# Conserved and specialized functions of Type VII secretion systems in non-tuberculous mycobacteria

Marion Lagune, Cecile Petit, Flor Vásquez Sotomayor, Matt D Johansen, Kathrine S H Beckham, Christina Ritter, Fabienne Girard-Misguich, Matthias Wilmanns, Laurent Kremer, Florian P Maurer, et al.

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Marion Lagune, Cecile Petit, Flor Vásquez Sotomayor, Matt D Johansen, Kathrine S H Beckham, et al.. Conserved and specialized functions of Type VII secretion systems in non-tuberculous mycobacteria. Microbiology, 2021, 167 (7), 10.1099/mic.0.001054. inserm-04842058

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# Conserved and specialized functions of Type VII secretion systems in non-tuberculous mycobacteria

Marion Lagune¹†, Cecile Petit²†, Flor Vásquez Sotomayor³†, Matt D. Johansen⁴†,‡, Kathrine S. H. Beckham²,
Christina Ritter², Fabienne Girard-Misguich¹,*, Matthias Wilmanns²,⁵,*, Laurent Kremer⁴,⁶,*, Florian P. Maurer³,⁷,\* and
Jean-Louis Herrmann¹,⁸,\*

## Abstract

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in *Mycobacterium smegmatis*, to ESX-5 structures in *Mycobacterium xenopi* and *Mycobacterium tuberculosis*. In addition, ESX-4, considered until recently as a non-functional system in both pathogenic and non-pathogenic mycobacteria, has been proposed to play an important role in the virulence of *Mycobacterium abscessus*; an increasingly recognized opportunistic NTM causing severe lung diseases. These major findings have led to important new insights into the functional mechanisms of these biological systems, their implication in virulence, nutrient acquisitions and cell wall shaping, and will be discussed in this review.

## INTRODUCTION

Secretion systems are an essential part of a bacterium's life,
allowing it to survive in a niche where all the conditions for
growth and multiplication are present. Type VII secretion
systems (T7SS) were first described in 2003 [1], although
it initially began with the discovery of the deletion region
1 (RD1) [2], thanks to the use of comparative genomics,
allowing genomes from pathogenic bacteria to be compared

with those from attenuated or non-pathogenic species. RD1
is absent in the non-virulent *Mycobacterium bovis* Bacillus
Calmette-Guérin vaccine strain and present in *Mycobacterium tuberculosis* (Mtb), a strict human pathogen responsible for tuberculosis (TB). Part of RD1 is also missing in *Mycobacterium microti* [3, 4], which is also used as a TB vaccine strain. The 'Type VII' nomenclature was then introduced in 2007 [5].

Received 01 February 2021; Accepted 06 April 2021; Published 05 July 2021

**Author affiliations:** ¹Université Paris-Saclay, UVSQ, Inserm, Infection et inflammation, 78180, Montigny-Le-Bretonneux, France; ²European Molecular Biology Laboratory, Hamburg Unit, Notkestraße 85, 22607 Hamburg, Germany; ³National and WHO Supranational Reference Center for Mycobacteria, Research Center Borstel, Leibniz Lung Center, Borstel, Germany; ⁴Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, 1919 route de Mende, 34293, Montpellier, France; ⁵University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20251 Hamburg, Germany; ⁶INSERM, IRIM, 34293 Montpellier, France; ⁷Institute of Medical Microbiology, Virology and Hospital Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; ⁸APHP, GHU Paris-Saclay, Hôpital Raymond Poincaré, Service de Microbiologie, Garches, France.

*Correspondence: Fabienne Girard-Misguich, fabienne.misguich@uvsq.fr; Matthias Wilmanns, matthias.wilmanns@embl-hamburg.de; Laurent Kremer, laurent.kremer@irim.cnrs.fr; Florian P. Maurer, fmaurer@fz-borstel.de; Jean-Louis Herrmann, jean-louis.herrmann@aphp.fr

**Keywords:** type VII secretion system; ESX; pathogenesis; non-tuberculous mycobacteria.

**Abbreviations:** Cryo-EM, cryo-electron microscopy; DCT, distributive conjugal transfer; DUF, domain of unknown function; Ecc, ESX conserved component; Esp, ESX secretion associated protein; ESX, early antigenic secretion systems; GDND, glycosyl diacylated nonadecyl diol lipid; NTD, N-terminal domain; NTM, non tuberculous mycobacteria; PDIM, phthiocerol dimicerosate; RD1, region of difference 1; RGM, rapidly-growing mycobacteria; SGM, slow-growing mycobacteria; TB, tuberculosis; TMH, transmembrane helix; TPR, tetratricopeptide repeats; T7SS, type VII secretion systems; Ubl, ubiquitin like.

‡Present address: Centre for Inflammation, Centenary Institute and University of Technology Sydney, Faculty of Science, Sydney, NSW, Australia.

†These authors contributed equally to this work
RD1 is a large genetic region encompassing two loci encoding small proteins, termed ESAT-6 and CFP-10 [6], known as important T-cell antigens and included in Interferon-Gamma Released Assays for the diagnosis of TB [7]. Both proteins play an important role in the virulence of *Mtb* [8–10]. Based on specific amino acid signatures within the N- and/or C-terminal domains of ESAT-6 and CFP-10 of the Early Antigenic Secretion-1 (ESX-1) machinery, additional ESX/T7SS have been discovered [11–13].

Type VII secretion systems are not specific to mycobacteria, as they are also found in other bacterial genera, such as the Gram-positive bacteria *Staphylococcus aureus* and *Listeria monocytogenes* [14], where they appear to be simpler than their mycobacterial counterparts, with only one ATPase containing FtsK/SpoIIIE domains and a few secreted substrates [14], hence their designation as Type VIb secretion systems [5, 15].

*Esx* loci appear to be involved in bacterial virulence [16], modulation of the host cell's response [17, 18], horizontal gene transfer of long DNA fragments [19], acquisition of nutrients [20–22], or in the maintenance of the bacterial cell wall integrity [23]. It is worth mentioning that these functions are allocated to ESX-1-3-4 and 5 and to date, no roles have been ascribed to ESX-2.

Major advances regarding the structure and function of the *esx* encoding loci, made it possible to elucidate the minimal structural arrangement required to generate a functional T7SS and to highlight the interactions between the different protein subunits composing these complex machineries [24–28]. On the other hand, recent discoveries have implicated a functional role of ESX-4 in the virulence of *Mycobacterium abscessus* (*Mab*) [29], an opportunistic rapidly-growing mycobacterium responsible for severe lung infections, particularly in patients with underlying pulmonary disorders, such as cystic fibrosis [30]. While the ESX systems in *Mtb* have been largely presented in previous reviews focusing mainly on ESX-1, the purpose of this review is to highlight the recent and unexpected findings on ESX systems in non-tuberculous mycobacteria (NTM).

## Evolution of *esx* loci in mycobacteria

The presence of ESX/T7SS on the mycobacterial chromosome arises from horizontal gene transfer from *esx* loci present on plasmids, with the exception of the ancestral locus *esx-4* and ‘*esx-4* like’ loci [31, 32]. The acquisition of the five loci, *esx-1* to *esx-5*, represents a major advance in the emergence and evolution of mycobacteria as strictly pathogenic to humans and animals [5, 11, 33, 34]. A comparative ‘pan-genome’ approach based on all currently available sequenced mycobacterial genomes identified five *esx* phylogenetic groups, confirming this ESX categorization. This underscored the ancestral nature of the *esx-4* locus, together with new *esx-4* like loci, and facilitated the discovery of new loci of unknown function harboured by mycobacterial mega-plasmids [31, 32].

Two phylogenetic studies on ESX reported the occurrence of *esx-4* loci, considered as the ancestral *esx* locus and several *esx-4* like loci, such as an *esx-4* bis locus [31, 32] (Fig. 1). Both studies describe an intact *esx-4* like locus that includes the essential elements for the constitution of the functional T7SS core required for the secretion of the specific substrates and found in *Nocardia* spp, a non-mycobacterial Actinobacterium [31, 32]. Comparatively, *esx-4*, referred to as the ancestral system, contains fewer genes [31, 32]. In fact, the likely scenario is the transfer of ancestral *esx-4*-like loci onto plasmids, which would have undergone major rearrangements leading to the different *esx* loci. These different types of *esx* would have been incorporated into the mycobacterial genomes, resulting in the four loci designated *esx-3*, *esx-1*, *esx-5* and *esx-2* [31, 32]. The acquisition patterns seem to differ between studies, with a first duplication resulting in the generation of *esx-1* [31, 35, 36] or *esx-3* [32], followed by the acquisition of *esx-2* and finally *esx-5* [31, 32, 35] (Fig. 1). Comparatively, *Mab* possess only two ESX (ESX-3 and ESX-4), while *Mycobacterium marinum* (*Mma*) and other strict human and animal pathogenic species, such as *Mtb*, harbour more ESX systems (Table 1).

The substrates secreted by T7SS have also evolved in parallel to the ESX systems, including genes encoding proteins called PE and PPE (discussed in further detail later) [35]. Their identification is facilitated by the presence of signatures within the N and/or C-terminal domains of these proteins. A phylogenetic study of the genes encoding these proteins confirms the evolutionary pathway of *esx* loci, with *esx-1* acquired first, followed by *esx-3*, *esx-2* and *esx-5*. The acquisition of the latter two *esx* loci coincides with the separation between slow- and rapidly-growing mycobacteria (SGM and RGM, respectively). In addition, the acquisition of *esx-5* led to the extensive duplication of *pe* and *ppe* genes throughout the mycobacterial genomes in pathogenic mycobacteria [32, 35]. The relationship between the number of *esx* loci and the growth rate is unknown. ESX acquisition likely contributes to the persistence within a dedicated ecological niche, as observed for *Mma*, a NTM pathogenic to humans and aquatic animals, which possesses four out of five ESX, while also missing ESX-2; the only ESX system so far with an unassigned function (Table 1) [37].

## Structural overview of the Type VII secretion machinery

Each *esx* locus encodes components required for the targeted transfer of folded heterodimeric substrates from the cytoplasm across the mycobacterial cell envelope. These components include membrane proteins that form the secretion machinery, soluble components and secretory proteins [34, 38]. The conserved membrane proteins encoded by the *esx* loci are *EccB* (ESX Conserved Component), *EccC*, *EccD*, *EccE* and *MycP*, which are found through all the ESX systems, with the exception of *EccE* that is absent in the ESX-4 system of most mycobacterial species, with the notable exception of *Mab* [29]. Here, we will present an overview of the structural
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Evolution of ESX systems (based on Newton *et al.*, 2016 [32] and Dumas *et al.*, 2016 [31]) ESX-4 and ESX-4 like are the ancestral ESX. ESX-4 like are the intermediate between ESX-4 and the other ESX duplications. ESX evolution is plasmid-mediated and leads to the insertion of ESX in mycobacterial genome with first ESX-1 or ESX-3 insertion, then ESX-2 and ESX-5 in slow growing mycobacteria.</figcaption></figure>

and functional data relating to the individual components and
how they assemble to form the T7SS complex.

T7SS subunits EccB, EccC, EccD, EccE and MycP

EccB is composed of a short cytosolic domain, a single trans-
membrane helix (TMH) and a large periplasmic domain.
A structure of the periplasmic domain of EccB revealed a
structure consisting of a central domain flanked by two repeat
domains [39]. Interestingly, EccB has homology with the
phage lysin PlyCB, that acts on bacterial cell walls indicating
a role in peptidoglycan binding in the mycobacterial cell
envelope [39].

EccC belongs to the superfamily of FtsK/SpoIIIIE AAA+ATPases
and is composed of two TMH connected by a domain of unknown
function (DUF), followed by three consecutive ATPase domains
[40]. Of note is the division of *eccC* from the *esx-1* locus into two
genes producing EccCa₁ and EccCb₁ which form a stable complex
[1]. The hydrolysis of ATP by EccC is responsible for driving the
secretion of substrates across the membrane. In particular, the
ATPase activity of the first ATPase domains is crucial for substrate
secretion [22]. The C-terminal ATPase domain and the linker
between the first and second ATPase domain is considered as
receptor for the secreted substrates [40, 41]. Perhaps key to the
function of EccC is its intrinsic flexibility, which presumably
enables the T7SS to accommodate a wide range of substrates
[24, 40].

EccD is composed of a small N-terminal cytosolic ubiquitin
like (Ubl) domain followed by an integral membrane region
consisting of 11 TMH [39]. These helices have been shown to

form a dimer around a central cavity [25]. The role of EccD as
a scaffold protein became apparent following the characteriza-
tion of the T7SS, described in further detail below.

EccE is composed of two TMH, a linker made of a single stalk
helix and a cytosolic domain made of a nine-stranded antipar-
allel β-sheet surrounded by six helices [25, 26]. EccE is found
in all ESX systems apart from the ESX-4 where it is notice-
ably absent, with the exception for the *Mab* ESX-4 secretion
complex. The presence of EccE is presumably responsible for
producing an active secretion system in *Mab* implying that
EccE is crucial for the overall function and stability of the
T7SS [29].

MycP or mycosin, is a subtilisin-like membrane anchored
protease, present in the periplasm. MycP is composed of
an N-terminal Sec secretory signal sequence, followed by
a putative propeptide sequence, and a catalytic domain
that is linked to a single TMH proposed to anchor MycP
to the inner membrane [42–44]. The X-ray structures of
the catalytic domain of *Mycobacterium thermoresistibile*
*MycP*₁, *Mycobacterium smegmatis* (*Msm*)-*MycP*₁, and of
*Msm*-*MycP*₃ revealed a seven-stranded-parallel β-sheet
core surrounded by eight α-helices [45–47]. The catalytic
domain of MycP displays three proline- or glycine-rich
loops that mediate MycP:MycP interactions. These loops
decorate the protease active site making a deep binding
pocket that is highly substrate specific and highly variable
between homologues [46, 48]. So far, only one target, ESX
secretion associated protein B (EspB), has been identified
as a MycP1 substrate and hence the significance of this
<table><caption>**Table 1.** The different ESX systems found in mycobacteria (table based on Newton *et al.*, 2016 [32]; Dumas *et al.*, 2016 [31]; Ummels *et al.*, 2014 [104]; Everall *et al.*, 2017 [109]; Kim *et al.*, 2019 [110]). Black circle=chromosomal ESX; Black square, ancestral ESX-2 that contains all of the ESX-2 components and precedes both the ESX-2 and ESX-5 systems; black triangle, ESX-4 lacking EccE₄.</caption><thead><tr><th></th><th>Species</th><th>ESX-1</th><th>ESX-2</th><th>ESX-3</th><th>ESX-4</th><th>ESX-5</th><th>espACD</th><th>Plasmid-encoded ESX</th><th>Pathogenicity</th></tr></thead><tbody><tr><td rowspan="10">RGM</td><td>M. abscessus complex</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>opportunistic</td></tr><tr><td>M. abscessus ATCC 19977</td><td></td><td></td><td>●</td><td>●</td><td></td><td></td><td></td><td>opportunistic</td></tr><tr><td>M. abscessus subsp. bolletii</td><td></td><td></td><td>●</td><td>●</td><td></td><td></td><td>pMBOL plasmid 2</td><td>opportunistic</td></tr><tr><td>M. abscessus subsp. massiliense</td><td></td><td></td><td>●</td><td>●</td><td></td><td></td><td>pMAB02</td><td>opportunistic</td></tr><tr><td>M. neoaurum</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td></td><td>saprophyte</td></tr><tr><td>M. gilvum</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td></td><td>saprophyte</td></tr><tr><td>M. vaccae ATCC 25954</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td></td><td>saprophyte</td></tr><tr><td>M. vanbaalenii</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td></td><td>saprophyte</td></tr><tr><td>M. thermoresistibile</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td></td><td>saprophyte</td></tr><tr><td>M. chubuense</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td>pMYCCH.01 pMYCCH.02</td><td>saprophyte</td></tr><tr><td>M. fortuitum</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td></td><td>opportunistic</td></tr><tr><td>M. smegmatis</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td>pMYCSM01 to pMYCSM03</td><td>saprophyte</td></tr><tr><td>M. sp. JLS</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td></td><td>saprophyte</td></tr><tr><td>M. phlei</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td></td><td>saprophyte</td></tr><tr><td>M. rhodesiae</td><td>●</td><td></td><td>●</td><td>▲</td><td></td><td></td><td></td><td>opportunistic</td></tr><tr><td>M. tusciae JS617</td><td>●</td><td>■</td><td>●</td><td>▲</td><td></td><td></td><td></td><td>saprophyte</td></tr><tr><td>M. terrae complex</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>opportunistic</td></tr><tr><td rowspan="10">SGM</td><td>M. sinense JDM601</td><td></td><td>●</td><td>●</td><td>▲</td><td>●</td><td></td><td></td><td>opportunistic</td></tr><tr><td>M. xenopi</td><td></td><td>●</td><td>●</td><td>▲</td><td>●</td><td></td><td></td><td>opportunistic</td></tr><tr><td>M. avium complex</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>opportunistic</td></tr><tr><td>M. avium</td><td></td><td>●</td><td>●</td><td>▲</td><td>●</td><td></td><td>pRAW-like pMAH135</td><td>opportunistic</td></tr><tr><td>M. intracellulare</td><td></td><td>●</td><td>●</td><td>▲</td><td>●</td><td></td><td></td><td>opportunistic</td></tr><tr><td>M. colombiense</td><td></td><td>●</td><td>●</td><td>▲</td><td>●</td><td></td><td></td><td>opportunistic</td></tr><tr><td>M. yongonense 05-1390</td><td></td><td>●</td><td>●</td><td>▲</td><td></td><td></td><td>pMyong1</td><td>opportunistic</td></tr><tr><td>M. indicus pranii</td><td></td><td>●</td><td>●</td><td>▲</td><td>●</td><td></td><td></td><td>saprophyte</td></tr><tr><td>M. sp. MOTT36Y</td><td></td><td>●</td><td>●</td><td>▲</td><td>●</td><td></td><td></td><td>opportunistic</td></tr><tr><td>M. paragordonae</td><td>●</td><td>●</td><td>●</td><td>▲</td><td>●</td><td></td><td>pMpg-1 and 2</td><td>saprophyte</td></tr><tr><td>M. kansasii</td><td>●</td><td>●</td><td>●</td><td>▲</td><td>●</td><td>✓</td><td>pMK12478</td><td>opportunistic</td></tr><tr><td>M. ulcerans</td><td></td><td></td><td>●</td><td>▲</td><td>●</td><td></td><td></td><td>pathogen</td></tr><tr><td>M. marinum</td><td>●</td><td></td><td>●</td><td>▲</td><td>●</td><td>✓</td><td>pRAW</td><td>pathogen</td></tr></tbody></table>

proteolytic activity in other ESX systems remains unknown [45, 46, 48, 49].

## ESX pore assembly

Recent studies on *Msm* ESX-3, *Mycobacterium xenopi* (*Mxe*) ESX-5 and *Mtb* ESX-5 have provided in-depth insights into the structural features of the membrane embedded secretion machineries built by the conserved membrane components *EccB*, *EccC*, *EccD* and *EccE* [24–28]. In addition, *MycP*, which appears to only transiently associate with the different ESX systems when being reconstituted, is present in the recent

structure of the *Mtb* ESX-5 system [27]. Common to all these structures is a three-layered overall organization, including a cytosolic segment, a TM segment and a periplasmic segment. While most of the TM segment and TM-proximal regions at the cytosolic face are relatively rigid and thus could be visualized at high resolution, the distal part of the cytosolic segment and the periplasmic segment tend to be more flexible.

Two *Msm* ESX-3 cryo-electron microscopy (cryo-EM) structures revealed the dimeric arrangement of two identical protomeric units built by the four conserved subunits *EccB*,
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Architecture of the T7SS. (a) Schematic arrangement of the conserved components EccB (orange), EccC (blue), EccD (green), EccE (purple) and MycP (red) within the membrane for the dimeric ESX-3 (Poweleit et al., 2019 [26] Famelis et al., 2019 [25]) and each of the two solved hexameric structures of ESX-5 (Beckham et al., 2020 [28]; Bunduc et al., 2020 [27]). Low resolution information is indicated by reduced opacity. As the structures on a protomeric level share high similarity, it seems plausible that a common T7SS architecture exists. (b) Side and top view of the protomeric unit of ESX-3 (6SGX), shown in surface representation.</figcaption></figure>

EccC, EccD and EccE in a 1:1:2:1 stoichiometry [25, 26] (Fig. 2a, b). These results were surprising, as they contradicted the nature of EccC as its homologue FtsK belongs to a family of hexameric ATPases and are in contrast with a previous low-resolution structure of *Mxe* ESX-5, that revealed a hexamer with a central pore [24]. Thus, both structures are considered as building blocks of a complete hexameric ESX-3 assembly.

In contrast, the recent ESX-5 structures from *Mtb* and *Mxe* both display a hexameric arrangement, built by six protomeric units similar to those in the dimeric ESX-3 structures [27, 28] (Fig. 2a). The similarity of the ESX-3 and ESX-5 structures from different mycobacterial species suggests that despite their diverse roles (see below) the ESX systems share a common architecture even across different mycobacterial species. Common to all ESX structures characterised to date, the TM segment includes an asymmetric EccD dimer central to the protomeric unit that forms a pore-like structure on its own. This pore has an approximate size of 2×4 nm a diameter and would, thus, be large enough to translocate folded substrates across the membrane, such as Esx, Esp and PE/PPE proteins (Fig. 2a). The absence of the central pore in the

dimeric assemblies led to the speculation that the EccD pore could have such a function [25, 26]. However, in most of the cryo-EM maps available at present, extra density within this cavity was observed that has been attributed to lipids plugging the cavity [26–28]. In addition, the two EccD subunits in each protomeric unit act as a scaffold, by interacting with EccB and EccE [25–28]. Given its distal location within the TM segment, it is not surprising that the EccE TMHs are considerably more flexible or even invisible in present structural data of ESX TM complexes [25–28].

The EccB TMH, together with the N-terminal cytosolic helix of EccB form an L-shape, as the N-terminal helix is positioned parallel to the membrane. This parallel helix forms electrostatic interactions with the EccC stalk domain that connects the EccC TMHs to the cytosolic DUF domain. In the cytosol, the DUF and stalk domain are encompassed by EccD. The stalk domain interacts with a loop originating from TMH 6 and 7 from EccD-1 and the cytoplasmic linker of EccD-2 that links the EccD-2 UbI domain with the TM domain. The DUF domain is surrounded by a dimer formed by the two UbI domains. On the opposite side of the two
UbI domains, the cytoplasmic domain of EccE (Fig. 2b) is situated.

New insights into possible conformations of EccC came from the *Mtb* ESX-5 structures, where low resolution density allowed approximate fitting of the three consecutive EccC ATPase domains [27], suggesting two possible conformations, e.g. contracted toward the cytosol and extended along the membrane. Additionally, the conformation of the central secretion pore differs substantially between *Mtb* and *Mxe* ESX-5, thus possibly representing different states in the secretion process. Remarkably, the pore in the *Mxe* ESX-5 complex is considerably more dynamic than most other parts of the TM segment, suggestive of a high level of structural dynamics required for pore opening. In addition, while the *Mxe* ESX-5 structure shows an extended cleft at the periplasmic face of the pore, formed by a dimer of trimers by the periplasmic EccB domains, the periplasmic EccB domains of the *Mtb* structure are locked into a trimer of dimers conformation by three MycP protease domains, that plug the pore (Fig. 2a, b) [27, 28].

In summary, although the very recent high-resolution structures of two related complete ESX complexes considerably advance our knowledge about the overall architecture of mycobacterial T7SS, many mechanistic questions still remain unresolved. Comparison of the structures provides first clues into level of structure dynamics required for ESX substrate gating, recognition, translocation, release and associated regulation of translocation activity through an opened secretion pore. The precise role of loosely associated MycP remains a major open question, especially as to how its structural and functional roles as a protease may be inter-connected. Overall, the present structural data indicate that diversity in ESX architecture may be dominated by different conformational states that range from an inactive closed state to an active, secretion-competent open state, rather than by differences originating from different ESX members and species. However, the present data are still too limited to draw definite conclusions on factors that lead to NTM diversity in different ESX machineries.

## The ESX substrates and cytosolic components

ESAT-6 and CFP-10, also designated EsxA and EsxB, were the first canonical Type VII secreted proteins described in *Mtb*. This family is characterized by their helix-turn-helix structure and a WxG motif located within the helix-turn-helix structure, and are comprised on average, 100 amino-acids [50]. The second family of ESX substrates is represented by PE and PPE proteins, characterized by the presence of Pro-Glu (PE) or Pro-Pro-Glu (PPE) motifs at the N-terminus, comprising 110 and 180 amino-acids, respectively [35]. A key element is the polycistronic organization of the different T7SS substrates, and often their co-transcription. These proteins form an anti-parallel heterodimeric four-helix bundle complex shown to be secreted fully folded [51]. Another key feature of T7SS substrates is the presence of a YxxxD/E motif at the C-terminus, as a secretion signal required to target T7SS,

and to modulate binding and activity of the key ATPase EccC [50, 52, 53]. The high degree of conservation of these motifs within the T7SSs' substrates indicates that they play an essential role by targeted T7SS secretion.

PE and PPE proteins represent a highly heterogenous group of proteins with diverse biological functions, modifications in the C-terminal domain due to acquisition of repetitive elements leading to the formation of PE-PGRS and four subfamilies within the PPE proteins, occurring at specific milestones in the evolution of mycobacteria, most notably the acquisition of ESX-5 in SGM [35]. Strikingly, the largest number of these PE and PPE proteins are found in strictly pathogenic mycobacteria such as *Mma* representing almost 10% of its coding capacity, or *Mycobacterium ulcerans* (*Mul*) comprising 21 and 121 genes (including pseudogenes) encoding PE and PE-PGRS proteins, respectively. In contrast, only ten genes encoding PE proteins and no genes encoding PE-PGRS proteins are found in *Mycobacterium avium* (*Mav*) [54]. Similarly, 81 genes (including pseudogenes) coding for PPE proteins are present in *Mul* or *Mma* versus 37 in *Mav* [35]. Correlatively, only three PE and six PPE proteins were reported in the opportunistic pathogen *Mab* [55]. EspG seems a key 'chaperone' for the secretion of PE and PPE proteins based on studies showing that the *EspG*~S~ or *EspG*~I~ binding domain controls PE and PPE secretion by directing the ESX specific substrates to the corresponding ESX complex [53, 56–58]. Regarding EspG, this cytosolic protein is conserved in all ESX systems with the exception of ESX-4 and acts as a molecular chaperone for PE and PPE proteins [59]. Structures of EspG have been solved either alone or in complex with PE/PPE proteins [59, 60].

Esp, are active members of the ESX-1 machinery and represent the third family of ESX-substrates. Esp genes are encoded within the *esx-1* locus, with the exception of *espA*, *espC* and *espD* located within the *espACD* locus. This locus, regulated by the EspR protein [61], is mostly encountered in strict pathogens, such as members of the *Mtb* complex, *Mycobacterium leprae*, *Mma* and opportunistic pathogens like *Mycobacterium kansasii* (*Mka*) [62] (Table 1). In *Mtb* ESX-1, EspA and EspC secretion is co-dependent on that of EsxA-B [63, 64]. EspB, in ESX-1 secretion, is essential for *Mma* intracellular survival in macrophages [65] and proper conjugation in the *Msm* [66] (see below). EspE, EspF and EspM have recently been demonstrated to negatively regulate *esx-1* gene expression through the WhiB6 transcription factor in *Mma* [67–69]. EspE and EspF proteins share a dual function in *Mma* by negatively regulating *esx-1* expression inside the cytoplasm and, after secretion, by their involvement in virulence and lytic activity unrelated to EsxA and EsxB [68]. EspH was shown to interact with EspE, while an *espH Mma* mutant abolished secretion of both EspE and EspF. These results suggest EspH may intervene as a chaperone-like protein [56]. An EspI-like protein, uncovered by a transposon library screening in *Mab*, appears to play a role in intra-amoebal and intra-macrophage survival [29].

Finally, EccA (65 kDa), is characterized by a C-terminal ATPase domain belonging to the CbxC/CfqX AAA⁺ ATPase superfamily
<table><caption>**Table 2.** Function of ESX and their substrates in various NTM</caption><thead><tr><th>NTM species</th><th>ESX</th><th>ESX substrates</th><th>ESX/ substrate function</th></tr></thead><tbody><tr><td rowspan="12">M. marinum</td><td rowspan="12">ESX-1</td><td>EsxA</td><td>EsxA is involved in haemolysis, cytotoxicity, cytolysis (Gao et al., 2004 [65]) host cell membrane lysis through pore formation (Smith et al., 2008 [80]) and in phagosomal rupture (Simeone et al., 2012 [79]). Controversial role of EsxA in virulence (Conrad et al., 2017 [77]; Lienard et al., 2019 [18]).</td></tr><tr><td>EspB</td><td>EspB is essential for intracellular living (Gao et al., 2004 [65]).</td></tr><tr><td>EspE, EspF</td><td>EspE and EspF negatively regulate the esx-1 locus through WhiB6 transcription factor (Chirakos et al., 2020 [68], Sanchez et al., 2020 [69]) inside cytoplasm and after secretion promote virulence and lytic activity (Chirakos et al., 2020 [68]).</td></tr><tr><td>EspM</td><td>EspM negatively regulate the esx-1 locus through WhiB6 transcription factor (Chirakos et al., 2020 [68], Sanchez et al., 2020 [69]).</td></tr><tr><td>EspH</td><td>EspH was shown to interact with EspE. EspH could undertake a chaperone-like role. Moreover, EspH is involved in haemolysis and growth in macrophage (Phan et al., 2018 [56])</td></tr><tr><td>MMAR_2894</td><td>MMAR_2894 is a PE involved in the haemolysis activity but not in the phagosomal membrane lysis. MMAR_2894 is also important for the secretion of other ESX-1 substrates (Bosserman et al., 2019 [84]).</td></tr><tr><td rowspan="4">ESX-5</td><td>PPE38</td><td>PPE38 is involved in host immune modulation and biofilm and cords formation (Dong et al., 2012 [95]; Wang et al., 2013 [96]).</td></tr><tr><td>PPE10</td><td>ESX-5 and its substrates PPE10 are involved in capsule integrity (Ates et al., 2016 [23]).</td></tr><tr><td>PecA</td><td>PecA is protease that cleve the PE motif of ESX substrates to the bacterial surface (Burggraaf et al., 2019 [94]).</td></tr><tr><td>LipY</td><td>LipY is a lipase possessing a PE motif and secreted by Mma ESX-5 (Daleke et al., 2011 [111]).</td></tr><tr><td rowspan="4">M. abscessus</td><td rowspan="4">ESX-4</td><td>EsxU and EsxT</td><td>ESX-4 is involved in phagosomal membrane damage and prevent acidification. EsxU and EsxT seems to be secreted by ESX-4 in the secretome analysis (Laencina et al., 2018 [29]).</td></tr><tr><td>/</td><td>ESX-3 seems to be involved in virulence (Kim et al., 2017 [106]), although this needs confirmation through complementation.</td></tr><tr><td>/</td><td>ESX-1 in the donor seems to negatively regulate DCT (Flint et al., 2004 [99]) while the ESX-1 in the recipient is essential for DCT (Coros et al., 2008 [66]).</td></tr><tr><td>EsxG and EsxH</td><td>esx-3 promoters are induced as a function of iron content (Fang et al., 2017 [112]). ESX-3 is involved in iron uptake via a mycobactin dependent mechanism (Siegrist et al., 2009 [20]). EsxG and EsxH are secreted by ESX-3 (Famelis et al., 2019 [25])</td></tr><tr><td rowspan="2">M. smegmatis</td><td>ESX-1</td><td>/</td><td>ESX-1 in the donor seems to negatively regulate DCT (Flint et al., 2004 [99]) while the ESX-1 in the recipient is essential for DCT (Coros et al., 2008 [66]).</td></tr><tr><td>Esx-3</td><td>EsxG and EsxH</td><td>esx-3 promoters are induced as a function of iron content (Fang et al., 2017 [112]). ESX-3 is involved in iron uptake via a mycobactin dependent mechanism (Siegrist et al., 2009 [20]). EsxG and EsxH are secreted by ESX-3 (Famelis et al., 2019 [25])</td></tr><tr><td>ESX-4</td><td>EsxU and EsxT</td><td>ESX-4 is involved in conjugation in the recipient strain. esxU and esxT transcripts are induced in the recipient strain but active secretion has not been proven (Gray et al., 2016 [100]; Clark et al., 2018 [101]).</td></tr><tr><td>M. avium</td><td>ESX-5</td><td>PPE25</td><td>PPE25 is involved in blocking the phagosomal acidification. It is an important factor for the growth in macrophages in vitro and in vivo in mice (Li et al., 2005 [54]). Moreover, PPE25 was shown to interact with an Esx protein (McNamara et al., 2012 [97]).</td></tr><tr><td>M. kansassii</td><td>ESX-1</td><td>/</td><td>ESX-1 is proposed to be associated with the espACD locus that was demonstrated to be involved in virulence (Guan et al., 2020 [87]).</td></tr></tbody></table>

(ATPase Associated to diverse cellular Activities) [70] and an N-terminal domain (NTD) composed of six tetratricopeptide repeats (TPR) interrupted between the second and third TPR by a β-finger [71]. To date, there is only one EccA structure, consisting of the NTD Mtb-EccA₁ (71). The role of EccA in secretion has been shown to be strain- and ESX-specific. Previous work attempting to identify EccA₃ interaction partners showed binding to the secreted substrates EsxG-H, EspC/EsxAB [72]. In addition, interactions between components of

the type II fatty acid synthase and EccA₁ hinted at EccA playing a regulatory role of mycolic acid synthesis in Mma [73]. Owing to the ATPase activity, it has been postulated that EccA may play a role in the dissociation of EspG-PE-PPE complexes [56, 74].

## Mycobacterial virulence

ESX-1 and ESX-5 have been the most studied ESX systems within the context of mycobacterial virulence. Recently,
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Synergistic action of ESX and lipids to achieve phagosomal membrane rupture in infected macrophages. (a) Combined action between the secretion system ESX-1 and the phthiocerol dimicerosate lipid (PDIM), secreted by Mmpl7, for the phagosomal escape of *M. marinum* (Osman *et al.* 2020 [83]). (b) Hypothetical combined action between the secretion system ESX-4 and the glycosyl diacylated nonadecyl diol lipid (GDND), secreted by Mmpl8<sup>MAB+</sup>, for the disruption of the phagosomal membrane by *M. abscessus*.</figcaption></figure>

ESX-4 was shown to play a similar role in *Mab* virulence [29] (Table 2). The biological function of ESX complexes, particularly ESX-1, have been extensively scrutinised for their association with virulence in the *Mtb* complex [2, 3, 75]. Among NTM, ESX-1-dependent virulence mechanisms have largely been explored in *Mma*, often used as a surrogate model to study *Mtb* pathogenesis [76]. *Mma* ESX-1 has been implicated in haemolysis [65, 77], phagosomal escape [18, 78, 79], cell-to-cell spread [65] and host cell death [79]. There are several reports on the pore-forming activity of EsxA, upon interaction with host cellular membranes, leading to cytosol access and cell lysis [80]. Moreover, the ESX-1 cell membrane lysis activity in *Mma* was demonstrated to be a contact-dependant process inducing membrane disruption, albeit the pore-forming and lytic activity of EsxA has been proposed to be due to residual detergent present after protein purification EsxA [77]. However, it remains to be determined whether EsxA on its own is involved in phagosomal escape, as many contradictory results are found in the literature [18, 65, 79]. Recent studies support the view that EsxA requires additional partners to trigger phagosomal escape of *Mtb*. For instance, EsxA membrane permeabilization is potentiated by the phthiocerol dimicerosate (PDIM), which are cell-surface associated lipids capable of disrupting

the biochemical properties of host membranes [81, 82]. The combined action of PDIM and ESX-1 in phagosomal membrane disruption was confirmed in *Mma* [83]. Indeed, a mutant defective in Mmpl7, the transporter of PDIM, demonstrated decreased phagosomal permeabilization similarly to the ESX-1 deletion mutant [83] (Fig. 3a). Moreover, the Mmpl7 mutant retains haemolytic activity, suggesting a different mechanism of membrane damage in red blood cells [83]. *Mma* haemolysis appears to imply another ESX-1 substrate; notably a *pe* mutant (*MMAR_2894*) encoding a PE protein secreted by ESX-1, displays a red blood cell lysis defect while maintaining cytolysis activity in macrophages [84]. Moreover, this PE is necessary for the secretion of other ESX-1 substrates, such as EsxA and possibly other proteins involved in *Mma* virulence [84].

Following access to the cytosolic compartment, several host cell responses are induced via an ESX-1-dependent mechanism. There is a type I interferon response after infection, which may be related to the release of mitochondrial and nuclear DNA in the cytosol [18]. The ESX-1 system from *Mma* also plays an important virulence function in zebrafish, promoting the formation of granulomas, which play a critical role in constraining mycobacterial dissemination [76, 85].
However, since ESX-1 is missing in *Mab*, a bacterium which can also have access to the cytosolic compartment of macrophages, it has been proposed that this may be achieved by ESX-4 in this species [29], although this remains to be addressed experimentally. Phagosomal escape has also been observed in other NTM species, such as *Mycobacterium szulgai*, as well as the opportunistic *Mka* type I strains, which has also been reported to secrete major ESX-1 virulence proteins EsxA and EsxB [78, 86]. The ESX-1 virulence seems to be associated with the *espACD* locus [33, 62, 79]. Indeed, the *espACD* operon is found in mycobacteria inducing a phagosomal rupture through an ESX-1 mechanism. The *espACD* operon is notably present in the *Mka* subtype I, the most frequently identified subtype in clinical isolates, while this operon is absent in other subtypes. The virulence function of the *espACD* operon was confirmed by the complementation of *Mka* subtype II, which does not harbour this operon [87], further suggesting that this operon may be a critical determinant of ESX-1 virulence. Interestingly, in the saprophyte *Msm* possessing ESX-1, *espACD* operon is absent which may explain differences in the pathogenicity of these species (Table 1).

## ESX-4

In *Mab*, the screening of a transposon library to identify the genes required for growth in amoeba and macrophages revealed the importance of the *esx-4* locus where 12 transposon insertions were identified [29]. Comparatively, no clones with a transposon insertion and attenuated growth were identified in the *Mab esx-3* locus, suggesting that ESX-3 is indispensable for growth in amoeba and macrophages. Deletion of the gene coding for an ESX-4 structural protein, *EccB*₄, led to a loss of the capacity of *Mab* to block acidification and phagosomal membrane disruption in infected macrophages [29], two intracellular survival strategies employed by wild-type *Mab* [88]. Importantly, an *eccB*₄ deletion mutant showed partial reduction in the secretion of ESX-4 effector proteins: EsxT and EsxU, which are respectively homologs to EsxB and EsxA of *Mtb* ESX-1 [29]. These results support the view that *Mab* ESX-4 functions as a secretion apparatus and importantly with respect to the phenotype conferred to the *eccB*₄ mutant, one may ascribe ESX-4 the same functionalities as those attributed to ESX-1 in strict pathogenic mycobacteria. While there is no reported virulence-associated function for ESX-4 in any other mycobacterial species, the identified function of *Mab* ESX-4 could reasonably be explained by the presence of *EccE*₄ found exclusively in the *M. abscessus-chelonae* complex [31]. This raises the question of whether ESX-4 is functional in the absence of *EccE*₄, or whether *EccE*₄ represents an essential ancestral component for the proper functioning of ESX-4.

Interestingly, another transposon resulting in defective phagosome membrane disruption was identified in *mmpL8*, encoding a transporter of a newly described lipid designated glycosyl diacylated nonadecyl diol (GDND) [89]. It has been hypothesized that there may be a cooperative mechanism in *Mab* between ESX-4 and GDND, similar to the one existing

in *Mma* between ESX-1 and PDIM, to promote phagosomal membrane disruption (Fig. 3a, b).

## ESX-5

The ESX-5 and ESX-2 systems are the two secretion systems restricted to the SGM (Table 1). Until now, no apparent function has been identified for ESX-2 [33, 38] while ESX-5 remains the major T7SS involved in PE/PPE secretion [90, 91]. *Pe* and *ppe* genes emerged from the *esx-1* duplication and are associated with *esx* loci, with the exception of *esx-4*. *Pe/ppe* genes can also be found outside the *esx* loci as in *Mab* genome [55], raising the question of whether ESX-3 and ESX-4 assists the secretion of these PE/PPE proteins in this species.

In *Mma*-infected macrophages, ESX-5 acts in association with ESX-1, playing an important role in virulence. The ESX-5 substrates trigger cytotoxicity and inflammasome activation after cytosol access by an ESX-1 dependent-mechanism [92]. Interestingly, ESX-5 seems to have a complex role during infection, as it also modulates the cell immune response via its substrates to escape the immune system and maintain a persistent infection.

PE/PPE proteins secreted by ESX-5 are hypothesized to be involved in antigenic variation due to their polymorphic C-terminal domain that could allow escape from the host immune system [93]. PE/PPE may also be required for virulence indirectly. For instance, a *pecA* (an aspartic protease) mutant in *Mma* only exhibits moderate virulence in zebrafish [94]. *PecA* processes other PE-PGRS, as well as itself by removing the PE domain of secreted protein at the bacterial surface. *PecA* is entailed in virulence by the cleavage and activation of virulence factor [94].

Moreover, comparison of the wild-type and an ESX-5 mutant strain indicates that *Mma* ESX-5 is involved in decreased antigen presentation, cytokine secretion and attenuates signalling through Toll-like receptors [17]. Other studies described a dual role for PPE38 inducing pro-inflammatory cytokines TNF and IL-6 in macrophages [95]. Cellular proteomic approaches on *Mma*-infected macrophages [96] demonstrate the potential role of PPE38 in preventing antigen presentation and major histocompatibility complex class I processing [96]. In the opportunistic *Mav*, a *ppe* mutant failed to block phagosome acidification, displayed a growth defect in macrophages and attenuated virulence in mice, supporting the idea that in this species lacking ESX-1 (Table 1), ESX-5 is able to fulfil the virulence function [54, 97].

## Mycobacterial gene flow

Compared to pathogenic mycobacteria, in the non-pathogenic NTM *Msm*, the *esx-1* locus is involved in conjugal DNA transfer, namely Distributive Conjugal Transfer (DCT). DCT is unidirectional from donor-to-recipient and is cell-cell contact dependent, with the capacity to transfer multiple segments that results in the transconjugants possessing a mosaic genome [98]. Thus, DCT seemingly requires the ESX-1 system in both donor and recipient strains. Indeed, an ESX-1 donor mutant leads to an hyper-conjugative phenotype [99], whereas an ESX-1 recipient
mutant expresses a hypo-conjugative phenotype in *Msm* [66]. Similarly, *Msm* ESX-4 appears to be involved in DCT, with a recipient ESX-4 mutant defective for conjugation. However, no role for ESX-4 in the donor strain was identified. Moreover, the recipient *esx-4* locus transcripts including *esxU* and *esxT* are induced only in co-culture with the donor by an ESX-1 dependent mechanism and a cell contact dependent process [100]. Furthermore, SigM, an extracytoplasmic transcription factor, was identified as the direct donor-recipient cell contact activator, which activates *esx-4* regulation and promotes *esxT* and *esxU* transcription [101]. Although ESX-4 plays an important function in the DCT, substrate secretion by ESX-4 was not demonstrated. It can be hypothesized that ESX-4 in *Msm* may have evolved to perform a different function from the virulence function observed in *Mab* to overcome the absence of the *eccE* gene. Overall, the role of ESX systems in DCT reflects the interaction and communication of two seemingly disparate secretion systems involved in the same processes with no functional redundancies.

DCT has not been described in other mycobacteria but some mosaic genome patterns have been found in the genome of *Mycobacterium canettii* where this DCT was experimentally demonstrated [102]. A mosaic genome is also observed in *Mab* [103] and some patterns similar to DCT in a 4 kb of *Mav* genome were found [19]. These hallmarks suggest that DCT or substantial horizontal gene transfer took place within mycobacteria and probably participated in the evolution and niche adaptation of mycobacteria [31]. Furthermore, we can also presume that DCT exists in other mycobacteria due to the conservation of ESX and their regulation, however this remains to be proven.

Finally, genes encoding an ESX system and a Type IV secretion system usually present in non-mycobacterial bacteria were detected in a conjugative plasmid found in SGM (pRAW). An *eccC* mutant from this plasmidic ESX impaired the plasmid transfer, suggesting that an ESX secretion system may be involved in DCT as well as in plasmid conjugation [104].

## Nutrient uptake

ESX systems may participate in the uptake of nutrients essential for the mycobacterial planktonic lifestyle and/or survival in phagocytes.

## ESX-3

The ESX-3 secretion system is widespread among mycobacteria, but its role has primarily been explored in *Mtb* where it is involved in iron and zinc homeostasis [20, 21] and in virulence [105]. The ESX-3 function in iron acquisition is conserved in *Msm* but has not been studied in other NTM [20]. While ESX-3 is essential in *Mtb*, it is dispensable in *Msm*, suggesting differential functions/requirements among mycobacterial species. Moreover, ESX-3 has been linked to virulence in *Mab*, in which an *esx-3* mutant exhibits a more attenuated phenotype *in vivo* with a decrease in the inflammatory cytokine response [106]. However, the role of *Mab* ESX-3 in contributing to virulence remains yet to be confirmed with functional complementation studies.

## ESX-5

Most studies relating the ESX-5 function in *Mma* include a mutant possessing a transposon in the cytosolic components, *eccA* and *espG* gene, while no transposons were identified in genes composing the core of the ESX-5 complex [90]. *Mma* ESX-5 has been shown to be essential *in vitro*, but it can be by-passed by disturbing the permeability of the mycomembrane or by the expression of a mycobacterial porin from *Msm*, *MspA*, only described in RGM [22]. In addition, cultures with different carbon sources unravelled the role of ESX-5 and its substrates in the uptake of fatty acids [22]. Since ESX-5 systems are a hallmark of SGM, considered to be the group in which most pathogenic mycobacteria are found, it can be inferred that ESX-5 has evolved towards a role dedicated to the transport of small nutrients. Moreover, increased permeability seems to disadvantage persistence of mycobacteria during infection, and thus ESX-5 could represent a niche adaptation of SGM to pathogenicity [22].

## Bacterial cell envelope homeostasis

In addition to virulence and fatty acid uptake, *Mma* ESX-5 and its PPE substrates are also involved in the mycobacterial capsule integrity. Indeed, an *esx-5* (*espG5::Tn*) and *ppe10* mutant showed an impairment in the integrity of the capsule, characterized by a decrease of capsular proteins and ESX-1 substrates to the bacterial surface leading to an attenuated virulence phenotype [23]. The link between mycomembrane homeostasis and PPE proteins was also demonstrated with *Mma* PPE38, for which the absence leads to deficiencies in the cell wall integrity, affecting cord and biofilm formation, ultimately resulting in defective phagocytosis and reduced virulence *in vivo* [95]. Both results demonstrate the importance of PPE in the mycobacterial membrane integrity.

In addition, in *Mma*, several ESX-1 substrate mutants, *espE*, *espG*, *espF* and *espH* exhibit a biofilm and sliding motility defect [107]. Mycobacterial biofilms are found in the environment, as well as on patient medical devices, and represents an advantage for persistent infections and antibiotic resistance [108]. The involvement of ESX in biofilm formation illustrates the role of ESX in the adaptation of mycobacteria and notably NTM to the various environments in which they are isolated.

## CONCLUSION

The last few years have been marked by key achievements in the study of ESX in mycobacteria. The structures of the ESX-3 and ESX-5 systems from two NTM species were solved, allowing a better understanding of the interaction of the core complex proteins, as well as their integration into the mycomembrane. The interaction of *EccE* with other ESX core components raises questions about the functionality of ESX-4 where *EccE* is absent, with the exception of *Mab* ESX-4. Recently, ESX-4 from *Mab* has been shown to be functional and involved in the resistance of this mycobacterium to phagocytic cells whereas, until recently, ESX-1 and ESX-5 were perceived as the major players in the
virulence of pathogenic and opportunistic mycobacteria. In addition to their role in virulence, ESX-4 and ESX-1 are also involved in DNA transfer in the saprophytic mycobacteria *Msm*, indicating that the pathogenicity of a strain cannot be explained by the sole presence of one ESX system. Plasmid-mediated evolution provides clues for understanding ESX acquisition in the different species, while ESX evolution correlates with the growth dichotomy typifying RGM and SGM, as illustrated by the emergence of ESX-2 and ESX-5 in the latter. Overall, ESX systems represent a powerful resource that allow mycobacteria to adapt to their various ecological niches.

## Funding information

This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement n°846476 (to C.P.) and from the Joachim Herz Stiftung, Hamburg, Germany (to M.W. and F.P.M. as members of the Infectrophysics Consortium). M.L. has received funding from the French associations 'Vaincre la mucoviscidose Association and Grégory Lemarchal Association' (grant agreement n° (RIF20190502522). M.D.J received a post-doctoral fellowship granted by Labex EpiGenMed, an «Investissements d'avenir» program (ANR-10-LABX-12-01).

## Author contributions

M.L., C.P., F.V., M.D.J. wrote the original manuscript, K.S.H.B. and C.R. contributed to the description of structural data; F.M., J-L.H., F.G-M. supervised the project. J-L.H., F.G-M., M.W., F.M. and L.K. participated in writing the manuscript and supported the project.

## Conflicts of interest

The authors declare that there are no conflicts of interest.

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