The Entner-Doudoroff pathway is obligatory for gluconate utilization and contributes to the pathogenicity of Vibrio cholerae.

The Entner-Doudoroff (ED) pathway has recently been shown to play an important role in sugar catabolism for many organisms although very little information is available on the functionality of this pathway in Vibrio cholerae, the causative agent of cholera. In this study, activation of the genes edd and eda, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, was used as a marker of a functional ED pathway in V. cholerae. Transcriptional activation analyses and gene silencing experiments with cells grown in sugar-supplemented M9 medium demonstrated that the ED pathway is functional in V. cholerae and is obligatory for gluconate catabolism. Importantly, selective activation of the ED pathway led to concurrent elevation of transcripts of prime virulence genes (ctxA and tcpA) and their regulator (toxT). Further, lowering of these transcript levels and cholera toxin production in vitro by an ED pathway-defective mutant (strain N16961 with a Δedd mutation [Δedd(N16961) strain]) suggested the importance of this pathway in regulating V. cholerae virulence. The in vivo relevance of these data was established as the mutant failed to colonize in suckling mice intestine or to induce fluid accumulation in ligated rabbit ileal loops. Activation of the ED pathway in V. cholerae was shown to inhibit biofilm formation in vitro that could be reversed in the mutant. As further support for these results, comparative transcriptome analysis with cells grown in the presence of glucose or gluconate revealed that a functional ED pathway led to activation of a subset of previously reported in vivo expressed genes. All of these results suggest the importance of the ED pathway in V. cholerae pathogenesis.

Lessons from cholera & Vibrio cholerae.

Cholera is an acute form of diarrhoeal disease that plagued human civilization over the centuries. The sudden and explosive onset of the disease in the form of an outbreak or epidemic, coupled with high mortality and morbidity rates, had a tragic impact on the personal as well as social life of people living in the affected areas. The enormity of human sufferings led clinicians and scientists to carry out extensive research on cholera and Vibrio cholerae (the causative bacterium of the disease) leading to major discoveries that opened up novel areas of research or new disciplines in biomedical sciences. An attempt is made here to summarize some of these breakthroughs and outline their significance in broader perspectives. Finally, the possible impact of the global socio-political scenario on the spread of cholera epidemics (pandemicity of cholera) is briefly discussed.

Lymph node cells from BALB/c mice with chronic visceral leishmaniasis exhibiting cellular anergy and apoptosis: involvement of Ser/Thr phosphatase.

Visceral leishmaniasis (VL) produced in BALB/c mice through intracardial administration of Leishmania donovani amastigotes was accompanied by hepatosplenomegaly with high organ parasite load and lymphadenopathy when followed up to 4-months or so. To elucidate the mechanism of immunosuppression associated with VL, we report here progressive impairment of the proliferative response of lymph node cells (lymphocytes) from infected animals (I-LNC) to in vitro stimulation with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) that could be related to the downregulation of PKC and MAP kinase (ERK 1/2) activation process. Further, pretreatment of I-LNC with the protein phosphatase inhibitor okadaic acid (OA), but not with calyculin A or sodium orthovanadate, significantly restored their proliferative response as well as PMA-induced activation of PKC. A population of LNC (primarily T-lymphocytes) from chronically infected animals was shown to undergo apoptosis, the number of which increased considerably following PMA+ Io stimulation. The apoptotic pathway, which was followed through binding of cells to Annexin V, activation of caspase-3 and fragmentation of DNA, involved destabilization of mitochondria, probably as a result of downregulation of PKC and Bcl-2. Interestingly, prior incubation of I-LNC with OA reversed the state of cell cycle arrest (anergy) and apoptosis through progression of cells from G0/G1 to S and G2/M phases with transcriptional activation of IL-2 and IL-2R genes. Our results suggest that the cellular (immune) dysfunction in VL could be attributed to dephosphorylation of key molecules in the T-lymphocyte signaling pathway by Ser/Thr phosphatase leading to their inactivation.

Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae.

The outer-membrane protein OmpW of Vibrio cholerae was studied with respect to its structure, functional properties and regulation of expression. On SDS-PAGE, the membrane-associated form of OmpW protein (solubilized by either 0.1 % or 2 % SDS at 25 degrees C) migrated as a monomer of 19 kDa that changed to 21 kDa on boiling. The protein was hyperexpressed in Escherichia coli in the histidine-tagged form and the purified His(6)-OmpW (heated or unheated) migrated as a 23 kDa protein on SDS-PAGE. Circular dichroism and Fourier-transform infrared spectroscopic analyses of the recombinant protein showed the presence of beta-structures ( approximately 40 %) with minor amounts (8-15 %) of alpha-helix. These results were consistent with those obtained by computational analysis of the sequence data of the protein using the secondary structure prediction program Jnet. The recombinant protein did not exhibit any porin-like property in a liposome-swelling assay. An antiserum to the purified protein induced a moderate level (66.6 % and 33.3 % at 1 : 50 and 1 : 100 dilutions, respectively) of passive protection against live vibrio challenge in a suckling mouse model. OmpW-deficient mutants of V. cholerae strains were generated by insertion mutagenesis. In a competitive assay in mice, the intestinal colonization activities of these mutants were found to be either only marginally diminished (for O1 strains) or 10-fold less (for an O139 strain) as compared to those of the corresponding wild-type strains. The OmpW protein was expressed in vivo as well as in vitro in liquid culture medium devoid of glucose. Interestingly, the glucose-dependent regulation of OmpW expression was less prominent in a ToxR(-) mutant of V. cholerae. Further, the expression of OmpW protein was found to be dependent on in vitro cultural conditions such as temperature, salinity, and availability of nutrients or oxygen. These results suggest that the modulation of OmpW expression by environmental factors may be linked to the adaptive response of the organism under stress conditions.

Immunosuppression in hamsters with progressive visceral leishmaniasis is associated with an impairment of protein kinase C activity in their lymphocytes that can be partially reversed by okadaic acid or anti-transforming growth factor beta antibody.

Progressive visceral infection of golden hamsters by Leishmania donovani amastigotes led to gradual impairment of the proliferative responses of their splenic or peripheral blood mononuclear cells (SPMC or PBMC, respectively) to in vitro stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io). Removal of macrophage-like adherent cells from SPMC or PBMC of infected animals (I-SPMC or I-PBMC) was earlier shown to restore almost completely their lymphoproliferative responses to PMA plus Io. The present study was directed to evaluate the status of protein kinase C (PKC), a molecule(s) known to play a key role in the lymphoproliferative process. Our results demonstrate that PKC activities (Ca(2+), phosphatidyl serine, and diacyl glycerol dependent) in the cytosolic fraction of untreated nonadherent I-SPMC or I-PBMC as well as in the membrane fraction of PMA-treated cells were decreased significantly relative to those for normal controls. However, removal of adherent cells from I-SPMC or I-PBMC and subsequent overnight in vitro cultivation of nonadherent cells (lymphocytes) resulted in significant restoration of PKC activity in the cytosolic or membrane fraction of untreated or PMA-treated cells, respectively. Partial, though significant, restoration of PKC activity could also be achieved in the membrane fraction of PMA-treated cells following overnight in vitro treatment of I-SPMC or I-PBMC with the Ser/Thr phosphatase inhibitor okadaic acid (OA) or an anti-transforming growth factor beta (anti-TGF-beta) neutralizing antibody. These results correlated well with the ability of OA or the anti-TGF-beta antibody to restore the lymphoproliferative response of I-SPMC or I-PBMC following stimulation with PMA plus Io. Interestingly enough, immunoblotting experiments failed to show any reduction in the level or translocation (following PMA treatment) of conventional PKC isoforms in the SPMC or PBMC of infected animals compared to those of normal controls. The results presented in this study suggest that the adherent cells generated in the SPMC or PBMC of infected animals exert a suppressive effect on the proliferative response of nonadherent cells (lymphocytes) which is likely to be mediated through the downregulation of the activation pathway involving PKC and its downstream molecules such as mitogen-activated protein kinases. Further, the observed suppression of PKC activity and subsequent lymphoproliferative responses can be attributed to alternations in the intracellular phosphorylation-dephosphorylation events. The relevance of these results is discussed in relation to the role of TGF-beta, levels of which are known to be elevated in visceral leishmaniasis.

Infection pattern and immune response in the spleen and liver of BALB/c mice intracardially infected with Leishmania donovani amastigotes.

Intracardial inoculation of BALB/c mice with Leishmania donovani amastigotes induced progressive visceral leishmaniasis (VL) with increasing splenic parasite load when followed upto 4-month postinfection period. In contrast, the liver parasite load reached maximum around 2-month postinfection period following which it started declining. The infection pattern differed somewhat from the earlier reports on mouse model of VL induced by intravenous inoculation of parasites with respect to the duration as well as magnitude of parasite burden in the organs (liver and spleen) and associated hepatosplenomegaly. Immunosuppression in mice with progressive VL was manifested in the form of impairment of proliferative response of the splenic mononuclear cells (SPMC) to in vitro stimulation with leishmanial antigen or the mitogen concanavalin A (ConA), although ConA stimulated cells were found to be capable of IL-2 and IFN-gamma synthesis. Differential expression of activating (IL-2, IFN-gamma and TNF-alpha) as well as deactivating (IL-4 and TGF-beta) cytokines was demonstrable in the spleen and liver of animals during the course of infection. Further, the synthesis of inducible nitric oxide synthase (iNOS) enzyme increased considerably in the liver as well as in the spleen of 4-month infected animal with parallel increase in the transcripts of the iNOS activating cytokines IFN-gamma and TNF-alpha. The temporal variation in the organ specific immune response could be related to the differential control of parasite burden in the liver and spleen of the infected host.

Purification and characterization of a potent hemolytic toxin with phospholipase A2 activity from the venom of Indian Russell's viper.

A potent toxin with phospholipase A2 (PLA2) and hemolytic activity in vitro was purified from the Russell's viper venom of eastern India (RVV-EI). The purified protein (RVV-PFIIc') of 15.3 kDa molecular weight, and a lethal toxicity dose (LD50i.p.) of 0.1 mg/kg body weight, was the most toxic PLA2 so far reported from the Indian subcontinent. The material also possessed anticoagulant activity as it enhanced the prothrombin induced plasma clotting time in vitro. The PLA2 toxin (RVV-PFIIc') was shown to be different from other PLA2s of RVV in respect to one or more of these parameters e.g. molecular weight, isoelectric pH, in vivo toxicity, specific activity of the enzyme and certain other biological activities. The first 19 amino terminal sequence (NLFQFAEMIVKMTGKEAVH) of RVV-PFIIc' showed variable degree of homology (42.1-94.7%) with those of other RVV-PLA2s described in the literature. Antisera raised against RVV-EI or RVV-PFIIc', though completely neutralized the in vivo lethal toxicity of RVV-EI or RVV-PFIIc', failed to inhibit their PLA2 activity in vitro thereby suggesting that in vivo toxicity and in vitro activity of the enzyme may not be directly related. Apart from RVV-PFIIc', at least two other PLA2 isozymes were found to be present in RVV-EI that were distinct from RVV-PFIIc' in respect to their molecular, biological as well as serological properties. The significance of these and related data in antivenom therapy is discussed.

Vibrio pathogenicity island and cholera toxin genetic element-associated virulence genes and their expression in non-O1 non-O139 strains of Vibrio cholerae.

A non-O1 non-O139 Vibrio cholerae strain, 10259, belonging to the serogroup O53 was shown to harbor genes related to the vibrio pathogenicity island (VPI) and a cholera toxin (CT) genetic element called CTX. While the nucleotide sequence of the strain 10259 tcpA gene differed significantly (26 and 28%) from those of O1 classical and El Tor biotype strains, respectively, partial sequence analysis data of certain other VPI-associated genes (aldA, tagA, tcpP/H, toxT, acfB/C, and int) and intergenic regions (tcpF to toxT and tcpH to tcpA) of the strain showed only minor variations (0.4 to 4.8%) from corresponding sequences in O1 strains. Strain 10259 also contained CTX element-associated toxin genes with sequences almost identical to those of O1 strains. Growth of the organism in Luria broth (LB) under ToxR inducing conditions (30 degrees C and pH 6.5) led to transcriptional activation of tcpP/H, toxR, toxT, and tcpA genes, but not of ctxA, as determined by reverse transcription-PCR (RT-PCR). Subsequent analysis revealed that strain 10259 possessed only two copies (instead of three or more copies found in epidemic-causing O1 or O139 strains) of the heptanucleotide (TTTTGAT) repeats in the intergenic region upstream of ctxAB. Therefore, a strain 10259 mutant was generated by replacement of this region with a homologous region (1.4 kb) derived from a V. cholerae O1 classical biotype strain (O395) that contained seven such repeats. The resultant recombinant strain (10259R) was found to be capable of coordinately regulated expression of toxT, ctxA, and tcpA when grown under the ToxR inducing conditions. Serological studies also demonstrated that the recombinant strain produced TcpA and a significantly ( approximately 1,000-fold) higher level of CT in vitro compared to that of the parent strain. Virulence gene expression in two other non-O1 non-O139 strains (serogroup O37) containing VPI and the CTX element was studied by RT-PCR and serological assay. One strain (S7, which was involved in an epidemic in Sudan in 1968) showed coordinately regulated expression of virulence genes leading to the production of both CT and TcpA in LB medium. However, the other strain, V2, produced RT-PCR-detectable transcripts of toxT, ctxA, or tcpA genes in the early phase (6 h), but not in the late phase (16 h) of growth in LB medium. These results are consistent with the low levels of production of CT and TcpA by the strain that were serologically detectable. The significance of these results is discussed in relation to the role of virulence genes and their expression to the pathogenic potential of V. cholerae strains belonging to non-O1 serogroups.

Methotrexate conjugate with branched polypeptide influences Leishmania donovani infection in vitro and in experimental animals.

Methotrexate (MTX) has been coupled to various structurally related, polycationic (poly[Lys(DL-Ala(m))] (AK), poly[Lys(Ser(i)-DL-Ala(m))] (SAK), poly[Lys(DL-Ala(m)-Leu(i))] (ALK)), or amphoteric (poly[Lys(Glu(i)-DL-Ala(m))] (EAK)) synthetic branched polypeptides containing poly[L-Lys] backbone by the aid of BOP reagent. The average degree of MTX incorporation was found to be dependent on the charge properties of the polymer. Under the experimental conditions used, the molar substitution ratio achieved was higher for polycations (25%) than for the amphoteric polypeptide (10%). We have studied the effect of polycationic polypeptides on Leishmania donovani infection. Results demonstrated that MTX conjugates in which the drug is covalently attached to carrier have pronounced leishmanicid activity. In this communication we showed that (a) a branched polypeptide-methotrexate conjugate with a polycationic carrier (ALK) increases the effect of MTX against Leishmania donovani infection in mice; (b) the covalent bond between the carrier and methotrexate is essential for both in vivo and in vitro activity; and (c) the number of Leishmania donovani parasites in infected macrophages are markedly reduced in conjugate treated animals. In vitro observation might also indicate that the MTX conjugate exhibits an effect through an uptake by macrophages which is different from that of the free drug.