Albert D. Beyers

The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria

BackgroundThe genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide.ResultsFinished and unfinished genome sequencing data of 98 publicly available microbial genomes has been analyzed for the presence of orthologs of the ESAT-6 loci. The multiple duplicates of the ESAT-6 gene cluster found in the genome of M. tuberculosis H37Rv are also conserved in the genomes of other mycobacteria, for example M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses of the resulting sequences have established the duplication order of the gene clusters and demonstrated that the gene cluster known as region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an ortholog could be found in the genomes of Corynebacterium diphtheriae and Streptomyces coelicolor.ConclusionsComparative genomic analysis revealed that the presence of the ESAT-6 gene cluster is a feature of some high-G+C Gram-positive bacteria. Multiple duplications of this cluster have occurred and are maintained only within the genomes of members of the genus Mycobacterium.

Can eradication of helminthic infections change the face of AIDS and tuberculosis

Abstract Helminth infections impair the hosts immune response to HIV and tuberculosis (TB) and might contribute to the spread of these diseases. Thus, eradication of helminth infections may have a major impact on both HIV and TB in the developing world.

Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages

BackgroundExported proteases are commonly associated with virulence in bacterial pathogens, yet there is a paucity of information regarding their role in Mycobacterium tuberculosis. There are five genes (mycP1-5) present within the genome of Mycobacterium tuberculosis H37Rv that encode a family of secreted, subtilisin-like serine proteases (the mycosins). The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORFs) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism.ResultsFull-length, 50 kDa mycosin-1 was observed in M. tuberculosis cellular lysates, whereas lower-molecular-weight species were detected in culture filtrates. A similar lower-molecular-weight species was also observed during growth in macrophages. Mycosin-1 was localized to the membrane and cell wall fractions in M. tuberculosis by Western blotting, and to the cell envelope by electron microscopy. Furthermore, M. tuberculosis culture filtrates were shown to contain a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca2+, features typical of the subtilisins.ConclusionsMycosin-1 is an extracellular protein that is membrane- and cell wall-associated, and is shed into the culture supernatant. The protein is expressed after infection of macrophages and is subjected to proteolytic processing. Although proteolytically active mycosin-1 could not be generated recombinantly, serine protease activity containing features typical of the subtilisins was detected in M. tuberculosis culture filtrates.

The mycosins of Mycobacterium tuberculosis H37Rv: a family of subtilisin-like serine proteases

There is little information regarding the role of proteolysis in Mycobacterium tuberculosis and no studies on the potential involvement of proteases in the pathogenesis of tuberculosis. We identified five M. tuberculosis genes (mycP1-5) that encode a family of serine proteases (mycosins-1 to 5), ranging from 36 to 47% identity. Each protein contains a catalytic triad (Asp, His, Ser) within highly conserved sequences, typical of proteases of the subtilisin family. These genes are also present in M. bovis BCG and other virulent mycobacteria, but only one homologue (mycP3) was detected in M. smegmatis. The mycosins have N-terminal signal sequences and C-terminal transmembrane anchors, and the localisation of the mycosins to the membrane/cell wall was verified by Western blot analysis of heterologously expressed proteins in cellular fractions of M. smegmatis. In M. tuberculosis, all the mycosins were expressed constitutively during growth in broth. Mycosins-2 and 3 were also expressed constitutively in M. bovis BCG, but no expression of mycosin-1 was detected. Mycosin-2 was modified by cleavage in all three mycobacterial species. The multiplicity and constitutive expression of these proteins suggests that they have an important role in the biology of M. tuberculosis.

Thymocyte activation induces the association of the proto-oncoprotein c-cbl and ras GTPase-activating protein with CD5.

Studies of knockout mice indicate that the glycoprotein CD5, which is expressed on T cells, most thymocytes and a subset of B cells, down‐regulates TCR‐ and B cell receptor (BCR)‐mediated signaling. CD5 is associated with the TCR and BCR, and is phosphorylated on cytoplasmic tyrosine residues following antigen receptor ligation. Cross‐linking of CD5 or pervanadate stimulation of thymocytes induces the association of a 120‐kDa tyrosine‐phosphorylated protein with CD5. The proto‐oncoprotein c‐cbl associates with CD5 in pervanadate‐stimulated thymocytes, and reprecipitation analysis demonstrates that the major proportion of CD5‐associated pp120 is c‐cbl. The GTPase‐activating protein for ras (ras GAP), which is not tyrosine phosphorylated following CD5 cross‐linking, associates with CD5 in pervanadate‐stimulated thymocytes. Using tyrosine‐phosphorylated peptides we show that ras GAP interacts in an SH2‐mediated manner with the phosphorylated Y429SQP sequence of CD5. Both c‐cbl and ras GAP have been proposed to suppress receptor‐mediated signaling, and may contribute to CD5‐mediated suppression of TCR or BCR signaling.

Thy-1 associated PP85-90 is a potential docking site for SH2 domain-containing signal transduction molecules.

Thy‐1, a glycosylphosphatidylinositol (GPI)‐anchored glycoprotein expressed at high levels on thymocytes, has been implicated in positive and negative signal transduction. We show that Thy‐1 associates with a protein of 85–90kDa, which is prominently phosphorylated in vitro as well as in vivo following the stimulation of thymocytes with pervanadate. pp85–90 is not identical to known proteins that are phosphorylated following T cell activation. The SH2 domains of fyn, csk, phosphatidylinositol 3′‐kinase, rasGAP, vav and lck bind to pp85–90 with varying affinities. The SH2 domains of ZAP70, SHP‐1 and PLCγ1 and the SH3 domains of lck, vav and HS1 did not bind to pp85–90. The molecular weight, iso‐electric point, efficient phosphorylation by fyn and lck and preferential binding to the SH2 domain of fyn compared to that of lck indicate that Thy‐1‐associated pp85–90 may be identical to a recently cloned, fyn‐associated transmembrane adaptor protein, PAG‐85.