated in a screen for V. cholerae genes that permitted growth of an Escherichia coli siderophore mutant in low-iron medium. The vciAB operon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation of E. coli and Shigella flexneri strains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen when vciB was expressed in an E. coli or S. flexneri strain defective for the ferrous iron transporter Feo. Supplying the E. coli feo mutant with a plasmid encoding either E. coli or V. cholerae Feo, or the S. flexneri ferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systems V. cholerae Fbp and Haemophilus influenzae Hit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. A V. cholerae vciB mutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen.

scholarly journals Characterization of the Yersinia pestisYfu ABC Inorganic Iron Transport System

2001 ◽  
Vol 69 (5) ◽  
pp. 2829-2837 ◽  
Author(s):  
Shimei Gong ◽  
Scott W. Bearden ◽  
Valerie A. Geoffroy ◽  
Jacqueline D. Fetherston ◽  
Robert D. Perry
Keyword(s):  
Transport System ◽  
Iron Transport ◽  
Iron Acquisition ◽  
Lethal Dose ◽  
In Vitro Transcription ◽  
Defined Medium ◽  
Transport Systems ◽  
Regulation Of Transcription ◽  
Iron Deficient

ABSTRACT In Yersinia pestis, the causative agent of plague, two inorganic iron transport systems have been partially characterized. The yersiniabactin (Ybt) system is a siderophore-dependent transport system required for full virulence. Yfe is an ABC transport system that accumulates both iron and manganese. We have identified and cloned aY. pestis yfuABC operon. The YfuABC system is a member of the cluster of bacterial ABC iron transporters that include Sfu ofSerratia, Hit of Haemophilus, and Yfu ofYersinia enterocolitica. The Y. pestis KIM6+ system is most homologous to that in Y. enterocolitica, showing identities of 84% for YfuA (periplasmic binding protein), 87% for YfuB (inner membrane permease), and 75% for YfuC (ATP hydrolase). We constructed a yfuABC promoter-lacZ fusion to examine regulation of transcription. This promoter contains a potential Fur binding sequence and is iron and Fur regulated. Significant expression from the yfuABC promoter occurred during iron-deficient growth conditions. In vitro transcription and translation of a recombinant plasmid encoding yfuABCindicates that YfuABC proteins are expressed. Escherichia coli 1017 (an enterobactin-deficient mutant) carrying this plasmid was able to grow in an iron-restrictive complex medium. We constructed a deletion encompassing the yfuABC promoter and most of yfuA. This mutation was introduced into strains with mutations in Ybt, Yfe, or both systems to examine the role of Yfu in iron acquisition in Y. pestis. Growth of theyfu mutants in a deferrated, defined medium (PMH2) at 26 and 37°C failed to identify a growth or iron transport defect due to the yfu mutation. Fifty percent lethal dose studies in mice did not demonstrate a role for the Yfu system in mammalian virulence.

scholarly journals ToxR-Independent Expression of Cholera Toxin from the Replicative Form of CTXφ

1998 ◽  
Vol 66 (1) ◽  
pp. 394-397 ◽  
Author(s):  
Sara Lazar ◽  
Matthew K. Waldor
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Toxin Production ◽  
Recipient Strain ◽  
Replicative Form ◽  
Filamentous Bacteriophage ◽  
Infant Mouse ◽  
Mouse Intestine

ABSTRACT The ctxAB operon, which encodes cholera toxin, resides in the genome of CTXφ, a filamentous bacteriophage. WithinVibrio cholerae cells, the CTXφ genome can exist either as a replicating plasmid or as a prophage integrated into the chromosome. Previous work established that ToxR is required for chromosomal ctxAB expression. We have learned that strains harboring the CTXφ replicative form produce cholera toxin under all conditions tested, independently of ToxR. During passage of CTXφ lysogens through the infant mouse intestine, transduction of CTXφ to a recipient strain can be detected, indicating that phage excision and replication occur in vivo. These results suggest that phage induction might provide a novel mechanism for the regulation of cholera toxin production.

scholarly journals DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains

2006 ◽  
Vol 188 (2) ◽  
pp. 745-758 ◽  
Author(s):  
Timothy J. Johnson ◽  
Kylie E. Siek ◽  
Sara J. Johnson ◽  
Lisa K. Nolan
Keyword(s):  
Escherichia Coli ◽  
Dna Sequence ◽  
Transport System ◽  
Iron Transport ◽  
Virulence Genes ◽  
Iron Acquisition ◽  
Transport Systems ◽  
E Coli ◽  
Virulence Traits ◽  
Associated Sequences

ABSTRACT ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains.

scholarly journals MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ryan W. Bogard ◽  
Bryan W. Davies ◽  
John J. Mekalanos
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Current Knowledge ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Pathogenic Potential ◽  
Content Type ◽  
Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

scholarly journals Identification of the Vibrio cholerae Enterobactin Receptors VctA and IrgA: IrgA Is Not Required for Virulence

2002 ◽  
Vol 70 (7) ◽  
pp. 3419-3426 ◽  
Author(s):  
Alexandra R. Mey ◽  
Elizabeth E. Wyckoff ◽  
Amanda G. Oglesby ◽  
Eva Rab ◽  
Ronald K. Taylor ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Mutant Strain ◽  
Genomic Sequence ◽  
Iron Acquisition ◽  
Lethal Dose ◽  
Single Mutation ◽  
Periplasmic Binding Protein ◽  
Infant Mouse ◽  
Abc Transport ◽  
Important Virulence Factor

ABSTRACT The gram-negative enteric pathogen Vibrio cholerae requires iron for growth. V. cholerae has multiple iron acquisition systems, including utilization of heme and hemoglobin, synthesis and transport of the catechol siderophore vibriobactin, and transport of several siderophores that it does not itself make. One siderophore that V. cholerae transports, but does not make, is enterobactin. Enterobactin transport requires TonB and is independent of the vibriobactin receptor ViuA. In this study, two candidate enterobactin receptor genes, irgA (VC0475) and vctA (VCA0232), were identified by analysis of the V. cholerae genomic sequence. A single mutation in either of these genes did not significantly impair enterobactin utilization, but a strain defective in both genes did not use enterobactin. When either irgA or vctA was supplied on a plasmid, the ability of the irgA vctA double mutant to use enterobactin was restored. This indicates that both VctA and IrgA transport enterobactin. We also identify the genes vctPDGC, which are linked to vctA and encode a periplasmic binding protein-dependent ABC transport system that functions in the utilization of both enterobactin and vibriobactin (VCA0227-0230). An irgA::TnphoA mutant strain, MBG40, was shown in a previous study to be highly attenuated and to have a strong colonization defect in an infant mouse model of V. cholerae infection (M. B. Goldberg, V. J. DiRita, and S. B. Calderwood, Infect. Immun. 58:55-60, 1990). In this work, a new irgA mutation was constructed, and this mutant strain was not significantly impaired in its ability to compete with the parental strain in infant mice and was not attenuated for virulence in an assay of 50% lethal dose. These data indicate that the virulence defect in MBG40 is not due to the loss of irgA function and that irgA is unlikely to be an important virulence factor.

scholarly journals The El Tor Biotype of Vibrio cholerae Exhibits a Growth Advantage in the Stationary Phase in Mixed Cultures with the Classical Biotype

2009 ◽  
Vol 192 (4) ◽  
pp. 955-963 ◽  
Author(s):  
Subhra Pradhan ◽  
Amit K. Baidya ◽  
Amalendu Ghosh ◽  
Kalidas Paul ◽  
Rukhsana Chowdhury
Keyword(s):  
Amino Acids ◽  
Stationary Phase ◽  
Vibrio Cholerae ◽  
Competitive Growth ◽  
Small Peptides ◽  
Ileal Loop ◽  
Infant Mouse ◽  
Culture Filtrates ◽  
El Tor

ABSTRACT Vibrio cholerae strains of the O1 serogroup that typically cause epidemic cholera can be classified into two biotypes, classical and El Tor. The El Tor biotype emerged in 1961 and subsequently displaced the classical biotype as a cause of cholera throughout the world. In this study we demonstrate that when strains of the El Tor and classical biotypes were cocultured in standard LB medium, the El Tor strains clearly had a competitive growth advantage over the classical biotype starting from the late stationary phase and could eventually take over the population. The classical biotype produces extracellular protease(s) in the stationary phase, and the amounts of amino acids and small peptides in the late stationary and death phase culture filtrates of the classical biotype were higher than those in the corresponding culture filtrates of the El Tor biotype. The El Tor biotype cells could utilize the amino acids more efficiently than the classical biotype under the alkaline pH of the stationary phase cultures but not in medium buffered to neutral pH. The growth advantage of the El Tor biotype was also observed in vivo using the ligated rabbit ileal loop and infant mouse animal models.

scholarly journals Spatiotemporal Analysis of Acid Adaptation-Mediated Vibrio cholerae Hyperinfectivity

2004 ◽  
Vol 72 (4) ◽  
pp. 2405-2407 ◽  
Author(s):  
Michael J. Angelichio ◽  
D. Scott Merrell ◽  
Andrew Camilli
Keyword(s):  
Small Intestine ◽  
Vibrio Cholerae ◽  
Spatiotemporal Analysis ◽  
Rapid Onset ◽  
Multiplication Rate ◽  
Acid Adaptation ◽  
Infant Mouse ◽  
Mouse Intestine ◽  
Kinetics Of ◽  
Transcriptional Induction

ABSTRACT Acid adaptation has previously been shown to increase the infectivity of Vibrio cholerae in the infant mouse model. To better understand this phenomenon, we monitored the spatial distribution and temporal changes in the ratios of acid-adapted cells to unadapted V. cholerae cells in the small intestine, as well as the timing of virulence factor expression. We found that the competitive advantage afforded by acid adaptation does not become manifest until greater than 3 h postinfection; thus, acid adaptation does not increase V. cholerae passage through the gastric acid barrier. Additionally, acid-adapted and unadapted V. cholerae cells colonize the same sections of the small intestine and show similar kinetics of transcriptional induction of the virulence genes tcpA and ctxA. These studies suggest that the increased infectivity of acid-adapted V. cholerae is due to a more rapid onset of multiplication and/or to an increased multiplication rate within the infant mouse intestine.

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