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Biomedical subjects

R A Finkelstein

Publications and source records attributed to R A Finkelstein.

At least 19 recordsLinked to original sources
PubMed2001

Environmental signals controlling production of hemagglutinin/protease in Vibrio cholerae.

Vibrio cholerae hemagglutinin/protease (Hap) was induced upon nutrient limitation and strongly repressed by glucose. Hap was not produced in a mutant defective in the cyclic AMP (cAMP) receptor protein, suggesting that intracellular cAMP levels mediate Hap expression. No difference was found in Hap production between an rpoS deletion mutant and its isogenic wild-type precursor, indicating that the alternate sigma(s) factor is not essential for Hap expression. Based on these and previous results, we discuss the role of Hap in the pathogenesis of cholera.

Bacterial Proteins↗
PubMed2000

Vibrio cholerae O139 conjugate vaccines: synthesis and immunogenicity of V. cholerae O139 capsular polysaccharide conjugates with recombinant diphtheria toxin mutant in mice.

Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to < or =50. Treatment with 2-ME reduced the titers of 17 of 20 patients to < or =50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned.

Animals↗
PubMed2000

Pseudomonas (Burkholderia) pseudomallei in Thailand, 1964-1967: geographic distribution of the organism, attempts to identify cases of active infection, and presence of antibody in representative sera.

The purpose of this study, initiated in 1964 and concluded in 1967, was to define the distribution of Pseudomonas (now Burkholderia) pseudomallei in Thailand, to evaluate its importance as an etiologic agent, and to survey the presence of antibody in people that might indicate prior infection and/or contact with the microorganism.

Animals↗
PubMed1999

Cholera toxin B subunit activates arachidonic acid metabolism.

Cholera toxin (CT) increases intestinal secretion of water and electrolytes and modulates the mucosal immune response by stimulating cellular synthesis of arachidonic acid (AA) metabolites (e.g., prostaglandin E2), as well as the intracellular second messenger cyclic AMP (cAMP). While much is known about the mechanism of CT stimulation of adenylate cyclase, the toxin's activation of phospholipase A2, which results in increased hydrolysis of AA from membrane phospholipids, is not well understood. To determine whether CT activation of AA metabolism requires CT's known enzymatic activity (i.e., ADP-ribosylation of GSalpha), we used native CT and a mutant CT protein (CT-2*) lacking ADP-ribose transferase activity in combination with S49 wild-type (WT) and S49 cyc- murine Theta (Th)1.2-positive lymphoma cells deficient in GSalpha. The experimental results showed that native CT stimulated the release of [3H[AA from S49 cyc- cells at a level similar to that for S49 WT cells, indicating that GSalpha is not essential for this process. Further, levels of cAMP in the CT-treated cyc- cells remained the same as those in the untreated control cells. The ADP-ribosyltransferase-deficient CT-2* protein, which was incapable of increasing synthesis of cAMP, displayed about the same capacity as CT to evoke the release of [3H]AA metabolites from both S49 WT and cyc- cells. We concluded that stimulation of arachidonate metabolism in S49 murine lymphoma cells by native CT does not require enzymatically functional CT, capable of catalyzing the ADP-ribosylation reaction. These results demonstrated for the first time that stimulation of adenylate cyclase by CT and stimulation of AA metabolism by CT are not necessarily coregulated. In addition, the B subunits purified from native CT and CT-2* both simulated the release of [3H]AA from S49 cyc- cells and murine monocyte/macrophage cells (RAW 264.7), suggesting a receptor-mediated cell activation process of potential importance in enhancing immune responses to vaccine components.

Animals↗
PubMed1998

The Vibrio cholerae mannose-sensitive hemagglutinin is the receptor for a filamentous bacteriophage from V. cholerae O139.

We previously isolated from a 1994 isolate of Vibrio cholerae O139 a filamentous lysogenic bacteriophage, choleraphage 493, which inhibits pre-O139 but not post-O139 El Tor biotype V. cholerae strains in plaque assays. We investigated the role of the mannose-sensitive hemagglutinin (MSHA) type IV pilus as a receptor in phage 493 infection. Spontaneous, Tn5 insertion, and mshA deletion mutants are resistant to 493 infection. Susceptibility is restored by mshA complementation of deletion mutants. Additionally, the 493 phage titer is reduced by adsorption with MSHA-positive strains but not with a DeltamshA1 strain. Monoclonal antibody against MSHA inhibits plaque formation. We conclude that MSHA is the receptor for phage 493. The emergence and decline of O139 in India and Bangladesh are correlated with the susceptibility and resistance of El Tor strains to 493. However, mshA gene sequences of post-O139 strains are identical to those of susceptible pre-O139 isolates, indicating that phage resistance of El Tor is not due to a change in mshA. Classical biotype strains are (with rare exceptions) hemagglutinin negative and resistant to 493 in plaque assays. Nevertheless, they express the mshA pilin gene. They can be infected with 493 and produce low levels of phage DNA, like post-O139 El Tor strains. Resistance to 493 in plaque assays is thus not equivalent to resistance to infection. The ability of filamentous phages, such as 493, to transfer large amounts of DNA provides them, additionally, with the potential for quantum leaps in both identity and pathogenicity, such as the conversion of El Tor to O139.

Bacterial Proteins↗
PubMed1997

Adherence of Vibrio cholerae to cultured differentiated human intestinal cells: an in vitro colonization model.

Choleragenic vibrios adhered to and multiplied on monolayers of the highly differentiated mucin-secreting cell line HT29-18N2. Their adherence followed first-order kinetics, was dependent on the concentration of vibrios, and was partially inhibited by lipopolysaccharide. Comparison of genetically modified vibrios showed that flagella, an active toxR gene, and the virulence cassette were not essential for initial binding. Inactivation of the hemagglutinin/protease increased binding. This highly differentiated human intestinal cell line provides a versatile new approach for studying major events occurring during intestinal colonization: adherence, multiplication, and detachment.

Bacterial Adhesion↗
PubMed1996

Tagging a Vibrio cholerae El Tor candidate vaccine strain by disruption of its hemagglutinin/protease gene using a novel reporter enzyme: Clostridium thermocellum endoglucanase A.

The celA gene encoding Clostridium thermocellum endoglucanase A was expressed in Vibrio cholerae on its own promoter and used to tag a candidate El Tor biotype cholera vaccine strain. Colonies of the tagged strain could be unequivocally distinguished by overlaying them with CM-cellulose indicator agar and Congo Red staining. Expression of celA did not affect growth of V. cholerae in vitro and in vivo. The celA gene was inserted in the chromosomal hap locus encoding V. cholerae hemagglutinin/protease, a putative "detachase", to create a hap- mutant that could be identified and scored by its halo of cellulolytic activity. The inactivation of hap had a positive effect on colonization in the infant mice model. The above results indicate that celA is a suitable marker gene for V. cholerae and hap is an appropriate locus for insertion of foreign DNA in vaccine development. Inactivation of hap, by increasing the duration of adherence, might decrease excretion of the resulting vaccine vector strain and thus increase its immunogenicity.

Animals↗
PubMed1996

Antibody against the capsule of Vibrio cholerae O139 protects against experimental challenge.

Antiserum to the capsular polysaccharide of an opaque variant of Vibrio cholerae O139 strain MDO-12 recognizes capsular antigen in three different colonial variants of the strain, although the amount of recognition varies with the extent of opacity. The anti-capsular-polysaccharide serum, at subagglutinating doses, protected suckling mice against challenge with both the most opaque variant and the most translucent variant. Further studies indicated that the protection was associated with inhibition of intestinal colonization by the vibrios. These results thus highlight the potential importance of the capsule in immunoprophylaxis against cholera caused by V. cholerae O139.

Animals↗
PubMed1996

A nontoxic cholera enterotoxin (CT) analog is chimeric with regard to both epitypes of CT-B subunits, CT-B-1 and CT-B-2.

The gene encoding a nontoxic analog, CT-2*, of cholera enterotoxin (CT) with attenuating codon substitutions in the A subunit was introduced into the attenuated Vibrio cholerae classical biotype mutant candidate vaccine strain CVD103, which produces the B subunit (but not the A subunit) of CT-1. The recombinant strain produces a chimeric nontoxic analog holotoxin containing both CT-B-1 and CT-B-2 subunits. This offers potential advantages over CVD103 in the induction of immunity against E1 Tor biotype and V. cholerae O139 strains which produce CT-B-2. The recombinant protein may also be useful in polysaccharide-protein conjugate vaccines against both O1 and O139 serovars of V. cholerae.

Cholera Toxin↗
PubMed1995

Why do we not yet have a suitable vaccine against cholera?

The available options, past and present, of cholera vaccines have been summarized above. It is saddening but clear that, more than a century after the introduction of the first cholera vaccine, we still do not have available a suitable vaccine against cholera. The currently raging and expanding new epidemic of cholera in the Western hemisphere dramatically illustrates anew the need, although a new illustration is not necessary if one but considers the innumerable, but unnecessary, victims of cholera of the past which could have been prevented had a suitable vaccine been available earlier. Indeed, it should also be clear from this review that a suitable vaccine against cholera still eludes us and will for an additional period of time. Cholera, a disease of humans only, can be controlled by prevention of human to human transmission, i.e., by universal availability of appropriate sewage disposal and clean water--an expensive solution, but one which will also reduce the toll of the other diseases transmitted by the fecal-oral route. Short of this, we should not be diverted by less than a highly effective, and economical vaccine. The "bottleneck" resides in the difficulty of getting from the laboratory to the field: the essential, and most efficient, intermediate step being the testing of candidate vaccines in volunteers--laboratory animal models do not suffice. Cholera is now, especially under controlled conditions, a perfectly treatable disease. Additional volunteer centers, and studies in volunteers, are essential to the solution of the cholera problem in the near future.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1994

Genetic characterization of mannose-sensitive hemagglutinin (MSHA)-negative mutants of Vibrio cholerae derived by Tn5 mutagenesis.

El Tor biotype Vibrio cholerae strains express a cell-associated mannose-sensitive hemagglutinin (MSHA) which is a putative attachment factor. Several MSHA-negative mutants from V. cholerae strain JBK70 were previously generated by Tn5 mutagenesis [Finn et al., Infect. Immun. 55 (1987) 942-946]. The chromosomal DNA regions containing the Tn5 insertions were isolated from eight strains for further analysis. Nucleotide sequencing of the insertional junctions and corresponding clones containing the intact chromosomal region from the parental strain revealed the presence of several contiguous ORFs. Only two ORFs of this region had received insertions, and these showed remarkable homology to genes involved in the general secretory pathway found in several Gram- bacterial species. Proteins corresponding to the observed ORFs were visualized with the T7 promoter/RNA polymerase expression system. Marker exchange mutagenesis was used to insert kanamycin-resistance cassettes and TnphoA insertions into different locations of this region in the chromosome of wild-type V. cholerae strains. The phenotypes of these mutants showed that this DNA region is involved in MSHA production, but is not required for general extracellular protein secretion.

Amino Acid Sequence↗
PubMed1994

Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.

The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera. Amino acids involved in the enzymatic activity of CT-A have previously been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity. The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2*. Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V. cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1*. These plasmid constructs were introduced into the CT-negative V. cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India. Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay. CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT. The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin bands with GM1-ganglioside in Ouchterlony tests. However, the reactions of the modified proteins with anti-A-subunit monoclonal antibodies were weaker than the reactions with wild-type holotoxins. V, cholerae strains carrying ctxA*, with either ctxB-1 or ctxB-2, and inactivated zot genes were created by homologous recombination. The recombinant strains and the purified toxin analogs were inactive in the infant rabbit animal model.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Checkerboard immunoblotting recognizes twenty epitopes among the B subunit proteins of the cholera enterotoxin family.

Checkerboard immunoblotting (CBIB) was used to analyze the reactions of a series of monoclonal antibodies with proteins of the cholera enterotoxin (CT) family, including heat labile enterotoxins (LTs) produced by diarrheagenic strains of Escherichia coli and genetically engineered chimeric proteins in which single amino acids of the CT-B subunit protein or human (H) LT-B subunit protein were substituted for corresponding residues in porcine (P) LT-B. The result indicated that there were at least twenty different patterns of reactivity suggesting that there are at least twenty recognizable epitopes among the proteins studied. An epitope which includes Ala46 appears to be particularly important. This epitope is common to CT and H-LT but not P-LT, and the epitope is not blocked by the Gm1 ganglioside. Human convalescent sera react with this epitope. CBIB is a versatile technique for epitope analysis.

Amino Acid Sequence↗
PubMed1993

Monoclonal immunoglobulin A antibodies directed against cholera toxin prevent the toxin-induced chloride secretory response and block toxin binding to intestinal epithelial cells in vitro.

Secretory immunoglobulin A (IgA) antibodies directed against cholera toxin (CT) are thought to be important in resistance to oral challenge with virulent Vibrio cholerae, although alternative mechanisms for protection of intestinal epithelia against CT-induced fluid secretion have been proposed. The ability of anti-CT IgA to block the effects of CT on human enterocytes has not been directly tested because of the lack of a well-defined in vitro intestinal epithelial cell system to directly measure toxin action and the limited availability of purified anti-CT IgA antibodies. We have generated hybridomas that produce monoclonal IgA and IgG antibodies directed against CT by fusion of Peyer's patch cells with mouse myeloma cells after oral-systemic immunization of mice with CT and CT B-subunit protein. All of the anti-CT antibodies recognized the B subunit. Three clones (designated anti-CTB IgA-1, IgA-2, and IgA-3) which produced IgA antibodies in dimeric and polymeric forms were selected. Checkerboard immunoblotting demonstrated that IgA-1 recognized an epitope distinct from that recognized by IgA-2 and IgA-3 and that none of the antibodies were directed against the binding site of GM1, the intestinal cell membrane toxin receptor. The protective capacity of these IgAs was tested in vitro with human T84 colon carcinoma cells grown on permeable supports as confluent monolayers of polarized enterocytes. When each anti-CTB IgA was mixed with 10 nM CT and applied to the apical surfaces of T84 cell monolayers, all three IgAs blocked CT-induced Cl- secretion in a dose-dependent manner and completely inhibited binding of rhodamine-labelled CT to apical cell membranes. Thus, monoclonal anti-CTB IgA antibodies are sufficient to protect human enterocytes in vitro against CT binding and action.

Animals↗
PubMed1993

Bacterial extracellular zinc-containing metalloproteases.

Extracellular zinc-containing metalloproteases are widely distributed in the bacterial world. The most extensively studied are those which are associated with pathogenic bacteria or bacteria which have industrial significance. They are found practically wherever they are sought in both gram-negative and gram-positive microorganisms, be they aerobic or anaerobic. This ubiquity in itself implies that these enzymes serve important functions for the organisms which produce them. Because of the importance of zinc to enzymatic activity, it is not surprising that there is a pervasive amino acid sequence homology in the primary structure of this family of enzymes regardless of their source. The evidence suggests that both convergent and divergent evolutionary forces are at work. Within the large family of bacterial zinc-containing metalloendopeptidases, smaller family units are observed, such as thermolysin-like, elastase-like, and Serratia protease-like metalloproteases from various bacterial species. While this review was in the process of construction, a new function for zinc-containing metalloproteases was discovered: the neurotoxins of Clostridium tetani and Clostridium botulinum type B have been shown to be zinc metalloproteases with specificity for synaptobrevin, an integral membrane protein of small synaptic vesicles which is involved in neurotransmission. Additional understanding of the mode of action of proteases which contribute to pathogenicity could lead to the development of inhibitors, such as chelators, surrogate substrates, or antibodies, which could prevent or interrupt the disease process. Further studies of this broad family of metalloproteases will provide important additional insights into the pathogenesis and structure-function relationships of enzymes and will lead to the development of products, including "designer proteins," which might be industrially and/or therapeutically useful.

Amino Acid Sequence↗
PubMed1992

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.

Animals↗