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 EccB5 and EccC5, a transmembrane protein (EccB5) and a membrane-bound ATPase (EccC5), both predicted to be building blocks of the M. tuberculosis ESX-5 membrane-associated complex. In vitro expression studies demonstrated that EccB5 and EccC5 encoding genes constitute an operon. The expression of this operon is essential for M. tuberculosis, since the deletion of the eccB5-eccC5 genomic segment at the ESX-5 locus is possible only after the integration of a second functional copy of eccB5-eccC5 genes into the M. tuberculosis chromosome. The characterization of two M. tuberculosis conditional mutant strains (MtbPptreccB5 and MtbPptreccC5), in which the eccB5-eccC5 operon or the eccC5 gene, respectively, were expressed under the control of an anhydrotetracycline-repressible promoter, confirmed that the repression of eccB5-eccC5 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 MtbPptreccB5-eccC5 and MtbPptreccC5 strains revealed that both EccB5 and EccC5 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.

The ESX-5 Associated eccB-eccC Locus Is Essential for Mycobacterium tuberculosis Viability

DI LUCA, MARIAGRAZIA
Primo
;
BOTTAI, DARIA
Co-primo
;
BATONI, GIOVANNA;COUNOUPAS, CLAUDIO;CAMPA, MARIO;ESIN, SEMIH
2012-01-01

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 EccB5 and EccC5, a transmembrane protein (EccB5) and a membrane-bound ATPase (EccC5), both predicted to be building blocks of the M. tuberculosis ESX-5 membrane-associated complex. In vitro expression studies demonstrated that EccB5 and EccC5 encoding genes constitute an operon. The expression of this operon is essential for M. tuberculosis, since the deletion of the eccB5-eccC5 genomic segment at the ESX-5 locus is possible only after the integration of a second functional copy of eccB5-eccC5 genes into the M. tuberculosis chromosome. The characterization of two M. tuberculosis conditional mutant strains (MtbPptreccB5 and MtbPptreccC5), in which the eccB5-eccC5 operon or the eccC5 gene, respectively, were expressed under the control of an anhydrotetracycline-repressible promoter, confirmed that the repression of eccB5-eccC5 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 MtbPptreccB5-eccC5 and MtbPptreccC5 strains revealed that both EccB5 and EccC5 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.
2012
DI LUCA, Mariagrazia; Bottai, Daria; Batoni, Giovanna; Orgeur, M; Aulicino, A; Counoupas, Claudio; Campa, Mario; Brosch, R; Esin, Semih
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/158410
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