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The ESX-5 associated eccB-EccC locus is essential for Mycobacterium tuberculosis viability.

ABSTRACT: The recently described ESX-5 secretion system of Mycobacterium tuberculosis is one of the most important modulators of host-pathogen interactions due to its crucial impact on PPE protein secretion, cell wall stability and virulence. Although various components of the ESX-5 secretion machinery have been defined, other ESX-5 core components still remain to be characterized. In this study, we focused on EccB(5) and EccC(5), a transmembrane protein (EccB(5)) and a membrane-bound ATPase (EccC(5)), both predicted to be building blocks of the M. tuberculosis ESX-5 membrane-associated complex. In vitro expression studies demonstrated that EccB(5) and EccC(5) encoding genes constitute an operon. The expression of this operon is essential for M. tuberculosis, since the deletion of the eccB(5)-eccC(5) genomic segment at the ESX-5 locus is possible only after the integration of a second functional copy of eccB(5)-eccC(5) genes into the M. tuberculosis chromosome. The characterization of two M. tuberculosis conditional mutant strains (Mtb(Pptr)eccB(5) and Mtb(Pptr)eccC(5)), in which the eccB(5)-eccC(5) operon or the eccC(5) gene, respectively, were expressed under the control of an anhydrotetracycline-repressible promoter, confirmed that the repression of eccB(5)-eccC(5) genes is detrimental for growth of M. tuberculosis both in vitro and in THP-1 human macrophage cell line. Moreover, analysis of the secretome of Mtb(Pptr)eccB(5)-eccC(5) and Mtb(Pptr)eccC(5) strains revealed that both EccB(5) and EccC(5) are required for secretion of ESX-5 specific substrates, thus confirming that they are indeed components of the ESX-5 secretion machinery. Taken together these findings demonstrate the importance of an intact and functional ESX-5 system for viability of M. tuberculosis, thus opening new interesting options for alternative antimycobacterial control strategies.

SUBMITTER: Di Luca M

PROVIDER: S-EPMC3524121 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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The ESX-5 associated eccB-EccC locus is essential for Mycobacterium tuberculosis viability.

Di Luca Mariagrazia M Bottai Daria D Batoni Giovanna G Orgeur Mickael M Aulicino Anna A Counoupas Claudio C Campa Mario M Brosch Roland R Esin Semih S

PloS one 20121217 12

The recently described ESX-5 secretion system of Mycobacterium tuberculosis is one of the most important modulators of host-pathogen interactions due to its crucial impact on PPE protein secretion, cell wall stability and virulence. Although various components of the ESX-5 secretion machinery have been defined, other ESX-5 core components still remain to be characterized. In this study, we focused on EccB(5) and EccC(5), a transmembrane protein (EccB(5)) and a membrane-bound ATPase (EccC(5)), bo ...[more]

PMID: 23284869

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Project description:Current models of horizontal gene transfer (HGT) in mycobacteria are based on "distributive conjugal transfer" (DCT), an HGT type described in the fast-growing, saprophytic model organism Mycobacterium smegmatis, which creates genome mosaicism in resulting strains and depends on an ESX-1 type VII secretion system. In contrast, only few data on interstrain DNA transfer are available for tuberculosis-causing mycobacteria, for which chromosomal DNA transfer between two Mycobacterium canettii strains was reported, a process which, however, was not observed for Mycobacterium tuberculosis strains. Here, we have studied a wide range of human- and animal-adapted members of the Mycobacterium tuberculosis complex (MTBC) using an optimized filter-based mating assay together with three selected strains of M. canettii that acted as DNA recipients. Unlike in previous approaches, we obtained a high yield of thousands of recombinants containing transferred chromosomal DNA fragments from various MTBC donor strains, as confirmed by whole-genome sequence analysis of 38 randomly selected clones. While the genome organizations of the obtained recombinants showed mosaicisms of donor DNA fragments randomly integrated into a recipient genome backbone, reminiscent of those described as being the result of ESX-1-mediated DCT in M. smegmatis, we observed similar transfer efficiencies when ESX-1-deficient donor and/or recipient mutants were used, arguing that in tubercle bacilli, HGT is an ESX-1-independent process. These findings provide new insights into the genetic events driving the pathoevolution of M. tuberculosis and radically change our perception of HGT in mycobacteria, particularly for those species that show recombinogenic population structures despite the natural absence of ESX-1 secretion systems.IMPORTANCE Data on the bacterial sex-mediated impact on mycobacterial evolution are limited. Hence, our results presented here are of importance as they clearly demonstrate the capacity of a wide range of human- and animal-adapted Mycobacterium tuberculosis complex (MTBC) strains to transfer chromosomal DNA to selected strains of Mycobacterium canettii Most interestingly, we found that interstrain DNA transfer among tubercle bacilli was not dependent on a functional ESX-1 type VII secretion system, as ESX-1 deletion mutants of potential donor and/or recipient strains yielded numbers of recombinants similar to those of their respective parental strains. These results argue that HGT in tubercle bacilli is organized in a way different from that of the most widely studied Mycobacterium smegmatis model, a finding that is also relevant beyond tubercle bacilli, given that many mycobacteria, like, for example, Mycobacterium avium or Mycobacterium abscessus, are naturally devoid of an ESX-1 secretion system but show recombinogenic, mosaic-like genomic population structures.

Dataset Information

The ESX-5 associated eccB-EccC locus is essential for Mycobacterium tuberculosis viability.

Publications

The ESX-5 associated eccB-EccC locus is essential for Mycobacterium tuberculosis viability.

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