Molecular cloning and expression of an alpha-mannosidase gene in Mycobacterium tuberculosis.

Mannose is a major component of glycolipids and glycoproteins of the cell envelope of M. tuberculosis (Mtb). However, the enzymes involved in the biosynthesis and catabolism of mannosylated glycans are largely unknown. We demonstrate alpha-mannosidase activity towards the fluorescent substrate 4-methylumberlliferyl-alpha-D-mannopyranoside (4MU-Man) in cell lysates of attenuated and virulent Mtb bacilli, with two-fold higher activity in the virulent strain Erdman. Mannosidase activity was optimal at pH 6.5, was not inhibited by deoxymannojirimycin (dMNJ), was mildly inhibited by swainsonine (SW) and stimulated two-fold by EDTA. GenBank BLAST analysis for sequences homologous to eukaryotic alpha-mannosidases revealed a 3.6 kb putative gene (Rv0648) in Mtb cosmid SCY20H10 (Acc# z92772), with strong homology (48%) to the rat ER/cytosolic alpha-mannosidase and containing signature sequences of class 2 mannosidases. By RT-PCR, gene Rv0648 was found differentially expressed, with lower expression during growth in A549 pneumocyte cultures. Gene Rv0648 was cloned, expressed in E. coli, and alpha-mannosidase activity in cell lysates determined. Expression of alphaMan-pET in E. coli cells resulted in an eight-fold increase in mannosidase activity toward 4-MU-Man, upon IPTG induction. Partial purification of the histidine-tagged Mtb mannosidase by metal chelation affinity chromatography, and analysis by SDS-PAGE, showed a protein with the predicted m.w. of 137.5 kDa. Enzyme assays of the column fractions showed alpha-mannosidase activity toward synthetic aryl-mannose substrates, in fractions enriched in the recombinant Mtb mannosidase. These results demonstrate that gene Rv0648 encodes an active alpha-mannosidase in Mtb.

Induction of MMP-9 mediated gelatinolytic activity in human monocytic cells by cell wall components of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) infection induces the expression of host matrix metalloIproteinases (MMPs) capable of tissue degradation. We show that infection of mice with Mtb results in differential expression of MMPs in the lung. MMP-9 activity increased by week 1 post-infection, while MMP-2 activity increased after week 2. RT-PCR analysis for gene expression of gelatinases and their respective inhibitors showed: a small increase in MMP-9 by week 1, no change in TIMP-1 and MMP-2, and a significant decrease in TIMP-2 by week 4. The increase in MMP-2 could be due to a decrease in TIMP-2 expression. Addition of 4-aminophenylmercuric acid to lung extracts increased MMP-9 activity, suggesting that its regulation could be due to endogenous activation by proteases. In vitro, attenuated and virulent Mtb strains equally induced MMP-9 expression in U937 monocytes. The inducer of MMP-9 in Mtb was present in culture filtrates, and was active after paraformaldehyde fixation. LAM stimulated MMP-9 expression in THP-1 cells, but not U937 cells. However, LAM-free extracts also induced MMP-9 activity in THP-1 cells. Fractionation of Mtb extracts by chromatography revealed fractions of 17 and 156 kDa with MMP-9 inducing activity. In conclusion, LAM and other components of Mtb induce the expression of MMP-9.

Identification of genes differentially expressed in Mycobacterium tuberculosis by differential display PCR.

RNA arbitrarily-primed differential display PCR (RAP-PCR) was used to identify and isolate genes differentially expressed between attenuated (H37Ra) and virulent (H37Rv, Erdman) laboratory strains of Mycobacterium tuberculosis (Mtb). Using this method, cDNA fragments showing homology to three known mycobacterial genes and six putative novel genes in mycobacterial cosmid vectors were identified. Among the putative novel Mtb genes identified, we found: (1) gene MTV041.29, containing multiple tandem repetitive sequences and encoding a putative Gly-, Ala, Asn-rich protein (PPE family); (2) gene MTV004.03, containing the AT10S repetitive gene sequence; (3) gene MTV028.09, encoding a hypothetical protein of unknown function; (4) genes MTCY78.20,21, possibly encoding two hypothetical proteins of unknown function; (5) gene MTCY01A6.09, encoding a putative novel ferrodoxin dependent glutamate synthase; and (6) gene MTCY31.20, encoding a putative cyclohexanone monooxygenase. Using gene specific primers in a second differential display PCR and by RT-PCR amplification, novel genes 1, 2, 3 and 4 were shown to be differentially up-regulated in the attenuated Mtb strain H37Ra compared to H37Rv and Erdman strain. Overall, we demonstrated that RAP-PCR, as a first step, is a quick and sensitive method for the identification and isolation of novel genes expressed in Mtb. Because of limitations inherent to the lack of specificity of arbitrary primers in the RAP-PCR method, a second differential display PCR and RT-PCR amplification with gene-specific primers was necessary in order to confirm differential expression of the identified genes.

Expression and purification of the alpha and beta subunits of Drosophila casein kinase II using a baculovirus vector.

Two recombinant baculoviruses that express the alpha and beta subunits of Drosophila melanogaster casein kinase II, respectively, have been constructed. The expressed proteins are similar to the authentic Drosophila subunits in size and are recognized by antisera raised against the Drosophila holoenzyme. Extracts derived from cells infected with the alpha subunit-expressing virus display elevated casein kinase II activity in vitro. This activity is markedly enhanced in extracts of cells infected with both viruses, or when alpha and beta subunit-containing extracts are mixed in vitro following lysis. Recombinant holoenzyme and the alpha subunit were purified to near homogeneity using phosphocellulose column chromatography. The specific activity of the purified recombinant holoenzyme was very similar to that of the native enzyme, and was fivefold higher than that of the purified free alpha subunit. The Stokes radius of the recombinant holoenzyme was estimated to be 50 A, a value similar to that reported for the native enzyme, whereas the alpha subunit demonstrated a Stokes radius of 26.5 A. Studies using sucrose density gradient centrifugation showed that, under conditions of high ionic strength, the quaternary structure of the purified holoenzyme was tetrameric (like the native enzyme), whereas the structure of the alpha subunit was monomeric. At lower ionic strength the recombinant holoenzyme had a significantly higher sedimentation coefficient, characteristic of the formation of filaments found for the native enzyme. Interestingly, the purified catalytic subunit also displayed a higher S value under conditions of low ionic strength, revealing the formation of alpha subunit aggregates.

Schistosoma mansoni contains a galactosyltransferase activity distinct from that typically found in mammalian cells.

It has been reported previously that some complex-type Asn-linked oligosaccharides contained in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal galactosyl residues. We report here that extracts from S. mansoni adult male and female worms contain a beta 1,4-galactosyltransferase activity that transfers galactose from the donor substrate UDP-galactose to the acceptor substrate N-acetylglucosamine in a beta 1,4-linkage position to form the disaccharide Gal beta 1,4GlcNAc. In this respect the schistosome-derived activity is similar to that commonly found in mammalian tissues. The kinetic properties, however, of the common beta 1,4-galactosyltransferase activity in mammalian tissues are dramatically altered in the presence of the modifier protein alpha-lactalbumin, whereas the beta 1,4-galactosyltransferase activities in adult male and female schistosomes are not altered by this modifier. Overall, our results demonstrate that adult schistosomes contain a beta 1,4-galactosyltransferase activity and that it is unlike that commonly found in mammalian tissues.

Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA.

We have previously isolated a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase (alpha(1,3)-GT) cDNA (Larsen, R. D., Rajan, V. P., Ruff, M. M., Kukowska-Latallo, J., Cummings, R. D., and Lowe, J. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8227-8231). This enzyme constructs the terminal alpha(1,3)-galactosyl linkage within the epitope Gal alpha 1----3Gal. This epitope is expressed by New World monkeys and many nonprimate mammals but generally not by Old World primates, anthropoid apes, or man. To investigate the molecular basis for the apparent species-specific absence of this enzyme and its oligosaccharide product, we have sequenced a human genomic DNA fragment homologous to the murine alpha(1,3)-GT cDNA. This fragment contains a 703-nucleotide region that shares 82% identity with a region of the murine cDNA encoding part of the enzyme's catalytic domain. The human sequence, however, has suffered deletion of single nucleotides at two separate positions, relative to the murine sequence. These frameshift mutations disrupt the translational reading frame that would otherwise maintain a 76% amino acid sequence identity between the human sequence and the murine alpha(1,3)-GT. Moreover, nonsense mutations exist within this disrupted reading frame that would truncate the human polypeptide, relative to the murine enzyme. We therefore propose that this human sequence represents a pseudogene and cannot determine expression of Gal alpha 1----3Gal epitopes on human cells.

Evaluation of immunodiagnostic antigens in the excretory-secretory products of Fasciola hepatica.

The metabolic antigens of F. hepatica have been shown to be a source of potential immunodiagnostic antigens. We have fractionated F. hepatica excretory-secretory (ES) antigens by conventional gel filtration and HPLC, analyzed these fractions in PAGE, and evaluated their immunogenicity by ELISA with sera from experimentally infected rabbits to identify potential serodiagnostic antigens for fascioliasis. A fraction enriched in high molecular weight components of ca. 150-160 kDa was found to be very reactive with sera from early fascioliasis. This fraction was successfully adapted to the DOT-ELISA, where titers up to 1:16,000 still appeared visually as positive. Both acute and chronic fascioliasis sera also recognized, in the enzyme-linked immunoelectrotransfer blot technique (EITB), prominent 25-30-kDa polypeptides that have previously been shown to be recognized by infected rabbits, cows, and sheep. We have therefore employed conventional gel filtration and HPLC gel exclusion chromatography as a 1-step procedure to obtain fractions enriched in antigens recognized in early fascioliasis. In addition, these antigens have been successfully applied to a sensitive, visual immunodiagnostic technique that can be easily employed in field studies.

Isolation and partial characterization of shared antigens of Biomphalaria glabrata and Schistosoma mansoni and their evaluation by the ELISA and the EITB.

Serum from humans infected with Schistosoma mansoni, when reacted with a Biomphalaria glabrata soluble hepatopancreas antigen extract (BgSHA) yields 2 lines of precipitation by gel diffusion and 1 by immunoelectrophoresis. The IgG from the serum of a human infected with S. mansoni was coupled to CNBr-activated Sepharose 4B. BgSHA (8.0 mg) was then filtered through the gel and the bound antigens, denoted BgSm, eluted with HCl-glycine, pH 2.6. These bound antigens comprised 2.8% of the total BgSHA. BgSm was then applied to an anti-Fasciola hepatica column as above. The drop through in PBS (38% yield) and containing the BgSm antigens depleted of cross-reactivity with F. hepatica was then tested by the ELISA to evaluate its serodiagnostic potential. These antigens detected a primary S. mansoni infection by 4 wk but were less sensitive than SmSEA in the detection of a primary infection with S. mansoni. However, the BgSm-specific antigens were more specific than SmSEA and showed less cross-reactivity with the serum of mice infected with F. hepatica. At least 16 peptides were seen by silver staining following SDS-PAGE with 5-20% gradient gels. The 2 more prominent bands obtained were estimated to have molecular weights of 62 and 66 kd. Nitrocellulose strips blotted with BgSHA were incubated with the serum of mice infected with S. mansoni for 12 wk and developed 6 bands with molecular weights of 66, 57, 55, 50, 48 and 32 kd.(ABSTRACT TRUNCATED AT 250 WORDS)