An intracellular replication niche for Vibrio cholerae in the amoeba Acanthamoeba castellanii

Vibrio cholerae is a human pathogen and the causative agent of cholera. The persistence of this bacterium in aquatic environments is a key epidemiological concern, as cholera is transmitted through contaminated water. Predatory protists, such as amoebae, are major regulators of bacterial populations in such environments. Therefore, we investigated the interaction between V. cholerae and the amoeba Acanthamoeba castellanii at the single-cell level. We observed that V. cholerae can resist intracellular killing. The non-digested bacteria were either released or, alternatively, established a replication niche within the contractile vacuole of A. castellanii. V. cholerae was maintained within this compartment even upon encystment. The pathogen ultimately returned to its aquatic habitat through lysis of A. castellanii, a process that was dependent on the production of extracellular polysaccharide by the pathogen. This study reinforces the concept that V. cholerae is a facultative intracellular bacterium and describes a new host–pathogen interaction.     

Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits

Vibrio nigripulchritudo is an emerging pathogen of farmed shrimp in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have suggested that pathogenicity is linked to particular lineages. Here, we performed high-throughput sequencing-based comparative genome analysis of 16 V. nigripulchritudo strains to explore the genomic diversity and evolutionary history of pathogen-containing lineages and to identify pathogen-specific genetic elements. Our phylogenetic analysis revealed three pathogen-containing V. nigripulchritudo clades, including two clades previously identified from New Caledonia and one novel clade comprising putatively pathogenic isolates from septicemic shrimp in Madagascar. The similar genetic distance between the three clades indicates that they have diverged from an ancestral population roughly at the same time and recombination analysis indicates that these genomes have, in the past, shared a common gene pool and exchanged genes. As each contemporary lineage is comprised of nearly identical strains, comparative genomics allowed differentiation of genetic elements specific to shrimp pathogenesis of varying severity. Notably, only a large plasmid present in all highly pathogenic (HP) strains encodes a toxin. Although less/non-pathogenic strains contain related plasmids, these are differentiated by a putative toxin locus. Expression of this gene by a non-pathogenic V. nigripulchritudo strain resulted in production of toxic culture supernatant, normally an exclusive feature of HP strains. Thus, this protein, here termed ‘nigritoxin'', is implicated to an extent that remains to be precisely determined in the toxicity of V. nigripulchritudo.     

Sexual selection drives rapid divergence in bowerbird display traits

Abstract.— Sexual selection driving display trait divergence has been suggested as a cause of rapid speciation, but there is limited supporting evidence for this from natural populations. Where speciation by sexual selection has occurred in newly diverged populations, we expect that there will be significant differences in female preferences and corresponding male display traits in the absence of substantial genetic and other morphological differentiation. Two allopatric populations of the Vogelkop bowerbird, Amblyornis inornatus , show large, qualitative differences in a suite of display traits including bower structure and decorations. We experimentally demonstrate distinct male decoration color preferences within each population, provide direct evidence of female preferences for divergent decoration and bower traits in the population with more elaborate display, and show that there is minimal genetic differentiation between these populations. These results support the speciation by sexual selection hypothesis and are most consistent with the hypothesis that changes in male display have been driven by divergent female choice.     

Requirement for Vibrio cholerae integration host factor in conjugative DNA transfer

The requirement for host factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether integration host factor (IHF) or Fis, two host-encoded nucleoid proteins, are required for transfer of SXT, a Vibrio cholerae-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from V. cholerae. In contrast, IHF proved to be required for V. cholerae to act as an SXT donor. In the absence of IHF, V. cholerae displayed a modest defect for serving as an SXT recipient. Surprisingly, SXT integration into or excision from the V. cholerae chromosome, which requires an SXT-encoded integrase related to lambda integrase, did not require IHF. Therefore, the defect in SXT transmission in the V. cholerae IHF mutant is probably not related to IHF's ability to promote DNA recombination. The V. cholerae IHF mutant was also highly impaired as a donor of RP4, a broad-host-range conjugative plasmid. Thus, the V. cholerae IHF mutant appears to have a general defect in conjugation. Escherichia coli IHF mutants were not impaired as donors or recipients of SXT or RP4, indicating that IHF is a V. cholerae-specific conjugation factor.     

Detection of an OmpA-like protein in Vibrio cholerae

Abstract Rhodopseudomonas marina/agilis was enriched from a natural microbial mat by using conditions that favor growth of anoxygenic photoheterotrophs able to fix N₂ rapidly. The isolated bacterium grows more readily on fructose or mannitol than on organic acid carbon sources, requires preformed biotin and thiamine as growth factors, and is extraordinarily motile; growth occurs up to a temperature of approx. 44°C. The photosynthetic pigments of R. marina/agilis are housed in intracytoplasmic lamellar membranes which show the in vivo absorbance characteristics of bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series. In common with other non-sulfur purple bacteria, R. marina/agilis can also grow as an aerobic heterotroph in darkness. Under these conditions, photopigment synthesis is severely repressed. R. marina/agilis requires 1–5% NaCl for optimal growth, and cells grown on N₂ showed nitrogenase activity of >1000 nmol acetylene reduced h/mg dry wt.     

Adaptation of an introduced host to an indigenous parasite

Introduction of striped bass to the west coast from the east coast of the U.S.A. provided the opportunity to study a recent host-parasite association in a marine system. An indigenous species of parasite was known to induce pathological changes in the introduced population. Because the west coast population has been in association with this pathogenic parasite more than 20 generations, we predicted that the host reaction of the west coast population would be less severe compared to that of the naive east coast stock of striped bass. This prediction was tested by conducting reciprocal infection experiments with east and west coast hosts and parasites. The group of west coast striped bass had a lower intensity of infection and exhibited less tissue damage compared to the group of east coast striped bass. We suggest that selection has acted only on the host and is driven by parasite-induced host mortality. This type of 1-sided selection is in contrast to present models of the evolution of host-parasite associations.     

Covariance of phenotypically plastic traits induces an adaptive shift in host selection behaviour

Flexibility in adult body size allows generalist parasitoids to use many host species at a cost of producing a range of adult sizes. Consequently, host selection behaviour must also maintain a level of flexibility as adult size is related to capture efficiency. In the present study, we investigated covariance of two plastic traits--size at pupation and host size selection behaviour-using Aphidius ervi reared on either Acyrthosiphon pisum or Aulacorthum solani, generating females of disparate sizes. Natal host was shown to change the ranking of perceived host quality with relation to host size. Parasitoids preferentially attacked hosts that corresponded to the size of the second instar of their natal host species. This resulted in optimal host selection behaviour when parasitoids were exposed to the same host species from which they emerged. Parasitoid size was positively correlated with host size preference, indicating that females use relative measurements when selecting suitable hosts. These coadapted gene complexes allow generalist parasitoids to effectively use multiple host species over several generations. However, the fixed nature of the behavioural response, within a parasitoid's lifetime, suggests that these traits may have evolved in a patchy host species environment.     

Quorum-sensing- and type VI secretion-mediated spatiotemporal cell death drives genetic diversity in Vibrio cholerae

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Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1

A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hlyA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.     

Adhesion of Vibrio cholerae to Granular Starches

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. Cholera can become epidemic and deadly without adequate medical care. Appropriate rehydration therapy can reduce the mortality rate from as much as 50% of the affected individuals to <1%. Thus, oral rehydration therapy (ORT) is an important measure in the treatment of this disease. To further reduce the symptoms associated with cholera, improvements in oral rehydration solution (ORS) by starch incorporation were suggested. Here, we report that V. cholerae adheres to starch granules incorporated in ORS. Adhesion of 98% of the cells was observed within 2 min when cornstarch granules were used. Other starches showed varied adhesion rates, indicating that starch source and composition play an important role in the interaction of V. cholerae and starch granules. Sugars metabolized by V. cholerae showed a repressive effect on the adhesion process. The possible mechanisms involved are discussed. Comparing V. cholerae adhesion with the adhesion of other pathogens suggests the involvement of starch degradation capabilities. This adhesion to granular starch can be used to improve ORT.     

Adhesion of Vibrio cholerae to granular starches

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. Cholera can become epidemic and deadly without adequate medical care. Appropriate rehydration therapy can reduce the mortality rate from as much as 50% of the affected individuals to <1%. Thus, oral rehydration therapy (ORT) is an important measure in the treatment of this disease. To further reduce the symptoms associated with cholera, improvements in oral rehydration solution (ORS) by starch incorporation were suggested. Here, we report that V. cholerae adheres to starch granules incorporated in ORS. Adhesion of 98% of the cells was observed within 2 min when cornstarch granules were used. Other starches showed varied adhesion rates, indicating that starch source and composition play an important role in the interaction of V. cholerae and starch granules. Sugars metabolized by V. cholerae showed a repressive effect on the adhesion process. The possible mechanisms involved are discussed. Comparing V. cholerae adhesion with the adhesion of other pathogens suggests the involvement of starch degradation capabilities. This adhesion to granular starch can be used to improve ORT.     

Toxins of Vibrio cholerae

Surveyed in the paper are published data on properties, biological activity, genetic determinants and action mechanisms of recently known toxins produced by different strains of Vibrio cholerae irrespectively of their capacity for the synthesis of choleric toxin--the main virulence factor. Their possible importance both for the general clinical pattern of cholera provoked by cholerogenic agents and as independent virulence factors causing diarrhea without cholera is elucidated. The sets and levels of expression of additional toxins can differ for different pathogenic clones and they can correspondingly condition degrees of their epidemic and etiological safety.     

The Zymovars of Vibrio cholerae: multilocus enzyme electrophoresis of Vibrio cholerae

Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several genetically diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.     

Outer Membrane Vesiculation Facilitates Surface Exchange and In Vivo Adaptation of Vibrio cholerae

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Vibrio cholerae and cholera: out of the water and into the host

The facultative human pathogen Vibrio cholerae can be isolated from estuarine and aquatic environments. V. cholerae is well recognized and extensively studied as the causative agent of the human intestinal disease cholera. In former centuries cholera was a permanent threat even to the highly developed populations of Europe, North America, and the northern part of Asia. Today, cholera still remains a burden mainly for underdeveloped countries, which cannot afford to establish or to maintain necessary hygienic and medical facilities. Especially in these environments, cholera is responsible for significant mortality and economic damage. During the last three decades, intensive research has been undertaken to unravel the virulence properties and to study the epidemiology of this significant human pathogen. More recently, researchers have been elucidating the environmental lifestyle of V. cholerae. This review provides an overview of the current knowledge of both the host- and environment-specific physiological attributes of V. cholerae.     

Vibrio cholerae cytolysin is composed of an alpha-hemolysin-like core

The enteric pathogen Vibrio cholerae secretes a water-soluble 80-kD cytolysin, Vibrio cholerae cytolysin (VCC) that assembles into pentameric channels following proteolytic activation by exogenous proteases. Until now, VCC has been placed in a unique class of pore-forming toxins, distinct from paradigms such as Staphyloccal alpha-hemolysin. However, as reported here, amino acid sequence analysis and three-dimensional structure modeling indicate that the core component of the VCC toxin is related in sequence and structure to a family of hemolysins from Staphylococcus aureus that include leukocidin F and alpha-hemolysin. Furthermore, our analysis has identified the channel-forming region of VCC and a potential lipid head-group binding site, and suggests a conserved mechanism of assembly and lysis. An additional domain in the VCC toxin is related to plant lectins, conferring additional target cell specificity to the toxin.