Showing papers on "El Tor published in 1990"

Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products.

Abstract: Vibrio cholerae O1 biotype El Tor produces and secretes a 65-kDa cytolysin/hemolysin into the culture medium. We cloned the structural gene (hlyA) for the cytolysin from the total DNA of a V. cholerae O1 El Tor strain, N86. Nucleotide sequence analysis of hlyA revealed an open reading frame consisting of 2,223 bp which can code for a protein of 741 amino acids with a molecular weight of 81,961. Consistent with this, a 79-kDa protein was identified as the product of hlyA by maxicell analysis in Escherichia coli. N-terminal amino acids of this 79-kDa HlyA protein and those of a 65-kDa El Tor cytolysin purified from V. cholerae were Asn-26 and Asn-158, respectively. The 82- and 79-kDa precursors of the 65-kDa mature cytolysin were found in V. cholerae by pulse-chase labeling and Western blot (immunoblot) analysis of hlyA products. Hemolytic activity of the 79-kDa HlyA protein from E. coli was less than 5% that for the 65-kDa cytolysin from V. cholerae. Our results suggest that in V. cholerae, the 82-kDa preprotoxin synthesized in the cytoplasm is secreted through the membranes into the culture medium as the 79-kDa inactive protoxin after cleavage of the signal peptide and is then further processed into the 65-kDa active cytolysin by release of the N-terminal 15-kDa fragment.

Characterization of the hlyB gene and its role in the production of the El Tor haemolysin of Vibrio cholerae O1.

Abstract: El Tor strains of Vibrio cholerae are capable of producing a haemolysin which they actively secrete into the growth medium. This requires translation to produce the protein at the surface of the cytoplasmic membrane and translocation across this membrane, the periplasmic space and the outer membrane. The mechanism by which this occurs is poorly understood. In addition to the structural gene for the haemolysin (hlyA), we have cloned a second adjacent gene, hlyB. By site-directed mutagenesis, specific hlyB mutants have been constructed. These mutants are defective in the secretion of HlyA in the early to mid-exponential phase of growth and the haemolysin becomes trapped within the cell and is only released in stationary phase. Nucleotide sequence analysis and cell fractionations reveal HlyB to be a 60.3 kD putative outer membrane-associated protein.

Purification of El Tor cholera enterotoxins and comparisons with classical toxin.

Abstract: Summary: In 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml−1 and 1 ng ml−1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3-5 μg ml−1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype-specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross-neutralizing activity.

Study of epitopes of cholera enterotoxin-related enterotoxins by checkerboard immunoblotting.

Abstract: Checkerboard immunoblotting, a versatile new technique for examining multiple antigen and antibody interactions simultaneously, was applied in studies of epitopes in the cholera enterotoxin (CT)-related heat-labile enterotoxin (LT) family. The purified antigens used included the following: the B-subunit proteins from two CTs (CT-B-1 and CT-B-2), from classical and El Tor biotype strains of Vibrio cholerae, respectively; human LT-B-1 (H-LT-B-1) and porcine LT-B (P-LT-B) derived from LTs produced by Escherichia coli strains of human (H) and porcine (P) origins, respectively; and genetically engineered chimeric P-LT-Bs with amino acid substitutions from H-LT-B-1. The antigens were used in native, partially denatured, and CNBr-fragmented forms. The antisera included a variety of mouse monoclonal antibodies against these proteins as well as polyclonal hyperimmune sera and sera from adult American volunteer vaccinees or convalescents from induced cholera. Rabbit antisera against synthetic peptides of the CT-B-1 subunit were also used. In some instances, the effect of GM1 ganglioside on antibody binding was evaluated. The reactivity of the monoclonal antibodies was directed primarily against conformational epitopes: some were specific for homologous antigen; some were promiscuously reactive; and some recognized particular related proteins. Individual amino acids (most notably amino acid 46) exerted a dominant effect on epitope formation--in some instances, in a complementary fashion. Epitope expression was also affected by distant amino acid residues (polar effects). Some reactions were blocked by GM1 treatment of the immobilized antigen, indicating that the epitope was involved in or affected by GM1 binding. Polyclonal antibody responses varied within and among animal species. Human serum antitoxic responses were higher in convalescents from induced cholera than in recipients of a genetically engineered live vaccine, and the convalescent sera (from El Tor biotype cholera patients) generally preferred CT-B-2 to CT-B-1. The results demonstrate the potential significance of the differences among these immunologically related enterotoxins and may help provide direction to further vaccine development.

Expression of virulence factors by classical and El Tor Vibrio cholerae in vivo and in vitro

Abstract: We examined the production of virulence factors of Vibrio cholerae O1 bacteria, and especially compared expression in vitro under near optimal growth conditions with that in vivo during experimental cholera infection. The results show that the in vitro formation of cholera toxin (CT), soluble hemagglutinin (SHA), colonizing pilus TCP, and the biotype associated hemagglutinins FSHA and MSHA, as well as of various cell envelope antigens often rather poorly reflected expression in vivo. For instance, production of CT by vibrios of classical biotype and of TCP by the El Tor biotype were enhanced in vivo, while production of SHA was instead suppressed. Likewise significant differences in cell envelope antigen composition were found between bacteria grown in vivo and in vitro. A more precise definition of the role of different postulated virulence factors in the processes of infection and immunity should include in vivo studies as outlined by this study.

Biotype-specific probe for Vibrio cholerae serogroup O1.

Abstract: The O1 serogroup of Vibrio cholerae can be divided into two biotypes, El Tor and Classical. Current tests to distinguish between these biotypes are often difficult to interpret. On the basis of the difference in sequence of the hlyA gene in these biotypes, we have developed a simple probe that can easily and reliably differentiate between the two biotypes. Images

Molecular cloning and nucleotide sequence analysis of cholera toxin genes of the CtxA- Vibrio cholerae strain Texas Star-SR.

Abstract: The ctx operons from the Vibrio cholerae El Tor strain 3083 and its CtxA- derivative Texas Star-SR were cloned, and their nucleotide sequences were compared. A single missense mutation in the Texas Star-SR ctxA cistron which results in the substitution of threonine for alanine at position 191 of the 258-amino-acid CtxA precursor was identified. Immunoblot analysis detected the mutant CtxA intracellularly early in the culture cycle but not extracellularly at any growth stage.

A cholera outbreak associated with eating uncooked pork in Thailand.

Abstract: In a village near Chiangmai, Thailand, during October 1987, there was an outbreak of cholera following a funeral in which 264 attendants were served food. The present article is a report of an epidemiological study performed to identify the source of infection and the mode of its transmission. All the attendants were screened for infection by bacteriological examination of their rectal swabs and were kept under diarrhoeal surveillance. Of them, 20 patients and 40 matched controls were interviewed about the details of their eating foods served at the funeral. Vibrio cholerae 01, Inaba, El Tor was detected from 24 persons (9.1%), 15 of whom suffered from mild diarrhoea and the rest 9 had inapparent infections. There was no death. Except one butcher whose rectal swab was positive for the same strain of V. cholerae, 3 other butchers and 4 women who had prepared food were free from the infection. Food remnants were not available for culture. The water used for cooking and the water from the cement well used for slaughter were negative for the organism. The only significant association (p less than .01, odds ratio = 15) was found between an attack of cholera and eating laebmoo--an uncooked pork preparation with Thai spices and chili. The transmission of cholera appeared to have occurred through eating the uncooked pork presumably due to its contamination with V. cholerae shed by the infected butcher. He was known to have earlier visits to Chiangmai where cholera epidemic was going on.

High-frequency spontaneous mutation of classical Vibrio cholerae to a nonmotile phenotype.

Abstract: The species Vibrio cholerae contains within it two biotypes, classical and El Tor, both of which are motile. Phenotypic expression of motility was unaffected by type of growth medium, salt concentration, pH, or temperature of incubation. However, seven strains of classical V. cholerae produced spontaneous nonmotile mutants at an unusually high frequency (ca. 10(-4)), while no mutants were detected for all three El Tor strains examined. No revertants of these nonmotile mutants were detected. Four independent mutants of classical strain 395 were isolated to characterize this phenomenon. By transmission electron microscopy, one of the nonmotile mutants was found to be flagellated, while the other three were found to be aflagellate. Chromosomal DNA from the mutants and parental wild-type strain 395 was examined by Southern blot analysis with, as probes, V. cholerae mutagenic prophages VcA-1 and VcA-2 and six cloned motility gene regions isolated from transposon insertion motility mutants of strains 395 and N16961 (El Tor, Inaba). The parental wild-type strain and all of the mutants exhibited the same pattern of bands when probed with VcA-1 and VcA-2 DNAs. Four of the cloned motility gene regions hybridized to the same fragments of DNA in both the wild-type and mutant isolates. However, two other probes detected a new fragment for a single aflagellate mutant. The observations that spontaneous nonmotile mutants occurred at a high frequency and that these mutants did not revert at a detectable frequency suggested that a genetic event is involved. The phenomenon appears to be limited to classical V. cholerae and may explain why classical V. cholerae is only sporadically associated with disease in the current pandemic.

Variation in epitopes of the B subunit of Vibrio cholerae non-O1 and Vibrio mimicus cholera toxins.

Abstract: Monoclonal antibodies reacting with the B subunit of Vibrio cholerae O1 strain 569B cholera toxin (CT-B) were used to identify unique and common epitopes of V. cholerae non-O1 and Vibrio mimicus CT-B. Vibrio cholerae non-O1 strains produced CT-B showing three monoclonal antibody reaction patterns (epitypes), which corresponded with epitypes described previously for V. cholerae O1 classical biotype CT-B (CT1), El Tor biotype CT-B (CT2), and a unique V. cholerae non-O1 CT-B (CT3), which lacked an epitope located in or near the GM1 ganglioside binding site of 569B CT-B. Vibrio mimicus CT-B was immunologically indistinguishable from 569B CT-B. These and previous results define six epitopes on 569B CT-B, and a fourth epitope in or near the GM1 ganglioside binding site. Key words: cholera toxin, epitopes, monoclonal antibodies.

Cholera epidemic in Solapur during July-August, 1988.

Abstract: Of the 77 Vibrio cholerae isolated during July to September, 1988, 76 were El Tor vibrios serotype Ogawa, 68 belonging to T2 phage (eight strains untypable). Peak incidence was noted in the month of August, 1988. Haemolytic and non-haemolytic colony variants of El Tor V. cholerae were encountered. All strains showed resistance to one or more antibiotics. No fatality was reported during the epidemic. The epidemiological data collected over the past three years (1986-88) show that cholera is prevalent in the local environment.

Cholera in Sarawak: a historical perspective (1873-1989).

Abstract: Cholera has been in existence in Sarawak for many years and since 1873 many major epidemics have occurred. The epidemics usually occur during the dry months of May, June and July and the population affected are those in coastal areas. As in other outbreaks the areas affected were those which had poor environmental sanitation, poor water supply, poor refuse disposal and indiscriminate disposal of faeces. Malays are more affected as in Peninsular Malaysia outbreaks. The classical biotype was common prior to 1961. In later years the El Tor (biotype) has been responsible for most outbreaks.

Epidemiological profile of outbreaks of cholera in India during 1975-1989.

Abstract: Cholera has been present in India since antiquity. Six pandemics originated in Indian subcontinent. The present seventh pandemic caused by El Tor Vibrio cholerae started from Indonesia (Sulawesi) in 1961 and entered India in 1964. By the end of 1965 it has replaced the age old classical V. cholerae. Many of the States which never had cholera or were free from it for a long time got infected and became endemic foci of El Tor infection. This article reviews the epidemiological features of important outbreaks reported after 1975 in India.

Vibrio cholerae serogroup O1 in northeast Thailand.

Abstract: Strains of Vibrio cholerae serogroup O1 biotype El Tor that are susceptible to Mukerjee cholera bacteriophage group IV (S. Mukerjee, Bull. W.H.O. 28:333-336, 1963) were found. Cholera vibrios isolated from epidemics in northeast Thailand were characterized, and 57 of 60 strains isolated in 1986 were susceptible to cholera phage IV. However, all 113 strains isolated in 1988 were not susceptible to the phage. All isolates in both epidemics revealed behaviors typical of El Tor vibrios, except phage IV susceptibility in the 57 strains. Although the plaques of phage IV were generally translucent, plaques on some isolates looked transparent, just like those on classical vibrios. The organisms grown in the plaques were lysogenized. If this kind of strain is frequently isolated, the biotype of V. cholerae O1 should be reconsidered.