The mycosins, a family of secreted subtilisin-like serine proteases associated with the immunologically-important ESAT-6 gene clusters of Mycobacterium tuberculosis
Date
2002-12
Authors
Gey van Pittius, Nicolaas Claudius
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Pathogenic organisms frequently utilize proteases to perform specific functions related to
virulence. There is little information regarding the role of proteolysis in Mycobacterium tuberculosis
and 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 wall
associated, subtilisin-like serine proteases (mycosins-1 to 5) of Mycobacterium tuberculosis. These
proteases were shown to be constitutively expressed in M. tuberculosis, to be located in the cell wall
of the organism and to be potentially shed (either actively or passively) from the wall. Relatively high
levels of gamma interferon secretion by T-cells in response to these proteases were observed in
Mantoux positive individuals. The absence of any detectable protease activity lead to a protein
sequence analysis which indicated that the mycosins are probable mycobacterial-specific proprotein
processing proteases.
To identify possible substrates for these proteases, the genome sequence regions
surrounding the mycosin genes were analyzed. This revealed that the mycosin genes are in fact part
of 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 antigen
family within this duplicated region, the five gene cluster regions were named the ESAT-6 loci. In
silico analysis of finished and unfinished genome sequencing data revealed the presence of
orthologues of the Mycobacterium tuberculosis H37Rv ESAT-6 loci in the genomes of other
mycobacteria, e.g. M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and
the avirulent strain M. smegmatis. Phylogenetic analyses done on the resulting sequences have
established 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 be
found in the genomes of Corynebacterium diptheriae and Streptomyces coelicoior. Thus, the
comparative genomic analyses revealed that the presence of the ESAT-6 gene cluster seems to be a
unique characteristic shared by members of the high G+C gram-positive bacteria and that multiple
duplications of this cluster have occurred and have been maintained only within the genomes of
members of the genus Mycobacterium. The ESAT-6 gene cluster regions were shown to consist of the members of the ESAT-6 gene
family (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 putative
ABC transporters, ATP-binding proteins, and other membrane-associated proteins. Thus, from the
observation that members of the ESAT-6 family are secreted without the normal sec-dependent
secretion signals, it was hypothesized that the membrane-associated and energy-providing proteins
function together to form a transport system for the secretion of the members of the ESAT-6 protein
family. Supporting this hypothesis, one of the ESAT-6 gene clusters was shown to be expressed as a
single polycistronic RNA, forming an operon structure. The promoter for this operon, P e s r e g 3. was
also identified and its activity characterized. Subsequent secretion analyses results have shown that
secretion of members of the ESAT-6 protein family is dependent on the presence of the proteins
encoded by the ESAT-6 gene cluster regions, confirming the putative transport-associated functions
of the ESAT-6 gene cluster-encoded proteins. The mycobacterial ESAT-6 gene clusters contain a
number of features of quorum sensing and lantibiotic operons, and an extensive review of the
literature have led to the hypothesis that the members of the ESAT-6 family may be secreted as
signaling molecules and may be involved in the regulation of expression of genes during intracellular
residence of the bacterium. In the final part of this study, the evolutionary history of the PE and PPE
gene 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 the
ESAT-6 gene clusters, which is supported by the absence of the multiple copies of the PE and PPE
families in the genome of the fast-growing mycobacterium M. smegmatis. Furthermore, dot blot
analyses showed that the PPE gene present in ESAT-6 gene cluster region 5 is able to distinguish
between mycobacteria belonging to the slow-growing or fast-growing species, indicating a function for
the genes of these two families and/or the ESAT-6 gene clusters in the phenotypical differences
distinguishing these two groups of mycobacteria.
In conclusion, this study has highlighted numerous important aspects of mycobacterial
genomics and has greatly contributed to the current body of knowledge concerning the role of
proteases, gene duplication and mechanisms of antigen expression and secretion in M. tuberculosis.
AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming
AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming
Description
Thesis (PhD)--Stellenbosch University, 2002.
Keywords
Mycobacterium tuberculosis, Proteolytic enzymes, Antigens, Gene duplication, Mycobacterial genomics, Dissertations -- Medicine