ESAT6 secretion (ESX; also known as type VII secre- tion) systems are bacterial secretion systems that are named after the first identified effector, the 6 kDa early secretory antigenic target (ESAT6; also known as EsxA), of Mycobacterium tuberculosis, the aetiological agent of human tuberculosis 1,2 . ESX systems are found in mycobacteria and various other genera in the phylum Actinobacteria 3,4 , such as Streptomyces, Corynebacterium, Nocardia or Gordonia, and more distantly related ESX- like systems also exist in Gram-positive bacteria in the phylum Firmicutes, including in Bacillus anthracis 5 , Bacillus subtilis 6,7 , Staphylococcus aureus 8,9 and Listeria monocytogenes 10 (BOX 1). In mycobacteria, ESX systems function as specialized secretion systems that enable the transport of selected substrates across the complex, thick mycobacterial cell envelope that forms a structural bar- rier to protein export 11 . The thickness and complexity of the envelope, which provides protection to myco- bacteria under harsh environmental conditions, are due to the presence of mycolic acids that are linked, usually covalently, to an arabinogalactan–peptidoglycan matrix, as well as various extractable lipids, polysaccharides, lipo- glycans and proteins that are not covalently attached to the matrix and that may vary among species 12 . Two characteristics unify all esx loci across the differ- ent phyla. First, the presence of genes that encode small secreted proteins of approximately 100 amino acids that have a conserved Trp-X-Gly (WXG) motif, which contributes to the formation of helix–turn–helix struc- tures 13 , in the centre of the polypeptide; and, second, the presence of genes that encode transmembrane proteins of the FtsK–SpoIIIE-like ATPase family 14 . The most well- known proteins that contain the WXG motif are EsxA of M. tuberculosis and its adjacently encoded hetero- dimerization partner EsxB (also known as CFP10) 15 . Apart from these core characteristics, ESX systems are quite diverse, which suggests that they have been shaped by a long evolutionary process that has involved gene duplication and diversification 3,16,17 , as well as horizontal gene transfer between chromosomes and plasmids of different bacterial species and genera 4,18 (BOX 2). Of the five ESX systems that have been described in M. tuberculosis (ESX-1, ESX-2, ESX-3, ESX-4 and ESX-5; FIG. 1a), at least three are required for full virulence. The first ESX system (ESX-1) was identified in parallel by different comparative and functional genomic studies that involved M. tuberculosis and the attenuated vaccine strains Mycobacterium bovis bacille Calmette–Guérin (BCG) and Mycobacterium microti 19–24 . The vaccine strains lack EsxA, owing to spontaneous deletions of dif- ferent sized portions of the esx‑1 locus, each of which is known as region of difference 1 (RD1) for the respective strain 25,26 (FIG. 1a). ESX-1 in M. tuberculosis has subse- quently been shown to be essential for resistance to, and evasion of, host responses. One of the key functions of ESX-1 is its role in the induction of phagosomal rupture, which releases bacteria and/or bacterial products into the cytosolic compartment of host phagocytes. The sens- ing of bacterial products, such as DNA, triggers a com- plex signalling cascade of the innate immune system, Actinobacteria A phylum of Gram-positive bacteria that is characterized by high GC content. Mycolic acids Long-chain (C 60 –C 90 ) fatty acids that are specifically found in the mycobacterial cell envelope; together with extractable lipids, mycolic acids form the mycobacterial outer membrane (also known as the mycomembrane). ESX secretion systems: mycobacterial evolution to counter host immunity Matthias I. Gröschel 1,2 , Fadel Sayes 1 , Roxane Simeone 1 , Laleh Majlessi 1 and Roland Brosch 1 Abstract | Mycobacterium tuberculosis uses sophisticated secretion systems, named 6 kDa early secretory antigenic target (ESAT6) protein family secretion (ESX) systems (also known as type VII secretion systems), to export a set of effector proteins that helps the pathogen to resist or evade the host immune response. Since the discovery of the esx loci during the M. tuberculosis H37Rv genome project, structural biology, cell biology and evolutionary analyses have advanced our knowledge of the function of these systems. In this Review, we highlight the intriguing roles that these studies have revealed for ESX systems in bacterial survival and pathogenicity during infection with M. tuberculosis. Furthermore, we discuss the diversity of ESX systems that has been described among mycobacteria and selected non-mycobacterial species. Finally, we consider how our knowledge of ESX systems might be applied to the development of novel strategies for the treatment and prevention of disease. 1 Institut Pasteur, Unit for Integrated Mycobacterial Pathogenomics, Paris 75724, Cedex 15, France. 2 Department of Pulmonary Diseases & Tuberculosis, University Medical Center Groningen, University of Groningen, 9700 RB Groningen, The Netherlands. Correspondence to R.B. roland.brosch@pasteur.fr doi:10.1038/nrmicro.2016.131 Published online 26 Sep 2016 NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 1 REVIEWS ©2016MacmillanPublishersLimited,partofSpringerNature.Allrightsreserved.