3.A.24 The Type VII or ESX Protein Secretion System (T7SS) Family
Mycobacteria possess several related protein secretion systems often referred to as Type VII Secretion Systems (T7SS) that may be distantly related to Type IV secretion systems (T4SS; TC# 3.A.7) (Bitter et al. 2009a; Bitter et al. 2009b). Virulent Mycobacterium tuberculosis (Mtb) possesses several regions of genetic virulence that are lacking in avirulent M. bovis BCG. One of these regions of difference, RD1, is present in all virulent strains and lacking in all avirulent strains tested. Deletion of this 9.5 kb region from Mtb yields attenuation of virulence, and this mutant can be complemented with the cloned region. This system is called the early secreted antigen 6 kilodaltons (ESAT-6) secretion system 1 (ESX1) or secretion in mycobacteria (Snm), and it delivers virulence factors into host macrophages during infection. Four proteins have been shown to be secreted via this system: ESAT-6, CFP-10, Rv3483c (MM1553 in M. marinum) and Rv3881c (Mb3881c in M. marinum) (Xu et al. 2007). Two characteristics unify all esx loci; first, the secretion of small (~100 aas) proteins with a conserved Trp-X-Gly (WXG) motif which contibutes to the formation of a helix-turn-hexix structure in the center of the protein, and second, the presence of a gene that encode a TM protein of the FtsK/SpoIIIE ATPase family (Gröschel et al. 2016). A phylogenetic tree of the various ESX components/systems has been published (Gröschel et al. 2016).
There are ten ORFs in RD1:
(1) Rv3870: an ATP-dependent chaperone (747 aas; 3 putative TMSs in N-terminal region) (Includes a region (467-702 aas) homologous to SpoIIIE of B. subtilis in the 5-DNA-T family) (TC #3.A.12)
(2) Rv3871: an integral membrane protein (591 aas; 2 or 3 putative TMS in C-terminal region) may form a functional complex with Rv3870 (slight sequence similarity to a region of SpoIIIE)
(3) Rv3872: a PE repetitive protein (111 aas)
(4) Rv3873: a PPE repetitive protein (368 aas)
(5) Rv3874(cfp-10): a 10 kDa cultured protein (100 aas)
(6) Rv3875(esat-6): a 6 kDa cultured protein (100 aas) that forms a tight 1:1 complex with Rv3874, a potent T-cell antigen; Rv3874 and Rv3875 are homologous
(7) Rv3876: a protein with a proline- + alanine-rich N-terminus and a C-terminal region homologous to a P. aeruginosa flagellar biosynthesis protein with a Walker A motif (666 aas)
(8) Rv3877: an integral membrane protein (511 aas; 11 putative TMSs)
(9) Rv3878 and 3879: nonessential for virulence.
(10) Rv3860, an essential component of the ESX-1 secretion system (Luthra et al. 2008).
CFP-10 and ESAT-6 are secreted via the RD1 system and may be part of it. Because Rv3870 and 3876 have motifs suggesting they are ATPases, the system may be ATP-dependent, but this has not been demonstrated. Deletion of any of the following genes blocks virulence and secretion: Rv3870, 71, 74, 75 and 76. The (CFP-10)(ESAT-6) dimer is stable, but the exported monomers may not be.
The ESX-1 system (ESAT-6 system-1) is required for controlling host-cell response to infection (Guinn et al. 2004; Hsu et al. 2003; Stanley et al. 2003). ESX-1 is encoded by genes in the RD1 (region of difference 1) locus of the genome that is missing in the M. bovis Bacille Calmette-Guérin vaccine strain (Behr et al. 1999; Mahairas et al. 1996). This system includes a multitransmembrane protein, Rv3877 (Snm4), and two putative SpoIIIE/FtsK adenosine triphosphatase (ATPase) family members, Rv3870 (Snm 1) and Rv3871 (Snm2). These three proteins are required for secretion of two virulence factors, ESAT-6 and CFP-10 (Stanley et al., 2003). ESAT-6 (product of the esxA gene) and CFP-10 (product of the esxB gene) interact to form a 1:1 dimer (Renshaw et al. 2002), and the stability of these proteins is interdependent in vivo. CFP-10, but not ESAT-6, interacts with the C-terminal domain of Rv3871, a cytosolic component of the ESX-1 system (Stanley et al., 2003). Although the secretion of ESAT-6 and CFP-10 is critical for M. tuberculosis virulence, the molecular mechanisms of ESX-1 substrate selection and secretion are unclear. The targetting sequence in CFP-10 is at its C-terminus (Champion et al. 2006).
RD1 appears to play a role in a novel type of DNA transfer (conjugation) in Mycobacterium smegmatis. DNA transfer requires prolonged cell-cell contact and occurs only from donors to recipients. There are multiple transfer initiations from a donor chromosome and, as a result, the inheritance of any gene is location-independent (Wang et al. 2005). Transfer is not contiguous; instead, multiple non-linked segments of DNA can be inherited in a recipient. However, with appropriate selection, segments of DNA at least 266 kb in length can be transferred. In contrast to Hfr transfer, transconjugants can become donors, suggesting that the recipient chromosome contains multiple cis-acting sequences required for transfer but lacks the trans-acting transfer functions. The mechanistic details of how RD1 plays a role in conjugation is not known and could be indirect (Wang et al. 2005). However, gene transfer from M. smegmatis to mammalian cells has also been demonstrated, possible via the same 'conjugation' system (Mo et al. 2007).
Two characteristics unify all esx loci across the different phyla. First, the presence of genes that encode small secreted proteins of approximately 100 aas that have a conserved Trp-X-Gly (WXG) motif that contributes to the formation of helix–turn–helix structures in the centres of these polypeptides; and, second, the presence of genes that encode transmembrane proteins of the FtsK–SpoIIIE-like ATPase family. The best characterized proteins that contain the WXG motif are EsxA of M. tuberculosis and its adjacently encoded hetero-dimerization partner EsxB (also known as CFP10). Apart from these core characteristics, ESX systems are quite diverse (Gröschel et al. 2016).
An ESX/Type VII secretion system in the plant pathogen, Streptomyces scabies, is required for normal sporulation development but not for virulence (Fyans et al. 2012). This system may export two sequence related proteins, EsxA and EsxB. ESX-1 is present in the avirulent species, Mycobacterium smegmatis, where it is involved in conjugation (Coros et al. 2008). ESX-1 uses the ESX-1-specific chaparone protein, EspG to interact with the secreted PE/PPE complex, while a homologous EspG specific for ESX-5 functions with the PE/PPE complex secreted by ESX-5. Thus, EspG proteins may be system-specific chaparones for T7SSs (Daleke et al. 2012).
Rv3869, Rv3870 and Rv3877, exhibit 1, 3 and 11 predicted TMSs, respectively. Together with Rv3871, a cytosolic component of the ESX-1 system, these proteins form the ESX-1 secretory complex, with protein export driven by ATP hydrolysis. A C-terminal signal sequence allows the unstructured C-terminus of CFP-10 to be recognized by Rv3871 that itself interacts with the membrane protein Rv3870. Point mutation in this signal sequence abolish binding of CFP-10 to Rv3871 and prevented secretion of ESAT-6 and CFP-10. As Rv3870 and Rv3871 both show similarity to proteins of the FtsK-SpoIIIE ATPase family, these proteins might perform an essential part of the work necessary to secrete ESX-1 substrates. This mechanism resembles type IV secretion system in Gram-negative bacteria, where a membrane-bound SpoIIIE/FtsK-like ATPase recognizes an unstructured C-terminal sequence and directs the secreted substrate to the cytoplasmic membrane (Simeone et al. 2009).
The M. tuberculosis rv3616c-rv3614c gene cluster is involved in the regulation of ESAT-6 secretion. This cluster is regulated at the expression level byPhoR/P and EspR (Simeone et al. 2009).
The ESX-3 cluster is controlled by the iron-dependent regulator IdeR and the zinc uptake regulator Zur (previously named FurB). This suggests that ESX-3 might be involved in fundamental biological processes such as metal ion homeostasis, consistent with the essentiality of ESX-3 for in vitro growth of M. tuberculosis and the conservation of orthologous ESX-3 systems in a wide range of mycobacterial species (Simeone et al. 2009).
Mycobacterial genomes contain two unique gene families, the so-called PE and PPE gene families, which are highly expanded in the pathogenic members of this genus. One of the PPE proteins, PPE41, is secreted by pathogenic mycobacteria. As PPE41 lacks a signal sequence, a dedicated secretion system must be involved. A gene was identified in Mycobacterium marinum that showed strongly reduced PPE41 secretion (Abdallah et al. 2006). It was located in a gene cluster whose predicted proteins encode components of an ESAT-6-like secretion system. This cluster, designated ESX-5, is conserved in various pathogenic mycobacteria, but not in the saprophytic species Mycobacterium smegmatis. Different regions of this cluster were introduced in M. smegmatis, and only introduction of the complete ESX-5 locus resulted in efficient secretion of heterologously expressed PPE41. This PPE secretion system is also involved in the virulence of pathogenic mycobacteria, as the ESX-5 mutant of M. marinum was affected in spreading to uninfected macrophages (Abdallah et al., 2006). Homologues are found in various Actinobacteria.
Gray et al. 2016 showed that the ESX secretion-system family member ESX-4 is essential for conjugal recipient activity in Mycobacterium smegmatis. Transcription of esx4 genes in the recipient requires coculture with a donor strain and a functional ESX-1 apparatus in the recipient. Conversely, mutation of the donor ESX-1 apparatus amplifies the esx4 transcriptional response in the recipient. The effect of ESX-1 on esx4 transcription correlates with conjugal DNA transfer efficiencies. The data show that intercellular communication via ESX-1 controls the expression of its evolutionary progenitor, ESX-4, to promote conjugation between mycobacteria (Gray et al. 2016).
Specialized protein transport systems, type VII secretion systems (T7SSs)-are central to the virulence of Mycobacterium tuberculosis, and they are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope. Bunduc et al. 2021 presented the structure of an intact T7SS inner-membrane complex of M. tuberculosis. They showed how the 2.32-MDa ESX-5 assembly, which contains 165 TMSs, is restructured and stabilized as a trimer of dimers by the MycP5 protease. A trimer of MycP5 caps a central periplasmic dome-like chamber that is formed by three EccB5 dimers, with the proteolytic sites of MycP5 facing the cavity. This chamber may be part of a central secretion and processing conduit. Complexes without MycP5 show disruption of the EccB5 periplasmic assembly and increased flexibility, which highlights the importance of MycP5 for complex integrity. Beneath the EccB5-MycP5 chamber, dimers of the EccC5 ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC5 domains adopt two distinctive conformations that probably reflect different secretion states. This suggests a previously undescribed mechanism of protein transport (Bunduc et al. 2021).
Transport reactions catalyzed by the RD1 complex include:
CFP-10in + ESAT-6in → (CFP-10)(ESAT-6)out.
The RD1 (ESX-1) protein secretion complex (Type VII protein secretion system, T7SS). EccA1 may be a secreted protein while ECCB1 - E1 may comprise the system (Houben et al. 2012). This system (ESX-1) is present in the avirulent species, Mycobacterium smegmatis, where it is involved in conjugation (Coros et al. 2008). ESX-1 uses the ESX-1-specific chaparone protein, EspG to interact with the secreted PE/PPE complex, while a homologous EspG specific for ESX-5 functions with the PE/PPE complex secreted by ESX-5. Thus, EspG proteins may be system-specific chaparones for T7SSs (Daleke et al. 2012). The main secreted virulence protein complex is a heterodimer: EsxA(ESAT-6)/EsxB(CFP-10) (Rosenberger et al. 2012). EccB, a periplasmic homoheximer with the ATP-binding active site shared by two adjacent subunits, may act as the energy provider in the transport of T7SS virulence factors and may be involved in the formation of a channel across the mycomembrane (Zhang et al. 2015). ESX-1 functions in resistance to and evasion of host responses. It induces phagosomal rupture which releases bacteria into the cytosol of the host phagocytes (Gröschel et al. 2016). ESX-1 secrete EsxA and EsxB, which form a heterodimer, seem to have differing functions as EsxA can disrupt lipid bilayers (RBC and artificial membranes. Thus EsxA may form pores as a prelude to membrane disruption (Gröschel et al. 2016).
The RD1 complex (Rv3868, Rv3870-3877 and Rv3615c and Rv3614c) of Mycobacterium tuberculosis
Rv3868 (EccA1) (O69733)
Rv3869 (EccB1) (O69734)
Rv3870 (EccCa1) (O69735)
Rv3871 (EccCb1) (O69736)
Rv3872 (EccC1a) (Q79F93)
Rv3873 (EccC1b) (Q79F92)
Rv3874 (Cfp-10) (P0A566)
Rv3875 (Esat-6) (P0A564)
Rv3876 (EspI) (O69740)
Rv3877 (EccD1) (O69741)
Rv1794 (EccE1) (O05462)
Rv3615c (Snm9) (P65087)
Rv3614c (Snm10) (O06269)
The Esx-2 type VII protein secretion system
Esx-2 protein secretion system of Mycobacterium tuberculosis
The Esx-3 type VII protein secretion system. Controlled by the iron-dependent regulator, IdeR and the Zn2+-uptake regulator, Zur. ESX-3 is involved in metal ion homeostasis, consistent with its essentiality for in vitro growth (Simeone et al. 2009). Exports the EsxG-EsxH complex which contains a Zn2+ binding site in EsxH and may function in Zn2+ acquisition (Ilghari et al. 2011). The system plays an essential role in siderophore-mediated iron uptake (Gröschel et al. 2016).
Esx-3 of Mycobacterium tuberculosis
The Type VII secretion system (T7SS or MPSS); the ESAT-6-like protein secretion system, ESX-5 (Abdallah et al., 2006; Sayes et al., 2012; Houben et al. 2012). Mediates the secretion of the EsxN, PPE [pro-pro-glu motif] and PE_PGRS proteins (Deng and Xie 2012). The EccB5, EccC5, EccD5 and EccE5 proteins form a membrane complex of about 1.5 MDa while EccA5 may be the channel-forming subunit that exports the substrate proteins (Bunduc et al. 2021). Thus, ESX-5 exports protein substrate (Houben et al. 2012). PE25/PPE41form a heterodimer that is targeted to the T7SS, ESX5, by a C-terminal signal in PE25 which is necessary but not sufficient for targeting to ESX5 (Daleke et al. 2012). An esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Possibly the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake (Ates et al. 2015). Mycobacterium tuberculosis releases membrane vesicles (MV) that modulate host immune responses and aid in iron acquisition, and the ESX-5 system seems to play a role, together with the SenX3-RegX3 complex which regulates the process (White et al. 2018). The 3-D structure of ESX-5 has been determined (Bunduc et al. 2021) (see ESX5 family description, paragraph 11 for details). The structure of the mycobacterial ESX-5 type VII secretion system pore complex has been determined (Beckham et al. 2021). The high-resolution structure of the 2.1-megadalton ESX-5 core complex captured a dynamic, secretion-competent conformation of the pore within a well-defined transmembrane section, sandwiched between two flexible protein layers at the cytosolic entrance and the periplasmic exit. Beckham et al. 2021 proposed that this flexibility endows the ESX-5 machinery with large conformational plasticity required to accommodate targeted protein secretion. A highly dynamic state of the pore may represent a fundamental principle of bacterial secretion machineries.
The potential 8 protein ESX5 system of Mycobacterium tuberculosis
Rv1782 (EccB5) (506 aas) (O53933)
Rv1783 (EccC5a) (435 aas; resembles Rv3870 under TC# 9.A.25.1.1) (O53934)
Rv1784 (EccC5b) (932 aas; resembles Rv3871 and regions of Rv3870 under TC# 9.A.25.1 as well as Spo and FtsK under TC# 3.A.12) (O53935)
Rv1794 (300aas) (O53943)
Rv1795 (EccD5) (503 aas; resembles Rv3877 under TC# 9.A.25) (O53944)
MycP5 (585 aas; resembles proteins of the AT family under TC# 1.B.12: TC# 1.B.12.5.1 and 1.B.12.9.1) (O53945)
Rv1797 (EccE5) (199aas) (O53946)
Rv1798 (EccA5) (610 aas; resembles ATPases) (P63744)
Esx-4 secretion system. Gray et al. 2016 showed that ESX-4 is essential for conjugal recipient activity in Mycobacterium smegmatis. Transcription of esx4 genes in the recipient requires coculture with a donor strain and a functional ESX-1 apparatus in the recipient. Conversely, mutation of the donor ESX-1 apparatus amplifies the esx4 transcriptional response in the recipient. The effect of ESX-1 on esx4 transcription correlates with conjugal DNA transfer efficiencies. Thus, intercellular communication via ESX-1 controls the expression of its evolutionary progenitor, ESX-4, to promote conjugation between mycobacteria. The eight proteins listed under this TC# are all encoded withing a single gene cluster (Gray et al. 2016).
Esx-4 of Mycobacterium smegmatis
Msmeg_1537 of 368 aas (A0QSN1)
EccB4 of 458 aas; Membrane protein with 1 N-terminal TMS (A0QSM7)
EccD4 of 437 aas; Membrane protein with 11 predicted TMSs (A0QSM9)
MycP4 of 442 aas with 2 TMSs (N- and C-terminal) as well as 7 central moderately hydrophobic peaks that could be, but were not predicted to be TMSs. Predicted to have serine endopeptidase activity. (A0QSM8)
EccA4 of 574 aas; an AAA ATPase (A0QNI9)
EccC4 of 1211 aas; FtsK/SpoIIIE ATPase (A0QSN0)
EsxU of 103 aas; ESAT-6-like protein (L8FHH3). May be a secreted pore-forming protein (see TC# 1.C.95.
EsxT of 98 aas; ESAT-6-like protein (L8FGI7). May be a secreted pore-forming protein (see TC# 1.C.95.