Mutually dependent secretion of proteins required for mycobacterial virulence

Abstract

The ESX-1 locus is a region critical for full virulence in Mycobacterium tuberculosis, which encodes two secreted proteins as well as other genes involved in their secretion. The mechanism of secretion of the two proteins, ESAT-6 and CFP-10, and their function remain unknown. Using proteomic methods to search for additional proteins secreted by the ESX-1 locus, we discovered that a protein encoded by a chromosomally unlinked gene, espA, is also secreted by strains that contain the ESX-1 locus but not by strains with ESX-1 deletions. Mutations in individual ESX-1 genes, including those that encode ESAT-6 and CFP-10, were found to block EspA secretion. Surprisingly, mutants that lack espA reciprocally failed to secrete ESAT-6 and CFP-10 and were as attenuated as ESX-1 mutants in virulence assays. The results indicate that secretion of these proteins, which are each critical for virulence of pathogenic mycobacteria, is mutually dependent. The results further suggest that discerning the nature of the interaction and the structure of macromolecular complexes will provide insights into both an alternative mechanism of protein secretion and mycobacterial virulence.ESAT-6 protein ͉ Mycobacterium tuberculosis ͉ virulence factor I nteractions between bacterial pathogens and their hosts occur at the surface of cells. Many pathogenic bacteria secrete proteins involved in virulence (1). These proteins often interfere with normal host defenses and allow bacteria to survive and multiply. Strikingly, many of these virulence factors are secreted by nonclassical secretory systems that, in some cases, facilitate translocation of proteins across host cell membranes (2, 3).During the evolution of Mycobacterium bovis Bacille Calmette-Guérin (BCG), the attenuated bacillus widely used as vaccine against tuberculosis, a major deletion removed a set of nine ORFs (4). Similar deletions are found in Mycobacterium microti (5) and the Dassie bacillus (6), other relatively avirulent mycobacteria. The BCG deletion, known as RD1, includes two genes that encode the abundantly secreted proteins, ESAT-6 and CFP-10. The RD1 deletion encompasses most but not all of a larger, 15-gene region known as ESX-1 (7), which is hypothesized to encode an alternative secretory system (8, 9).The proteins encoded by the ESX-1 locus clearly play an important role in virulence. The secreted proteins encoded within the locus are potent T cell antigens (7, 10, 11). More significantly, Mycobacterium tuberculosis strains with RD1 deletions are far less virulent in mice than strains that are otherwise isogenic (12-14). They replicate poorly and cause less pathology in both immunocompetent and immunocompromised mice, although the latter ultimately succumb to infection. RD1-deleted strains of Mycobacterium marinum elicit only very modest macrophage aggregation and granuloma formation in zebrafish (15). Additional work suggests the RD1-encoded proteins may disrupt host cell membranes. In M. tuberculosis, these proteins may be required for lysis of infected cells (13) and c...

General secretion signal for the mycobacterial type VII secretion pathway

“…This secretion defect was not accompanied by an increase in cellular levels of PE25 and PPE41. This phenomenon is often observed for ESX-1 and ESX-5 substrates and indicates that the stability of the substrates depends on the presence of the T7S systems in the bacterial cell (5,11,13,33). PPE41 was detected in two forms, a 25-kDa band, which represents full-length PPE41, and a smaller form of ∼19 kDa, which probably represents degraded PPE41 (20).…”

“…The archetypical T7S system, ESX-1, is responsible for the secretion of several proteins, including the ESX-1-encoded T-cell antigens ESAT-6 (early secreted antigenic target of 6 kDa) and CFP-10 (culture filtrate protein of 10 kDa) (7,(11)(12)(13)(14)(15). ESX-1 and its substrates play an important, albeit not yet fully understood role in M. tuberculosis virulence.…”

Dissecting virulence pathways of Mycobacterium tuberculosis through protein–protein association

“…As a result, one of the central aims of this work was to thoroughly test the capacity of M-PFC to reveal undefined Mtb virulence mechanisms. Several studies (16,17,(20)(21)(22) have shown that proteins in and outside of the Mtb RD1 region are involved in the secretion of the immunogenic antigens, Esat-6 and Cfp-10. An important feature of this yet-undefined specialized secretion system is that these small effector proteins contain no signal peptide.…”

Activation of the eis gene in a W-Beijing strain of Mycobacterium tuberculosis correlates with increased SigA levels and enhanced intracellular growth

“…These genes are components of the ESX-1 secretion system, which is primarily associated with mycobacterial virulence (Fortune et al, 2005;MacGurn et al, 2005;Raghavan et al, 2008), although in M. smegmatis, the ESX-1 system is also involved in DNA conjugation (Coros et al, 2008;Flint et al, 2004). The basis for the downregulation of the RecA-NDp and ESX-1 genes is unknown, but we speculate that these genes are regulated by an alternative sigma factor that is at a competitive disadvantage in the presence of increased levels of SigA, or has reduced expression in TB294-pSigA.…”