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.
Highlights
Throughout evolution, numerous bacterial pathogens have acquired specialized protein secretion pathways to deliver effector proteins to host cells
It was reported to be encoded by a single non-divided eccC5 gene in several mycobacterial species such as Mycobacterium leprae, Mycobacterium bovis and Mycobacterium marinum, as well as in other M. tuberculosis strains (Figure 1B) [29,30,31,32]
In previous studies we demonstrated that selected ESX-5 encoded genes had an impact on secretion of ESX-5 specific substrates, cell wall integrity and virulence of M. tuberculosis [20,21]
Summary
Throughout evolution, numerous bacterial pathogens have acquired specialized protein secretion pathways to deliver effector proteins to host cells. Each ESX cluster typically carries a pair of esx genes flanked by esx conserved components (ecc) genes coding for predicted core components of the ESX secretion machineries responsible for the ATP-dependent transport of the corresponding ESX substrates outside the cell [5,7]. By the characterization of several M. tuberculosis knock-out mutants for ESX-5 components, we recently demonstrated that ESX-5 plays a crucial role in host pathogen interaction in M. tuberculosis. By constructing/characterizing multi-copy gene variants and conditional mutants, in which the eccB5-eccC5 genes were deleted or expressed under the control of an anhydrotetracycline-repressible promoter, we demonstrate that an intact eccB5-eccC5 locus is essential for M. tuberculosis and that disruption/repression of single core components of the ESX5 secretion machinery strongly impacts the M. tuberculosis in vitro growth properties. The results obtained demonstrated the importance of an intact and functional ESX-5 for M. tuberculosis viability, emphasizing the key role of this secretion system in the biology of this human pathogen
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