Aimed at identification and structural characterization of novel putative therapeutic targets in H. pylori, the etiological agent of numerous gastrointestinal diseases including peptic ulcer and gastric cancer, the present study comprised of three phases. First, through subtractive analysis of metabolic pathways of Helicobacter pylori HPAG1 and human, as documented in the KEGG database, 11 pathogen-specific pathways were identified. Next, all proteins involved in these pathogen-specific pathways were scrutinized in search of promising targets and the study yielded 25 candidate target proteins that are likely to be essential for the pathogen viability, but have no homolog in human. The lipopolysaccharide (LPS) biosynthesis pathway was found to be the largest contributor (nine proteins) to this list of candidate proteins. Considering the importance of LPS in H. pylori virulence, 3D structural models of three predicted target enzymes of this pathway, namely 2-dehydro-3-deoxy-phosphooctonate aldolase, UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase and Phosphoheptose isomerase, were then built up using the homology modeling approaches. Binding site analysis and docking of the known biological substrate PEP to 2-dehydro-3-deoxyphosphooctonate aldolase revealed the potential binding pocket present in the single monomeric form of the enzyme and identified 11 amino acid residues that might play the key roles in this protein-ligand interaction.

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A sialic acid binding lectin, AchatininH, was purified in single step from the hemolymph of the land snail, Achatina fulica, by the affinity chromatography on sheep submaxillary mucin coupled to Sepharose 4B. The yield of the lectin was found to be 3 mg from 100 ml of hemolymph. The homogeneity of the lectin was established by alkaline gel electrophoresis, immunodiffusion, immunoelectrophoresis and analytical isoelectrophoresis. The molecular weight of the native protein was 242,000, having identical subunits of Mr 15,000. The lectin agglutinated rabbit erythrocytes in the presence of Ca2+. The inhibition study clearly suggests that the binding site of the lectin recognizes sialic acid as the immunodominant sugar. This was further confirmed by the observation that there was a marked decrease of agglutinating activity of the lectin with neuraminidase treated rabbit erythrocytes and asialofetuin was unable to inhibit the activity of AchatininH. Among the inhibitors used the glycoconjugate containing alpha 2----6 linkages of N-acetylneuraminic acid with subterminal galactopyranose or 2-acetamido-2-deoxy-galactopyranose residue was found to be better inhibitor than that containing alpha 2----3 linkages of N-acetyl neuraminic acid. Besides that sialoglycoprotein containing both N and O type of glycosidic linkages plays an important role in binding with the lectin. Fetuin was found to be the best inhibitor.

Toxoplasmosis is a well-documented cause of bad obstetric history (BOH) and a major reason of congenitally-acquired infection. The study was conducted to determine the seropositivity of toxoplasmosis in women with BOH, attending the antenatal clinic of the Mamata General Hospital, Khammam, Andhra Pradesh, India. The study subjects included 105 antenatal women with BOH and 105 antenatal women who had previous normal deliveries. A serological evaluation was carried out to determine the presence of Toxoplasma gondii-specific IgG and IgM antibodies, using commercial diagnostic kits, by the enzyme-linked immunosorbent assay method. The seropositivity for Toxoplasma was 49.52% in the study group compared to 12.38% in the control group. The difference in seropositivity was significant (p=0.00). The seroprevalence gradually increased with advancing age. Abortion (51.92%) was the commonest form of pregnancy wastage, followed by stillbirths (36.53%) and premature deliveries (7.69%). The seropositivity of toxoplasmosis was significantly higher in the study group than that in the control group, and the seropositivity played an important role in determining the foetal outcome. Considering the subclinical pattern of infection, routine serological test is recommended for all pregnant women for both IgG and IgM antibodies.

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that infects neutrophils and neutrophil precursors. Bacterial recognition of P-selectin glycoprotein ligand-1 (PSGL-1) and the ␣2,3-sialylated-and ␣1,3-fucosylated-moiety sialyl-Lewis x (sLe x ), which modifies the PSGL-1 N terminus, is important for adhesion to and invasion of myeloid cells. We have previously demonstrated that A. phagocytophilum organisms of the NCH-1 strain that utilize an sLe x -modified PSGL-1-independent means of entry can be enriched for by cultivation in undersialylated HL-60 cells that are unable to construct sLe x . Because it was unknown whether other A. phagocytophilum isolates share this ability, we extended our studies to the geographically diverse strains HZ and HGE1. HL-60 A2 is a clonal cell line that is defective for sialylation and ␣1,3-fucosyltransferase. HL-60 A2 cell surfaces, therefore, not only lack sLe x but also are virtually devoid of any other sialic acid-and/or ␣1,3-fucose-modified glycan. By cultivating HZ and HGE1 in HL-60 A2 cells, we enriched for bacterial subpopulations (termed HZA2 and HGE1A2) that bind and/or infect myeloid cells in the absence of sialic acid and ␣1,3-fucose and in the presence of antibody that blocks the N terminus of PSGL-1. Thus, multiple A. phagocytophilum isolates share the ability to use sLe xmodified PSGL-1-dependent and -independent routes of entry into myeloid cells. HZA2 and HGE1A2 represent enriched bacterial populations that will aid dissection of the complexities of the interactions between A. phagocytophilum and host myeloid cells.

Coordinated expression and upregulation of interleukin‐1α, interleukin‐1β, tumor necrosis factor‐α, interleukin‐6, granulocyte–macrophage colony‐stimulating factor, interleukin‐8, monocyte chemotactic protein‐1 (MCP‐1) and epithelial cell derived neutrophil activator‐78, with chemoattractant and proinflammatory properties of various cytokine families, were obtained in the intestinal epithelial cell line Int407 upon Vibrio cholerae infection. These proinflammatory cytokines also showed increased expression in T84 cells, except for interleukin‐6, whereas a striking dissimilarity in cytokine expression was observed in Caco‐2 cells. Gene expression studies of MCP‐1, granulocyte–macrophage colony‐stimulating factor, interleukin‐1α, interleukin‐6 and the anti‐inflammatory cytokine transforming growth factor‐β in Int407 cells with V. cholerae culture supernatant, cholera toxin, lipopolysaccharide and ctxA mutant demonstrated that, apart from cholera toxin and lipopolysaccharide, V. cholerae culture supernatant harbors strong inducer(s) of interleukin‐6 and MCP‐1 and moderate inducer(s) of interleukin‐1α and granulocyte–macrophage colony‐stimulating factor. Cholera toxin‐ or lipopolysaccharide‐induced cytokine expression is facilitated by activation of nuclear factor‐κB (p65 and p50) and cAMP response element‐binding protein in Int407 cells. Studies with ctxA mutants of V. cholerae revealed that the mutant activates the p65 subunit of nuclear factor‐κB and cAMP response element‐binding protein, and as such the activation is mediated by cholera toxin‐independent factors as well. We conclude that V. cholerae elicits a proinflammatory response in Int407 cells that is mediated by activation of nuclear factor‐κB and cAMP response element‐binding protein by cholera toxin, lipopolysaccharide and/or other secreted products of V. cholerae.

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Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis. MSP2(P44), the bacterium's major surface protein, is encoded by a paralogous gene family and has been implicated in a variety of pathobiological processes, including antigenic variation, host adaptation, adhesion, porin activity, and structural integrity. The consensus among several studies performed at the DNA and RNA levels is that a heterogeneous mix of a limited number of msp2(p44) transcripts is expressed by A. phagocytophilum during in vitro cultivation. Such analyses have yet to be extended to the protein level. In this study, we used proteomic and molecular approaches to determine that MSP2(P44)-18 is the predominant if not the only paralog expressed and is modified into multiple 42-to 44-kDa isoforms by A. phagocytophilum strain HGE1 during infection of HL-60 cells. The msp2(p44) expression profile was homogeneous for msp2(p44)-18. Thus, MSP2 (P44)-18 may have a fitness advantage in HL-60 cell culture in the absence of selective immune pressure. Several novel 22-to 27-kDa MSP2 isoforms lacking most of the N-terminal conserved region were also identified. A. phagocytophilum MSP2(P44) orthologs expressed by other pathogens in the family Anaplasmataceae are glycosylated. Gas chromatography revealed that recombinant MSP2(P44)-18 is modified by glucose, galactose, xylose, mannose, and trace amounts of other glycosyl residues. These data are the first to confirm differential modification of any A. phagocytophilum MSP2(P44) paralog and the first to provide evidence for expression of truncated versions of such proteins.