Identification of a Family of Vibrio Type III Secretion System Effectors That Contain a Conserved Serine/Threonine Kinase Domain.

Vibrio parahaemolyticus is a marine Gram-negative bacterium that is a leading cause of seafood-borne gastroenteritis. Pandemic strains of V. parahaemolyticus rely on a specialized protein secretion machinery known as the type III secretion system 2 (T3SS2) to cause disease. The T3SS2 mediates the delivery of effector proteins into the cytosol of infected cells, where they subvert multiple cellular pathways. Here, we identify a new T3SS2 effector protein encoded by VPA1328 (VP_RS21530) in V. parahaemolyticus RIMD2210633. Bioinformatic analysis revealed that VPA1328 is part of a larger family of uncharacterized T3SS effector proteins with homology to the VopG effector protein in Vibrio cholerae AM-19226. These VopG-like proteins are found in many but not all T3SS2 gene clusters and are distributed among diverse Vibrio species, including V. parahaemolyticus, V. cholerae, V. mimicus, and V. diabolicus and also in Shewanella baltica. Structure-based prediction analyses uncovered the presence of a conserved C-terminal kinase domain in VopG orthologs, similar to the serine/threonine kinase domain found in the NleH family of T3SS effector proteins. However, in contrast to NleH effector proteins, in tissue culture-based infections, VopG did not impede host cell death or suppress interleukin 8 (IL-8) secretion, suggesting a yet undefined role for VopG during V. parahaemolyticus infection. Collectively, our work reveals that VopG effector proteins, a new family of likely serine/threonine kinases, is widely distributed in the T3SS2 effector armamentarium among marine bacteria. IMPORTANCE Vibrio parahaemolyticus is the leading bacterial cause of seafood-borne gastroenteritis worldwide. The pathogen relies on a type III secretion system to deliver a variety of effector proteins into the cytosol of infected cells to subvert cellular function. In this study, we identified a novel Vibrio parahaemolyticus effector protein that is similar to the VopG effector of Vibrio cholerae. VopG-like effectors were found in diverse Vibrio species and contain a conserved serine/threonine kinase domain that bears similarity to the kinase domain in the enterohemorrhagic Escherichia coli (EHEC) and Shigella NleH effectors that manipulate host cell survival pathways and host immune responses. Together our findings identify a new family of Vibrio effector proteins and highlight the role of horizontal gene transfer events among marine bacteria in shaping T3SS gene clusters.

Comprehensive identification of Vibrio vulnificus genes required for growth in human serum.

Vibrio vulnificus can be a highly invasive pathogen capable of spreading from an infection site to the bloodstream, causing sepsis and death. To survive and proliferate in blood, the pathogen requires mechanisms to overcome the innate immune defenses and metabolic limitations of this host niche. We created a high-density transposon mutant library in YJ016, a strain representative of the most virulent V. vulnificus lineage (or phylogroup) and used transposon insertion sequencing (TIS) screens to identify loci that enable the pathogen to survive and proliferate in human serum. Initially, genes underrepresented for insertions were used to estimate the V. vulnificus essential gene set; comparisons of these genes with similar TIS-based classification of underrepresented genes in other vibrios enabled the compilation of a common Vibrio essential gene set. Analysis of the relative abundance of insertion mutants in the library after exposure to serum suggested that genes involved in capsule biogenesis are critical for YJ016 complement resistance. Notably, homologues of two genes required for YJ016 serum-resistance and capsule biogenesis were not previously linked to capsule biogenesis and are largely absent from other V. vulnificus strains. The relative abundance of mutants after exposure to heat inactivated serum was compared with the findings from the serum screen. These comparisons suggest that in both conditions the pathogen relies on its Na+ transporting NADH-ubiquinone reductase (NQR) complex and type II secretion system to survive/proliferate within the metabolic constraints of serum. Collectively, our findings reveal the potency of comparative TIS screens to provide knowledge of how a pathogen overcomes the diverse limitations to growth imposed by serum.

Host-specific differences in the contribution of an ESBL IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4.

Objectives: To assess stability and contribution of a large ESBL-encoding IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4 in two different mammalian hosts. Methods: Specific-pathogen-free 3-4-day-old New Zealand White rabbits and conventionally reared 6-week-old weaned lambs were orally infected with WT E. coli O104:H4 or the ESBL-plasmid-cured derivative, and the recovery of bacteria in intestinal homogenates and faeces monitored over time. Results: Carriage of the ESBL plasmid had differing impacts on E. coli O104:H4 colonization of the two experimental hosts. The plasmid-cured strain was recovered at significantly higher levels than WT during late-stage colonization of rabbits, but at lower levels than WT in sheep. Regardless of the animal host, the ESBL plasmid was stably maintained in virtually all in vivo passaged bacteria that were examined. Conclusions: These findings suggest that carriage of ESBL plasmids has distinct effects on the host bacterium depending upon the animal species it encounters and demonstrates that, as for E. coli O157:H7, ruminants could represent a potential transmission reservoir.

Horizontal transfer of Shiga toxin and antibiotic resistance genes among Escherichia coli strains in house fly (Diptera: Muscidae) gut.

Whether the house fly, Musca domestica L., gut is a permissive environment for horizontal transfer of antibiotic resistance and virulence genes between strains of Escherichia coli is not known. House flies were immobilized and force fed suspensions of defined, donor strains of E. coli containing chloramphenicol resistance genes on a plasmid, or lysogenic, bacteriophage-born Shiga toxin gene stx1 (bacteriophage H-19B::Ap1). Recipient strains were E. coli lacking these mobile elements and genes but having rifampicin as a selectable marker. Plasmid transfer occurred at rates of 10(-2) per donor cell in the fly midgut and 10(-3) in the fly crop after 1 h of incubation postfeeding. Bacteriophage transfer rate was approximately 10(-6) per donor cell without induction, but induction with mitomycin C increased rates of transfer to 10(-2) per donor cell. These findings show that genes encoding antibiotic resistance or toxins will transfer horizontally among bacteria in the house fly gut via plasmid transfer or phage transduction. The house fly gut may provide a favorable environment for the evolution and emergence of pathogenic bacterial strains through acquisition of antibiotic resistance genes or virulence factors.

Comparison of SXT and R391, two conjugative integrating elements: definition of a genetic backbone for the mobilization of resistance determinants.

The SXT element (SXT) is becoming an increasingly prevalent vector for the dissemination of antibiotic resistances in Vibrio cholerae. SXT is a member of a larger family of elements, formerly defined as IncJ plasmids, that are self-transmissible by conjugation and integrate site-specifically into the host chromosome. Comparison of the DNA sequences of SXT and R391, an IncJ element from Providencia rettgeri, indicate that these elements consist of a conserved backbone that mediates the regulation, excision/integration and conjugative transfer of the elements. Both elements have insertions into this backbone that either confer the element-specific properties or are of unknown function. Interestingly, the conserved SXT and R391 backbone apparently contains hotspots for insertion of additional DNA sequences. This backbone represents a scaffold for the mobilization of genetic material between a wide range of gram-negative bacteria, allowing for rapid adaptation to changing environments.

Molecular analysis of antibiotic resistance gene clusters in vibrio cholerae O139 and O1 SXT constins.

Many recent Asian clinical Vibrio cholerae E1 Tor O1 and O139 isolates are resistant to the antibiotics sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm), and streptomycin (Sm). The corresponding resistance genes are located on large conjugative elements (SXT constins) that are integrated into prfC on the V. cholerae chromosome. We determined the DNA sequences of the antibiotic resistance genes in the SXT constin in MO10, an O139 isolate. In SXT(MO10), these genes are clustered within a composite transposon-like structure found near the element's 5' end. The genes conferring resistance to Cm (floR), Su (sulII), and Sm (strA and strB) correspond to previously described genes, whereas the gene conferring resistance to Tm, designated dfr18, is novel. In some other O139 isolates the antibiotic resistance gene cluster was found to be deleted from the SXT-related constin. The El Tor O1 SXT constin, SXT(ET), does not contain the same resistance genes as SXT(MO10). In this constin, the Tm resistance determinant was located nearly 70 kbp away from the other resistance genes and found in a novel type of integron that constitutes a fourth class of resistance integrons. These studies indicate that there is considerable flux in the antibiotic resistance genes found in the SXT family of constins and point to a model for the evolution of these related mobile elements.

Evidence for a rolling-circle mechanism of phage DNA synthesis from both replicative and integrated forms of CTXphi.

The genes encoding cholera toxin, the principal virulence factor of Vibrio cholerae, are part of the circular single-stranded DNA genome of CTXphi. In toxigenic V. cholerae strains, the CTXphi genome is typically found in integrated arrays of tandemly arranged CTX prophages. Infected cells that lack a chromosomal integration site harbour the CTXphi genome as a plasmid (pCTX). We studied the replication of pCTX and found several indications that this plasmid replicates via a rolling-circle (RC) mechanism. The initiation and termination sites for pCTX plus-strand DNA synthesis were mapped to a 22 bp sequence that contains inverted repeats and a nonanucleotide motif found in the plus-strand origins of several RC replicons. Furthermore, similar to other RC replicons, replication of plasmids containing duplicated pCTX origins resulted in the deletion of sequences between the two origins and the formation of a single chimeric origin. Our previous work revealed that CTX prophage arrays give rise to hybrid CTX virions that contain sequences derived from two adjacent prophages. We now report that the boundaries between the sequences contributed to virions by the upstream and the downstream prophages in an array correspond to the site at which synthesis of plus-strand pCTX DNA is initiated and terminated. These data support the model that plus-strand CTXphi DNA is generated from chromosomal prophages via a novel process analogous to RC replication.

Formation of chromosomal tandem arrays of the SXT element and R391, two conjugative chromosomally integrating elements that share an attachment site.

The SXT element, a conjugative, self-transmissible, integrating element (a constin) originally derived from a Vibrio cholerae O139 isolate from India, and IncJ element R391, originally derived from a South African Providencia rettgeri isolate, were found to be genetically and functionally related. Both of these constins integrate site specifically into the Escherichia coli chromosome at an identical attachment site within the 5' end of prfC. They encode nearly identical integrases, which are required for chromosomal integration, excision, and extrachromosomal circularization of these elements, and they have similar tra genes. Therefore, these closely related constins have virtually identical mechanisms for chromosomal integration and dissemination. The presence of either element in a recipient cell did not significantly reduce its ability to acquire the other element, indicating that R391 and SXT do not encode surface exclusion determinants. In cells harboring both elements, SXT and R391 were integrated in tandem fashion on the chromosome, and homologous recombination appeared to play little or no role in the formation of these arrays. Interference between R391 and SXT was detected by measuring the frequency of loss of an unselected resident element upon introduction of a second selected element. In these assays, R391 was found to have a stronger effect on SXT stability than vice versa. The level of expression and/or activity of the donor and recipient integrases may play a role in the interference between these two related constins.

Human neutrophils and their products induce Shiga toxin production by enterohemorrhagic Escherichia coli.

The Shiga toxins (Stx) are critical virulence factors for Escherichia coli O157:H7 and other serotypes of enterohemorrhagic E. coli (EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H2O2), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H2O2 and also human neutrophils, an important endogenous source of H2O2, induced Stx2 expression by an EHEC clinical isolate. Both H2O2 and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.

Role for a phage promoter in Shiga toxin 2 expression from a pathogenic Escherichia coli strain.

Shiga toxins (Stxs), encoded by the stxA and stxB genes, are important contributors to the virulence of Escherichia coli O157:H7 and other Stx-producing E. coli (STEC) strains. The stxA and stxB genes in STEC strains are located on the genomes of resident prophages of the lambda family immediately downstream of the phage late promoters (p(R')). The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognate p(R') promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of p(R') as well as terminator kilobases distal to p(R'). To test if this Q-directed processive transcription plays a role in stx(2)AB expression, we constructed a mutant prophage in an O157:H7 clinical isolate from which p(R') and part of Q were deleted but which has an intact pStx, the previously described stx(2)AB-associated promoter. We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the p(R') promoter. Since transcription initiating at p(R') ultimately requires activation of the phage lytic cascade, expression of stx(2)AB in STEC depends primarily on prophage induction. By showing this central role for the prophage in stx(2)AB expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.

CTX prophages in classical biotype Vibrio cholerae: functional phage genes but dysfunctional phage genomes.

CTXphi is a filamentous, lysogenic bacteriophage whose genome encodes cholera toxin, the primary virulence factor produced by Vibrio cholerae. CTX prophages in O1 El Tor and O139 strains of V. cholerae are found within arrays of genetically related elements integrated at a single locus within the V. cholerae large chromosome. The prophages of O1 El Tor and O139 strains generally yield infectious CTXphi. In contrast, O1 classical strains of V. cholerae do not produce CTXphi, although they produce cholera toxin and they contain CTX prophages integrated at two sites. We have identified the second site of CTX prophage integration in O1 classical strains and characterized the classical prophage arrays genetically and functionally. The genes of classical prophages encode functional forms of all of the proteins needed for production of CTXphi. Classical CTX prophages are present either as solitary prophages or as arrays of two truncated, fused prophages. RS1, a genetic element that is closely related to CTXphi and is often interspersed with CTX prophages in El Tor strains, was not detected in classical V. cholerae. Our model for CTXphi production predicts that the CTX prophage arrangements in classical strains will not yield extrachromosomal CTX DNA and thus will not yield virions, and our experimental results confirm this prediction. Thus, failure of O1 classical strains of V. cholerae to produce CTXphi is due to overall deficiencies in the structures of the arrays of classical prophages, rather than to mutations affecting individual CTX prophage genes.

Molecular analyses of a putative CTXphi precursor and evidence for independent acquisition of distinct CTX(phi)s by toxigenic Vibrio cholerae.

The genes encoding cholera toxin (ctxA and ctxB) are encoded in the genome of CTXphi, a filamentous phage that infects Vibrio cholerae. To study the evolutionary history of CTXphi, we examined genome diversity in CTX(phi)s derived from a variety of epidemic and nonepidemic Vibrio sp. natural isolates. Among these were three V. cholerae strains that contained CTX prophage sequences but not the ctxA and ctxB genes. These prophages each gave rise to a plasmid form whose genomic organization was very similar to that of the CTXphi replicative form, with the exception of missing ctxAB. Sequence analysis of these three plasmids revealed that they lacked the upstream control region normally found 5' of ctxA, as well as the ctxAB promoter region and coding sequences. These findings are consistent with the hypothesis that a CTXphi precursor that lacked ctxAB simultaneously acquired the toxin genes and their regulatory sequences. To assess the evolutionary relationships among additional CTX(phi)s, two CTXphi-encoded genes, orfU and zot, were sequenced from 13 V. cholerae and 4 V. mimicus isolates. Comparative nucleotide sequence analyses revealed that the CTX(phi)s derived from classical and El Tor V. cholerae isolates comprise two distinct lineages within otherwise nearly identical chromosomal backgrounds (based on mdh sequences). These findings suggest that nontoxigenic precursors of the two V. cholerae O1 biotypes independently acquired distinct CTX(phi)s.

Sunlight-induced propagation of the lysogenic phage encoding cholera toxin.

In toxigenic Vibrio cholerae, the cholera enterotoxin (CT) is encoded by CTXPhi, a lysogenic bacteriophage. The propagation of this filamentous phage can result in the origination of new toxigenic strains. To understand the nature of possible environmental factors associated with the propagation of CTXPhi, we examined the effects of temperature, pH, salinity, and exposure to direct sunlight on the induction of the CTX prophage and studied the transmission of the phage to potential recipient strains. Exposure of cultures of CTXPhi lysogens to direct sunlight resulted in approximately 10,000-fold increases in phage titers. Variation in temperature, pH, or salinity of the culture did not have a substantial effect on the induction of the prophage, but these factors influenced the stability of CTXPhi particles. Exposure of mixed cultures of CTXPhi lysogens and potential recipient strains to sunlight significantly increased both the in vitro and in vivo (in rabbit ileal loops) transduction of the recipient strains by CTXPhi. Included in these transduction experiments were two environmental nontoxigenic (CTXPhi(-)) strains of V. cholerae O139. These two O139 strains were transduced at high efficiency by CTXPhi, and the phage genome integrated into the O139 host chromosome. The resulting CTXPhi lysogens produced biologically active CT both in vitro and in rabbit ileal loops. This finding suggests a possible mechanism explaining the origination of toxigenic V. cholerae O139 strains from nontoxigenic progenitors. This study indicates that sunlight is a significant inducer of the CTX prophage and suggests that sunlight-induced transmission of CTXPhi may constitute part of a natural mechanism for the origination of new toxigenic strains of V. cholerae.

CTXphi contains a hybrid genome derived from tandemly integrated elements.

CTXphi is a filamentous, temperate bacteriophage whose genome includes ctxAB, the genes that encode cholera toxin. In toxigenic isolates of Vibrio cholerae, tandem arrays of prophage DNA, usually interspersed with the related genetic element RS1, are integrated site-specifically within the chromosome. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. Coding sequences are always derived from the 5' prophage whereas most of an intergenic sequence, intergenic region 1, is always derived from the 3' element. The presence of tandem elements is required for production of virions: V. cholerae strains that contain a solitary prophage rarely yield CTX virions, and the few virions detected result from imprecise excision of prophage DNA. Thus, generation of the replicative form of CTXphi, pCTX, a step that precedes production of virions, does not depend on reversal of the process for site-specific integration of CTXphi DNA into the V. cholerae chromosome. Production of pCTX also does not depend on RecA-mediated homologous recombination between adjacent prophages. We hypothesize that the CTXphi-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of pCTX and CTXphi does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA while still enabling dissemination of CTXphi to new hosts.

Molecular cloning and expression of a gene encoding Cryptosporidium parvum glycoproteins gp40 and gp15.

Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host cell interactions and the molecular mechanisms involved in the pathogenesis of cryptosporidiosis are unknown. In this study we have shown that gp40, a mucin-like glycoprotein, is localized to the surface and apical region of invasive stages of the parasite and is shed from its surface. gp40-specific antibodies neutralize infection in vitro, and native gp40 binds specifically to host cells, implicating this glycoprotein in C. parvum attachment to and invasion of host cells. We have cloned and sequenced a gene designated Cpgp40/15 that encodes gp40 as well as gp15, an antigenically distinct, surface glycoprotein also implicated in C. parvum-host cell interactions. Analysis of the deduced amino acid sequence of the 981-bp Cpgp40/15 revealed the presence of an N-terminal signal peptide, a polyserine domain, multiple predicted O-glycosylation sites, a single potential N-glycosylation site, and a hydrophobic region at the C terminus, a finding consistent with what is required for the addition of a GPI anchor. There is a single copy of Cpgp40/15 in the C. parvum genome, and this gene does not contain introns. Our data indicate that the two Cpgp40/15-encoded proteins, gp40 and gp15, are products of proteolytic cleavage of a 49-kDa precursor protein which is expressed in intracellular stages of the parasite. The surface localization of gp40 and gp15 and their involvement in the host-parasite interaction suggest that either or both of these glycoproteins may serve as effective targets for specific preventive or therapeutic measures for cryptosporidiosis.

Convergence of the secretory pathways for cholera toxin and the filamentous phage, CTXphi.

Virulence of Vibrio cholerae depends on secretion of cholera toxin (CT), which is encoded within the genome of a filamentous phage, CTXphi. Release of CT is mediated by the extracellular protein secretion (eps) type II secretion system. Here, the outer membrane component of this system, EpsD, was shown to be required for secretion of the phage as well. Thus, EpsD plays a role both in pathogenicity and in horizontal transfer of a key virulence gene. Genomic analysis suggests that additional filamentous phages also exploit chromosome-encoded outer membrane channels.

Mobilization of plasmids and chromosomal DNA mediated by the SXT element, a constin found in Vibrio cholerae O139.

The Vibrio cholerae SXT element encodes resistance to multiple antibiotics and is a conjugative, self-transmissible, and chromosomally integrating element (a constin). Excision and self-transfer of the SXT element require an element-encoded integrase. We now report that the SXT element can also mobilize the plasmids RSF1010 and CloDF13 in trans as well as chromosomal DNA in an Hfr-like manner. SXT element-mediated mobilization of plasmids and chromosomal DNA, unlike its self-transfer, is not dependent upon excision of the element from the chromosome. These results raise the possibility that the SXT element and other constins play a general role in horizontal gene transfer among gram-negative bacteria.

Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice.

Shiga toxin-producing Escherichia coli (STEC) cause significant disease; treatment is supportive and antibiotic use is controversial. Ciprofloxacin but not fosfomycin causes Shiga toxin-encoding bacteriophage induction and enhanced Shiga toxin (Stx) production from E. coli O157:H7 in vitro. The potential clinical relevance of this was examined in mice colonized with E. coli O157:H7 and given either ciprofloxacin or fosfomycin. Both antibiotics caused a reduction in fecal STEC. However, animals treated with ciprofloxacin had a marked increase in free fecal Stx, associated with death in two-thirds of the mice, whereas fosfomycin did not. Experiments that used a kanamycin-marked Stx2 prophage demonstrated that ciprofloxacin, but not fosfomycin, caused enhanced intraintestinal transfer of Stx2 prophage from one E. coli to another. These observations suggest that treatment of human STEC infection with bacteriophage-inducing antibiotics, such as fluoroquinolones, may have significant adverse clinical consequences and that fluoroquinolone antibiotics may enhance the movement of virulence factors in vivo.

Infectious CTXPhi and the vibrio pathogenicity island prophage in Vibrio mimicus: evidence for recent horizontal transfer between V. mimicus and V. cholerae.

Vibrio mimicus differs from Vibrio cholerae in a number of genotypic and phenotypic traits but like V. cholerae can give rise to diarrheal disease. We examined clinical isolates of V. mimicus for the presence of CTXPhi, the lysogenic filamentous bacteriophage that carries the cholera toxin genes in epidemic V. cholerae strains. Four V. mimicus isolates were found to contain complete copies of CTXPhi. Southern blot analyses revealed that V. mimicus strain PT5 contains two CTX prophages integrated at different sites within the V. mimicus genome whereas V. mimicus strains PT48, 523-80, and 9583 each contain tandemly arranged copies of CTXPhi. We detected the replicative form of CTXPhi, pCTX, in all four of these V. mimicus isolates. The CTX prophage in strain PT5 was found to produce infectious CTXPhi particles. The nucleotide sequences of CTXPhi genes orfU and zot from V. mimicus strain PT5 and V. cholerae strain N16961 were identical, indicating contemporary horizontal transfer of CTXPhi between these two species. The receptor for CTXPhi, the toxin-coregulated pilus, which is encoded by another lysogenic filamentous bacteriophage, VPIPhi, was also present in the CTXPhi-positive V. mimicus isolates. The nucleotide sequences of VPIPhi genes aldA and toxT from V. mimicus strain PT5 and V. cholerae N16961 were identical, suggesting recent horizontal transfer of this phage between V. mimicus and V. cholerae. In V. mimicus, the vibrio pathogenicity island prophage was integrated in the same chromosomal attachment site as in V. cholerae. These results suggest that V. mimicus may be a significant reservoir for both CTXPhi and VPIPhi and may play an important role in the emergence of new toxigenic V. cholerae isolates.