No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1
Abstract
Vibrio cholerae serogroup O139 Bengal is the first documented serogroup other than O1 to cause epidemic cholera. The O139 Bengal strains are very similar to V. cholerae serogroup O1 biotype El Tor strains. The major differences between the two serogroups are that O139 Bengal contains a distinct O antigen and produces a polysaccharide capsule. We previously described three TnphoA mutants of O139 strain AI1837 which abolish both O antigen and capsule production. These TnphoA insertions were mapped to a 21.5 kb EcoRI fragment of the O139 chromosome. We describe here the cloning and mapping of this 21.5 kb EcoRI fragment and it was shown to complement each of the mutants in trans to produce O antigen and capsule. The EcoRI fragment contains 13 kb of DNA that is specific to O139 and 8.5 kb of DNA that is common to O1 and O139. Sequence analysis of the 13 kb of O139-specific DNA revealed that it contains 11 open reading frames all of which are transcribed in the same direction. Eight of the 11 open reading frames are homologous to sugar biosynthesis genes from other organisms. Using extended polymerase chain reactions, we show that the extent of the DNA region in O139 that is not present in O1 is approximately 35 kb. The site of insertion of this O139-specific DNA in the O1 chromosome was mapped to the rfbO1 region. We also demonstrate that O139 Bengal strain AI1837 contains a deletion of 22 kb that in serogroup O1 strains contains the rfb region. Therefore, O139 Bengal probably arose from an O1 strain that had undergone genetic rearrangements including deletion of the O1 rfb region and acquisition of a 35 kb region of DNA which encodes O139 surface polysaccharide.
... When they first emerged in India and Bangladesh, O139 isolates were shown to be virtually identical to O1 El Tor isolates, with the substitution of a 35-kb region of DNA encoding the O139 surface polysaccharide for a 22-kb region that included the rfb region in O1 isolates (7). O139 isolates carried all "standard" virulence factors found in O1 isolates, including the elements of CTX prophage and Vibrio seventh pandemic island I (VSP-I) and VSP-II. ...
... It is possible that clinical O139 isolates lost virulence elements such as the CTX prophage when introduced into the environment; however, based on our observations, they would also have had to have lost VSP-I and VSP-II and undergone substantial changes in housekeeping genes, as reflected in the diversity of STs noted. Alternatively, nontoxigenic/nonvirulent environmental isolates, from diverse sources, may have acquired the genetic cassette responsible for biosynthesis of the O139 surface polysaccharide (capsule and O antigen) (7). In keeping with the latter hypothesis, we know that the O139 biosynthetic cassette is movable, as its introduction into a "standard" El Tor O1 isolate appears to have been responsible for the original emergence of the epidemic O139 V. cholerae (25). ...
Vibrio cholerae serogroup O139 was first identified in 1992 in India and Bangladesh, in association with major epidemics of cholera in both
countries; cases were noted shortly thereafter in China. We characterized 211 V. cholerae O139 isolates that were isolated at multiple sites in China between 1993 and 2012 from patients (n = 92) and the environment (n = 119). Among clinical isolates, 88 (95.7%) of 92 were toxigenic, compared with 47 (39.5%) of 119 environmental isolates.
Toxigenic isolates carried the El Tor CTX prophage and toxin-coregulated pilus A gene (tcpA), as well as the Vibrio seventh pandemic island I (VSP-I) and VSP-II. Among a subset of 42 toxigenic isolates screened by multilocus sequence typing
(MLST), all were in the same sequence type as a clinical isolate (MO45) from the original Indian outbreak. Nontoxigenic isolates,
in contrast, generally lacked VSP-I and -II, and fell within13 additional sequence types in two clonal complexes distinct
from the toxigenic isolates. In further pulsed-field gel electrophoresis (PFGE) (with NotI digestion) studies, toxigenic isolates
formed 60 pulsotypes clustered in one group, while the nontoxigenic isolates formed 43 pulsotypes which clustered into 3 different
groups. Our data suggest that toxigenic O139 isolates from widely divergent geographic locations, while showing some diversity,
have maintained a relatively tight clonal structure across a 20-year time span. Nontoxigenic isolates, in contrast, exhibited
greater diversity, with multiple clonal lineages, than did their toxigenic counterparts.
... Traditional typing of bacterial pathogens by serological or other methods (antibiotic resistance, phage typing, etc.) may be relevant to disease treatment and to vaccine development but is increasingly unsuitable for modern epidemiology because it is based on variable phenotypes that are not necessarily related to genetic descent. Horizontal genetic exchange leads to the acquisition of genes encoding foreign lipopolysaccharides (LPS) (Reeves, 1993; Comstock et al., 1996; Davies et al., 1997; Mooi and Bik, 1997; Shepherd et al., 2000) or capsular polysaccharides (Kroll and Moxon, 1990; Swartley et al., 1997, 1998; Morona et al., 1999). Antibiotic resistance is also spread by horizontal genetic exchange (Dowson et al., 1994; Reichmann et al., 1997; Bootsma et al., 2000) as well as by mobile genetic elements (Hall and Collis, 1995). ...
... Recent pandemics have been caused by biotypes called`classical' (fifth and sixth pandemics) and`El Tor' (seventh pandemic) strains. The O139 bacteria from recent epidemics in India arose from El Tor strains through horizontal genetic exchange by substitution of a 35 kb region encoding O139 LPS for a 22 kb region encoding O1 LPS (Comstock et al., 1996; Sozhamannan et al., 1999). MLEE, ribotyping, RAPD and sequencing of housekeeping genes have shown that El Tor, Classical V. cholerae strains and V. cholerae O1 strains isolated in the Gulf states of the USA are closely related clonal groupings that differ at only a few loci (Karaolis et al., 1994a; Karaolis et al., 1995; Byun et al., 1999). ...
This chapter presents a phylogenetic perspective of molecular bacterial epidemiology. It summarizes approaches that are informative from the phylogenetic viewpoint, and describes results from selected pathogenic species where the available information seems to be somewhat more complete. Horizontal genetic exchange leads to the acquisition of genes encoding foreign lipopolysaccharides (LPS) or capsular polysaccharides. Antibiotic resistance is also spread by horizontal genetic exchange as well as by mobile genetic elements. Selectively neutral markers that are not under selection are more likely to index the genetic relatedness between isolates and less likely to be imported by gene transfer than are serological markers. A classification scheme that is based on selectively neutral markers would reflect the population biology of the species under investigation and can be used for phylogenetic analyses. Analysis of the sequence variation of multiple genes encoding housekeeping enzymes is probably the most reliable method for the classification of clonal bacteria into genetically related groupings. Sequence variation in housekeeping genes is usually neutral, that is, not subject to natural selection, and therefore the differences between alleles of housekeeping genes are more likely to reflect the time since phylogenetic descent than are those of genes whose products are under selection.
... Previous infection with V. cholerae O1 does not provide protection against O139 and vice versa (12), despite the fact that V. cholerae O139 evolved from V. cholerae O1 and expresses identical proteins including cholera toxin (11). The major difference between V. cholerae O139 and O1 is in the rfb genes involved in OSP synthesis (21,30,31) (18,19,34). The capsule of V. cholerae O139 contains a flexible, complex, and branched polymer of this hexasaccharide (18,19,34). ...
Cholera caused by Vibrio cholerae O139 emerged in the early 1990s and spread rapidly to 11 Asian countries before receding for unclear reasons. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). V. cholerae O139 also expresses the OSP-capsule. We, therefore, assessed antibody responses targeting V. cholerae O139 OSP, LPS, capsule, and vibriocidal responses in patients in Bangladesh with cholera caused by V. cholerae O139. We compared these responses to those of age-gender-blood group-matched recipients of the bivalent oral cholera vaccine (OCV O1/O139). We found prominent OSP, LPS, and vibriocidal responses in patients, with a high correlation between these responses. OSP responses primarily targeted the terminal tetrasaccharide of OSP. Vaccinees developed OSP, LPS, and vibriocidal antibody responses, but of significantly lower magnitude and responder frequency (RF) than matched patients. We separately analyzed responses in pediatric vaccinees born after V. cholerae O139 had receded in Bangladesh. We found that OSP responses were boosted in children who had previously received a single dose of bivalent OCV 3 yr previously but not in vaccinated immunologically naïve children. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 despite the presence of capsules, that vaccination with bivalent OCV is poorly immunogenic in the short term in immunologically naïve individuals, but that OSP-specific immune responses can be primed by previous exposure, although whether such responses can protect against O139 cholera is uncertain.
IMPORTANCE
Cholera is a severe dehydrating illness in humans caused by Vibrio cholerae serogroups O1 or O139. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) of V. cholerae LPS. Yet, little is known about immunity to O139 OSP. In this study, we assessed immune responses targeting OSP in patients from an endemic region with cholera caused by V. cholerae O139. We compared these responses to those of the age-gender-blood group-matched recipients of the bivalent oral cholera vaccine. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 and that the OSP responses primarily target the terminal tetrasaccharide of OSP. Our results further suggest that vaccination with the bivalent vaccine is poorly immunogenic in the short term for inducing O139-specific OSP responses in immunologically naïve individuals, but OSP-specific immune responses can be primed by previous exposure or vaccination.
... The current circulating pandemic strain belongs to serogroup O1 (biotype El Tor) and has been attributed to global outbreaks since 1961 (4,5), while epidemics of O139 have arisen from the Bay of Bengal beginning in 1992 (6). Serogroup O139 was initially discovered based on its inability to agglutinate with O1 antisera (7) and was subsequently shown to contain differences in O-antigen biosynthesis regions (8). Furthermore, serogroup O139 was determined to have emerged from the O1 El Tor biotype as a result of serogroup conversion and genetic exchange of O-antigen DNA (4,(8)(9)(10)(11). Because serogroup O139 is recently descended from O1 El Tor, it harbors the genetic backbone of seventh pandemic O1 El Tor strains, including many of the same virulence genes, such as the cholera toxin genes (12). ...
Toxigenic Vibrio cholerae serogroup O1 is the etiologic agent of the disease cholera, and strains of this serogroup are responsible for pandemics. A few other serogroups have been found to carry cholera toxin genes-most notably, O139, O75, and O141-and public health surveillance in the United States is focused on these four serogroups. A toxigenic isolate was recovered from a case of vibriosis from Texas in 2008. This isolate did not agglutinate with any of the four different serogroups' antisera (O1, O139, O75, or O141) routinely used in phenotypic testing and did not display a rough phenotype. We investigated several hypotheses that might explain the recovery of this potential nonagglutinating (NAG) strain using whole-genome sequencing analysis and phylogenetic methods. The NAG strain formed a monophyletic cluster with O141 strains in a whole-genome phylogeny. Furthermore, a phylogeny of ctxAB and tcpA sequences revealed that the sequences from the NAG strain also formed a monophyletic cluster with toxigenic U.S. Gulf Coast (USGC) strains (O1, O75, and O141) that were recovered from vibriosis cases associated with exposures to Gulf Coast waters. A comparison of the NAG whole-genome sequence showed that the O-antigen-determining region of the NAG strain was closely related to those of O141 strains, and specific mutations were likely responsible for the inability to agglutinate. This work shows the utility of whole-genome sequence analysis tools for characterization of an atypical clinical isolate of V. cholerae originating from a USGC state. IMPORTANCE Clinical cases of vibriosis are on the rise due to climate events and ocean warming (1, 2), and increased surveillance of toxigenic Vibrio cholerae strains is now more crucial than ever. While traditional phenotyping using antisera against O1 and O139 is useful for monitoring currently circulating strains with pandemic or epidemic potential, reagents are limited for non-O1/non-O139 strains. With the increased use of next-generation sequencing technologies, analysis of less well-characterized strains and O-antigen regions is possible. The framework for advanced molecular analysis of O-antigen-determining regions presented herein will be useful in the absence of reagents for serotyping. Furthermore, molecular analyses based on whole-genome sequence data and using phylogenetic methods will help characterize both historical and novel strains of clinical importance. Closely monitoring emerging mutations and trends will improve our understanding of the epidemic potential of Vibrio cholerae to anticipate and rapidly respond to future public health emergencies.
... Although there were high GIs and PIs amongst the clinical and environmental isolates, it only indicates possession of additional unidentified fitness factors which may be involve in other virulence indices (Hasan et al. 2012a, b, c). Other studies also corroborates these reports (Yamasaki et al. 1999;Comstock et al. 1996;Bik et al. 1995;Stroeher et al. 1995;Aydanian et al. 2015). These reports confirm the genealogical and phylogenetic relationship of both SAAT-1/139-Vc and SANAS/NSAG/NAG-Vc as members of the family Vibrionaceae. ...
Somatic antigen agglutinable type Vibrio cholerae of 1/139 (SAAT-Vc-1/139) members or O1/O139 Vibrio cholerae have been described by various investigators as choleragenic due to their increase pathogenic potential and production of choleragen. Reported cholera outbreak cases around the World have been associated with these choleragenic Vibrio cholerae with high case fatality involving various educational, human, governmental, animal and financial resources. These Vibrio members have shown genealogical and phylogenetic relationship with the somatic antigen non-agglutinable strains of 1/139 Vibrio cholerae (SANAS- Vc -1/139) or O1/O139 non-agglutinating Vibrio cholerae (O1/O139-NAG-Vc). The O1/O139-NAGVc members have been reported to be implicated in most cholera/cholera-like cases, sporadic cases, diarrhea, production of cholera toxin and transmitted via consumption and/or contact with contaminated water/seafood. Some reported cases of cholera outbreaks, sporadic cases and observed change in nature has also been tracable to these non-agglutinable Vibrio members (O1/O139-NAGVc) yet there is a sustained paucity of reports on the non-agglutinable V. cholerae members implication in cholera outbreak. The emergence of fulminating extraintestinal and systemic Vibriosis is another aspect of SANAS- Vc -1/139 involvement which has received low attention in terms of research driven interest. This review addresses the need to appraise and continue in research based studies on the somatic antigen non-serogroup agglutinable type-1/139 Vibrio cholerae members which are currently prevalent in water bodies, fruits/vegetables, foods and terrestrial environment. Our opinion is a summative of interest in integrated surveillance studies, management/control of cholera outbreaks as well as diarrhea and other disease related cases both in the rural, suburban and urban metropolis.
... Although there were high GIs and PIs amongst the clinical and environmental isolates, it only indicates possession of additional unidentified fitness factors which may be involve in other virulence indices (Hasan et al. 2012a, b, c). Other studies also corroborates these reports (Yamasaki et al. 1999;Comstock et al. 1996;Bik et al. 1995;Stroeher et al. 1995;Aydanian et al. 2015). These reports confirm the genealogical and phylogenetic relationship of both SAAT-1/139-Vc and SANAS/NSAG/NAG-Vc as members of the family Vibrionaceae. ...
Somatic antigen agglutinable type-1/139 Vibrio cholerae (SAAT-1/139-Vc) members or O1/O139 V. cholerae have been described by various investigators as pathogenic due to their increasing virulence potential and production of choleragen. Reported cholera outbreak cases around the world have been associated with these choleragenic V. cholerae with high case fatality affecting various human and animals. These virulent Vibrio members have shown genealogical and phylogenetic relationship with the avirulent somatic antigen non-agglutinable strains of 1/139 V. cholerae (SANAS-1/139- Vc) or O1/O139 non-agglutinating V. cholerae (O1/O139-NAG-Vc). Reports on implication of O1/O139-NAGVc members in most sporadic cholera/cholera-like cases of diarrhea, production of cholera toxin and transmission via consumption and/or contact with contaminated water/seafood are currently on the rise. Some reported sporadic cases of cholera outbreaks and observed change in nature has also been tracable to these non-agglutinable Vibrio members (O1/O139-NAGVc) yet there is a sustained paucity of research interest on the non-agglutinable V. cholerae members. The emergence of fulminating extraintestinal and systemic vibriosis is another aspect of SANAS-1/139- Vc implication which has received low attention in terms of research driven interest. This review addresses the need to appraise and continually expand research based studies on the somatic antigen non-serogroup agglutinable type-1/139 V.cholerae members which are currently prevalent in studies of water bodies, fruits/vegetables, foods and terrestrial environment. Our opinion is amassed from interest in integrated surveillance studies, management/control of cholera outbreaks as well as diarrhea and other disease-related cases both in the rural, suburban and urban metropolis.
... The two serogroups have very high homology, including for cholera toxin. The primary difference between the two serogroups is in the rfb genes encoding the O-specific polysaccharide and the presence of a capsule in V. cholerae O139 that is absent from V. cholerae O1 (16,23,24). The OSP of V. cholerae O1 is a repetitive structure containing approximately 10 to 20 units of (1!2)-a-linked (4-N-3-deoxy-L-glycero-tetronyl)-perosamine, with or without a methyl group at O-2 on the terminal perosamine, distinguishing the Ogawa and Inaba serotypes, respectively. ...
Cholera is a severe dehydrating illness of humans caused by Vibrio cholerae serogroup O1 or O139. Protection against cholera is serogroup specific, and serogroup specificity is defined by O-specific polysaccharide (OSP).
... The O-PS gene cluster from O1 consists of five regions whose genes encode perosamine biosynthesis, O-antigen transport, tetronate biosynthesis, O-antigen modification, and some additionally required genes [13]. Gene cluster comparisons suggest that O139 probably resulted from a precise 22-kb deletion of the wbe region of O1, with replacement by a 35-kb wbf region (wbfA through wbfX) encoding the O139 O-antigen [14], possibly involving a homologous recombination event [15]. ...
Background
Of the hundreds of Vibrio cholerae serogroups, O1 and O139 are the main epidemic-causing ones. Although non-O1/non-O139 serogroups rarely cause epidemics, the possibility exists for strains within them to have pathogenic potential.
Results
We selected 25 representative strains within 16 V. cholerae serogroups and examined their genomic and functional characteristics. We tentatively constructed a gene pool containing 405 homologous gene clusters, which is well organized and functions in O-antigen polysaccharide (O-PS) synthesis. Our network analysis indicate that great diversity exists in O-PS among the serogroups, and several serogroup pairs share a high number of homologous genes (e.g., O115 and O37; O170 and O139; O12 and O39). The phylogenetic analysis results suggest that a close relationship exists between serogroups O170, O89 and O144, based on neighbor-joining (NJ) and gene trees, although serogroup O159 showed an inconsistent phylogenetic relationship between the NJ tree and the gene tree, indicating that it may have undergone extensive recombination and horizontal gene transfer. Different phylogenetic structures were observed between the core genes, pan genes, and O-PS genes. The virulence gene analysis indicated that the virulence genes from all the representative strains may have their sources from four particular bacteria (Pseudomonas aeruginosa, V. vulnificus, Haemophilus somnus and H. influenzae), which suggests that V. cholerae may have exchanged virulence genes with other bacterial genera or species in certain environments. The mobile genetic element analysis indicated that O159 carries nearly complete VSP-II and partial VPI-1 and VPI-2, O170 carries partial VPI-1 and VPI-2, and several non-O1/non-O139 strains contain full or partial VPI-1 and VPI-2. Several genes showing evidence of positive selection are involved in chemotaxis, Na + resistance, or cell wall synthesis, suggestive of environmental adaptation.
Conclusions
This study reports on the newly sequenced O159 and O170 genomes and their comparisons with other V. cholerae serogroups. The complicated O-PS network of constituent genes highlights the detailed recombination mechanisms that have acted on the serogroups’ genomes. The serogroups have different virulence-related gene profiles, and there is evidence of positive selection acting on other genes, possibly during adaptation to different environments and hosts.
Electronic supplementary material
The online version of this article (10.1186/s12864-019-5603-7) contains supplementary material, which is available to authorized users.
... It was concluded that transition from sixth to seventh cholera pandemic resulted in a change from V. cholerae O1 classical to O1 El Tor biotype (8). Several evidences suggest that O139 is closely related to and is deriveed from the El Tor biotype of V. cholerae O1 by the replacement of genes encoding the O139 antigen and acquisition of the ability to produce a capsule (9)(10)(11)(12)(13). ...
... Vibrio cholerae O139 Bengal was recognized as a second V. cholerae serotype capable of causing epidemic cholera in 1992, after emerging in India, and rapidly spreading across multiple Asian countries [1,2]. Genetic characterization of V. cholerae O139 isolates revealed that they were identical to V. cholerae O1 El Tor isolates, except for the substitution of a 35-kb region of DNA encoding the O139 surface polysaccharide for the 22-kb rfb region in O1 isolates [3]. Thus, V. cholerae O139 carried the same virulence factors found in O1, including the CTX prophage, toxin co-regulated pilus A (tcpA) and Vibrio seventh pandemic island I (VSP-I) and VSP-II [4]. ...
Vibrio cholerae
O139 emerged as a causative agent of epidemic cholera in 1992 in India and Bangladesh, and was subsequently reported in China in 1993. The genetic relatedness and molecular characteristics of
V. cholerae
O139 in Guangdong Province, located in the southern coastal area of China, remains undetermined. In this study, we investigated 136 clinical
V. cholerae
O139 isolates from 1993 to 2013 in Guangdong. By conventional PCR, 123 (90·4%) isolates were positive for
ctxB, ace
and
zot
. Sequencing of the positive amplicons indicated 113 (91·7%) isolates possessed the El Tor allele of
ctxB
(genotype 3); seven carried the classical
ctxB
type (genotype 1) and three harboured a novel
ctxB
type (genotype 5). With respect to
tcpA
, 123 (90·4%) isolates were positive for the El Tor allele. In addition, pulsed-field gel electrophoresis (with
Not
I digestion) differentiated the isolates into clusters A and B. Cluster A contained seven of the non-toxigenic isolates from 1998 to 2000; another six non-toxigenic isolates (from 1998 and 2007) and all of the toxigenic isolates formed cluster B. Our results suggest that over a 20-year period, the predominant O139 clinical isolates have maintained a relatively tight clonal structure, although some genetic variance and shift has occurred. Our data highlight the persistence of toxigenic
V. cholerae
O139 in clinical settings in the southern coastal area of China.
... The sxt element harbors several antibiotic resistance genes that provide selective advantages to the host and is present in almost all post-1990 V. cholerae O1 clinical isolates (Waldor et al., 1996). The third selective sweep is considered to have been triggered by replacement of V. cholerae O1 serogroup encoding genes with those for O139, resulting in an outbreak of cholera across the Indian subcontinent (Bik et al., 1995;Comstock et al., 1996;Mooi and Bik, 1997). The fourth, or most recent selective sweep, is proposed to have been initiated by replacement of the El Tor ctx allele with the classical ctx allele in an El Tor background (Raychoudhuri et al., 2009), presumed to be associated with the acute form of cholera (Nair et al., 2002). ...
Vibrio cholerae, the etiological agent of cholera, has been a scourge for centuries. Cholera remains a serious health threat for developing countries and has been responsible for millions of deaths globally over the past 200 years. Identification of V. cholerae has been accomplished using a variety of methods, ranging from phenotypic strategies to DNA based molecular typing and currently whole genomic approaches. This array of methods has been adopted in epidemiological investigations, either singly or in the aggregate, and more recently for evolutionary analyses of V. cholerae. Because the new technologies have been developed at an ever increasing pace, this review of the range of fingerprinting strategies, their relative advantages and limitations, and cholera case studies was undertaken. The task was challenging, considering the vast amount of the information available. To assist the study, key references representative of several areas of research are provided with the intent to provide readers with a comprehensive view of recent advances in the molecular epidemiology of V. cholerae. Suggestions for ways to obviate many of the current limitations of typing techniques are also provided. In summary, a comparative report has been prepared that includes the range from traditional typing to whole genomic strategies.
... The O139 serogroup strains of V. cholerae are postulated to derive from a serogroup O1 biotype El Tor strain by a genetic re-arrangement event including the acquisition of the 35 kb wbf locus which encodes the O139 surface polysaccharide and capsule biosynhesis genes in place of a 22 kb deletion at the rfb locus (38,95). The insertion of the acquired locus partially replaced the rfb region which encodes the O1 serogroup antigen (38). ...
... It has been suggested that the new epidemic strain V. cholerae O139 emerged from the pandemic O1 biotype El Tor through a genetic rearrangement, involving the horizontal transfer of genes encoding enzymes involved in O-specific polysaccharide (O-SP) biosynthesis. [2][3][4] Indeed, two important differences exist between V. cholerae O1 and V. cholerae O139 Bengal strains, both reside in their cell surface components. Manning et al. (1993) 5) have demonstrated that V. cholerae O139 possesses a truncated LPS O-side chain, which is un-reactive with O1-specific antiserum. ...
A glycoconjugate construct was based on attachment of V. cholerae O139 hydrazine-treated lipopolysaccharide (LPS) to carboxylated bovine serum albumin (CBSA) via its amino group. The immunological properties of the glycoconjugate were tested using BALB/c mice, injected subcutaneously without any adjuvant three times at 2 weeks interval. The immunogenicity of the conjugate was estimated by enzyme-linked immunosorbent assay, testing of anti-LPS IgG, IgM, and IgA antibodies. The conjugate elicited a statistically significant increase of LPS-specific IgG levels in mice (p < 0.001). The specific anti-LPS IgG and IgA response after the second booster dose was significantly higher compared with reference and unconjugated detoxified LPS response. Antibodies elicited by the dLPS-CBSA conjugate were vibriocidal.
... The typing of strains of V. cholerae is important for an exact diagnosis and subsequent treatment. Molecular methods for gene-typing of organisms including bacteria are widely used in last two decades and they have many advantages (8,13,20,21,23,24,26,30,31). Vital microorganisms are not needed and very small amounts of DNA is enough for application of amplification techniques such PCR (Polymerase Chain Reaction). ...
Cholera is an epidemic life threatening disease caused by V. cholerae. The main symptoms of this acute disease are vomiting, profuse watery diarrhea, and severe dehydration. The infection is transmitted by fecal-oral mechanism, food consumption and V. cholerae contaminated water (10,15). Often outbreaks are related to wars or natural disasters like hurricanes and floods when water and food sources become contaminated with V. cholerae in areas with bad living conditions and poor sanitation. Although cholera is a rare disease eradicated in developed countries it is still common in some parts of the world like Latin America, Africa, India and Asia. Its potential to cause large outbreaks and pandemics with lethal exit creates the need of a better epidemic prevention and an early response (25, 27). In the last decades the cholera is not common disease in our country but the increasing number of refugees from the Middle East and Africa makes it potential threat for Bulgarian population and requests the health authorities to react in case of possible outbreak.
... However, there are important differences between the O139 and O1 strains (Stroeher, 1995). The major differences between the two serogroups are that O139 Bengal contains a distinct O antigen and produces a polysaccharide capsule (Comstock et al., 1996). Bik et al. (1995) suggested that O139 arose from a strain closely related to the V. cholerae O1 El Tor by acquisition of novel DNA which was inserted into, and replaced part of, the O antigen gene cluster of the recipient strain. ...
... Among the mobile elements are pathogenic islands (described below) and the serotype: although often thought as a phylogenetic marker and by definition not subject to lateral gene transfer, the first evidence that it might be mobile came from the discovery of the O139 morph of the O1-O139 lineage. It has been shown by three groups that the O1 encoding genes were replaced by O139 encoding genes (Bik, Bunschoten et al. 1995;Comstock, Johnson et al. 1996;Mooi and Bik 1997). Subsequently, the O1 encoding genes were shown to transfer between SNP-defined lineages, and furthermore the "jump start" sequence (Hobbs and Reeves 1994) was identified as the junction point and shown to be similar to a DNA uptake sequence leading to the suggestion that serotype genes are mechanistically prone to being mobile (Gonzalez-Fraga, Pichel et al. 2008 Salim et al. 2013). ...
Cholera Cholera is still a major public health problem. The underlying bacterial pathogen Vibrio cholerae Vibrio cholerae (V. cholerae) is evolving and some of its mutations have set the stage for outbreaks outbreaks . After V. cholerae acquired the mobile elements VSP I & II, the El Tor pandemic began and spread across the tropics. The replacement of the O1 serotype encoding genes with the O139 encoding genes triggered an outbreak that swept across the Indian subcontinent. The sxt element generated a third selective sweep and most recently a fourth sweep was associated with the exchange of the El Tor ctx allele for a classical ctx allele in the El Tor background. In Kenya, variants of this fourth selective sweep have differentiated and become endemic residing in and emerging from environmental reservoirs. On a local level, studies in Bangladesh have revealed that outbreaks may arise from a nonrandom subset of the genetic lineages in the environment and as the population of the pathogen expands, many novel mutations may be found increasing the amount of genetic variation genetic variation , a phenomenon known as a founder flush. In Haiti, after the initial invasion and expansion of V. cholerae in 2010, a second outbreak occurred in the winter of 2011-2012 driven by natural selection of specific mutations.
... Genetic changes in this region are correlated with specific somatic antigens which are serologically different. The serogroup O139 resulted from a 22 kb deletion of the rfb region of an O1 El Tor strain, with replacement by a 35 kb wbf region encoding the O139 specific O antigen [15]. ...
Vibrio cholerae serogroup O1 has two major serotypes, Ogawa and Inaba, which may alternate among cholera epidemics. The rfbT gene is responsible for the conversion between the two serotypes. In this study, we surveyed the sequence variance of rfbT in the Ogawa and Inaba strains in China over a 48-year (1961-2008) period in which serotype shifts occurred among epidemic years.
Various mutation events including single nucleotide, short fragment insertions/deletions and transposases insertions, were found in the rfbT gene of the Inaba strains. Ectopically introducing an intact rfbT could overcome the mutations by converting the Inaba serotype to the Ogawa serotype, suggesting the effects of these mutations on the function of RfbT. Characteristic rfbT mutations were recognized in the Inaba strains among Inaba serotype dominant epidemic years which were separate from the Ogawa dominant epidemics. Three distinguishable mutation sites in rfbT between the classical and the El Tor biotype strains were identified and could serve as biotype-specific biomarkers.
Our results provide a comprehensive picture of the rfbT gene mutations among the V. cholerae O1 strains in different epidemic periods, which could be further used as the tracing markers in clonality analysis and dissemination surveillance of the epidemic strains.
... The spread of CT genes in the environment can be facilitated by the exposure of CTXФ positive strains to sunlight (Faruque et al. 2000a). Genetic and phenotypic evidence strongly suggests that the O139 strain arose from a V. cholerae O1 strain by horizontal gene transfer (Bik et al. 1995;Comstock et al. 1996;Waldor and Mekalanos 1994;Faruque et al. 2000b). Similarly, V. cholerae non-O1/ non-O139 strains can also acquire toxigenic genes for toxin production by transduction and therefore might be the source of new epidemics. ...
Water and post-larvae samples from black tiger (Penaeus monodon) shrimp hatcheries; pond water, pond sediment and shrimp from aquaculture farms were screened for the presence of V. cholerae. A V. cholerae-duplex PCR method was developed by utilizing V. cholerae species specific sodB primers and ctxAB genes specific primers. Incidence of V. cholerae was not observed in shrimp hatchery samples but was noticed in aquaculture samples. The incidence of V. cholerae was higher in pond water (7.6%) than in pond sediment (5.2%). Shrimp head (3.6%) portion had relatively higher incidence
than shrimp muscle (1.6%). All the V. cholerae isolates (n = 42) belonged to non-O1/non-O139 serogroup, of which 7% of the V. cholerae isolates were potentially cholera-toxigenic (ctx positive). All the ctx positive V. cholerae (n = 3) were isolated from the pond water. Since, cholera toxin (CT) is the major contributing factor for cholera gravis, it is proposed that the mere presence of non-O1/non-O139 V. cholerae need not be the biohazard criterion in cultured black tiger shrimp but only the presence of ctx carrying non-O1/non-O139 V. cholerae may be considered as potential public health risk.
... Analysis of the DNA sequence extending from the transposon insertions that affected resistance to PM in pJG02 showed the insertions to be located in pagA (now called pmrE ), a previously identified acid-regulated PhoP-PhoQ and PmrA-PmrB-activated gene predicted to be a UDP glucose dehydrogenase (Miller et al., 1989;Soncini and Groisman, 1996;Valdivia and Falkow, 1996). UDP glucose dehydrogenases in other organisms such as Streptococcus pneumoniae, Vibrio cholerae and Shigella flexneri are involved in capsule or complex carbohydrate synthesis (Morona et al., 1994;Dillard et al., 1995;Comstock et al., 1996). Mass spectrometry of a S. typhimurium pmrE mutant showed that this strain was unable to add aminoarabinose to LPS, and that the inability to add this modification could be complemented with the plasmid carrying the wild-type gene. ...
Antimicrobial peptides are distributed throughout the animal kingdom and are a key component of innate immunity. Salmonella typhimurium regulates mechanisms of resistance to cationic antimicrobial peptides through the two-component systems PhoP–PhoQ and PmrA–PmrB. Polymyxin resistance is encoded by the PmrA–PmrB regulon, whose products modify the lipopolysaccharide (LPS) core and lipid A regions with ethanolamine and add aminoarabinose to the 4′ phosphate of lipid A. Two PmrA–PmrB-regulated S. typhimurium loci (pmrE and pmrF ) have been identified that are necessary for resistance to polymyxin and for the addition of aminoarabinose to lipid A. One locus, pmrE, contains a single gene previously identified as pagA (or ugd ) that is predicted to encode a UDP-glucose dehydrogenase. The second locus, pmrF, is the second gene of a putative operon predicted to encode seven proteins, some with similarity to glycosyltransferases and other complex carbohydrate biosynthetic enzymes. Genes immediately flanking this putative operon are also regulated by PmrA–PmrB and/or have been associated with S. typhimurium polymyxin resistance. This work represents the first identification of non-regulatory genes necessary for modification of lipid A and subsequent antimicrobial peptide resistance, and provides support for the hypothesis that lipid A aminoarabinose modification promotes resistance to cationic antimicrobial peptides.
... The O139 serogroup remains present in India and Bangladesh since last outbreak in 1996 and requires careful monitoring. It has been suggested that the emergence of V. cholerae O139 is the result of a complex chromosomal rearrangement involving the horizontal transfer of genes encoding enzymes involved in O-specific polysaccharide biosynthesis (Comstock et al. 1996;Mukhopadhyay et al. 1998). However, choleralike diseases are also caused by other non-O1 serotypes (Janda et al. 1988; Kerketta et al. 2002). ...
O135 serotype Vibrio cholerae isolated from Slovak river was used as a source of surface polysaccharide antigens. Following detoxification procedure, fractions of polysaccharides were separated by size exclusion chromatography. Two resultant fractions were the capsular polysaccharide (M
w ∼ 197,000 Da) and the lipopolysaccharide fragment (M
w ∼ 13,300 Da). These materials were used for preparation of four novel glycoconjugates. Two of them containing detoxified lipopolysaccharide as antigen were prepared by original chemical method using the new biocompatible polymer as carrier of antigen. Additionally, other two conjugates were prepared by direct linking of capsular and detoxified lipopolysaccharide antigens to the protein carrier using adipic acid dihydrazide spacer. The immunogenicities (induced IgM, IgG, IgA antibodies) of all conjugates were determined by enzyme-linked immunosorbent assay. Polymer containing conjugates elicited higher levels of specific anti-lipopolysaccharide IgM and IgG antibodies in comparison with other conjugates without polymer carrier. Enhanced IgM vibriocidal activity of mice antisera was also evident here.
This book, originally published in 2004, is concerned with the links between human evolution and infectious disease. It has long been recognised that an important factor in human evolution has been the struggle against infectious disease and, more recently, it was revealed that complex genetic polymorphisms are the direct result of that struggle. As molecular biological techniques become more sophisticated, a number of breakthroughs in the area of host-pathogen evolution led to an increased interest in this field. From the historical beginnings of J. B. S. Haldane's original hypothesis to more recent research, this book strives to evaluate infectious diseases from an evolutionary perspective. It provides a survey of information regarding host-pathogen evolution related to major infectious diseases and parasitic infections, including malaria, influenza and leishmaniasis. Written by leading authorities in the field, and edited by a former pupil of Haldane, Infectious Disease and Host-Pathogen Evolution will be valuable for those working in related areas of microbiology, parasitology, immunology and infectious disease medicine, as well as genetics, evolutionary biology and epidemiology.
The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374–10378, 1995) and designated IS 1358 O139 . We studied the distribution of the IS 1358 element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS 1358 was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS 1358 from V. cholerae O22 and one copy from V. cholerae O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS 1358 to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 × 10 ⁻⁸ . Sequence analysis revealed that IS 1358 duplicates 10 bp at its insertion site.
In the US and Canada in August–December 2014 there was an epidemic of enterovirus infection caused
by EV-D68, with the prevalence of clinical forms of severe respiratory disease (1374 cases, including 17
deaths). In 93 patients, the disease was accompanied by the development of acute flaccid paralysis. The
etiological role of EV-D68 in their development continues to be studied. Outbreaks have occurred in European
countries (France, UK, Netherlands, Norway). This indicates a growing epidemic and pathogenic potential
EV-D68, previously caused only sporadic cases of respiratory disease, and suggests the EV-D68 as reemergent exciter and requires the implementation of proper monitoring viral circulation EV-D68.
Key words: D enterovirus type 68, severe acute respiratory disease, acute flaccid paralysis, epidemic
spread.
Three pentasaccharides, two tetrasaccharides, and a trisaccharide fragment of the O-specific antigen of Vibrio cholerae O139 were synthesized by applying 1 + 1, 2 + 1, 3 + 1, and 4 + 1 coupling strategies. The most challenging tasks involved were the synthesis of the 1,2-cis-glycosidic linkage between galactose and the linker (spacer) molecule and final purification of the target multicharged substances. Difficulties with final deprotection by hydrogenation/hydrogenolysis caused by the presence of galacturonic acid were overcome by protecting the acid with a group inert to the treatment with hydrogen. Some intermediates described previously as incompletely characterized amorphous materials were obtained in the crystalline condition and were fully characterized for the first time.
Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host, pill are post-translationally modified by addition of an O-linked trisaccharide, Gal(beta 1-4)Gal(alpha 1-3)2,4-diacetimido-2,4,6-trideoxyhexose. In a previous study the authors identified and characterized a gene, pglA, encoding a galactosyltransferase involved in pilin glycosylation. In this study a set of random genomic sequences from N. meningitidis strain MC58 was used to search for further genes involved in pilin glycosylation. Initially, an open reading frame was identified, and designated pglD (pilin glycosylation gene D), which was homologous to genes involved in polysaccharide biosynthesis, The region adjacent to this gene was cloned and nucleotide sequence analysis revealed two further genes, pglB and pglC, which were also homologous with genes involved in polysaccharide biosynthesis, Insertional mutations were constructed in pglB, pglC and pglD in N, meningitidis C311#3, a strain with well-defined LPS and pilin-linked glycan structures, to determine whether these genes had a role in the biosynthesis of either of these molecules. Analysis of these mutants revealed that there was no alteration in the phenotype of LPS in any of the mutant strains as judged by SDS-PAGE gel migration. In contrast, increased gel migration of the pilin subunit molecules of pglB, pglC and pglD mutants by Western analysis was observed, Pilin from each of the pglB, pglC and pglD mutants did not react with a terminal-galactose-specific stain, confirming that the gel migration differences were due to the alteration or absence of the pilin-linked trisaccharide structure in these mutants. In addition, antisera specific for the C311#3 trisaccharide failed to react with pilin from the pglB, pglC, pglD and galE mutants. Analysis of nucleotide sequence homologies has suggested specific roles for pglB, pglC and pglD in the biosynthesis of the 2,4-diacetimido-2,4,6-trideoxyhexose structure.
Vib' ri.o . L. v. vibrio move rapidly back and forth, vibrate; M.L. masc. n. Vibrio the vibrating, darting organism.
Proteobacteria / Gammaproteobacteria / “Vibrionales” / Vibrionaceae / Vibrio
Small, straight, slightly curved, curved, or comma‐shaped rods , 0.5–0.8 × 1.4–2.6 µm. Involution forms often occur in old cultures and are formed under adverse growth conditions. Do not form endospores or microcysts. Gram negative. In liquid media, motile by monotrichous or multitrichous polar flagella enclosed in a sheath continuous with the outer membrane of the cell wall. On solid media, some species synthesize numerous lateral flagella with a wavelength shorter than that of the sheathed polar flagellum. Facultative anaerobes capable of both fermentative and respiratory metabolism . Molecular oxygen is a universal electron acceptor. Most do not denitrify or fix molecular nitrogen. All are chemoorganotrophs; most are able to grow in a mineral medium containing D ‐glucose as the sole carbon source and NH ⁺ as the sole nitrogen source . A few strains have organic growth factor requirements. Na 4 ⁺ stimulates growth of all species and is an absolute requirement for most ; the minimal concentration necessary for optimal growth ranges from 5 to 700 mM (0.029–4.1%). Most species grow well in media containing a seawater base. All ferment D ‐glucose producing acid but rarely gas ; several species produce acetoin and acetyl methyl carbinol ( positive Voges–Proskauer test ). Most ferment and utilize D ‐fructose, maltose, and glycerol; are oxidase positive and reduce nitrate to nitrite . Several grow at 4°C; all grow at 20°C; most grow at 30°C; many grow at 35–37°C. A few species are bioluminescent , as are a few strains of normally nonluminescent species. Primarily aquatic ; species distribution is usually dependent on Na ⁺ and nutrient content of the water as well as its temperature. Very common in marine and estuarine environments and on the surfaces and in the intestinal contents of marine animals. Species with a low Na ⁺ requirement are also found in freshwater habitats. Twelve species occur in human clinical specimens; 11 of these are apparently pathogenic for humans, causing diarrhea or extraintestinal infections. Several species cause diseases of other vertebrates and invertebrates .
The mol % G + C of the DNA is : 38–51.
Type species : Vibrio cholerae Pacini 1854, 411.
Vibrio cholerae and V. anguillarum are recognized as aquatic-borne human and fish pathogens, respectively. Based upon analyses of several genes and the presence of novel genetic elements it seems that these two species are very closely related. Studies in this laboratory have identified an association of IS1358 with rfb and capsule loci in these two species. The most recent findings suggest that IS1358 is associated with the rfb region in V. cholerae O1 and O139 and in V. anguillarum O1 and O2. In addition, the rfb region in both V. cholerae serogroups and in V. anguillarum O1 is limited at one end by gmhD. These features make it feasible to envisage a mechanism by which the evolution of new rfb genes is taking place involving IS1358 and the region around gmhD. Furthermore, it is possible to envisage that there is or has been an exchange of genetic material between these species leading to new rfb/capsule regions. This review examines the genetics and biosynthesis of the O-antigen and capsule of V. cholerae O1 and O139, as well as the V. anguillarum serogroup O1 and the role of IS1358. Throughout this review we have used the new nomenclature for rfb genes proposed by Reeves et al. (1996).
This chapter discusses the mobile genetic elements and the evolution of new epidemic strains of Vibrio cholera. All epidemic strains of V. cholerae produce cholera toxin. The toxin, which consists of five copies of the B protein bound to a single copy of the A protein, is encoded by the genes, ctxA and ctxB. The B subunits bind to the surface of target cells and facilitate the entry of the A subunit into the cytoplasm. The A subunit catalyzes the ADP-ribosylation of the eukaryotic protein resulting in marked increases in cellular cyclic AMP levels. The ctxAB genes lie within a region on the V. cholerae chromosome, which is referred to as the “CTX element.” Adjacent to the structural genes for cholera toxin are two genes— ace and zot—which had been postulated to encode proteins with entertoxic activity. Many chromosomal elements contain two different RS elements, which are tandemly arranged and are not identical. Bacteriophage genomes that have been studied to date all contain the RS element, which is adjacent to the core genes present in the bacterial chromosome. It is found that RS1, which does not appear to be incorporated into the phage particle, contains rstR, rstA, and rstB genes that are quite similar to those in RS2 and an additional open reading frame, rstC.
Vibrio cholerae and V. anguillarum are recognized as aquatic-borne human and fish pathogens, respectively. Based upon analyses of several genes and the presence of novel genetic elements it seems that these two species are very closely related. Studies in this laboratory have identified an association of IS1358 with rfb and capsule loci in these two species. The most recent findings suggest that IS1358 is associated with the rfb region in V. cholerae O1 and O139 and in V. anguillarum O1 and O2. In addition, the rfb region in both V. cholerae serogroups and in V. anguillarum O1 is limited at one end by gmhD. These features make it feasible to envisage a mechanism by which the evolution of new rfb genes is taking place involving IS1358 and the region around gmhD. Furthermore, it is possible to envisage that there is or has been an exchange of genetic material between these species leading to new rfb/capsule regions. This review examines the genetics and biosynthesis of the O-antigen and capsule of V. cholerae O1 and O139, as well as the V. anguillarum serogroup O1 and the role of IS1358. Throughout this review we have used the new nomenclature for rfb genes proposed by Reeves et al. (1996).
A second aetiological agent of cholera, Vibrio cholerae O139 Bengal was identified in late 1992 when it caused large outbreaks of diarrhoea in India and Bangladesh. The new strain probably arose as a result of lateral transfer of genes encoding a novel somatic antigen and a capsule from an unknown bacterium to an O1 El Tor strain with the subsequent loss of genes encoding O1 somatic antigen. O139 produces a semi-rough type colony with a truncated lipopolysaccharide which contains a unique sugar, colitose. Otherwise, O139 and O1 are strikingly similar. The diseases produced by the two serogroups are indistinguishable. There is a moderate inflammatory response in cholera, and in spite of the possession of a capsule by O139, it is no more inflammatory than O1. A number of diagnostic tests have been developed for O139 that are modelled after tests for O1 including rapid tests for field use. O139 has spread to countries of south and south-east Asia, China and Russia. Currently, cholera is caused by both these serogroups in these countries with the O1 serogroup predominant. O139 continues to cause occasional local outbreaks in India and Bangladesh. Genotyping of isolates has shown circulation of multiple clones and their derivation from multiple progenitors. Although there have been changes in antibiogram with the resistance genes being carried on a constin, the strains remain susceptible to tetracycline, a preferred antibiotic for treatment. O139 carries a number of filamentous lysogenic and lytic phages and the latter have been utilized to develop a phage typing scheme. A number of environmental variables have been linked to the occurrence of cholera and lytic phages may determine the course of epidemics. Although the capsule confers serum resistance, vibriocidal antibody assay has been developed for O139. However, unlike in O1 cholera, serum vibriocidal antibody does not seem to correlate with protection against O139 infection. Promising O139 vaccines have been developed or are in the pipeline. It is too early to predict whether O139 will become the causative agent of 'the eighth pandemic of cholera'; it will require many years of monitoring and reporting from regions prone to cholera to find the answer.
The fragments described contain a galactose residue with cyclic 4,6-phosphate and are equipped with an amino-functionalized spacer, allowing further derivatization or directconjugation to suitable carriers. The core Gal-(1→3)-GlcNAc disaccharide was obtained by condensation of 8-azido-3,6-dioxaoctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside with 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide under Helferich conditions. Reductive opening of the benzylidene acetal, followed by deacetylation and selective benzylation gave 8-azido-3,6-dioxaoctyl 2-acetamido-6-O-benzyl-3-O-(3-O-benzyl-β-D-galactopyranosyl)-2-deoxy-β-D-glucopyranoside, which was regioselectively phosphorylated to give two isomeric 4,6-cyclic 2,2,2-trichloroethyl phosphates. The (R)-phosphate was subjected to catalytic hydrogenation/hydrogenolysis to give the fully deprotected title disaccharide fragment. Glycosylation of the (S)-phosphate diol with 2,4-di-O-benzyl-3,6-dideoxy-α-L-xylo-hexopyranosyl bromide under halide-assisted conditions yielded a mixture of the tetrasaccharide and a trisaccharide, which were readily separable by chromatography. Their hydrogenation/hydrogenolysis effected global deprotection as well as reduction of the azide, to furnish the deprotected title tri- and tetrasaccharide.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)
The title disaccharide, 2-{2-{2-[(2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino]ethoxy}ethoxy}ethyl 2-O-(3,6-dideoxy--L-xylo-hexopyranosyl)-β-d-galactopyranoside cyclic 4,6-(potassium phosphate) (2), was synthesized from the two isomeric linker-equipped galactose acceptors 9 and 10, obtained by phosphorylation of 2-[2-(2-azidoethoxy)ethoxy]ethyl 3-O-benzyl-β-d-galactopyranoside (8), which were glycosylated with ethyl 2,4-di-O-benzyl-3,6-dideoxy-1-thio-β-l-xylo-hexopyranoside (12; Scheme). Mainly the fully protected -(1 → 2)-linked products 13 and 14 were formed. Catalytic hydrogenolysis/hydrogenation effected global deprotection, thereby removing the chirality at the P-atom, and simultaneously converted the azido group in the linker to an amino group (→ 15). Final treatment with diethyl squarate (= 3,4-diethoxycyclobut-3-ene-1,2-dione) gave target compound 2, amenable for conjugation to proteins.
Cholera clearly knows well the role of the grim reaper. It is one of the first diseases most people think of when calamity strikes and civilization breaks down. We have seen this happen several times in the last decade, with the most dramatic example being the cholera deaths of 80 000 people in the Rwandan refugee camps of Goma, Zaire, in 1994.
While cholera as a disease is probably not as old as others like tuberculosis or smallpox, it clearly has been around for at least a thousand years. Although there are many diarrheal diseases, the intensity of cholera and the explosiveness of cholera epidemics clearly is unmistakable and must have impressed ancient observers. For example, there are descriptions that could only be cholera in ancient Greek and Chinese literature. As noted by Pollitzer, the translation by Schmidt of a Sanskrit work whose origin is believed to be Tibet around 802 AD clearly suggests cholera:
“When the strength of virtues and merits decreases on earth, there appears amongst the people, first among those living on the shores of big rivers, various ailments which gives no time for treatment, but prove fatal immediately after they appear.… The various vessels secrete water so that the body becomes empty.… Its first signs are dizziness, a numb feeling in the head, and then the most violent purging and vomiting (Trans.)” [1].
Pollitzer also noted that in 1543 the explorer Gaspar Correa described a cholera victim in an outbreak in India in which the fatality rate exceeded the survivors' ability to bury the dead:
“So grievous was the throe, and so bad a sort that the very worst poison seemed to take effect, as proved by vomiting, with drought of water accompanying it, as if the stomach were parched up, and cramps that fixed …
Analysis of the nucleotide sequence of a 1592 bp region of Acetobacter xylinum genomic DNA involved in acetan biosynthesis
revealed the presence of an open-reading frame (aceM) encoding a protein of 449 amino acids with a molecular weight of 48.5
kDa. The deduced amino acid sequence of aceM displayed high homology to the protein sequences of genes encoding UDP-glucose
dehydrogenase (UGDH) activities from other organisms. AceM is likely to encode the UGDH involved in the biosynthesis of UDP-glucuronic
acid required for acetanbiosynthesis.
Vibrio cholerae elaborates three types of polysaccharide structures: lipopolysaccharide (LPS), a component of which is the O-polysaccharide or O-antigen, capsular polysaccharide (CPS) or K-antigen, and “rugose” polysaccharide also known as exopolysaccharide (EPS) or Vibrio polysaccharide (VPS). The major protective antigen for V. cholerae is the O-antigen. A strain typing scheme based on the somatic O-antigen has been in use for a number of years. There are 206 serogroups identified so far and of these only O1 and O139 are known to cause epidemic/pandemic cholera, although a handful of non-O1/non-O139 strains are known to possess the major virulence factors. The O-antigen diversity is due to the number and composition of monosaccharide components, linkages, addition of non-sugar moieties, modal length of the polysaccharide chain, and biosynthesis mechanisms. The genetic basis of this diversity is just beginning to be understood with the sequencing of a number of gene clusters that encode O-polysaccharide (OPS)/capsule structures. In this review, we summarize our current knowledge on the biochemical composition and structure of some of the O-polysaccharides, genes involved in their biosynthesis, and touch upon the role of horizontal gene transfer in creating this diversity and possible mechanisms that may be operative in this process. We highlight the fact that the distinction between OPS and CPS seems to be less evident in V. cholerae than in other species since the genes encoding these structures are shared and map in the same region of the genome. We also describe our current understanding of the genetics and regulation of EPS/VPS synthesis and its role in biofilm formation and environmental survival of V. cholerae.
Vibrio cholerae and campylobacters are recognized as important water-borne and food-borne pathogens, respectively, associated with acute
gastroenteritis. In this article, we describe the recent scenario of cholera on the basis of active surveillance conducted
at the National Institute of Cholera and Enteric diseases, Kolkata, India with special reference to a newly emerged V. cholerae O1 El Tor variant and the recent molecular epidemiologic investigation related with Campylobacter and related organisms in Japan, Thailand and the People’s Republic of China.
Several studies have shown that the emergence of the O139 serogroup of Vibrio cholerae is a result of horizontal gene transfer of a fragment of DNA from a serogroup other than O1 into the region responsible for O-antigen biosynthesis of the seventh pandemic V. cholerae O1 biotype El Tor strain. In this study, we show that the gene cluster responsible for O-antigen biosynthesis of the O139 serogroup of V. cholerae is closely related to those of O22. When DNA fragments derived from O139 O-antigen biosynthesis gene region were used as probes, the entire O139 O-antigen biosynthesis gene region could be divided into five classes, designated as I–V based on the reactivity pattern of the probes against reference strains of V. cholerae representing serogroups O1–O193. Class IV was specific to O139 serogroup, while classes I–III and class V were homologous to varying extents to some of the non-O1, non-O139 serogroups. Interestingly, the regions other than class IV were also conserved in the O22 serogroup. Long and accurate PCR was employed to determine if a simple deletion or substitution was involved to account for the difference in class IV between O139 and O22. A product of approx. 15 kb was amplified when O139 DNA was used as the template, while a product of approx. 12.5 kb was amplified when O22 DNA was used as the template, indicating that substitution but not deletion could account for the difference in the region between O22 and O139 serogroups. In order to precisely compare between the genes responsible for O-antigen biosynthesis of O139 and O22, the region responsible for O-antigen biosynthesis of O22 serogroup was cloned and analyzed. In concurrence with the results of the hybridization test, all regions were well conserved in O22 and O139 serogroups, although wbfA and the five or six genes comprising class IV in O22 and O139 serogroups, respectively, were exceptions. Again the genes in class IV in O22 were confirmed to be specific to O22 among the 155 ‘O’ serogroups of V. cholerae. These data suggest that the gene clusters responsible for O139 O-antigen biosynthesis are most similar to those of O22 and genes within class IV of O139, and O22 defines the unique O antigen of O139 or O22.
This report completes a preliminary analysis of the sequence of the 330,740-bp chlorella virus PBCV-1 genome, the largest virus genome to be sequenced to date. The PBCV-1 genome is 57% the size of the genome from the smallest self-replicating organism,Mycoplasma genitalium.Analysis of 74 kb of newly sequenced DNA, from the right terminus of the PBCV-1 genome, revealed 153 open reading frames (ORFs) of 65 codons or longer. Eighty-five of these ORFs, which are evenly distributed on both strands of the DNA, were considered major ORFs. Fifty-nine of the major ORFs were separated by less than 100 bp. The largest intergenic distance was 729 bp, which occurred between two ORFs located in the 2.2-kb inverted terminal repeat region of the PBCV-1 genome. Twenty-seven of the 85 major ORFs resemble proteins in databases, including the large subunit of ribonucleotide diphosphate reductase, ATP-dependent DNA ligase, type II DNA topoisomerase, a helicase, histidine decarboxylase, dCMP deaminase, dUTP pyrophosphatase, proliferating cell nuclear antigen, a transposase, fungal translation elongation factor 3 (EF-3), UDP glucose dehydrogenase, a protein kinase, and an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease. Seventeen of the 153 ORFs resembled other PBCV-1 ORFs, suggesting that they represent either gene duplications or gene families.
Vibrio cholerae O1 exists as two major serotypes, Inaba and Ogawa, which are associated with the O antigen of the lipopolysaccharide and are capable of unequal reciprocal interconversion. The 20-kilobase rfb regions encoding O-antigen biosynthesis in strains 569B (Inaba) and O17 (Ogawa) have been cloned in Escherichia coli K-12 and the nucleotide sequences have been determined. Besides several base substitutions and a small deletion in the 569B sequence relative to O17, there is a single nucleotide change resulting in a TGA stop codon within the gene for the 32-kDa RfbT protein. We have demonstrated that rfbT is responsible for serotype conversion (Inaba to Ogawa). The construction of a specific rfbT mutation in the Ogawa strain O17, and the ability of the gene from O17 to complement Inaba strains to Ogawa, confirmed rfbT as the gene required for the Ogawa serotype. By Southern hybridization and sequencing of PCR products of a number of strains, we have shown that the changes observed in one Inaba strain (569B) are not conserved in other Inaba strains. This may explain why some Inaba strains are able to convert to Ogawa whereas others are not. The protein encoded by rfbT has been identified and expressed in E. coli K-12 using a phage T7 expression system. Amino-terminal analysis of partially purified protein has identified the translational start of the protein. Primer extension studies have enabled the 5' end of the mRNA to be defined. It exists as a separate transcript from the rest of the rfb region, and the distinctive G + C content of rfbT suggests that it has been acquired from a non-Vibrio source.
Vibrio cholerae serogroup O139 emerged on the Indian subcontinent in October 1992 to become the first non-O1 V. cholerae serogroup documented to cause epidemic cholera. Although related to V. cholerae El Tor O1 strains, O139 strains have unique surface structures that include a capsular surface layer and lipopolysaccharide (LPS). Immunoblot analysis of either whole-cell lysates or LPS preparations revealed three electrophoretic forms of the O139 antigen: two slowly migrating forms and one rapidly migrating form that appeared identical to O139 LPS. All three forms of the antigen shared an epitope defined by an O139-specific monoclonal antibody. A serum-sensitive nonencapsulated mutant was isolated that lacks only the slow migrating forms. The slow migrating forms did not stain with silver whereas the rapidly migrating form did, suggesting that the former might constitute highly polymerized O-antigen side-chain molecules that were not covalently bound to core polysaccharide and lipid A (an "O-antigen capsule"). A single transposon insertion resulted in the loss of immunoreactivity of both the LPS and the O-antigen capsule, implying that there are genes common to the biosynthesis of both these macromolecules. The O139 LPS and O-antigen capsule were both important for colonization of the small intestine of the newborn mouse and for serum resistance, demonstrating that both of these forms of the O139 serogroup antigen are virulence factors.
The galE gene from Haemophilus influenzae was used as a hybridization probe for the galE gene of Neisseria meningitidis Group B, identifying two different homologous loci. Each of the loci was cloned and nucleotide sequence analysis revealed that both loci contained sequences similar to galE. One contained a functional galE gene and mapped to the capsule biosynthetic locus. The second contained only a partial galE-coding sequence, which did not express a functional gene product. A galE mutant meningococcal strain was constructed by transformation with an inactivated galE gene. Analysis of the LPS from the galE mutant strain revealed an apparent reduction in molecular weight and a loss of reactivity with monoclonal antibodies specific for structures known to contain galactose. These results are consistent with an essential role for galE in the incorporation of galactose into meningococcal lipopolysaccharide.
We showed previously that a mutant strain of group B Streptococcus (GBS) defective in capsule production was avirulent. This study describes the derivation of an unencapsulated mutant from a highly encapsulated wild-type strain of type III GBS, COH1, by transposon mutagenesis with Tn916ΔE. The mutant, COH1-13, was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat sepsis model compared with the wild-type strain. No capsular polysaccharide was evident in the cytoplasm or on the cell surface of the mutant strain. The Tn916ΔE insertion site in COH1-13 was mapped to the same chromosomal location as the Tn916 insertion site in the unencapsulated type III mutant COH31-15 reported previously. Nucleotide sequencing of DNA flanking the insertion site in COH1-13 revealed an open reading frame, designated cpsD, with significant homology to the rfbP gene of Salmonella typhimurium. RfbP encodes a galactosyl transferase enzyme that catalyses the transfer of galactose to undecaprenol phosphate, the initial step in O-polysaccharide synthesis. A particulate fraction of a lysate of wild-type strain GBS COH1 mediated the transfer of galactose from UDP-galactose to an endogenous acceptor. The galactose–acceptor complex partitioned into organic solvents, suggesting it is lipid in nature or membrane-associated. Galactosyl transferase activity was significantly reduced in the unencapsulated mutant strain COH1-13. These results, together with the similarity in deduced amino acid sequence between cpsD and rfbP suggest that cpsD encodes a galactosyl transferase essential for assembly of the GBS type III capsular polysaccharide.
The rfb gene cluster of Salmonella LT2 has been cloned and sequenced. The genes rfbA, rfbB, rfbD, rfbF, rfbG, rfbK, rfbM and rfbP were located individually and the gene rfbL was located outside the cluster. Approximately 16 open reading frames were found in the region which is essential for the expression of O antigen. The gene products of rfbB and rfbG were found to have homology with the group of dehydrogenase and related enzymes described previously. Analysis of the G + C ratio of the rfb cluster extended the area of low-G + C composition previously found in the sequence of rfbJ to the whole rfb gene cluster. Three to five segments with discrete G + C contents and codon adaptation indices are present in the rfb region, indicating a heterogeneous origin of these segments. Potential promoters were found near the start of the rfb region, supporting the possibility that the rfb gene cluster is an operon.
Zymovar analysis of 260 strains of Vibrio cholerae plus 3 reference strains of V. mimicus, using 13 structural loci, led to the grouping of strains in 73 zymovars (strain or group of strains sharing the same alleles). Effective separation of strains, distinction of V. cholerae strains from closely related V. mimicus and the detection of 2 vibrio strains, including one with two O1 serovars, in supposedly pure collection cultures, illustrate the potential of zymovar analysis in the identification of V. cholerae isolates. Two El Tor strains from USA, one CT+ and the other CT-, shared the same zymovar 71, while 127 typical El Tor strains belonged to zymovar 14.
Phosphomannose isomerase (PMI) has been proposed to catalyze the first step of the alginic acid biosynthetic pathway in Pseudomonas aeruginosa. The nucleotide sequence of the P. aeruginosa pmi gene contained on a 2.0-kb BamHI-SstI DNA fragment has been determined. The gene was defined by the start and stop codons and by in vitro disruption of an open reading frame of 1440 bp corresponding to a polypeptide product with a predicted Mr of 52 860. This polypeptide displayed an apparent Mr of approx. 56 000 upon electrophoresis of a maxicell extract on sodium dodecyl sulfate-polyacrylamide gels. The codon utilization of the pmi gene was distinct in the wobble base preference and influenced by the high G + C content (66 mol%) of the P. aeruginosa DNA. Computer assisted matching analysis failed to demonstrate any significant homology at the nucleotide level between the P. aeruginosa pmi and Escherichia coli manA (pmi) genes. However, sequences homologous to the P. aeruginosa pmi gene were found in other Pseudomonas species, such as P. putida and P. mendocina, and in Azotobacter vinelandii, all capable of producing alginic acid.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The production and use of the 6 kb cosmid pHC79, a derivative of pBR322, is described. It can be used for cloning of fragments cleaved by EcoRI, ClaI, BamHI (also BglII, BclI, Sau3A and MboI), SalI (also XhoI and AvaI), EcaI and PstI. Hybrid cosmids containing inserts in the size range of 40 kb are packaged in vitro and transduced with an efficiency of 5 X 10(4) - 5 X 10(5) clones/microgram of insert DNA. Prefractionation of the DNA fragments to be cloned into 40 kb sized fragments ensures the cloning of contiguous stretches of DNA. Proteins produced in vitro by the cosmid pHC79 are identical to the ones produced by its pBR322 parent.
Lipopolysaccharide (LPS) from Vibrio cholerae O139 Bengal contained colitose (3,6-dideoxy-L-galactose) in addition to glucose, L-glycero-D-manno-heptose, fructose, glucosamine and quinovosamine in its polysaccharide and only glucosamine in lipid A, while perosamine, a characteristic component sugar of V. cholerae O1 LPS, was absent. 3-Hydroxydodecanoic, tetradecanoic and hexadecanoic acids as ester-bound fatty acids and 3-hydroxytetradecanoic acid as amide-bound fatty acid were identified in the lipid A. A very high serological specificity of O139 LPS distinct from that of O1 V. cholerae was demonstrated by passive hemolysis and passive hemolysis inhibition tests by using the LPS either as antigen for sensitizing sheep red blood cells or as inhibitor in the latter test.
We report the identification and sequence from Escherichia coli and Salmonella enterica strains of the cld gene, encoding the chain-length determinant (CLD) which confers a modal distribution of chain length on the O-antigen component of lipopolysaccharide (LPS). The distribution of chain lengths in the absence of this gene fits a model in which as the chain is extended there is a constant probability of 0.165 of transfer of growing chain to LPS core, with termination of chain extension. The data for E. coli O111 fit a model in which the CLD reduces this probability for short chains and increases it to 0.4 for longer chains, leading to a reduced number of short chain molecules but an increase in numbers of longer molecules and transfer of essentially all molecules by chain length 21. We put forward a model for O-antigen polymerase which resembles the ribosome and fatty acid synthetase in having two sites, with the growing chain being transferred from a D site onto the new unit at the R site to extend the chain and then back to the D site to repeat the process. It is proposed that the CLD protein and polymerase form a complex which has two states: 'E' facilitating extension and 'T' facilitating transfer to core. The complex is postulated to enter the E state as O-antigen polymerization starts, and to shift to the T state after a predetermined time, the CLD acting as a molecular clock. The CLD is not O-antigen or species-specific but the modal value does depend on the source of the cld gene.
A newly described Vibrio cholerae serogroup--O139 Bengal, the causative agent of the recent large epidemics of cholera-like disease in the Indian subcontinent and neighbouring countries--possesses a high molecular weight capsular polysaccharide (CPS) that can be visualized by electron microscopy and in composition differs from the lipopolysaccharide (LPS). The CPS and LPS can be separated from each other by a two-step extraction procedure, a phenol-water extraction in order to extract all polysaccharides from the bacterial suspension followed by a phenol-chloroform-petroleum ether (PCP) extraction. The CPS is mainly composed of 3,6-dideoxyhexose (abequose or colitose), quinovosamine and glucosamine. The LPS of the O139 Bengal strain appears to possess a short polysaccharide which contains glucose, galactose, glucosamine and heptose. Both the LPS and CPS are immunogenic. They react in an enzyme immunoassay with rabbit antibodies generated against whole heat-killed bacteria. By analogy with other capsulated bacteria, the possession of a capsule may confer increased virulence of O139 Bengal.
Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and an important virulence factor of many pathogens, such as Neisseria gonorrhoeae. We have cloned the gonococcal galE gene which was found to be located in the gonococcal homologue of the meningococcal capsule gene complex region D. Sequence alignment indicated extensive homology with the Escherichia coli and Salmonella GalE proteins. Mutants with insertions in the galE gene were used as a tool to characterize the structure and function of gonococcal lipopolysaccharide. They displayed deep rough phenotypes, and chemical analysis confirmed the loss of galactose from the mutant lipopolysaccharide. Functional analysis indicated that the terminal oligosaccharides contain galactose and that these are lost in galE mutants. The importance of these oligosaccharides in gonococcal biology is clear from the fact that they contain the epitopes that are the targets for killing by normal human serum, and the acceptor site for sialic acid, which acts to protect the gonococcus from this killing. Furthermore, infection experiments in vitro indicate that the galE mutants exhibit unaltered intergonococcal adhesion as well as adhesion to, and invasion of, epithelial cells.