The mycosins, a family of secreted subtilisin-like serine proteases associated with the immunologically-important ESAT-6 gene clusters of Mycobacterium tuberculosis

[摘要] ENGLISH ABSTRACT:Pathogenic organisms frequently utilize proteases to perform specific functions related tovirulence. There is little information regarding the role of proteolysis in Mycobacterium tuberculosisand no studies on the potential involvement of these enzymes in the pathogenesis of tuberculosis.The present study initially focused on the characterization of a family of membrane anchored, cell wallassociated, subtilisin-like serine proteases (mycosins-1 to 5) of Mycobacterium tuberculosis. Theseproteases were shown to be constitutively expressed in M. tuberculosis, to be located in the cell wallof the organism and to be potentially shed (either actively or passively) from the wall. Relatively highlevels of gamma interferon secretion by T-cells in response to these proteases were observed inMantoux positive individuals. The absence of any detectable protease activity lead to a proteinsequence analysis which indicated that the mycosins are probable mycobacterial-specific proproteinprocessing proteases.To identify possible substrates for these proteases, the genome sequence regionssurrounding the mycosin genes were analyzed. This revealed that the mycosin genes are in fact partof a cluster of 6 to 12 genes which have been duplicated multiple times in the genome of M.tuberculosis. Due to the presence of members of the previously described ESAT-6 T-cell antigenfamily within this duplicated region, the five gene cluster regions were named the ESAT-6 loci. Insilico analysis of finished and unfinished genome sequencing data revealed the presence oforthologues of the Mycobacterium tuberculosis H37Rv ESAT-6 loci in the genomes of othermycobacteria, e.g. M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, andthe avirulent strain M. smegmatis. Phylogenetic analyses done on the resulting sequences haveestablished the duplication order of the gene clusters and demonstrated that gene cluster region 4(Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an orthologue could befound in the genomes of Corynebacterium diptheriae and Streptomyces coelicoior. Thus, thecomparative genomic analyses revealed that the presence of the ESAT-6 gene cluster seems to be aunique characteristic shared by members of the high G+C gram-positive bacteria and that multipleduplications of this cluster have occurred and have been maintained only within the genomes ofmembers of the genus Mycobacterium. The ESAT-6 gene cluster regions were shown to consist of the members of the ESAT-6 genefamily (encoding secreted T-cell antigens that lack detectable secretion signals), the mycosins(secreted, cell wall-associated subtilisin-like serine proteases) as well as genes encoding putativeABC transporters, ATP-binding proteins, and other membrane-associated proteins. Thus, from theobservation that members of the ESAT-6 family are secreted without the normal sec-dependentsecretion signals, it was hypothesized that the membrane-associated and energy-providing proteinsfunction together to form a transport system for the secretion of the members of the ESAT-6 proteinfamily. Supporting this hypothesis, one of the ESAT-6 gene clusters was shown to be expressed as asingle polycistronic RNA, forming an operon structure. The promoter for this operon, P e s r e g 3. wasalso identified and its activity characterized. Subsequent secretion analyses results have shown thatsecretion of members of the ESAT-6 protein family is dependent on the presence of the proteinsencoded by the ESAT-6 gene cluster regions, confirming the putative transport-associated functionsof the ESAT-6 gene cluster-encoded proteins. The mycobacterial ESAT-6 gene clusters contain anumber of features of quorum sensing and lantibiotic operons, and an extensive review of theliterature have led to the hypothesis that the members of the ESAT-6 family may be secreted assignaling molecules and may be involved in the regulation of expression of genes during intracellularresidence of the bacterium. In the final part of this study, the evolutionary history of the PE and PPEgene families (members of which is found situated in the ESAT-6 gene clusters) were investigated.This investigation revealed that the expansion of these families are linked to the duplications of theESAT-6 gene clusters, which is supported by the absence of the multiple copies of the PE and PPEfamilies in the genome of the fast-growing mycobacterium M. smegmatis. Furthermore, dot blotanalyses showed that the PPE gene present in ESAT-6 gene cluster region 5 is able to distinguishbetween mycobacteria belonging to the slow-growing or fast-growing species, indicating a function forthe genes of these two families and/or the ESAT-6 gene clusters in the phenotypical differencesdistinguishing these two groups of mycobacteria.In conclusion, this study has highlighted numerous important aspects of mycobacterialgenomics and has greatly contributed to the current body of knowledge concerning the role ofproteases, gene duplication and mechanisms of antigen expression and secretion in M. tuberculosis.

[发布机构] Stellenbosch University