Disruption of the ESX‐5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation

Abstract

SummaryThe chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX-1-ESX-5). While the role of the ESX-1 and ESX-3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX-5 system came from data obtained in Mycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX-5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX-5 knockout/deletion mutants, inactivating eccA 5, eccD5, rv1794 and esxM genes or the ppe25-pe19 region. Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX-5-encoded EsxN and PPE41, a representative member of the large PPE protein family. Strikingly, the MtbeccD5ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics. When the virulence of the five mutants was evaluated, the MtbeccD5ko and MtbDppe25-pe19 mutants were found attenuated both in macrophages and in the severe combined immune-deficient mouse infection model. Altogether these findings indicate an essential role of ESX-5 for transport of PPE proteins, cell wall integrity and full virulence of M. tuberculosis, thereby opening interesting new perspectives for the study of this human pathogen.

General secretion signal for the mycobacterial type VII secretion pathway

“…S4, lanes 1-8). M. tuberculosis expresses and secretes endogenous PPE41 (30), and consequently PPE41 was detected in the supernatant of strains with and without plasmids. However, introduction of the WT operon on a plasmid resulted in elevated PPE41 levels in the culture supernatant, whereas in strains harboring plasmids with truncated forms of PE25, the PPE41 levels in the culture supernatant were reduced to background levels.…”

“…Previously, PPE41 was shown to be exported by ESX-5, both in its native host M. tuberculosis (30) and in M. marinum upon heterologous expression (20). The PPE41 gene forms an operon with PE25 (31) and both proteins form a heterodimer when expressed in Escherichia coli (32).…”

Multivalent TB vaccines targeting the esx gene family generate potent and broad cell-mediated immune responses superior to BCG

“…Finally, although originally the esx family was considered to be as early secreted antigens, expressed only during the early stage of Mtb infection, new evidence suggests that this family of genes may be secreted and expressed at different stages of the life cycle of TB. [48][49][50] Accordingly these studies suggest that the RSQ-15 vaccine may not only be studied as a prophylactic vaccine, but may also be examined as a post-exposure vaccine against latent TB.…”

Structure of a PE–PPE–EspG complex fromMycobacterium tuberculosisreveals molecular specificity of ESX protein secretion

“…These structures define the key elements of the PPE-EspG interaction and, coupled with bioinformatics and biochemical interaction studies, suggest that EspGs function as adaptors to deliver PE-PPE complexes to their cognate ESX system for translocation across the plasma membrane. Moreover, our work shows that the vast majority of PE-PPE proteins in Mtb interact with EspG from the ESX-5 secretion system, which has been hypothesized to play a major role in PE-PPE protein secretion (9,20,23).…”