Efficacy of the phosphorylation of synthetic peptides by purified catalytic subunit of PKA (PKAcat) from bovine lens depends on the amino acid sequence of the peptides.

Protein kinase (PK) A catalytic (PKAcat) subunit was purified to homogeneity from bovine lens using a 100-kDa cut-off membrane filtration followed by different chromatographic procedures. The molecular weight of PKAcat was found to be 41 kDa. The kinase phosphorylates histone IIIs and other synthetic modified peptides of VRKRTLRRL with different amino acid environment. The extent of phosphorylation depends not only on the presence of Ser or Thr (phosphorylating residues) but also on other surrounding amino acid residues. Although some peptides compete in phosphorylating histone, they are not very significant. The result suggests that the extent of phosphorylation depends on the amino acid residue(s) surrounding phosphorylable residue(s) on the peptide.

Modification of the multiplex PCR for unambiguous differentiation of the El Tor & classical biotypes of Vibrio cholerae O1.

BACKGROUND & OBJECTIVES: Biotyping of Vibrio cholerae O1 using multiplex PCR (ctxA-tcpA) exploits the nucleotide sequence differences of the major subunit protein of the toxin co-regulated pilus (TCP) gene (tcpA) to differentiate between the classical and El Tor biotypes. However, the presence of classical biotype specific tcpA amplicon with the El Tor strains often complicates the interpretation. The effect of PCR variables on the amplification of biotype specific tcpA in the multiplex PCR has been investigated. METHODS: Reference strains of toxigenic V. cholerae O1 belonging to classical and El Tor biotypes were selected to optimize the PCR variables for the unambiguous biotype determination by multiplex PCR. RESULTS: In the multiplex PCR assay, a reduction in the reaction volume from 100 microliters to 25 microliters and the annealing temperature of 64 degrees C, the El Tor strain produced ctxA amplicon (302 bp) along with tcpA amplicons of 618 bp and 472 bp which are specific for classical and El Tor tcpA respectively. The simplex PCR with biotype specific tcpA primer pairs showed the amplification of either 472 bp or 618 bp tcpA amplicon with El Tor template. With the classical biotype strain, the specific primer pair yielded tcpA amplicon of the expected size. Lowering of PCR annealing temperature from 64 to 60 degrees C resulted in the elimination of the amplification of the nonspecific tcpA amplicon with El Tor strain. INTERPRETATION & CONCLUSION: A comparison of the theoretical melting temperature (Tm) values of the reacting primers, and their alignment to the biotype specific tcpA revealed the basis of unambiguous biotyping of V. cholerae O1 at a PCR annealing temperature of 60 degrees C.

Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW.

The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 ( approximately 98%) of 233 were positive for toxR. None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW- and toxR-specific probes in DNA dot blot assay. While the V. cholerae strains reacted with ompW probe, only one (V. mimicus) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus, V. splendidus, V. alginolyticus, V. fluvialis, V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.

Antibodies (IgG) to lipopolysaccharide of Vibrio cholerae O1 mediate protection through inhibition of intestinal adherence and colonisation in a mouse model.

An antiserum raised against purified lipopolysaccharide (LPS) of a Vibrio cholerae O1 strain (Co366) induced passive protection against challenge with the parent as well as other O1 organisms but not against O139 or non-O1/non-O139 organisms. A considerable level of protection against O1 strains was also observed with the IgG fraction of the antiserum which inhibited intestinal adherence and colonisation. The monovalent Fab(IgG) fragment, on the other hand, showed only a low level of protection. Interestingly, purified LPS failed to inhibit intestinal colonisation by the parent strain (Co366), thereby suggesting that the cell surface LPS moieties of vibrios may not be directly involved in the colonisation process. It may be concluded that the anti-LPS antibodies induce passive protection through microagglutination and/or immobilisation of vibrios which do not allow the organisms to adhere to and colonise the intestine.

Molecular characterization of a new variant of toxin-coregulated pilus protein (TcpA) in a toxigenic non-O1/Non-O139 strain of Vibrio cholerae.

A toxigenic non-O1/non-O139 strain of Vibrio cholerae (10259) was found to contain a new variant of the toxin-coregulated pilus (TCP) protein gene (tcpA) as determined by PCR and Southern hybridization experiments. Nucleotide sequence analysis data of the new tcpA gene in strain 10259 (O53) showed it to be about 74 and 72% identical to those of O1 classical and El Tor biotype strains, respectively. The predicted amino acid sequence of the 10259 TcpA protein shared about 81 and 78% identity with the corresponding sequences of classical and El Tor TcpA strains, respectively. An antiserum raised against the TCP of a classical strain, O395, although it recognized the TcpA protein of strain 10259 in an immunoblotting experiment, exhibited considerably less protection against 10259 challenge compared to that observed against the parent strain. Incidentally, the tcpA sequences of two other toxigenic non-O1/non-O139 strains (V2 and S7, both belonging to the serogroup O37) were determined to be almost identical to that of classical tcpA. Further, tcpA of another toxigenic non-O1/non-O139 strain V315-1 (O nontypeable) was closely related to that of El Tor tcpA. Analysis of these results with those already available in the literature suggests that there are at least four major variants of the tcpA gene in V. cholerae which probably evolved in parallel from a common ancestral gene. Existence of highly conserved as well as hypervariable regions within the sequence of the TcpA protein would also predict that such evolution is under the control of considerable selection pressure.

Antibodies to the truncated (short) form of 'O' polysaccharides (TFOP) of Vibrio cholerae O139 lipopolysaccharides protect mice against experimental cholera induced by encapsulated O139 strains and such protection is mediated by inhibition of intestinal colonization of vibrios.

An antiserum raised against the lipopolysaccharides (LPS) of an encapsulated Vibrio cholerae O139 strain was shown to induce passive protection against challenge with O139, but not O1, organisms. Subsequent experiments, however, revealed that the purified LPS, obtained by the conventional phenol-water extraction method, contained capsular polysaccharide (CPS) material. Therefore, another antiserum was raised by immunization with electrophoresed gel-cut material containing only the truncated (short) form of 'O' polysaccharides (TFOP) linked to the core of O139 LPS. Anti-TFOP antibodies and their Fab (IgG) fragments induced passive protection against challenge with colonial variants of encapsulated O139 strains and such protection was mediated by inhibition of intestinal colonization. These results suggest that it is possible to engender protection against encapsulated O139 strains by using TFOP material (devoid of CPS) as the immunogen.

Immunosuppression in hamsters with progressive visceral leishmaniasis: an evaluation of the role of nitric oxide toward impairment of the lymphoproliferative response.

The progressive visceral infection caused in golden hamsters by Leishmania donovani amastigotes led to gradual impairment of the proliferative response of their splenic (SPMC) or peripheral blood (PBMC) mononuclear cells to in vitro stimulation with leishmanial antigen, with mitogen (concanavalin A), and even with a combination of phorbol myristate acetate (PMA) and ionomycin (Io). Removal of macrophage-like adherent cells from SPMC or PBMC of infected animals, however, almost completely restored their proliferative response to PMA + Io, thus ruling out the possibility of any intrinsic defect in the signal-transduction pathways of lymphocyte activation and proliferation. Subsequent studies demonstrated that the generation of soluble mediators such as nitric oxide by these adherent cells is responsible, albeit partially, for the down-regulation of the lymphoproliferative response in hamsters with visceral leishmaniasis.

Therapeutic and prophylactic uses of protein A in the control of Leishmania donovani infection in experimental animals.

The role of the immunomodulator Protein A (PA) (from Staphylococcus aureus, Cowan I strain) in the control of leishmanial infection was studied in experimental animals. Treatment of Leishmania donovani infected hamsters with PA led to a moderate level of reduction of parasite load in their spleen (68%) and liver (46%). However, combination therapy of PA with the antileishmanial drug stibanate induced a more marked reduction of the spleen (88%) and liver (85%) parasitemia compared to that induced by PA or drug treatment alone. Similar results were also obtained with L. donovani infected BALB/c mice as the combination therapy of PA and stibanate led to a significant reduction (84%) of liver parasite load in comparison to that induced by PA (38%) or drug (61%) treatment alone. Apart from its therapeutic use, PA could also be used as a prophylactic agent in the control of leishmanial infection. Thus, treatment of hamsters with PA before leishmanial challenge significantly reduced their organ parasite load (by 59-78%) compared to that observed in infected controls without prior PA treatment. The antileishmanial effect of PA was likely to be mediated through the activation of macrophages leading to an enhancement of their phagocytic as well as leishmaniacidal activities. Subsequent studies demonstrated that PA treatment led to an increased production of nitric oxide by macrophages which could primarily be responsible for their enhanced parasite killing ability.

Characterization of a 20-kDa pilus protein expressed by a diarrheogenic strain of non-O1/non-O139 Vibrio cholerae.

A diarrheogenic strain of non-O1/non-O139 Vibrio cholerae (10,325) belonging to serogroup O34 was earlier shown to express a new type of pilus composed of a 20-kDa subunit protein. Amino-terminal sequence data (determined up to 20 amino acid residues) of this protein showed it to be different from the subunit proteins of other known types of pili of V. cholerae. On the other hand, it showed complete homology with the corresponding sequence of a 22-kDa outer membrane protein (OmpW) of V. cholerae. Expression of 10,325 pili was favored in AKI rather than in NB medium and at 30 degrees C rather than at 37 degrees C. Further, cultural conditions favoring pilus expression also enhanced autoagglutination and adherence properties of strain 10,325. An antiserum to the 20-kDa protein induced passive protection against challenge with the parent organism 10,325, but not against V. cholerae O1 strains. Such protection was shown to be mediated by inhibition of intestinal colonization in vivo.

A search for cholera toxin (CT), toxin coregulated pilus (TCP), the regulatory element ToxR and other virulence factors in non-01/non-0139 Vibrio cholerae.

Twenty-four selected non-O1/non-O139 Vibrio cholerae strains were examined for the presence of virulence associated genes like ctxA, tcpA, toxR and the repetitive sequence (RS element). Seventeen of these were isolated from diarrhoeal stool samples while the remaining seven were of local environmental origin. Nine and four respectively of these strains were positive for ctxA and tcpA by Multiplex PCR analysis. The majority (16 out of 18 tested) of the strains (including the four tcpA + strains) contained toxR sequences as determined by another PCR assay. The presence of RS element was demonstrable in ctxA+ strains only. Interestingly, three of these non-O1/non-O139 strains were shown to contain all the three virulence associated genes (ctxA, tcpA and toxR) as well as the RS element. Two of these belonged to serogroups 037 (V2) and 064 (CG15) while the third one (V315-1) was untypable. These three strains also produced cholera toxin, expressed toxin coregulated pilus (TCP) and/or TcpA related antigens when grown under appropriate culture conditions. Southern hybridization analysis of their chromosomal DNA fragments using DNA probes representing ctxA, zot, ace and RS element revealed that the strains V2 and CG15 contained, at least, two complete copies of the CTX genetic element, while the strain V315-1 had three or more copies of the same. Presence of the RS element in these strains led to tandem duplication of the CTX genetic element in the chromosome of V2 and V315-1, but not in CG15 where the copies were likely to be present at different loci. These results also indicate the presence of additional copies of incomplete "core region' with zot and ace genes, but not ctxA, in strains V2 and CG15. The significance of these results in terms of the pathogenic and epidemic potential of V. cholerae strains is discussed.

Subpopulations of T lymphocytes in the peripheral blood and lymph nodes of Indian kala-azar patients.

Distribution of T cells in the peripheral blood and lymph nodes of Indian kala-azar (KA) patients was studied by using appropriate phenotypic markers for CD2+, CD4+ and CD8+ cells. Significant reduction in the CD2+, CD4+ cell numbers as well as CD4+/CD8+ cell ratio was noted in the peripheral blood of active KA cases. Such alteration in the T cell population appeared to be a manifestation of the disease process as it showed a tendency to return close to normalcy several months after successful chemotherapy. Histopathological studies of KA patients with lymphadenopathy demonstrated gradual destruction of lymph node follicular architecture which correlated well with the severity and duration of illness. Massive infiltration of CD2+ cells in the cortical region of lymph node was evident. The observed preponderance of CD4+ cells over CD8+ ones in these infiltrates was in sharp contrast to the distribution pattern of these cells in the periphery. Significance of these findings is discussed in relation to the current concepts on the immunology of leishmaniasis and related diseases.

Serum antibacterial and antitoxin responses in clinical cholera caused by Vibrio cholerae O139 Bengal and evaluation of their importance in protection.

Vibrio cholerae O139 Bengal strain was the causative agent of the recent epidemics of cholera in India and Bangladesh. We studied antibacterial and antitoxin immune responses in acute and convalescent phase paired sera collected from seven of these cholera patients. Significant rise in the levels of both antibacterial and antitoxin antibodies was demonstrable in the sera of convalescent cholera patients. Antibacterial antibodies, directed primarily against O139 lipopolysaccharides (LPS), belonged to IgM class, while antitoxin antibodies were of IgG and IgA class and neutralized cholera toxin. The convalescent sera, however, showed no increase in the reactivity towards V. cholerae O1 whole cells or their LPS preparation. Immunoblotting experiments revealed that the convalescent, but not the acute, phase serum recognized the truncated form of LPS characteristics of O139 strains. Convalescent serum also induced definite protection against O139, but not O1, challenge in experimental animal model. Further studies showed that such protection was probably mediated by antibodies inhibiting intestinal colonization of O139 organisms. These results suggest that critical difference(s) exists between the immunogenic somatic components of V. cholerae O1 and O139 organisms that are of considerable importance in protection against cholera.

Adherence & colonization properties of Vibrio cholerae & diarrhoeagenic Escherichia coli.

Bacterial adherence to host cells is the initial key step towards colonization and establishment of infection within the host. The adherence process requires the participation of two components: an 'adhesin' (adherence or colonization factor) of bacteria and a 'receptor' on the host (eucaryotic) cell surface. Many bacteria express several distinct and alternative mechanisms of cell adherence depending on the environmental conditions and nature of the adhesins as well as receptors. Bacteria causing gastrointestinal infection need to penetrate the mucous layer before attaching themselves to epithelial and other absorptive cells in the intestine. This attachment is usually mediated by fimbriae or pilus structures although other cell surface components of bacteria may also take part in the process. Adherent bacteria colonize intestinal epithelium by multiplication and initiation of a series of biochemical reactions inside the target cell through signal transduction mechanisms (with or without the help of toxins). Alternatively, adherent bacteria induce extensive rearrangement of the cytoskeletal structure of the epithelial cell thereby making more intimate contact with the cell or even forcing their entry into it. This is followed by bacterial multiplication and intercellular spread leading to eventual death of the target cell. Available information on the adherence and colonization properties of V. cholerae and E. coli, the two important causative agents of gastrointestinal illness in man, is discussed and summarized in this article.

Phenotypic expression of a mannose-sensitive hemagglutinin by a Vibrio cholerae O1 E1Tor strain and evaluation of its role in intestinal adherence and colonization.

A Vibrio cholerae O1 strain (1150) of the EIT or biotype and Ogawa serotype with haemagglutination (HA) activity was subjected to TnphoA mutagenesis. Out of several mutants isolated, one HA- and another HA+ mutant were further characterised. The HA- mutant showed about 50% reduction in its intestinal adherence capacity in vitro and about 9-fold decrease of its colonisation ability in vivo, as compared to the wild-type strain. Subsequent studies showed that the HA activity of strain 1150 was mediated by a mannose-sensitive haemagglutinin (MSHA). Thus, the phenotypic expression of MSHA appears to be partly responsible for the intestinal adherence and colonisation properties of strain 1150.

Immunoglobulin G subclass-specific antileishmanial antibody responses in Indian kala-azar and post-kala-azar dermal leishmaniasis.

Antileishmanial antibody responses in the sera of Indian kala-azar (KA) and post-KA dermal leishmaniasis (PKADL) patients were analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments using immunoglobulin G (IgG) class- and subclass-specific reagents. All sera showed antileishmanial reactivities in IgG ELISA which followed the order IgG1 > IgG2 > IgG3, with very little IgG4. Immunoblot analysis with IgG class-specific reagents revealed variable patterns of reactivity by KA and PKADL sera, although certain common bands around the 60- to 63-kDa regions were discernible. Sera from antimony-unresponsive KA cases, on the other hand, strongly recognized two bands at around 20 to 22 kDa, in addition to other bands in the high-molecular-mass region. Further analysis showed that the 28-kDa band was preferentially recognized by the IgG2 isotype, while 20- to 22-kDa and 60- to 63-kDa bands were recognized by the IgG1 isotype. Antibodies belonging to the IgG3 isotype reacted to antigens primarily in the region of 14 to 34 kDa and persisted in patients even several months after cure. Immunoblot studies also revealed the presence of a nonspecific band which arose as a result of binding between a 66-kDa leishmanial antigen and streptavidin. Finally, the results presented in this study suggest that certain leishmanial antigens preferentially stimulate the synthesis of a particular IgG subclass(es), depending on the nature of such antigens or their epitopes.

A comparative study of the properties of Vibrio cholerae O139, O1 and other non-O1 strains.

Vibrio cholerae O139 organisms isolated from different parts of India and from Bangladesh were characterised with respect to their haemagglutination (HA) activity, plasmid content, cholera toxin (CT) production, cell surface protein and lipopolysaccharide (LPS) profiles, and antigenic properties. Of 28 V. cholerae O139 isolates tested, 14 (50%) were shown to agglutinate chicken erythrocytes; the HA activity was sensitive to D-mannose 0.1%. In parallel experiments, 12 (92.3%) of 13 V. cholerae O1 (El Tor) and 12 (75%) of 16 non-O1, non-O139 strains agglutinated chicken erythrocytes. Plasmid analysis of 32 O139 isolates showed that 12 (37.5%) carried one or more plasmids of 35.8-2.6 MDa. Plasmids were not detected in any of the V. cholerae O1 strains, although plasmids were demonstrable in 35% of the non-O1, non-O139 strains tested. V. cholerae O139 isolates showed an ability to produce CT that depended on media composition and other cultural conditions. A comparison of envelope and outer-membrane protein profiles between O1 and O139 isolates failed to show any significant differences. LPS analysis of O139 isolates revealed that these organisms were devoid of long "O" side-chain polysaccharides. Some of the non-O1, non-O139 strains also showed similar LPS profiles whereas others showed the presence of long repetitive "O" side-chain polysaccharides similar to those seen in O1 organisms. An antiserum raised against V. cholerae O1 strain O395 did not show any significant reactivity towards O139 and non-O1, non-O139 strains although it reacted with other O1 strains. Furthermore, the anti-O1 serum induced marked protection against challenge with an O1 strain but not with an O139 strain in passive protection experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Subpopulations of T lymphocytes in the peripheral blood, dermal lesions and lymph nodes of post kala-azar dermal leishmaniasis patients.

Distribution of different subpopulations of T cells in the dermal lesions, lymph nodes and peripheral blood of post kala-azar dermal lesihmaniasis (PKADL) patients was studied by using appropriate phenotypic markers for CD2+, CD4+ and CD8+ cells. Histopathological studies of skin lesions showed marginal to massive infiltration of mononuclear cells depending upon the duration of illness and type of lesions. Thus, while the hypopigmented patches were represented by small focal collections of lymphocytes with scanty parasites in the dermis, these were replaced at the nodular stage with massive granulomas consisting of lymphocytes, plasma cells and histiocytes with numerous amastigotes. The involvement of CD4+ and CD8+ cell types in these lesions also showed a gradual change from the appearance of a few cells of both the phenotypes in early hypopigmented type to massive accumulation of cells, primarily of CD8+ phenotype, in the granuloma of nodular type. However, the observed preponderance of CD8+ cells at the lesion site of chronic PKADL patients is in contrast to their peripheral blood CD4+/CD8+ cell ratio (1.9:1) which remained within the normal limits. Similar studies of lymph nodes from PKADL patients with lymphadenopathy revealed infiltration of the cortical areas by T cells which were more of CD8+ than CD4+ phenotypes. All these results document the involvement of CD8+ cells in leishmanoid lesions. Thus, it is likely that these cells, in association with appropriate subpopulations of CD4+ cells, play a profound role in the evolution of dermal pathology in PKADL.

Expression of antigenically distinct fimbriae with hemagglutination and HeLa cell adherence properties by an enteroaggregative Escherichia coli strain belonging to the enteropathogenic serogroup.

An enteroaggregative Escherichia coli (EAggEC) strain (DS92), isolated from a case of infantile diarrhea, was shown to express mannose-resistant hemagglutination and HeLa cell adhering properties when grown at 37 degrees C but not at 28 degrees C. Cellular adherence properties of DS92, which belonged to enteropathogenic serogroup 0125, were shown to correlate well with the expression of fimbriae that were encoded by a 112 kb plasmid. The fimbriae of the EAggEC strain DS92 were composed of 20 kDa subunit proteins and were serologically distinct from fimbrial or non-fimbrial cell surface antigen(s) of other diarrheagenic E. coli strains including the reference EAggEC strain 17-2. Interestingly, the 20-kDa fimbrial protein was found to be antigenically related to 18- and 14.5-kDa cell surface proteins of two other locally isolated EAggEC strains belonging to the enteropathogenic serogroup 086.

Epidemic isolates of Vibrio cholerae 0139 express antigenically distinct types of colonization pili.

Vibrio cholerae belonging to the recently described serogroup 0139, which are responsible for the current cholera epidemics in India and Bangladesh, were shown to express pilus-like structures partially cross-reacting with the toxin-coregulated pilus of V. cholerae strain (0395) belonging to the 01 serogroup and classical biotype. The 0139 pili were composed of 20 kDa subunit proteins which were antigenically related to the 20 kDa pilus protein of another diarrhoeagenic non-01 V. cholerae strain (serogroup 034) isolated earlier. The pili described in this study were found to be involved in the intestinal colonization process and, therefore, may contribute towards the virulence of the 0139 epidemic isolates.

A 20-kDa pilus protein with haemagglutination and intestinal adherence properties expressed by a clinical isolate of non-01 Vibrio cholerae.

A clinical isolate of non-01 V. cholerae (10325) was shown to exhibit higher haemagglutination and intestinal adherence activities in vitro when grown in enriched media, such as trypticase soy broth (TSB) as compared to those of cells grown in a synthetic Tris-buffered or 'T'-medium. A comparison of their cell-surface protein and lipopolysaccharide profiles suggested the involvement of a 20-kDa protein in the cellular adherence process. An antiserum, raised specifically against the 20-kDa protein, recognised pilus structures on the surface of TSB grown cells. Further studies showed that the pilus was morphologically as well as antigenically distinct from toxin coregulated pilus (TCP) or other types of pili expressed by both 01 and non-01 organisms. Inhibition data established the involvement of the 20-kDa protein in haemagglutination as well as intestinal tissue adherence activities of the parent organism.