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

<jats:title>Summary</jats:title><jats:p>The chromosome of <jats:italic>Mycobacterium tuberculosis</jats:italic> encodes five type VII secretion systems (ESX‐1–ESX‐5). While the role of the ESX‐1 and ESX‐3 systems in <jats:italic>M. tuberculosis</jats:italic> has been elucidated, predictions for the function of the ESX‐5 system came from data obtained in <jats:italic>Mycobacterium marinum</jats:italic>, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX‐5 system in <jats:italic>M. tuberculosis</jats:italic>, in this study, we have constructed five <jats:italic>M. tuberculosis</jats:italic> H37Rv ESX‐5 knockout/deletion mutants, inactivating <jats:italic>eccA<jats:sub>5</jats:sub></jats:italic>, <jats:italic>eccD<jats:sub>5</jats:sub></jats:italic>, <jats:italic>rv1794</jats:italic> and <jats:italic>esxM</jats:italic> genes or the <jats:italic>ppe25‐pe19</jats:italic> region. Whereas the Mtb<jats:italic>rv1794</jats:italic>ko 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 Mtb<jats:italic>eccD<jats:sub>5</jats:sub></jats:italic>ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics. When the virulence of the five mutants was evaluated, the Mtb<jats:italic>eccD<jats:sub>5</jats:sub></jats:italic>ko and MtbΔ<jats:italic>ppe25‐pe19</jats:italic> 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 <jats:italic>M. tuberculosis</jats:italic>, thereby opening interesting new perspectives for the study of this human pathogen.</jats:p>