Biomedical subjects
R A Finkelstein
Publications and source records attributed to R A Finkelstein.
Vibrio cholerae hemagglutinin/protease, colonial variation, virulence, and detachment.
The structural gene, hap, for the secreted hemagglutinin/protease (HA/protease), a putative virulence factor of Vibrio cholerae, has recently been cloned and sequenced (C. C. Häse and R. A. Finkelstein, J. Bacteriol. 173:3311-3317, 1991). The availability of the null mutant, HAP-1, and HAP-1 complemented with pCH2 (which expresses HA/protease), enabled an examination of the role of HA/protease in the virulence of V. cholerae in an animal model. However, the mutants exhibited reversible colonial variation similar but not identical to that which was previously associated with dramatic changes in virulence of parental strain 3083. Regardless of colonial morphology, the mutants were found to be fully virulent in infant rabbits. Thus, the HA/protease is not a primary virulence factor (for infant rabbits). Observations using cultured human intestinal cells indicated, instead, that the HA/protease is responsible for detachment of the vibrios from the cultured cells by digestion of several putative receptors for V. cholerae adhesins.
Synthesis, characterization, and some immunological properties of conjugates composed of the detoxified lipopolysaccharide of Vibrio cholerae O1 serotype Inaba bound to cholera toxin.
Protection against cholera has been correlated with the level of serum vibriocidal antibodies. The specificity of these vibriocidal antibodies was mostly to the lipopolysaccharide (LPS). We synthesized conjugates of detoxified LPS with cholera toxin (CT) and other proteins in order to elicit serum LPS antibodies with vibriocidal activity. Treatment with hydrazine (deacylated LPS) reduced the endotoxic properties of the LPS to clinically acceptable levels and resulted in a molecule larger and more antigenic than the saccharide produced by acid hydrolysis. More immunogenic conjugates resulted from multipoint compared with single-point attachment of the deacylated LPS to the protein. The conjugates containing CT had low levels of pyrogen and no toxic activity upon Chinese hamster ovary cells and elicited booster responses of vibriocidal and CT antibodies when injected subcutaneously as saline solutions into mice; the vibriocidal titers were similar to those elicited by comparable doses of cellular vaccines. We suggest how serum vibriocidal antibodies might prevent cholera.
Bovine lactogenic immunity against pediatric enteropathogens.
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Identification of epitopes of the receptor binding subunit of cholera toxin by synthetic peptide and CBIB approaches.
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Mapping epitopic regions of cholera toxin B-subunit protein.
Continuous overlapping synthetic hexapeptides representing the entire 103 amino acid sequence of the immunodominant B-subunit protein of cholera enterotoxin were used to examine reactivities of a variety of antisera in attempts to detect and define sequence-related (continuous) antigenic regions. The validity of the methods was established by the reactions of polyclonal antisera raised against longer synthetic peptides with appropriate synthetic hexapeptides. An unexpected cross-reaction is attributed to the presence of three identical amino acids (Gln16-Ile17-His18)--although in different order (Gln56-His57-Ile58)--in two parts of the B-subunit chain. Adsorption studies using polyclonal rabbit antisera revealed that, in many instances, denatured B-subunit protein more effectively removed reactivity with hexapeptides than did the native protein. Native holotoxin was more effective than native B-subunit. Sera from human cholera convalescents gave diffuse patterns of reactivity with synthetic hexapeptides--primarily against regions of reactive hexapeptides rather than with clearly defined continuous epitopes. Among many epitopic regions encountered, a strongly reactive tetramer, Ser-Gln-His-Ile (SQHI), was discovered in a highly conserved region, residues 55-58, of the B-subunit amino acid sequence. Adsorption studies revealed that this epitope is apparently exposed on the surface of the native protein. Amino acid substitution revealed the essentiality of Gln and His residues to this epitope. Gly54 was not part of the epitope but substitution of acidic residues Glu and Asp for Gly eliminated reactivity with antibody. The results suggest that continuous epitopes may contribute to the antigenicity of the native toxin protein and may be potentially useful for development of a peptide vaccine.
Heterogeneity of immunotypes of heat-labile enterotoxins of enterotoxigenic Escherichia coli of human origin.
A new technique, checkerboard immunoblotting (CBIB), has been applied to detect and to differentiate heat-labile enterotoxins, (LTs), from enterotoxigenic strains of Escherichia coli of human origin using polyclonal and monoclonal antibodies. Optimal conditions of production and release of LTs were defined using CBIB. LT release was enhanced when E. coli cells were treated with 8 M urea. LT production was highest when E. coli strains were incubated with shaking (200 rpm) at 37 degrees C for 12 h in CAYE-2 medium. Two hundred and five strains of E. coli, isolated from patients with diarrhea in Japan, Thailand, the United States, Mexico, and Brazil, were examined for LT. Of 133 LT-positive strains, 4 (3%) produced an LT that reacted like H-LT-1 (originally isolated from E. coli strain H-74-114) while 126 strains (94.7%) produced LT that reacted like H-LT-2 (originally isolated from strain H-10407) or H-LT-3 (from strain H-240-3). Three strains of human origin (2.3%) produced an LT that reacted like P-LT (produced by E. coli strains of porcine origin). This study shows that CBIB, a simple, efficient, and practical assay, might be useful for epidemiologic surveys and for evaluation of serologic responses to LTs and antitoxic vaccines.
Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain.
The structural gene hap for the extracellular hemagglutinin/protease (HA/protease) of Vibrio cholerae was cloned and sequenced. The cloned DNA fragment contained a 1,827-bp open reading frame potentially encoding a 609-amino-acid polypeptide. The deduced protein contains a putative signal sequence followed by a large propeptide. The extracellular HA/protease consists of 414 amino acids with a computed molecular weight of 46,700. In the absence of protease inhibitors, this is processed to the 32-kDa form which is usually isolated. The deduced amino acid sequence of the mature HA/protease showed 61.5% identity with the Pseudomonas aeruginosa elastase. The cloned hap gene was inactivated and introduced into the chromosome of V. cholerae by recombination to construct the HA/protease-negative strain HAP-1. The cloned fragment containing the hap gene was then shown to complement the mutant strain.
Urea-induced release of heat-labile enterotoxin from Escherichia coli.
Urea induces the release of heat-labile enterotoxin (LT) from cells of LT-producing Escherichia coli strains. Optimal conditions were defined by using the checkerboard immunoblotting system. LT release was highest when E. coli cells were incubated in 8 M urea, pH 8.0, at 37 degrees C in a water bath for 30 min. Urea was more effective than polymyxin B in inducing the release of LT antigen from E. coli; the activity of LT from urea-treated cells was seven times that of LT from polymyxin B-treated cells. Urea also increased the antigenic and biological reactivities of purified LT. This procedure is potentially applicable for the detection of LT-producing E. coli strains in the clinical laboratory.
Checkerboard immunoblotting (CBIB): an efficient, rapid, and sensitive method of assaying multiple antigen/antibody cross-reactivities.
A simple technique, checkerboard immunoblotting (CBIB), is described, which facilitates the examination of multiple antigen/antibody interactions, conveniently and reproducibly, using minimal amounts of reactants. Antigens, immobilized on a solid-phase membrane in parallel lanes, are allowed to react with primary antibodies, applied in lanes perpendicular to the antigens, and the reactions are developed with appropriately labeled secondary antibody and substrate. Positive reactions, at the intersections of antigen/antibody lanes, are small squares, giving a checkerboard appearance to the blot. The results are easily read visually and presented in the form of a permanent record. CBIB has wide range of applications, including the screening of hybridomas for monoclonal antibody production. With the cholera toxin (CT)-related antigens used, homologous reactions were markedly stronger then heterologous reactions.
Comparison of the Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase.
The soluble hemagglutinin/protease (HA/protease) produced by Vibrio cholerae and the elastase of Pseudomonas aeruginosa are both zinc/calcium-dependent proteases. In the present study the two enzymes are compared immunologically and functionally. The N-terminal amino acid sequences of the proteins had 65% identity within the first 20 amino acids. Polyclonal antisera against each purified protein recognized the enzyme of the other species in enzyme-linked immunosorbent assay, checkerboard immunoblot, and Western blot analyses and inhibited the protease activity of both enzymes in milk and elastin agars. Like the HA/protease, the elastase hemagglutinated "responder" but not "nonresponder" chicken erythrocytes, degraded ovomucin, lactoferrin, and fibronectin, and nicked the A subunit of the cholera toxin-related heat-labile enterotoxin from Escherichia coli. Whereas none of the three proteases tested (elastase, HA/protease, or pronase E) had any obvious effect in ileal loop tests in rabbits at doses up to 50 micrograms, all three produced some detectable skin reactions at a dose of 0.1 micrograms and necrosis at a higher dose (i.e., 5 micrograms). We conclude that the V. cholerae HA/protease and the P. aeruginosa elastase are structurally, functionally, and immunologically related.
Molecular cloning and nucleotide sequence analysis of cholera toxin genes of the CtxA- Vibrio cholerae strain Texas Star-SR.
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.
Study of epitopes of cholera enterotoxin-related enterotoxins by checkerboard immunoblotting.
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.
Passive oral immunisation of children.
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Inhibition of enteropathogenic bacteria by human milk whey in vitro.
Diarrhea caused by enteropathogenic bacteria is a leading cause of childhood mortality world-wide, particularly in less developed regions. Breast-feeding has been advocated to protect infants and children from infectious illnesses. We examined the antibacterial activity of human whey in vitro against multiple strains of the following species of enteropathogenic bacteria, Campylobacter jejuni, Escherichia coli, Salmonella typhimurium, Shigella flexneri, Shigella sonnei and Vibrio cholerae, all isolated from humans with diarrheal illness. In vitro human whey inhibited the growth of most but not all of the enteropathogenic strains tested. The test strains multiplied in both bacterial growth medium and commercial infant-feeding formula controls, with the exception of the Campylobacter strains, which were markedly inhibited in formula. These results confirm and extend previous observations that human whey contains components capable of inhibiting enteropathogenic bacteria and which may be associated with the protective effects of breast feeding. The precise mechanisms of these inhibitory effects merit further study.
Bovine lactogenic immunity against cholera toxin-related enterotoxins and Vibrio cholerae outer membranes.
The newly parturient cow secretes large quantities of immunoglobulin G1, a relatively protease- and heat-resistant immunoglobulin, in its colostrum and milk. This study establishes the feasibility of producing protective colostral immunoglobulins by immunizing pregnant cows with cholera toxin (CT), a CT-related enterotoxin from Escherichia coli, and Vibrio cholerae outer membranes (OMs). The OMs were prepared from bacteria grown under iron-replete or iron-deficient (to simulate the in vivo environment) conditions. Immunoglobulins were purified from the colostrum of newly parturient control and immunized cows. The bovine anti-CT and anti-H-LT (CT-related heat-labile enterotoxin produced by diarrheogenic E. coli strains of human origin) antibodies were quantitated by enzyme-linked immunosorbent assays and by neutralization of toxin activity in both Y-1 adrenal cell and infant rabbit assays. The bovine anti-OM antibodies from both high-iron-grown and low-iron-grown vibrios were assessed by bacterial agglutination and by Western blot (immunoblot) analysis of polyacrylamide gel electrophoresis of high-iron-grown and low-iron-grown OMs. To test their protective effect, immunoglobulin preparations were administered orally in infant feeding formula to 6-day-old rabbits. Anti-CT and anti-OM immunoglobulins elicited statistically significant protection against diarrhea in infant rabbits challenged intraintestinally with virulent cholera vibrios.
Purification and characterization of a protease produced by Vibrio cholerae non-O1 and comparison with a protease of V. cholerae O1.
A protease produced by a clinical isolate of Vibrio cholerae non-O1 was purified to apparent homogeneity by ammonium sulfate fractionation and successive column chromatography on DEAE-Sephadex A25, Sephadex G100, Mono Q, and Phenyl Superose. Like the hemagglutinin-protease of V. cholerae O1, the purified protease had both hemagglutinating and proteolytic activities. The protease was heat labile, and in contrast to crude preparations, no Arrhenius effect was observed with the purified protein. Immunological analyses indicated that the proteases (or hemagglutinins) derived from V. cholerae O1 and non-O1 are identical.
Anti-idiotypic antibodies as probes of protein active sites: application to cholera toxin subunit B.
Since Jerne proposed a "network" theory of immune regulation, the properties of anti-idiotypic antibodies (anti-IdAb) have been investigated widely. Anti-IdAb raised against antibodies to a variety of ligands have been shown to bind the ligands' receptors. Thus, the combining site of an anti-IdAb may contain information regarding the three-dimensional structure of an antigen. However, this remarkable property of "internal imagery" has not been exploited for structural investigation at the molecular level. In the present report, a monoclonal "auto"-anti-IdAb was raised against ganglioside GM1 (a cell-surface glycolipid that binds cholera toxin) and was shown to crossreact with the B subunit of cholera toxin. This antibody was presumed to recognize amino acid residues located within the GM1 binding domain. To identify these residues, the antibody was screened against homologous toxins purified from enterotoxigenic strains of Escherichia coli and chimeric peptides produced by recombinant methods. Amino acid variation at position 4 from the N terminus of these proteins was found to disrupt antibody binding. Since the toxins and chimera are all closely related in structure and function, the residue at position 4 (an asparagine in cholera toxin B subunit) appears to be in the epitope of the antibody and, by implication, in the GM1 binding site. Of particular significance, this structural detail could not be deduced with GM1 alone. It would seem that ligand and anti-ligand anti-IdAb encode similar stereochemical information but do so with different "chemical alphabets," giving rise to distinct binding specificities.