10.1371/journal.pgen.1005190 Louis S. Ates Louis S. Ates Roy Ummels Roy Ummels Susanna Commandeur Susanna Commandeur Robert van der Weerd Robert van der Weerd Marion Sparrius Marion Sparrius Eveline Weerdenburg Eveline Weerdenburg Marina Alber Marina Alber Rainer Kalscheuer Rainer Kalscheuer Sander R. Piersma Sander R. Piersma Abdallah M. Abdallah Abdallah M. Abdallah Moataz Abd El Ghany Moataz Abd El Ghany Alyaa M. Abdel-Haleem Alyaa M. Abdel-Haleem Arnab Pain Arnab Pain Connie R. Jiménez Connie R. Jiménez Wilbert Bitter Wilbert Bitter Edith N.G. Houben Edith N.G. Houben Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria Public Library of Science 2015 membrane ATPase EccC 5 pe substrate T 7S systems Outer Membrane Permeability carbon sources cell envelope fractions type VII secretion Subsequent proteomic analysis cell envelope proteins heterologous porin MspA ppe esx Pathogenic Mycobacteria Mycobacteria 2015-05-04 03:25:37 Dataset https://plos.figshare.com/articles/dataset/_Essential_Role_of_the_ESX_5_Secretion_System_in_Outer_Membrane_Permeability_of_Pathogenic_Mycobacteria_/1402948 <div><p>Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that <i>esx-5</i> is essential for growth of both <i>Mycobacterium marinum</i> and <i>Mycobacterium bovis</i>. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC<sub>5</sub> prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type <i>M</i>. <i>marinum</i> cells and cells expressing <i>mspA</i> to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of <i>esx-5</i>, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an <i>esx-5</i> mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.</p></div>