Cloning and genetic analysis of the Vibrio cholerae aminopeptidase gene.

The structural gene for the Vibrio cholerae leucine aminopeptidase (lap) was cloned and sequenced. The cloned DNA fragment contained a 1,503-bp open reading frame potentially encoding a 501-amino-acid polypeptide with a calculated molecular mass of 54,442 Da. The deduced amino acid sequence of the entire protein showed high homology with the sequence of Vibrio proteolyticus leucine aminopeptidase. The residues potentially involved in binding the zinc ions were completely conserved in the V. cholerae aminopeptidase as well as in the V. proteolyticus aminopeptidase. The recombinant protein was partially purified and characterized. The molecular mass was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 34 kDa, suggesting a processing of the protein to acquire the mature form. The protease showed maximum activity at pH 9.0 and was thermostable at 70 degrees C. The substrate leucyl-p-nitroanilide was cleaved by the protease, and its activity was inhibited by EDTA and bestatin. These results suggested that the protein was a leucine aminopeptidase. The PCR analysis of lap gene distribution showed that it was widely distributed among the V. cholerae strains. It was not present in the other species examined.     

Characterization of the lipopolysaccharide from Vibrio cholerae 395 (Ogawa).

The chemical structure and biological properties of the lipopolysaccharide (LPS) from Vibrio cholerae 395 (Ogawa), isolated by the phenol-water procedure, were studied. Upon acid hydrolysis, the LPS was split into its polysaccharide and lipid A moieties. The polysaccharide contained both neutral (glucose, heptose, fructose) and amino (glucosamine, quinovosamine) sugars. The LPS contained the acid-labile amino sugar, 4-amino-arabinose, which was absent in the Inaba serotype of V. cholerae. The LPS differed from the LPSs of Enterobacteriaceae by the absence of 2-keto-3-deoxyoctonate and the presence of fructose. Analysis of the methylated polysaccharide by gas-liquid chromatography and mass spectrometry showed that it had a branched structure with glucose and heptose residues primarily appearing at the nonreducing-end groups. Interactions with lectins, concanavalin A. and wheat germ agglutinin suggested that terminal glucose residues were alpha linked, whereas terminal glucosamine residues were connected by alpha-1,3 linkages. The major fatty acids of the LPS were C14:0, C16:0, C12h:0, and C14h:0 compounds, of which only the C14h:0 were amide linked, the remainder being ester linked to the backbone. Biological studies showed that the LPS possessed endotoxic properties such as lethality, pyrogenicity, limulus lysate gelation, and ability to induce non-specific resistance to infection. Thus, the LPS from V. cholerae 395 (Ogawa) possessed both common and distinct features as compared with the LPSs from the Enterobacteriaceae.     

Immunological characterization of Vibrio cholerae O:1 lipopolysaccharide, O-side chain, and core with monoclonal antibodies.

Lipopolysaccharide (LPS) was extracted from Vibrio cholerae O:1 strains of the serotypes Ogawa, Inaba, and Hikojima and delipidated by mild-acid hydrolysis. Two polysaccharide fragments with the molecular weights of approximately 9,000 and 900, respectively, were isolated by gel permeation chromatography. The LPS preparations and the polysaccharide fragments were studied in enzyme-linked immunosorbent assay inhibition, rocket immunoelectrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroblotting with monoclonal antibodies directed against the group-specific antigen A, the type-specific antigens B (Ogawa) and C (Inaba), and the core region. Antigen A was demonstrated in all LPS preparations and all 9,000-molecular-weight fragments tested. The type-specific antigens B and C were demonstrated in LPSs and 9,000-molecular-weight fragments from Ogawa and Inaba, respectively. Furthermore, antigens B and C were both demonstrated in LPSs and 9,000-molecular-weight fragments from two of four Hikojima strains tested. Core antigen was demonstrated in the LPS and in the 9,000- and 900-molecular-weight fragments. The results indicate that the 9,000-molecular-weight fragment represents the complete polysaccharide chain, including group- and type-specific antigens as well as core antigens, whereas the 900-molecular-weight fragment constitutes the main part of the core region.     

Comparative study of Vibrio cholerae non-O1 protease and soluble hemagglutinin with those of Vibrio cholerae O1.

Protease and soluble hemagglutinating activities produced by a non-O1 Vibrio cholerae strain isolated from a patient with diarrhea were compared with similar activities produced by V. cholerae O1. The soluble protease activities were indistinguishable in heat stability, immunodiffusion, inhibition by antiserum, and electrophoretic analysis. On the other hand, the soluble hemagglutinating activities of both strains were not completely identical. The hemagglutinating activity of the non-O1 V. cholerae strain was not inhibited by Zincov; it was more sensitive to inhibition by normal serum, and it had an unusual pattern of heat stability. Heating at 100 degrees C resulted in some recovery of activity of a sample previously inactivated by heating at 60 degrees C.     

Characterization of monoclonal antibodies with specificity for the core oligosaccharide of Shigella lipopolysaccharide

Monoclonal antibodies (MAbs) were prepared against different strains of Shigella, following immunization of BALB/c mice with a heat-killed preparation of Shigella. Antibody-producing hybridomas were screened in an indirect enzyme-linked immunoadsorbent assay (ELISA) and epitope specificity determined using chemically defined lipopolysaccharide, lipid, and KDO fragments. Five MAbs were characterized and the following specificities identified: 2C32E6 and 4D64B9 (reactive to S. flexneri and S. boydii), 5E45D8 (reactive with S. flexneri), 4B33D10 and 1B52F10 (all species of Shigella). The properties of 1B52F10 revealed its potential importance in immunological detection of Shigella from unknown samples, as it was able to bind to all strains of Shigella.     

A murine monoclonal antibody specific for the outer core oligosaccharide of Salmonella lipopolysaccharide.

Immunoglobulin G3 murine monoclonal antibody T6 specific for the lipopolysaccharide of Salmonella O serogroups A to E was established. By using R mutants of Salmonella spp., Escherichia coli, and Shigella spp., the major reactive epitope with T6 was tentatively identified as the terminal disaccharide, N-acetylglucosamine 1.2----alpha glucose, of the core oligosaccharide. T6 was reactive with 10 clinical isolates of each of the Salmonella O serogroups A to E but not with 58 isolates of other gram-negative bacteria. Its selective reactivity against Salmonella spp. renders T6 a potentially more useful reagent than the conventional polyvalent serum for the identification of Salmonella spp. It may also serve as a useful molecular tool for the study of the outer core structure of all Salmonella and related species.     

Expression of the Escherichia coli lamB gene in Vibrio cholerae

A phage lambda mediated transduction system was devised to facilitate molecular analysis of Vibrio cholerae. A lamB expression plasmid, pAMH62 was introduced into Vibrio cholerae by conjugation. The resulting V. cholerae derivatives harboring pAMH62 produced substantial amounts of the LamB protein. This protein was properly inserted into the outer membrane, as suggested by (i) its localization into the cell envelope, (ii) its association with the peptidoglycan layer of the cell wall, and (iii) its function as receptor for phage lambda. In vivo packaged cosmids were efficiently transduced into these strains of V. cholerae.     

Monoclonal antibodies against group- and type-specific lipopolysaccharide antigens of Vibrio cholerae O:1

Hybrid cell lines producing monoclonal antibodies against the O-antigenic determinants of Vibrio cholerae O:1 have been established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay inhibition experiments by using lipopolysaccharides from V. cholerae O:1 strains and type strains of groups O:2 and O:21. The anti-A antibody was of the immunoglobulin M (IgM) class, whereas the anti-B and -C antibodies were IgG3. The antibodies had a good agglutinating capacity when tested against V. cholerae O:1 strains in the slide agglutination test.     

Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae.

Live oral candidate cholera vaccines have previously been constructed by deletion of Vibrio cholerae sequences encoding the enzymatically active A subunit of the cholera toxin. However, volunteer studies have shown that these non-cholera toxin-producing strains still provoke mild to moderate diarrhea in some individuals. We recently reported the identification of a second toxin produced by V. cholerae which may be responsible for this residual diarrhea (A. Fasano, B. Baudry, D. W. Pumplin, S. S. Wasserman, B. D. Tall, J. M. Ketley, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 88:5242-5246, 1991). This new toxigenic factor increases the permeability of rabbit ileal mucosa by affecting the structure of the intercellular tight junctions (zonula occludens). We now report the identification and cloning of the gene encoding this new toxin. This gene, named zot (for zonula occludens toxin), consists of a 1.3-kb open reading frame which could potentially encode a 44.8-kDa polypeptide. The location of the zot gene encoding the new toxin is immediately upstream of the ctx operon encoding cholera toxin.     

Presence of CTX gene cluster in environmental non-O1/O139 Vibrio cholerae and its potential clinical significance

Purpose: The aim of this study was to understand the epidemiological linkage of clinical and environmental isolates of Vibrio cholerae and to determine their genotypes and virulence genes content. Materials and Methods: A total of 60 V. cholerae strains obtained from clinical specimens (n = 40) and surface waters (n = 20) were subjected to genotyping using PFGE and determination of their virulence-associated gene clusters. Result: PCR analysis showed the presence of chromosomally located hly and RTX genetic elements in 100% and 90% of the environmental isolates, respectively. The phage-mediated genetic elements such as CTX, TLC and VPI were detected in 5% of the environmental isolates suggesting that the environmental isolates cannot acquire certain mobile gene clusters. A total of 4 and 18 pulsotypes were obtained among the clinical and environmental V. cholerae isolates, respectively. Non-pathogenic environmentally isolated V. cholerae constituted a distinct cluster with one single non-O1, non-O139 strain (EP6) carrying the virulence genes similar to the epidemic strains. This may suggest the possible potential of conversion of non-pathogenic to a pathogenic environmental strain. Conclusions: The emergence of a single environmental isolate in our study containing the pathogenicity genes amongst the diverse non-pathogenic environmental isolates needs to be further studied in the context of V. cholerae pathogenicity sero-coversion.     

Identification and occurrence of Vibrio cholerae flagellar core proteins in isolated outer membrane

Sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of outer membranes from a flagellated and an isogenic nonflagellated strain of Vibrio cholerae (classical, Inaba) suggested that two proteins were absent from the nonflagellated strain. Immunoblot examination of such preparations demonstrated that two proteins, present only in outer membrane from the flagellated strain, were associated with flagella. Analysis of purified flagellar cores from strains CA401 and N16961 (El Tor, Inaba) by electron microscopy, sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, and immunoblotting showed that these two proteins, with apparent molecular weights of 47,000 and 49,000, composed the flagellar core. Antiserum specific for flagellar core proteins did not agglutinate or inhibit the motility of intact V. cholerae. These latter findings suggested that, for intact cells, the flagellar core proteins are not accessible to antibody.     

Effect of phage on the infectivity of Vibrio cholerae and emergence of genetic variants

Seasonal epidemics of cholera in Bangladesh are self-limited in nature, presumably due to phage predation of the causative Vibrio cholerae during the late stage of an epidemic, when cholera patients excrete large quantities of phage in their stools. To further understand the mechanisms involved, we studied the effect of phage on the infectivity and survival of V. cholerae shed in stools. The 50% infectious dose of stool vibrios in infant mice was ~10-fold higher when the stools contained a phage (1.8 × 10³ to 5.7 × 10⁶ PFU/ml) than when stools did not contain a detectable phage. In competition assays in mice using a reference strain and phage-negative cholera stools, the infectivity of biofilm-like clumped cells was 3.9- to 115.9-fold higher than that of the corresponding planktonic cells. However, the difference in infectivity of these two cell populations in phage-positive stools was significantly less than that in phage-negative stools (P = 0.0006). Coculture of a phage and V. cholerae or dilutions of phage-positive cholera stools in nutrient medium, but not in environmental water, caused rapid emergence of phage-resistant derivatives of the bacteria, and these derivatives lost their O1 antigen. In cholera stools and in intestinal contents of mice prechallenged with a mixture of V. cholerae and phage, the bacteria remained completely phage susceptible, suggesting that the intestinal environment did not favor the emergence of phage-resistant derivatives that lost the O1 antigen. Our results indicate that phages lead to the collapse of epidemics by modulating the required infectious dose of the bacteria. Furthermore, the dominance of phage-resistant variants due to the bactericidal selective mechanism occurs rarely in natural settings, and the emerging variants are thus unable to sustain the ongoing epidemic.     

Identification and isolation of a putative permease gene in the quinic acid utilization (QUT) gene cluster of Aspergillus nidulans

Summary Mutations in the qutD gene of Aspergillus nidulans cause the loss of ability to grow upon quinic acid as sole carbon source in media at normal pH 6.5 and failure to induce three enzyme activities specifically required for metabolism to protochatechuic acid. All 9 qutD mutants recovered are recessive and have been found to be pH sensitive, growing strongly on quinic acid media at pH 3.5 and producing significant induced enzyme acitivities. These properties are consistent with the hypothesis that the QUTD gene encodes an essential component of a permease required for transport of quinate ion into mycelium at pH 6.5. The QUTD gene has been located within the cloned QUT gene cluster of A. nidulans by transformation of qutD mutants with fragments of cloned sequences from phage -Q1. The QUTD locus is in a region distinct from other QUT genes and which contains sequences homologous to the QA-Y gene in the corresponding QA gene cluster of Neurospora crassa.     

The cytolysin gene of Vibrio vulnificus: sequence and relationship to the Vibrio cholerae E1 Tor hemolysin gene.

A cytolysin of ca. 56 kilodaltons has been suggested as a possible virulence factor in Vibrio vulnificus infections. We sequenced the DNA encoding cytolytic activity and found that the sequence contained two open reading frames, vvhA and vvhB. vvhA encoded the structural gene for the cytolysin and contained the N-terminal amino acid sequence previously reported for the protein. Regions of the vvhA gene showed homology to the structural gene for the Vibrio cholerae E1 Tor hemolysin.     

Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae

The Vibrio cholerae genome contains a 5.4-kb pil gene cluster that resembles the Aeromonas hydrophila tap gene cluster and other type IV-A pilus assembly operons. The region consists of five complete open reading frames designated pilABCD and yacE, based on the nomenclature of related genes from Pseudomonas aeruginosa and Escherichia coli K-12. This cluster is present in both classical and El Tor biotypes, and the pilA and pilD genes are 100% conserved. The pilA gene encodes a putative type IV pilus subunit. However, deletion of pilA had no effect on either colonization of infant mice or adherence to HEp-2 cells, demonstrating that pilA does not encode the primary subunit of a pilus essential for these processes. The pilD gene product is similar to other type IV prepilin peptidases, proteins that process type IV signal sequences. Mutational analysis of the pilD gene showed that pilD is essential for secretion of cholera toxin and hemagglutinin-protease, mannose-sensitive hemagglutination (MSHA), production of toxin-coregulated pili, and colonization of infant mice. Defects in these functions are likely due to the lack of processing of N termini of four Eps secretion proteins, four proteins of the MSHA cluster, and TcpB, all of which contain type IV-A leader sequences. Some pilD mutants also showed reduced adherence to HEp-2 cells, but this defect could not be complemented in trans, indicating that the defect may not be directly due to a loss of pilD. Taken together, these data demonstrate the effectiveness of the V. cholerae genome project for rapid identification and characterization of potential virulence factors.     

Synergistic protective effect in rabbits of immunization with Vibrio cholerae lipopolysaccharide and toxin/toxoid.

Subcutaneous immunization of rabbits with a combination of Vibrio cholerae lipopolysaccharide (LPS) and enterotoxin induced a more than 100-fold-higher degree of protection against intestinal challenge with live cholera vibrios than did vaccination with either of the two antigens alone. Such a synergistic effect was also obtained by immunization with a combination of LPS and choleragenoid. The immunization with LPS and toxin (or toxoid) in combination did not enhance the reistance to toxin challenge above that induced by the toxin component alone. This, together with data from titrations of anti-LPS and antitoxin antibodies in serum and in intestinal washings, contradicts enhanced immune responses due to adjuvant action of the two antigens as the explanation for the synergistic effect of the combined vaccines. A more likely explanation would be that the antibacterial and antitoxic immune responses, without being increased in themselves, function synergistically by interfering with two separate events in cholera pathogensis.     

Nucleotide sequence variations within the lipopolysaccharide biosynthesis gene gseA (Kdo transferase) among the Chlamydia trachomatis serovars

The gene gseA, involved in the expression of the genus-specific epitope of chlamydial lipopolysaccharide (LPS), was analyzed by temperature gradient gel electrophoresis (TGGE) to visualize nucleotide sequence variations among the 15 serovars of Chlamydia trachomatis. Sequence analysis showed that the TGGE melting-profile patterns were able to detect single nucleotide variations within gseA and allowed the arrangement of the serovars in groups of both identical nucleotide sequences and sequences containing identical sites of nucleotide substitutions. Compared to serovar L2, four types of patterns were obtained: (i) serotypes A and Ba; (ii) B and C (causing endemic trachoma); (iii) D through K (causing sexually transmitted oculo-genital infections); (iv) L1 through L3 (the causative agents of lymphogranuloma venereum). A total of 58 isolates of C. trachomatis of genital or conjunctival origin were tested by this method in comparison to reference strains. Forty-eight isolates (13 of type E, 16 of type F, nine of type G, and ten of type K) yielded the same melting profile as the corresponding type strain, independent of whether they were isolated from genital or ocular infections. However, ten B serotype strains of genital origin behaved in TGGE like a typical genital strain and not a trachoma strain. Thus, although gseA was found to be highly conserved among C. trachomatis, the obtained TGGE profiles of the tested strains tended to correlate with their specific site of infection.     

多重聚合酶链式反应（MPCR）用于霍乱弧菌检测的研究

本文首先从实用的角度探讨用三对特异的引物对不同血清群霍乱弧菌进行ＭＰＣＲ检测的可能性。这三对引物具有很大的应用价值，从一次扩增反应中可以得出有关菌株的毒力因子和血清群的信息。检测的敏感性可达到１０ＣＦＵ。实验还提示将引物数量改为五条寡核苷酸链来进行ＭＰＣＲ能得出同样的结果。对Ｐ３、Ｐ４引物扩增形成的６１７ｂｐ的ＤＮＡ片段进行酶切分析表明，埃尔托型霍乱弧菌Ｏ１３９群霍乱弧菌的ｔｃｐＡ基因有很大的相似性，至少在ＰｓｔⅠ和ＢｇｌⅡ酶切位点上表现相同，但两者均与古典型霍乱弧菌不同。     

Genovariants of Vibrio cholerae biovar El Tor: Construction,molecular-genetic,and proteomic analyses

Experimental modeling of the emergence of virulent Vibrio cholerae El Tor genovariants is presented. It has been shown that the obtained genovariants differed neither in phenotypic or genotypic traits from natural genetically altered strains that emerged in populations of wild-type strains. It has been established, using the PCR and sequencing methods, that the genovariants formed in the process of conjugation carried in their genome a fragment of the CTX^Classφ prophage genome with the ctxB1 gene of classical-type cholera vibrios. It has been shown that changes in the prophage’s structure led to higher levels of toxigenicity and virulence in the genovariants compared to a typical recipient strain. A proteomic analysis has also revealed changes in the expression of 26 proteins performing various functions in the cell, such as metabolism, energy exchange, transport of amino acids, etc.). These data are indicative of the effect produced by the new DNA region in the genome of the genovariants on the expression level of some house-keeping genes. The obtained results confirm the idea that horizontal gene transfer is one of the mechanisms leading to the emergence of genovariants in the populations of wild-type strains.     

Hemagglutination is a novel biological function of lipopolysaccharide (LPS), as seen with the Vibrio cholerae O139 LPS.

It has been generally thought that the polysaccharide moiety of lipopolysaccharide (LPS) maintains only serological specificity, while the lipid A portion determines various biological functions. However, we found that hemagglutination was a common function of the polysaccharide moiety of LPSs from important human enteropathogenic bacteria. Of the LPSs examined, Vibrio cholerae O139 LPS showed the highest hemagglutinating activity. Glycoproteins, such as mucin and fetuin, showed efficient inhibition of the hemagglutinating ability. Since cell-mediated hemagglutination is known to be correlated with bacterial adherence, hemagglutination induced by the polysaccharide moiety is interpreted to indicate that cell-surface LPS is a potential adhesin.