RESUMO
The bacterial type 6 secretion system (T6SS) functions as a virulence factor capable of attacking both eukaryotic and prokaryotic target cells by a process that involves protein transport through a contractile bacteriophage tail-like structure. The T6SS apparatus is composed, in part, of an exterior sheath wrapped around an interior tube. Here, we report that in living cells the cytoplasmic adenosine triphosphatase called ClpV specifically recognizes the contracted T6SS sheath structure, causing its disassembly within seconds. ClpV imaging allowed spatial and temporal documentation of cell-cell interactions (termed T6SS dueling) that likely mark the location of repeated T6SS-mediated protein translocation events between bacterial cells.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Pseudomonas aeruginosa/fisiologia , Vibrio cholerae/fisiologia , Alanina/genética , Alanina/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Mutação , Transporte Proteico , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tirosina/genética , Tirosina/metabolismo , Vibrio cholerae/metabolismoRESUMO
Type VI secretion systems are bacterial virulence-associated nanomachines composed of proteins that are evolutionarily related to components of bacteriophage tails. Here we show that protein secretion by the type VI secretion system of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheath. Time-lapse fluorescence light microscopy reveals that sheaths of the type VI secretion system cycle between assembly, quick contraction, disassembly and re-assembly. Whole-cell electron cryotomography further shows that the sheaths appear as long tubular structures in either extended or contracted conformations that are connected to the inner membrane by a distinct basal structure. These data support a model in which the contraction of the type VI secretion system sheath provides the energy needed to translocate proteins out of effector cells and into adjacent target cells.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Sistemas de Secreção Bacterianos/fisiologia , Bacteriófagos/química , Vibrio cholerae/química , Vibrio cholerae/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Microscopia de Fluorescência , Vibrio cholerae/citologia , Vibrio cholerae/ultraestruturaRESUMO
Toxigenic Vibrio cholerae strains are lysogens of CTX(Phi), a filamentous bacteriophage which encodes cholera toxin (CT). Following infection of recipient V. cholerae cells by CTX(Phi), the phage genome either integrates into the host chromosome at a specific attachment site (attRS) or exists as a replicative-form (RF) plasmid. We infected naturally occurring attRS-negative nontoxigenic V. cholerae or attenuated (CTX(-) attRS negative) derivatives of wild-type toxigenic strains with CTX(Phi) and examined the diarrheagenic potential of the strains carrying the RF of the CTX(Phi) genome using the adult rabbit diarrhea model. Under laboratory conditions, strains carrying the RF of CTX(Phi) produced more CT than corresponding lysogens as assayed by a G(M1)-based enzyme-linked immunosorbent assay and by fluid accumulation in ligated ileal loops of rabbits. However, when tested for diarrhea in rabbits, the attRS-negative strains (which carried the CTX(Phi) genome as the RF) were either negative or produced mild diarrhea, whereas the attRS-positive strains with integrated CTX(Phi) produced severe fatal diarrhea. Analysis of the strains after intestinal passage showed that the attRS-negative strains lost the phage genome at approximately a fivefold higher frequency than under in vitro conditions, and 75 to 90% of cells recovered from challenged rabbits after 24 h were CT negative. These results suggested that strains carrying the RF of CTX(Phi) are unable to cause severe disease due to rapid loss of the phage in vivo, and the gastrointestinal environment thus provides selection of toxigenic strains with an integrated CTX(Phi) genome. These results may have implications for the development of live V. cholerae vaccine candidates impaired in chromosomal integration of CTX(Phi). These findings may also contribute to understanding of the etiology of diarrhea occasionally associated with nontoxigenic V. cholerae strains.
Assuntos
Bacteriófagos/genética , Diarreia/fisiopatologia , Vibrio cholerae/virologia , Replicação Viral/genética , Animais , Sítios de Ligação Microbiológicos , Toxina da Cólera/biossíntese , Vacinas contra Cólera , Diarreia/prevenção & controle , Modelos Animais de Doenças , Genoma Viral , Intestinos , Lisogenia , Coelhos , Vibrio cholerae/patogenicidade , VirulênciaRESUMO
To study the utility of in vitro-polarized intestinal cell monolayers for modeling Vibrio cholerae-host cell interactions, we added live V. cholerae bacteria to the apical surfaces of polarized T84 cell monolayers and monitored changes in electrical properties. We found that both classical and El Tor strains produce cholera toxin after addition to the monolayer, but induction is most likely due to medium components rather than bacterium-cell interactions. We also found that the RTX toxin is produced by El Tor strains. This toxin caused a loss of the barrier function of the paracellular tight junction that was measured as a decrease in transepithelial resistance. This decrease occurred when bacteria were added to either the apical or basolateral surfaces, indicating that the RTX toxin receptor is expressed on both surfaces. These results are discussed with regard to the applicability of the polarized T84 cell monolayers as an in vitro model of host-pathogen interactions.
Assuntos
Toxina da Cólera/metabolismo , Vibrio cholerae , Polaridade Celular , Meios de Cultura , Endotoxinas , Células Epiteliais , Guanilato Ciclase/metabolismo , Humanos , Mucosa Intestinal/citologia , Metaloendopeptidases/metabolismo , Metaloendopeptidases/fisiologia , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Receptores de Peptídeos/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/fisiologia , Células Tumorais CultivadasRESUMO
TolC and its homologues are outer membrane proteins that are essential for the transport of many molecules across the cell envelope. In this study we characterized the gene encoding Vibrio cholerae TolC. V. cholerae tolC mutants failed to secrete the RTX cytotoxin, were hypersensitive to antimicrobial agents, and were deficient in intestinal colonization.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Bile/imunologia , Vibrio cholerae/crescimento & desenvolvimento , Animais , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Ácidos e Sais Biliares/farmacologia , Resistência Microbiana a Medicamentos , Proteínas de Escherichia coli , Proteínas Hemolisinas , Humanos , Proteínas de Membrana Transportadoras , Camundongos , Mutagênese Insercional , Células Tumorais Cultivadas , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/imunologia , Vibrio cholerae/patogenicidadeRESUMO
We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments.
Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Helicobacter pylori/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Western Blotting , Análise Mutacional de DNA , Helicobacter pylori/patogenicidade , Mutagênese , Transposases , Virulência/genéticaRESUMO
Transposons are a powerful tool for identifying genes essential for bacterial viability. The availability of many bacterial genome sequences and the large number of genes of unknown function therein have inspired the generation of a variety of different approaches. These methods are described and their advantages and disadvantages are discussed.
Assuntos
Bactérias/genética , Elementos de DNA Transponíveis , Genes Essenciais , Genoma Bacteriano , Mutagênese Insercional , Bactérias/crescimento & desenvolvimento , Genes BacterianosRESUMO
Recent efforts to develop a vaccine against the diarrheal disease cholera have focused on the use of live attenuated strains of the causative organism, Vibrio cholerae. The Ogawa lipopolysaccharide phenotype is expressed by many epidemic strains, and motility defects reduce the risk of reactive diarrhea in vaccine recipients. We therefore converted a motile Inaba(+) vaccine candidate, Peru-2, to a nonmotile Ogawa(+) phenotype using a mariner-based transposon carrying rfbT, the gene required for expression of the Ogawa phenotype. Analysis of 22 nonmotile Peru-2 mutants showed that two were Ogawa(+), and both of these strains had insertions in the flgE gene. It was possible to convert these strains to antibiotic sensitivity by introducing a recombinase that acts on sites flanking the antibiotic marker on the transposon. The resulting strains are competent for colonization in infant mice and may therefore be suitable as vaccine candidates for use either independently or in a combination with strains of different biotypes and serotypes.
Assuntos
Vacinas contra Cólera , DNA Nucleotidiltransferases/fisiologia , Elementos de DNA Transponíveis , Vibrio cholerae/imunologia , Animais , Proteínas de Bactérias/genética , Cloranfenicol/farmacologia , Teste de Complementação Genética , CamundongosRESUMO
Culture supernatants prepared from reactogenic strains of Vibrio cholerae cause a decrease in the transcellular epithelial resistance of T84 intestinal cells. This decrease correlates with the presence of hemagglutinin/protease but not with the presence of other potential accessory toxins or proteases. These data suggest a possible role for hemagglutinin/protease in reactogenicity, although other factors may also contribute.
Assuntos
Vacinas contra Cólera , Endopeptidases/fisiologia , Mucosa Intestinal/fisiologia , Vibrio cholerae/fisiologia , Aminopeptidases/fisiologia , Linhagem Celular , Polaridade Celular , Toxina da Cólera/biossíntese , Endotoxinas , Hemaglutininas/fisiologiaRESUMO
Enteric pathogens often export toxins that elicit diarrhea as a part of the etiology of disease, including toxins that affect cytoskeletal structure. Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated rtxA. Here we investigate the association of rtxA with the cell-rounding effect. We find that V. cholerae exports a large toxin, RTX (repeats-in-toxin) toxin, to culture supernatant fluids and that this toxin is responsible for cell rounding. Furthermore, we find that cell rounding is not due to necrosis, suggesting that RTX toxin is not a typical member of the RTX family of pore-forming toxins. Rather, RTX toxin causes depolymerization of actin stress fibers and covalent cross-linking of cellular actin into dimers, trimers and higher multimers. This RTX toxin-specific cross-linking occurs in cells previously rounded with cytochalasin D, indicating that G-actin is the toxin target. Although several models explain our observations, our simultaneous detection of actin cross-linking and depolymerization points toward a novel mechanism of action for RTX toxin, distinguishing it from all other known toxins.
Assuntos
Actinas/metabolismo , Toxinas Botulínicas , Toxina da Cólera/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Citotoxinas/metabolismo , Vibrio cholerae/metabolismo , ADP Ribose Transferases/metabolismo , Actinas/química , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Anticorpos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Western Blotting , Permeabilidade da Membrana Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Toxina da Cólera/química , Reagentes de Ligações Cruzadas/química , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Citocalasina D/farmacologia , Citotoxinas/química , Dimerização , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Fibras de Estresse/efeitos dos fármacos , Células Tumorais Cultivadas , Vibrio cholerae/química , Vibrio cholerae/patogenicidade , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The complete DNA sequence of Pseudomonas aeruginosa provides an opportunity to apply functional genomics to a major human pathogen. A comparative genomics approach combined with genetic footprinting was used as a strategy to identify genes required for viability in P. aeruginosa. Use of a highly efficient in vivo mariner transposition system in P. aeruginosa facilitated the analysis of candidate genes of this class. We have developed a rapid and efficient allelic exchange system by using the I-SceI homing endonuclease in conjunction with in vitro mariner mutagenesis to generate mutants within targeted regions of the P. aeruginosa chromosome for genetic footprinting analyses. This technique for generating transposon insertion mutants should be widely applicable to other organisms that are not naturally transformable or may lack well developed in vivo transposition systems. We tested this system with three genes in P. aeruginosa that have putative essential homologs in Haemophilus influenzae. We show that one of three H. influenzae essential gene homologs is needed for growth in P. aeruginosa, validating the practicality of this comparative genomics strategy to identify essential genes in P. aeruginosa.
Assuntos
Cromossomos Bacterianos , Pegada de DNA , Proteínas de Ligação a DNA/genética , Pseudomonas aeruginosa/genética , Alelos , Mapeamento Cromossômico , Conjugação Genética , Elementos de DNA Transponíveis , Escherichia coli/genética , Haemophilus influenzae/genética , Humanos , Mutagênese Insercional , Pseudomonas aeruginosa/crescimento & desenvolvimento , TransposasesRESUMO
Here we determine the complete genomic sequence of the gram negative, gamma-Proteobacterium Vibrio cholerae El Tor N16961 to be 4,033,460 base pairs (bp). The genome consists of two circular chromosomes of 2,961,146 bp and 1,072,314 bp that together encode 3,885 open reading frames. The vast majority of recognizable genes for essential cell functions (such as DNA replication, transcription, translation and cell-wall biosynthesis) and pathogenicity (for example, toxins, surface antigens and adhesins) are located on the large chromosome. In contrast, the small chromosome contains a larger fraction (59%) of hypothetical genes compared with the large chromosome (42%), and also contains many more genes that appear to have origins other than the gamma-Proteobacteria. The small chromosome also carries a gene capture system (the integron island) and host 'addiction' genes that are typically found on plasmids; thus, the small chromosome may have originally been a megaplasmid that was captured by an ancestral Vibrio species. The V. cholerae genomic sequence provides a starting point for understanding how a free-living, environmental organism emerged to become a significant human bacterial pathogen.
Assuntos
Cromossomos Bacterianos , DNA Bacteriano , Vibrio cholerae/genética , Sequência de Bases , Transporte Biológico , Cólera/microbiologia , Reparo do DNA , Metabolismo Energético , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Humanos , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Vibrio cholerae/classificação , Vibrio cholerae/patogenicidadeRESUMO
Identification of genes that encode essential products provides a promising approach to validation of new antibacterial drug targets. We have developed a mariner-based transposon, TnAraOut, that allows efficient identification and characterization of essential genes by transcriptionally fusing them to an outward-facing, arabinose-inducible promoter, PBAD, located at one end of the transposon. In the absence of arabinose, such TnAraOut fusion strains display pronounced growth defects. Of a total of 16 arabinose-dependent TnAraOut mutants characterized in Vibrio cholerae, four were found to carry insertions upstream of known essential genes (gyrB, proRS, ileRS, and aspRS) whereas the other strains carried insertions upstream of known and hypothetical genes not previously shown to encode essential gene products. One of the essential genes identified by this analysis appears to be unique to V. cholerae and thus may represent an example of a species-specific drug target.
Assuntos
Análise Mutacional de DNA/métodos , Elementos de DNA Transponíveis/genética , Genes Bacterianos/genética , Arabinose/farmacologia , Técnicas de Cultura de Células/métodos , Divisão Celular/genética , Relação Dose-Resposta a Droga , Modelos Genéticos , Mutagênese , Mutação , Fenótipo , Regiões Promotoras Genéticas , Fatores de Tempo , Transcrição Gênica , Vibrio cholerae/genética , beta-Galactosidase/metabolismoRESUMO
We have developed a method for isolation of random peptides that inhibit intracellular processes in bacteria. A library of random peptides expressed as fusions to Escherichia coli thioredoxin (aptamers) were expressed under the tight control of the arabinose-inducible P(BAD) promoter. A selection was applied to the library to isolate aptamers that interfered with the activity of thymidylate synthase (ThyA) in vivo. Expression of an aptamer isolated by this method resulted in a ThyA(-) phenotype that was suppressed by simultaneous overexpression of ThyA. Two-hybrid analysis showed that this aptamer is likely to interact with ThyA in vivo. The library also was screened for aptamers that inhibited growth of bacteria expressing them, and five such aptamers were characterized. Four aptamers were bacteriostatic when expressed, whereas one showed a bactericidal effect. Introduction of translational stop codons into various aptamers blocked their activity, suggesting that their biological effects were likely to be due to protein aptamer rather than RNA. Combinatorial aptamers provide a new genetic and biochemical tool for identifying targets for antibacterial drug development.
Assuntos
Antibacterianos/farmacologia , Peptídeos/isolamento & purificação , Timidilato Sintase/antagonistas & inibidores , Sequência de Aminoácidos , Clonagem Molecular , Desenho de Fármacos , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Dados de Sequência Molecular , Biossíntese Peptídica , Terminação Traducional da Cadeia Peptídica , Peptídeos/farmacologia , Tiorredoxinas/genética , Timidilato Sintase/genéticaRESUMO
Novel methods for adapting DNA vaccine technology to the prevention of mucosal diseases are greatly needed. Here we show that regulated expression of phage lambda lysis genes S and R causes dramatic lysis of both Vibrio cholerae and Salmonella enterica serovar Typhimurium cells with concomitant release of plasmid DNA into the surrounding media. We also used single and double DNase mutant strains to show that secreted V. cholerae DNases can adversely affect the integrity of DNA molecules released upon lysis. These results suggest that incorporation of lambda SR-mediated lysis constructs and DNA stabilizing mutations into candidate live attenuated bacterial vaccines offers a promising approach for the development of effective mucosal DNA delivery vectors for humans.
Assuntos
Vacinas Bacterianas/administração & dosagem , Bacteriófago lambda/genética , Vacinas contra Cólera/administração & dosagem , Salmonella typhimurium/imunologia , Vacinas de DNA/administração & dosagem , DNA Bacteriano/metabolismo , Desoxirribonucleases/farmacologia , PlasmídeosRESUMO
In vitro assays contribute greatly to our understanding of bacterial pathogenesis, but they frequently cannot replicate the complex environment encountered by pathogens during infection. The information gained from such studies is therefore limited. In vivo models, on the other hand, can be difficult to use, and this has to some extent diminished the incentive to perform studies in living animals. However, several recently developed techniques permit in vivo examination of many genes simultaneously. Most of these methods fall into two broad classes: in vivo expression technology and signature-tagged mutagenesis. In vivo expression technology is a promoter-trap strategy designed to identify genes whose expression is induced in a specific environment, typically that encountered in a host. Signature-tagged mutagenesis uses comparative hybridization to isolate mutants unable to survive specified environmental conditions and has been used to identify genes critical for survival in the host. Both approaches have so far been used exclusively for investigating pathogen-host interactions, but they should be easily adaptable to the study of other processes.
Assuntos
Bactérias/genética , Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Técnicas Genéticas , Animais , Perfilação da Expressão Gênica , Mutagênese Insercional , Virulência/genéticaRESUMO
The filamentous bacteriophage CTXPhi, which encodes cholera toxin (CT) in toxigenic Vibrio cholerae, is known to propagate by infecting susceptible strains of V. cholerae by using the toxin coregulated pilus (TCP) as its receptor and thereby causing the origination of new strains of toxigenic V. cholerae from nontoxigenic progenitors. Besides V. cholerae, Vibrio mimicus strains which are normally TCP negative have also been shown to occasionally produce CT and cause diarrhea in humans. We analyzed nontoxigenic V. mimicus strains isolated from surface waters in Bangladesh for susceptibility and lysogenic conversion by CTXPhi and studied the expression of CT in the lysogens by using genetically marked derivatives of the phage. Of 27 V. mimicus strains analyzed, which were all negative for genes encoding TCP but positive for the regulatory gene toxR, 2 strains (7.4%) were infected by CTX-KmPhi, derived from strain SM44(P27459 ctx::km), and the phage genome integrated into the host chromosome, forming stable lysogens. The lysogens spontaneously produced infectious phage particles in the supernatant fluids of the culture, and high titers of the phage could be achieved when the lysogens were induced with mitomycin C. This is the first demonstration of lysogenic conversion of V. mimicus strains by CTXPhi. When a genetically marked derivative of the replicative form of the CTXPhi genome carrying a functional ctxAB operon, pMSF9.2, was introduced into nontoxigenic V. mimicus strains, the plasmid integrated into the host genome and the strains produced CT both in vitro and inside the intestines of adult rabbits and caused mild-to-severe diarrhea in rabbits. This suggested that in the natural habitat infection of nontoxigenic V. mimicus strains by wild-type CTXPhi may lead to the origination of toxigenic V. mimicus strains which are capable of producing biologically active CT. The results of this study also supported the existence of a TCP-independent mechanism for infection by CTXPhi and showed that at least one species of Vibrio other than V. cholerae may contribute to the propagation of the phage.
Assuntos
Bacteriófagos/genética , Toxina da Cólera/genética , Lisogenia , Vibrio/virologia , Animais , Fímbrias Bacterianas/fisiologia , Regulação Bacteriana da Expressão Gênica , RNA Ribossômico/genética , Coelhos , Vibrio/genética , Vibrio/patogenicidade , VirulênciaRESUMO
Mariner-family transposable elements are active in a wide variety of organisms and are becoming increasingly important genetic tools in species lacking sophisticated genetics. The Himar1 element, isolated from the horn fly, Haematobia irritans, is active in Escherichia coli when expressed appropriately. We used this fact to devise a genetic screen for hyperactive mutants of Himar1 transposase that enhance overall transposition from approximately 4- to 50-fold as measured in an E. coli assay. Purified mutant transposases retain their hyperactivity, although to a lesser degree, in an in vitro transposition assay. Mutants like those described herein should enable sophisticated analysis of the biochemistry of mariner transposition and should improve the use of these elements as genetic tools, both in vivo and in vitro.
Assuntos
Elementos de DNA Transponíveis , Muscidae/genética , Transposases/genética , Animais , DNA/metabolismo , MutaçãoRESUMO
The expression of several virulence factors of Vibrio cholerae is coordinately regulated by the ToxT molecule and the membrane proteins TcpP/H and ToxR/S, which are required for toxT transcription. To identify proteins that negatively affect toxT transcription, we screened transposon mutants of V. cholerae carrying a chromosomally integrated toxT::lacZ reporter construct for darker blue colonies on media containing 5-bromo-4-chlor-3-indolyl beta-D galactoside (X-gal). Two mutants had transposon insertions in a region homologous to the nqr gene cluster of Vibrio alginolyticus, encoding a sodium-translocating NADH-ubiquinone oxidoreductase (NQR). In V. alginolyticus, NQR is a respiration-linked Na+ extrusion pump generating a sodium motive force that can be used for solute import, ATP synthesis, and flagella rotation. Inhibition of NQR enzyme function in V. cholerae by the specific inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) resulted in elevated toxT::lacZ activity. Increased toxT::lacZ expression in an nqr mutant strain compared with the parental strain was observed when the TcpP/H molecules alone were strongly expressed, suggesting that the negative effect of the NQR complex on toxT transcription is mediated through TcpP/H. However, the ability of the TcpP/H proteins to activate the toxT::lacZ reporter construct was greatly diminished in the presence of high NaCl concentrations in the growth medium. The flagellar motor of V. cholerae appears to be driven by a sodium motive force, and modulation of flagella rotation by inhibitory drugs, high media viscosity, or specific mutations resulted in increases of toxT::lacZ expression. Thus, the regulation of the main virulence factors of V. cholerae appears to be modulated by endogenous and exogenous sodium levels in a complex way.
Assuntos
Proteínas de Bactérias/genética , Cólera/microbiologia , Regulação Bacteriana da Expressão Gênica , Sódio/metabolismo , Fatores de Transcrição/genética , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidade , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Cólera/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Transporte de Íons , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Fatores de Transcrição/metabolismo , Vibrio cholerae/genética , Virulência/genética , beta-GalactosidaseRESUMO
We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.