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Shelley M Payne

Publications and source records attributed to Shelley M Payne.

16 recordsLinked to original sources
PubMed2007

Enterohaemorrhagic Escherichia coli O157 and non-O157 serovars differ in their mechanisms for iron supply.

Clinical isolates of enterohaemorrhagic Escherichia coli, both O157 and non-O157 serotypes, were investigated for siderophore production, for growth promotion by haem and esculetin in iron-restricted conditions, for production of enterohaemolysin and esculin hydrolase, and for the presence of the chuA and ehx genes by PCR. As expected, all the strains produced enterobactin, but the prevalence of other factors varied among the serovars tested. None of the O157 and O26 strains produced aerobactin or "colibactin", whereas among other enterohaemorrhagic E. coli non-O157 serovars the frequencies of aerobactin and "colibactin" production were similar to those of commensal E. coli strains. The ability to use ferric esculetin for growth in iron-limited media was markedly more prevalent among non-O157 serovars and less prevalent among O157 strains compared with commensal E. coli strains. Almost all O157, O26 and O103 strains expressed enterohaemolysin, compared with only 50% of other non-O157 strains. Similarly, almost all O157 and O26 strains utilised haem as a host iron source; the frequency of haem use by other non-O157 strains was generally lower and variable among serovars, such that none of the O103:H2 isolates tested used haem as an iron source. The gene chuA, which encodes the haem transport protein ChuA and which is prevalent in O157:H7 strains, was only rarely noted among non-O157 serovars of enterohaemorrhagic E. coli, even among isolates that could use haem as an iron source. Overall our data demonstrate that O157:H7 and non-O157 serovars, in particular O26:H(-)/H11 and O103:H2, use distinctly different strategies for obtaining iron, and suggest two evolutionary distinct lines of enterhaemorrhagic E. coli.

Antigens, Bacterial↗
PubMed2007

Iron acquisition in Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron and must obtain this element in the human host as well as in its varied environmental niches. It has multiple systems for iron acquisition, including the TonB-dependent transport of heme, the endogenous siderophore vibriobactin and several siderophores that are produced by other microorganisms. There is also a Feo system for the transport of ferrous iron and an ABC transporter, Fbp, which transports ferric iron. There appears to be at least one additional high affinity iron transport system that has not yet been identified. In iron replete conditions, iron acquisition genes are repressed by Fur. Fur also represses the synthesis of a small, regulatory RNA, RyhB, which negatively regulates genes for iron-containing proteins involved in the tricarboxylic acid cycle and respiration as well as genes for motility and chemotaxis. The redundancy in iron transport systems has made it more difficult to determine the role of individual systems in vivo and in vitro, but it may reflect the overall importance of iron in the growth and survival of V. cholerae.

ATP-Binding Cassette Transporters↗
PubMed2006

Iron and pathogenesis of Shigella: iron acquisition in the intracellular environment.

Shigella species are able to grow in a variety of environments, including intracellularly in host epithelial cells. Shigella have a number of different iron transport systems that contribute to their ability to grow in these diverse environments. Siderophore iron uptake systems, heme transporters, and ferric and ferrous iron transport systems are present in these bacteria, and the genes encoding some of these systems appear to have spread among the Shigella species by horizontal transmission. Iron is not only essential for growth of Shigella but also plays an important role in regulation of metabolic processes and virulence determinants in Shigella. This regulation is mediated by the repressor protein Fur and the small RNA RyhB.

Bacterial Proteins↗
PubMed2006

Induction of expression of hfq by DksA is essential for Shigella flexneri virulence.

Hfq plays an important role in cellular physiology by regulating the expression of several genes. Hfq synthesis in Escherichia coli is subject to auto-repression at translational level. Studies with Shigella flexneri show that hfq transcription is regulated by a pleiotropic regulator, DksA. Comparison of gene expression profiles of wild type and dksA mutant S. flexneri determined that hfq expression was reduced in the dksA mutant. As DksA is required for stress resistance and plaque formation in cultured cell monolayers, a measure of virulence, we assessed the role of Hfq in the dksA virulence phenotype. Expression of hfq in the dksA mutant restored plaque formation, and an hfq mutant failed to form plaques. Thus, DksA plays a role in regulating hfq gene expression and this regulation is important for S. flexneri virulence. In an in vitro transcription assay, addition of DksA increased transcription of hfq and this effect was greatest with one of the known hfq promoters. Addition of ppGpp, a stringent response molecule, along with DksA in the in vitro transcription assay resulted in a further increase in transcription of hfq, indicating that DksA is required for maximal transcription of hfq during both exponential and stringent response growth conditions.

Bacterial Proteins↗
PubMed2006

Characterization of ferric and ferrous iron transport systems in Vibrio cholerae.

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.

ATP-Binding Cassette Transporters↗
PubMed2006

Endogenous MMTV proviruses induce susceptibility to both viral and bacterial pathogens.

Most 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.

Animals↗
PubMed2005

Fur regulates acid resistance in Shigella flexneri via RyhB and ydeP.

Shigella flexneri requires iron for survival, and the genes for iron uptake and homeostasis are regulated by the Fur protein. Microarrays were used to identify genes regulated by Fur and to study the physiological effects of iron availability in S. flexneri. These assays showed that the expression of genes involved in iron acquisition and acid response was induced by low-iron availability and by inactivation of fur. A fur null mutant was acid sensitive in media at pH 2.5, and acid sensitivity was also observed in the wild-type strain grown under iron-limiting conditions. Acid resistance of the fur mutant in minimal medium was restored by addition of glutamate during acid challenge, indicating that the glutamate-dependent acid resistance system was not defective. Inactivation of ryhB, a small regulatory RNA whose expression is repressed by Fur, restored acid resistance in the fur mutant, while overexpressing ryhB increased acid sensitivity in the wild-type strain. RyhB-regulated genes were identified by microarray analysis. The expression of one of the RyhB-repressed genes, ydeP, which encodes a putative oxidoreductase, suppressed acid sensitivity in the fur mutant. Furthermore, an S. flexneri ydeP mutant was defective for both glutamate-independent and glutamate-dependent acid resistance. The repression of ydeP by RyhB may be indirect, as real time polymerase chain reaction (PCR) experiments indicated that RyhB negatively regulates evgA, which encodes an activator of ydeP. These results demonstrate that the acid sensitivity defect of the S. flexneri fur mutant is due to repression of ydeP by RyhB, most likely via repression of evgA.

Bacterial Proteins↗
PubMed2005

Iron and fur regulation in Vibrio cholerae and the role of fur in virulence.

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.

Bacterial Proteins↗
PubMed2005

Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of ryhB in biofilm formation.

Vibrio cholerae encodes a small RNA with homology to Escherichia coli RyhB. Like E. coli ryhB, V. cholerae ryhB is negatively regulated by iron and Fur and is required for repression of genes encoding the superoxide dismutase SodB and multiple tricarboxylic acid cycle enzymes. However, V. cholerae RyhB is considerably longer (>200 nucleotides) than the E. coli RNA (90 nucleotides), and it regulates the expression of a variety of genes that are not known to be regulated by RyhB in E. coli, including genes involved in motility, chemotaxis, and biofilm formation. A mutant with a deletion in ryhB had reduced chemotactic motility in low-iron medium and was unable to form wild-type biofilms. The defect in biofilm formation was suppressed by growing the mutant in the presence of excess iron or succinate. The wild-type strain showed reduced biofilm formation in iron-deficient medium, further supporting a role for iron in normal biofilm formation. The ryhB mutant was not defective for colonization in a mouse model and appeared to be at a slight advantage when competing with the wild-type parental strain. Other genes whose expression was influenced by RyhB included those encoding the outer membrane porins OmpT and OmpU, several iron transport systems, and proteins containing heme or iron-sulfur clusters. These data indicate that V. cholerae RyhB has diverse functions, ranging from iron homeostasis to the regulation of biofilm formation.

Bacterial Proteins↗
PubMed2005

Shigella dysenteriae ShuS promotes utilization of heme as an iron source and protects against heme toxicity.

Shigella dysenteriae serotype 1, a major cause of bacillary dysentery in humans, can use heme as a source of iron. Genes for the transport of heme into the bacterial cell have been identified, but little is known about proteins that control the fate of the heme molecule after it has entered the cell. The shuS gene is located within the heme transport locus, downstream of the heme receptor gene shuA. ShuS is a heme binding protein, but its role in heme utilization is poorly understood. In this work, we report the construction of a chromosomal shuS mutant. The shuS mutant was defective in utilizing heme as an iron source. At low heme concentrations, the shuS mutant grew slowly and its growth was stimulated by either increasing the heme concentration or by providing extra copies of the heme receptor shuA on a plasmid. At intermediate heme concentrations, the growth of the shuS mutant was moderately impaired, and at high heme concentrations, shuS was required for growth on heme. The shuS mutant did not show increased sensitivity to hydrogen peroxide, even at high heme concentrations. ShuS was also required for optimal utilization of heme under microaerobic and anaerobic conditions. These data are consistent with the model in which ShuS binds heme in a soluble, nontoxic form and potentially transfers the heme from the transport proteins in the membrane to either heme-containing or heme-degrading proteins. ShuS did not appear to store heme for future use.

Aerobiosis↗
PubMed2005

Mouse mammary tumor virus encodes a self-regulatory RNA export protein and is a complex retrovirus.

Mouse mammary tumor virus (MMTV) has been classified as a simple retrovirus with two accessory genes, dut and sag. Cloned MMTV proviruses carrying a trimethoprim (trim) cassette in the envelope gene were defective for Gag protein production and the nuclear export of unspliced gag-pol RNA. Complementation experiments indicated that a trans-acting product was responsible for the Gag defect of such mutants. Analysis of MMTV-infected cells revealed the presence of a novel, doubly spliced RNA that encodes a putative product of 301 amino acids. Overexpression of cDNA from this RNA increased Gag levels from env mutant proviruses or reporter gene expression from unspliced mRNAs and allowed detection of a 33-kDa protein product, which has been named regulator of export of MMTV mRNA, or Rem. The Rem N terminus has motifs similar to the Rev-like export proteins of complex retroviruses, and mutation of the nuclear localization signal (NLS) abolished RNA export and detection within the nucleus. The Rem C terminus has few identifiable features, but removal of this domain increased Rem-mediated export, suggesting an autoregulatory function. A reporter vector developed from the 3' end of the MMTV provirus was Rem responsive and required both the presence of the MMTV env-U3 junction and a functional Crm1 pathway. The identification of a third accessory protein from a doubly spliced transcript suggests that MMTV is the first murine complex retrovirus to be documented. Manipulation of the MMTV genome may provide mouse models for human retroviral diseases, such as AIDS.

Animals↗
PubMed2004

HutZ is required for efficient heme utilization in Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, requires iron for growth. One mechanism by which it acquires iron is the uptake of heme, and several heme utilization genes have been identified in V. cholerae. These include three distinct outer membrane receptors, two TonB systems, and an apparent ABC transporter to transfer heme across the inner membrane. However, little is known about the fate of the heme after it enters the cell. In this report we show that a novel heme utilization protein, HutZ, is required for optimal heme utilization. hutZ (open reading frame [ORF] VCA0907) is encoded with two other genes, hutW (ORF VCA0909) and hutX (ORF VCA0908), in an operon divergently transcribed from the tonB1 operon. A hutZ mutant grew poorly when heme was provided as the sole source of iron, and the poor growth was likely due to the failure to use heme efficiently as a source of iron, rather than to heme toxicity. Heme oxygenase mutants of both Corynebacterium diphtheriae and C. ulcerans fail to use heme as an iron source. When the hutWXZ genes were expressed in the heme oxygenase mutants, growth on heme was restored, and hutZ was required for this effect. Biochemical characterization indicated that HutZ binds heme with high efficiency; however, no heme oxygenase activity was detected for this protein. HutZ may act as a heme storage protein, and it may also function as a shuttle protein that increases the efficiency of heme trafficking from the membrane to heme-containing proteins.

Bacterial Proteins↗
PubMed2003

Analysis of residues determining specificity of Vibrio cholerae TonB1 for its receptors.

In gram-negative organisms, high-affinity transport of iron substrates requires energy transduction to specific outer membrane receptors by the TonB-ExbB-ExbD complex. Vibrio cholerae encodes two TonB proteins, one of which, TonB1, recognizes only a subset of V. cholerae TonB-dependent receptors and does not facilitate transport through Escherichia coli receptors. To investigate the receptor specificity exhibited by V. cholerae TonB1, chimeras were created between V. cholerae TonB1 and E. coli TonB. The activities of the chimeric TonB proteins in iron utilization assays demonstrated that the C-terminal one-third of either TonB confers the receptor specificities associated with the full-length TonB. Single-amino-acid substitutions near the C terminus of V. cholerae TonB1 were identified that allowed TonB1 to recognize E. coli receptors and at least one V. cholerae TonB2-dependent receptor. This indicates that the very C-terminal end of V. cholerae TonB1 determines receptor specificity. The regions of the TonB-dependent receptors involved in specificity for a particular TonB protein were investigated in experiments involving domain switching between V. cholerae and E. coli receptors exhibiting different TonB specificities. Switching the conserved TonB box heptapeptides at the N termini of these receptors did not alter their TonB specificities. However, replacing the amino acid immediately preceding the TonB box in E. coli receptors with an aromatic residue allowed these receptors to use V. cholerae TonB1. Further, site-directed mutagenesis of the TonB box -1 residue in a V. cholerae TonB2-dependent receptor demonstrated that a large hydrophobic amino acid in this position promotes recognition of V. cholerae TonB1. These data suggest that the TonB box -1 position controls productive interactions with V. cholerae TonB1.

Amino Acid Sequence↗
PubMed2002

Shigella flexneri DegP facilitates IcsA surface expression and is required for efficient intercellular spread.

A degP mutant of Shigella flexneri was identified in a screen for insertion mutants that invaded cultured cells but did not form wild-type plaques in monolayers. The degP mutant SM1100 invaded Henle cells at wild-type levels and induced apoptosis in macrophages but formed smaller plaques than those formed by wild-type S. flexneri in confluent monolayers of Henle and Caco-2 cells. The proportion of SM1100 bacteria with IcsA localized to the bacterial pole, a process required for actin polymerization into actin "tails," was reduced compared to results with wild-type bacteria. The reduction in proper IcsA localization may account for the reduced plaque size of the degP mutant. Although DegP is a protease, the protease activity of S. flexneri DegP was not required for IcsA localization or the formation of plaques in Henle cell monolayers. DegP was also required for efficient polar IcsA localization in E. coli expressing icsA. In addition, the growth or survival of SM1100 was compromised compared to that of the wild type at elevated temperatures and in acidic conditions.

Actins↗
PubMed2002

Identification of the Vibrio cholerae enterobactin receptors VctA and IrgA: IrgA is not required for virulence.

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.

ATP-Binding Cassette Transporters↗