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3.A.24 Type VII or ESX Protein Secretion System (T7SS) Family

Mycobacteria possess several related protein secretion systems often referred to as Type VII Scretion Systems (T7SS) that may be distantly related to Type IV secretion systems (T4SS; TC# 3.A.7) (Bitter et al. 2009aBitter 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).

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. 2004Hsu et al. 2003Stanley 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. 1999Mahairas 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).

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).

Transport reactions catalyzed by the RD1 complex include:

CFP-10in + ESAT-6in → (CFP-10)(ESAT-6)out.

This family belongs to the: AAA-ATPase Superfamily.

References associated with 3.A.24 family:

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Ates, L.S., R. Ummels, S. Commandeur, R. van der Weerd, M. Sparrius, E. Weerdenburg, M. Alber, R. Kalscheuer, S.R. Piersma, A.M. Abdallah, M. Abd El Ghany, A.M. Abdel-Haleem, A. Pain, C.R. Jiménez, W. Bitter, and E.N. Houben. (2015). Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria. PLoS Genet 11: e1005190. 25938982
Behr, M.A., M.A. Wilson, W.P. Gill, H. Salamon, G.K. Schoolnik, S. Rane, and P.M. Small. (1999). Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284: 1520-1523. 10348738
Bitter, W., E.N. Houben, D. Bottai, P. Brodin, E.J. Brown, J.S. Cox, K. Derbyshire, S.M. Fortune, L.Y. Gao, J. Liu, N.C. Gey van Pittius, A.S. Pym, E.J. Rubin, D.R. Sherman, S.T. Cole, and R. Brosch. (2009). Systematic genetic nomenclature for type VII secretion systems. PLoS Pathog 5: e1000507. 19876390
Bitter, W., E.N. Houben, J. Luirink, and B.J. Appelmelk. (2009). Type VII secretion in mycobacteria: classification in line with cell envelope structure. Trends Microbiol. 17: 337-338. 19660950
Champion, P.A., S.A. Stanley, M.M. Champion, E.J. Brown, and J.S. Cox. (2006). C-terminal signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis. Science 313: 1632-1636. 16973880
Coros, A., B. Callahan, E. Battaglioli, and K.M. Derbyshire. (2008). The specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis. Mol. Microbiol. 69: 794-808. 18554329
Daleke, M.H., A.D. van der Woude, A.H. Parret, R. Ummels, A.M. de Groot, D. Watson, S.R. Piersma, C.R. Jiménez, J. Luirink, W. Bitter, and E.N. Houben. (2012). Specific chaperones for the type VII protein secretion pathway. J. Biol. Chem. 287: 31939-31947. 22843727
Fyans JK., Bignell D., Loria R., Toth I. and Palmer T. (2013). The ESX/type VII secretion system modulates development, but not virulence, of the plant pathogen Streptomyces scabies. Mol Plant Pathol. 14(2):119-30. 23009676
Gray, T.A., R.R. Clark, N. Boucher, P. Lapierre, C. Smith, and K.M. Derbyshire. (2016). Intercellular communication and conjugation are mediated by ESX secretion systems in mycobacteria. Science 354: 347-350. 27846571
Guinn, K.M., M.J. Hickey, S.K. Mathur, K.L. Zakel, J.E. Grotzke, D.M. Lewinsohn, S. Smith, and D.R. Sherman. (2004). Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol. Microbiol. 51: 359-370. 14756778
Hsu, T., S.M. Hingley-Wilson, B. Chen, M. Chen, A.Z. Dai, P.M. Morin, C.B. Marks, J. Padiyar, C. Goulding, M. Gingery, D. Eisenberg, R.G. Russell, S.C. Derrick, F.M. Collins, S.L. Morris, C.H. King, and W.R. Jacobs, Jr. (2003). The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc. Natl. Acad. Sci. USA 100: 12420-12425. 14557547
Luthra, A., A. Mahmood, A. Arora, and R. Ramachandran. (2008). Characterization of Rv3868, an essential hypothetical protein of the ESX-1 secretion system in Mycobacterium tuberculosis. J. Biol. Chem. 283: 36532-36541. 18974091
Mahairas, G.G., P.J. Sabo, M.J. Hickey, D.C. Singh, and C.K. Stover. (1996). Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178: 1274-1282. 8631702
Mo, Y., N.M. Quanquin, W.H. Vecino, U.D. Ranganathan, L. Tesfa, W. Bourn, K.M. Derbyshire, N.L. Letvin, W.R. Jacobs, Jr, and G.J. Fennelly. (2007). Genetic alteration of Mycobacterium smegmatis to improve mycobacterium-mediated transfer of plasmid DNA into mammalian cells and DNA immunization. Infect. Immun. 75: 4804-4816. 17664267
Renshaw, P.S., K.L. Lightbody, V. Veverka, F.W. Muskett, G. Kelly, T.A. Frenkiel, S.V. Gordon, R.G. Hewinson, B. Burke, J. Norman, R.A. Williamson, and M.D. Carr. (2005). Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6. EMBO. J. 24: 2491-2498. 15973432
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Simeone, R., D. Bottai, and R. Brosch. (2009). ESX/type VII secretion systems and their role in host-pathogen interaction. Curr. Opin. Microbiol. 12: 4-10. 19155186
Stanley, S.A., S. Raghavan, W.W. Hwang, and J.S. Cox. (2003). Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc. Natl. Acad. Sci. USA 100: 13001-13006. 14557536
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