The plasmid-mediated evolution of the mycobacterial ESX (Type VII) secretion systems

Abstract

Background: The genome of Mycobacterium tuberculosis contains five copies of the ESX gene cluster, each encoding a dedicated protein secretion system. These ESX secretion systems have been defined as a novel Type VII secretion machinery, responsible for the secretion of proteins across the characteristic outer mycomembrane of the mycobacteria. Some of these secretion systems are involved in virulence and survival in M. tuberculosis; however they are also present in other non-pathogenic mycobacteria, and have been identified in some non-mycobacterial actinomycetes. Three components of the ESX gene cluster have also been found clustered in some gram positive monoderm organisms and are predicted to have preceded the ESX gene cluster.

Results: This study used in silico and phylogenetic analyses to describe the evolution of the ESX gene cluster from the WXG-FtsK cluster of monoderm bacteria to the five ESX clusters present in M. tuberculosis and other slow-growing mycobacteria. The ancestral gene cluster, ESX-4, was identified in several nonmycomembrane producing actinobacteria as well as the mycomembrane-containing Corynebacteriales in which the ESX cluster began to evolve and diversify. A novel ESX gene cluster, ESX-4EVOL, was identified in some non-mycobacterial actinomycetes and M. abscessus subsp. bolletii. ESX-4EVOL contains all of the conserved components of the ESX gene cluster and appears to be a precursor of the mycobacterial ESX duplications. Between two and seven ESX gene clusters were identified in each mycobacterial species, with ESX-2 and ESX-5 specifically associated with the slow growers. The order of ESX duplication in the mycobacteria is redefined as ESX-4, ESX-3, ESX-1 and then ESX-2 and ESX-5. Plasmid-encoded precursor ESX gene clusters were identified for each of the genomic ESX-3, -1, -2 and -5 gene clusters, suggesting a novel plasmid-mediated mechanism of ESX duplication and evolution.

Conclusions: The influence of the various ESX gene clusters on vital biological and virulence-related functions has clearly influenced the diversification and success of the various mycobacterial species, and their evolution from the non-pathogenic fast-growing saprophytic to the slow-growing pathogenic organisms.

Keywords: ESAT-6; ESX; Evolution; Mycobacterium; Plasmid; Type VII secretion system.

Fig. 1

The phylogeny of the ESX gene cluster. Maximum likelihood phylogeny of representative ESX gene clusters describing the evolution of the ESX gene cluster from its WXG-FtsK cluster progenitor. The ESX gene clusters form five groups, ESX-4, ESX-3, ESX-1, ESX-2 and ESX-5. The plasmid located and ancestral ESX gene clusters form subgroups of each genomic ESX gene cluster. The ESX gene clusters have evolved divergently from a single duplication of ESX-4 to ESX-1 and ESX-3 and then ESX-2 and ESX-5. One hundred subsets were generated for bootstrapping resampling of the data

Fig. 2

Model of ESX evolution based on plasmid-mediated duplication and evolution. The ancestral ESX-4 gene cluster evolved from the WXG-FtsK cluster via the incorporation of additional genes, eccB, eccD, mycP and rv3446c. ESX-4 was duplicated into plasmid DNA, into which additional ESX genes, eccA, eccE, espI, espG, pe and ppe, were incorporated. The plasmid ancestor (ESX-P_AN) was reinserted into the genomes of various Mycolata, generating ESX-4_EVOL. Continuous evolution generated the operonic structure of the plasmid ESX gene cluster. Divergent evolution of the plasmid ESX generated several plasmid ESX (ESX-P1, -P3, -P2’, -P2 and -P5) which were inserted into the mycobacterial genome to generate ESX-3, ESX-1, ESX-2 and ESX-5. An earlier version of ESX-P1 was inserted into the genomes of some actinomycetes as ESX-1_AN and a precursor of ESX-P2’ was inserted into the M. tusciae genome as ESX-2_AN. Red arrows represent genome insertions

Fig. 3

The phylogeny of the mycobacteria based on ESX duplication and evolution. Maximum likelihood phylogeny describing the evolution of the mycobacteria based on the concatenated ESX gene cluster amino acid sequences from each species. ESX duplication and deletion events influenced the evolution and diversification of the mycobacteria as described in the text. Species which contain plasmid ESX gene clusters are underlined. One thousand subsets were generated for bootstrapping resampling of the data