Role of Temperate Bacteriophage 139 in Changing Cholera Toxin Production in Vibrio cholerae Classical Biovar

New data were obtained concerning cell sensitivity of pathogenic strains of cholera vibrions, which belong to the serogroup O1 of classical biovar, to the temperate bacteriophage 139, the native host of which is Vibrio cholerae O139. Molecular–genetic and biochemical studies showed that phage 139 integrated into the chromosome of strains V. cholerae O1 can change their toxigenic properties. A change in the production of cholera toxin (CT) in lysogens is associated both with an increase in the activity of the toxR regulatory gene and with an alteration of the structure of a chromosomal DNA region that contains a copy of the operon ctxAB encoding CT biosynthesis.     

Complete Genome Sequence of Vibrio parahaemolyticus Bacteriophage vB_VpaM_MAR

Vibrio parahaemolyticus is a major pathogen that is mainly associated with seafood and is a global food safety issue. Our objective was to isolate and completely sequence a specific phage against this bacterium. Phage vB_VpaM_MAR is able to lyse 76% of the V. parahaemolyticus strains tested. MAR belongs to the Myoviridae family and has a genome comprised of double-stranded DNA with a size of 41,351 bp, a G+C content of 51.3%, and 62 open reading frames (ORFs). Bioinformatic analysis showed that phage MAR is closely related to Vibrio phages VHML, VP58.5, and VP882 and Halomonas aquamarina phage ΦHAP-1.     

Incidence of Vibrio parahaemolyticus Bacteriophages and Other Vibrio Bacteriophages in Marine Samples

The distribution of the aerobic spirochetes Leptospira in surface waters, soil, and aquatic animals was investigated. Isolates from water and soil exhibited physiological characteristics common to members of the "biflexa complex," none were capable of infecting experimental animals, and leptospires could not be isolated from the eight genera of aquatic animals examined. The isolation frequencies from surface waters were: stream, 100%; lake, 65%; spring, 28%; bog lake, 5%; and marsh, 0%. With the exception of the stream, more isolations were obtained from the soil adjacent to the water than from the water. Leptospires were most frequently associated with soils of high moisture and organic matter content.     

Somatic O Antigen Relationship of Brucella and Vibrio cholerae

The antigenic relationship between Brucella species and Vibrio cholerae was examined by agglutinin and agglutinin-absorption tests by using rabbit antisera. Brucella antisera agglutinated only the Inaba serotype of V. cholerae and at low titer. Inaba-reactive antibody was absorbed by either heat-stable (100 C, 2 hr) Ogawa or Inaba O antigens. Cholera antisera from rabbits immunized with either O or HO antigens of either Ogawa or Inaba serotypes contained brucella agglutinins. This activity was absorbed completely from Ogawa antisera by either Ogawa or Inaba O antigens but only partially from Inaba antisera by Ogawa O antigen. These findings support the claim of Gallut that the cross-reaction is due to heat-stable O antigens of V. cholerae rather than heat-labile flagellar antigens as described in many text books. The cross-reactive component is more dominant in the Inaba than in the Ogawa serotype of V. cholerae.     

Characterization of Vibrio cholerae Bacteriophages Isolated from the Environmental Waters of the Lake Victoria Region of Kenya

Over the last decade, cholera outbreaks have become common in some parts of Kenya. The most recent cholera outbreak occurred in Coastal and Lake Victoria region during January 2009 and May 2010, where a total of 11,769 cases and 274 deaths were reported by the Ministry of Public Health and Sanitation. The objective of this study is to isolate Vibrio cholerae bacteriophages from the environmental waters of the Lake Victoria region of Kenya with potential for use as a biocontrol for cholera outbreaks. Water samples from wells, ponds, sewage effluent, boreholes, rivers, and lakes of the Lake Victoria region of Kenya were enriched for 48 h at 37 °C in broth containing a an environmental strain of V. cholerae. Bacteriophages were isolated from 5 out of the 42 environmental water samples taken. Isolated phages produced tiny, round, and clear plaques suggesting that these phages were lytic to V. cholerae. Transmission electron microscope examination revealed that all the nine phages belonged to the family Myoviridae, with typical icosahedral heads, long contractile tails, and fibers. Head had an average diameter of 88.3 nm and tail of length and width 84.9 and 16.1 nm, respectively. Vibriophages isolated from the Lake Victoria region of Kenya have been characterized and the isolated phages may have a potential to be used as antibacterial agents to control pathogenic V. cholerae bacteria in water reservoirs.     

Bacteriophage P70: Unique Morphology and Unrelatedness to Other Listeria Bacteriophages

Listeria monocytogenes is an important food-borne pathogen, and its bacteriophages find many uses in detection and biocontrol of its host. The novel broad-host-range virulent phage P70 has a unique morphology with an elongated capsid. Its genome sequence was determined by a hybrid sequencing strategy employing Sanger and PacBio techniques. The P70 genome contains 67,170 bp and 119 open reading frames (ORFs). Our analyses suggest that P70 represents an archetype of virus unrelated to other known Listeria bacteriophages.     

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.     

Sequence Analyses of Type IV Pili from Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus

Bacterial surface structures called pili have been studied extensively for their role as possible colonization factors. Most sequenced Vibrio genomes predict a variety of pili genes in these organisms, including several types of type IV pili. In particular, the mannose-sensitive hemagglutinin (MSHA) and the PilA pili, also known as the chitin-regulated pilus (ChiRP), are type IVa pili commonly found in Vibrio genomes and have been shown to play a role in the colonization of Vibrio species in the environment and/or host tissue. Here, we report sequence comparisons of two type IVa pilin subunit genes, mshA and pilA, and their corresponding amino acid sequences, for several strains from the three main human pathogenic Vibrio species, V. cholerae, V. parahaemolyticus, and V. vulnificus. We identified specific groupings of these two genes in V. cholerae, whereas V. parahaemolyticus and V. vulnificus strains had no apparent allelic clusters, and these genes were strikingly divergent. These results were compared with other genes from the MSHA and PilA operons as well as another Vibrio pili from the type IVb group, the toxin co-regulated pilus (TCP) from V. cholerae. Our data suggest that a selective pressure exists to cause these strains to vary their MSHA and PilA pilin subunits. Interestingly, V. cholerae strains possessing TCP have the same allele for both mshA and pilA. In contrast, V. cholerae isolates without TCP have polymorphisms in their mshA and pilA sequences similar to what was observed for both V. parahaemolyticus and V. vulnificus. This data suggests a possible linkage between host interactions and maintaining a highly conserved type IV pili sequence in V. cholerae. Although the mechanism underlying this intriguing diversity has yet to be elucidated, our analyses are an important first step towards gaining insights into the various aspects of Vibrio ecology.     

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.     

In silico comparative study of the genomic islands of Vibrio cholerae MJ1236 with those of Classical and El Tor N16961 strains of Vibrio cholerae

The evolution of microbial genomes is greatly influenced by horizontal gene transfer (HGT), where large blocks of horizontally acquired foreign sequences, often encoding virulence determinants, occur in chromosomes of pathogenic bacteria. A program design-island developed in our laboratory was used on three completely sequenced Vibrio cholerae genomes, V. cholerae Classical O395, El Tor N16961 and MJ1236, in order to identify the putative horizontally acquired regions. The putative genomic islands (GIs) were graphically represented and analyzed. The study identified distinct regions in the GIs of V. cholerae MJ1236 which were shared either with the Classical or the El Tor strain of V. cholerae. A cluster comprising of 38 ORFs was common to V. cholerae strains of MJ1236 and Classical O395 but absent in El Tor N16961. About 5% of the predicted GIs of V. cholerae MJ1236 were unique to itself. Among these unique ORFs, a region of mostly hypothetical genes was identified, where the ORFs were present in a large cluster. The results show that the HGT had played a significant role in the evolution and the differentiation of V. cholerae MJ1236.     

Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae

Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.The type II secretion (T2S) pathway is widely used by pathogenic gram-negative bacteria for delivery of virulence factors into the extracellular milieu (11, 17, 46). Proteins destined for release through this pathway are first translocated across the cytoplasmic membrane via the Sec (24, 42) or Tat (59) machinery. Following folding and assembly in the periplasm, the proteins are transported across the outer membrane via the T2S machinery, a complex composed of 12 to 16 different gene products, depending on the species. In Vibrio cholerae, the elements of the T2S apparatus are encoded by the extracellular protein secretion (eps) genes, epsC through epsN and pilD (vcpD) (18, 31, 39, 49, 50). Together these proteins coordinate the outer membrane translocation of the major virulence factor, cholera toxin, as well as chitinase, lipase, hemagglutinin/protease, and other proteases (12, 27, 49). Our studies are focused on better understanding how the T2S complex assembles in the cell envelope of V. cholerae to begin to elucidate the mechanism by which extracellular secretion is accomplished.The T2S apparatus is modeled as an envelope-spanning complex with subcomplexes in the inner and outer membranes (see Fig. S1 in the supplemental material). The precise stoichiometry and juxtaposition of the Eps proteins are not known, but accumulating biochemical, genetic, and molecular studies continue to refine our understanding of complex assembly and function (for a review, see reference 25). A trimolecular complex consisting of cytoplasmic protein EpsE and inner membrane proteins EpsL and EpsM has been identified. EpsL and EpsM have been shown to coimmunoprecipitate and participate in mutual stabilization interactions in vivo by protecting each other from proteolysis (34, 41, 43, 48). Homologs of inner membrane protein EpsC have been implicated in interactions with the aforementioned inner membrane subcomplex (20, 29, 57), as well as homologs of outer membrane protein EpsD, which form oligomeric rings through which the secreted substrates, it is hypothesized, exit the cell (1, 10, 36, 38). More specifically, EpsC homologs in Pseudomonas aeruginosa and Klebsiella oxytoca are sensitive to proteolysis or unable to oligomerize in the absence of EpsD homologs (2, 40); however, direct interactions between these two proteins in their full-length forms have not been shown by coimmunoprecipitation or copurification. Although yeast two-hybrid analysis of the periplasmic domains of the Erwinia chrysanthemi EpsC and EpsD homologs also did not reveal interaction (15), recently it was shown that periplasmic subdomains of EpsC and EpsD homologs of Vibrio vulnificus copurified (28). It seems likely that EpsC, having interactions with both inner and outer membrane subcomplexes, plays a crucial role in complex function by connecting the inner membrane components to the outer membrane EpsD pore. Furthermore, it has been speculated that EpsC homologs impart specificity to the various T2S systems by directly interacting with proteins to be secreted (3).We have taken a cell biology approach to characterizing Eps protein interactions, observing the dynamics of green fluorescent protein (GFP)-tagged components of the Eps complex in live cells by fluorescence microscopy. This method permits study of Eps protein assembly in the context of the complete apparatus, situated in both membranes, without the disruptive procedures required for many in vitro molecular and biochemical analyses of protein-protein interactions. Here we present data illustrating the importance of expressing GFP fusions for localization studies with all other interacting components, preserving wild-type stoichiometry and expression levels. In particular, we note that GFP-EpsM does not appear to be focused at the polar membrane as previously described (53), when expressed in balance with its interacting proteins. Chromosomal replacement of epsM and epsC with gfp-tagged versions instead reveals a more distributed pattern, with punctate fluorescent foci along the full length of the cell. We have exploited these chromosomal gfp-eps strains to further dissect the interactions and requirements for localization of EpsC and EpsM by systematically deleting other eps genes in the operon.     

Relationship between structure and antigenicity of O1 Vibrio cholerae lipopolysaccharides

The relationship between the release of fructose from O1 Vibrio cholerae lipopolysaccharides (LPS) by dilute acetic acid hydrolysis and the decrease in their antigenicity was examined. Decrease in the antigenicity of LPS was not parallel with the release of fructose, and occurred very much later than the latter. Periodate oxidation of LPS resulted in the total elimination of the fructose and glucose, and two-thirds of the heptose constituents, but no difference in the antigenicity of LPS was observed before and after oxidation. These findings indicate that the fructose present in O1 V. cholerae LPS is not substantially involved in their specific antigenicity. In the O1 V. cholerae LPS, the fructose is in the branch structure, most probably in the core region.     

Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.     

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.     

PCR-based identification of Vibrio cholerae and the closely related species Vibrio mimicus using the large chromosomal ori sequence of Vibrio cholerae

The bacterial chromosomal replication origin (ori) sequences are a highly conserved essential genetic element. In this study, the large chromosomal replication origin sequence of Vibrio cholerae (oriCIVC) has been targeted for identification of the organism, including the biotypes of serogroup O1. The oriCIVC sequence-based PCR assay specifically amplified an 890 bp fragment from all the V. cholerae strains examined. A point mutation in the oriCIVC sequence of the classical biotype of O1 serogroup led to the loss of a BglII site, which was utilized for differentiation from El Tor vibrios. Interestingly, the PCR assay amplified a similarly sized ori segment, designated as oriCIVM, from V. mimicus strains, but failed to produce any amplicon with other strains. Cloning and sequencing of the oriCIVM revealed high sequence similarity (96%) with oriCIVC. The results indicate that V. mimicus is indeed very closely related to V. cholerae. In addition, the BglII restriction fragment length polymorphism (RFLP) between oriCIVM and oriCIVC sequences allowed us to differentiate the two species. The ori sequence-based PCR-RFLP assay developed in this study appears to be a useful method for rapid identification and differentiation of V. cholerae and V. mimicus strains, as well as for the delineation of classical and El Tor biotypes of V. cholerae O1.