Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions

Abstract

Background: The PE and PPE multigene families of Mycobacterium tuberculosis comprise about 10% of the coding potential of the genome. The function of the proteins encoded by these large gene families remains unknown, although they have been proposed to be involved in antigenic variation and disease pathogenesis. Interestingly, some members of the PE and PPE families are associated with the ESAT-6 (esx) gene cluster regions, which are regions of immunopathogenic importance, and encode a system dedicated to the secretion of members of the potent T-cell antigen ESAT-6 family. This study investigates the duplication characteristics of the PE and PPE gene families and their association with the ESAT-6 gene clusters, using a combination of phylogenetic analyses, DNA hybridization, and comparative genomics, in order to gain insight into their evolutionary history and distribution in the genus Mycobacterium.

Results: The results showed that the expansion of the PE and PPE gene families is linked to the duplications of the ESAT-6 gene clusters, and that members situated in and associated with the clusters represent the most ancestral copies of the two gene families. Furthermore, the emergence of the repeat protein PGRS and MPTR subfamilies is a recent evolutionary event, occurring at defined branching points in the evolution of the genus Mycobacterium. These gene subfamilies are thus present in multiple copies only in the members of the M. tuberculosis complex and close relatives. The study provides a complete analysis of all the PE and PPE genes found in the sequenced genomes of members of the genus Mycobacterium such as M. smegmatis, M. avium paratuberculosis, M. leprae, M. ulcerans, and M. tuberculosis.

Conclusion: This work provides insight into the evolutionary history for the PE and PPE gene families of the mycobacteria, linking the expansion of these families to the duplications of the ESAT-6 (esx) gene cluster regions, and showing that they are composed of subgroups with distinct evolutionary (and possibly functional) differences.

Figure 1

Genomic organization of the PE and PPE genes associated with the Mycobacterium tuberculosis ESAT-6 (esx) gene clusters. Open reading frames are represented by blocked arrows indicating direction of transcription, with the different colors reflecting specific gene families and the length of the arrow reflecting the relative lengths of the genes. (A) Schematic representation of the PE and PPE genes situated within the ESAT-6 (esx) gene cluster regions. The vertical arrow indicates the direction of duplication of the ESAT-6 (esx) gene cluster regions, from region 4, 1, 3, 2 and lastly 5 in descending order. The positions of the PE (small arrow in light green) and PPE (larger arrow in yellow) genes are blocked, (B) Schematic representation of the PE and PPE genes duplicated from the ESAT-6 (esx) gene cluster regions, with the positions of the ESAT-6 and CFP-10 genes indicated, (C) Schematic representation of the PE and PPE genes associated with the ESAT-6 (esx) gene cluster regions (''associated with'' denotes genes which are hypothesized to have been duplicated from ESAT-6 (esx) gene cluster regions, as they are very homologous to their paralogues within the ESAT-6 (esx) gene clusters and have the same paired genomic orientation – see also Table 2).

Figure 2

PE/PPE gene structure. (A) Diagrammatic representation of the gene structure of the members of the PE and PPE gene family, showing conserved N-terminal domains, motif positions and differences between different subfamilies found in the two families [12,16]. (B) Alignment of the region surrounding the SVP motif Gly-X-X-Ser-Val-Pro-X-X-Trp in the members of the PPE-SVP subfamily. (C) Alignment of the region surrounding the GFGT motif (Gly-Phe-X-Gly-Thr) and PPW motif (Pro-X-X-Pro-X-X-Trp) in the members of the PPE-PPW subfamily.

Figure 3

Phylogenetic tree of all the members of the genus Mycobacterium. Strict consensus of the 230 most parsimonious trees using Paup 4.0b10 (heuristic search, gaps = fifth state) [89] from the 1286 aligned nucleotides of the 16S rRNA DNA sequence of 80 species of the genus Mycobacterium with the sequence of the species Gordonia aichiensis as the outgroup. Sequenced genomes are highlighted in yellow. The division between fast and slow-growing species is indicated by a dotted line. Underlined species are considered pathogens [62]. The members of the M. tuberculosis complex and the M. avium complex are indicated. The divisions between the normal helix 18, long helix 18 and extended helix 18 of the 16S rRNA gene sequence are indicated [94,95]. * = M. farcinogenes is a slow growing mycobacterium.

Figure 4

Phylogeny of the PE and PPE protein families present within the ESAT-6 (esx) gene clusters in M. tuberculosis H37Rv. Phylogenetic trees of the PE and PPE proteins, respectively, present within the ESAT-6 (esx) gene clusters in M. tuberculosis H37Rv, demonstrating a duplication order similar to that observed with other genes in the M. tuberculosis ESAT-6 (esx) gene cluster regions [1]. (A) PE proteins, (B) PPE proteins and (C) ESAT-6/CFP-10 proteins.

Figure 5

Phylogenetic reconstruction of the evolutionary relationships between the members of the PE protein family. The phylogenetic tree was constructed from the phylogenetic analyses done on the 110 aa N-terminal domains of the PE proteins. The tree was rooted to the outgroup, Rv3872 (PE35), shown to be the first PE insertion into the ESAT-6 (esx) gene clusters (region 1). The genes highlighted in purple, green and blue are present in ESAT-6 (esx) gene cluster region 1, 3 and 2, respectively. Genes highlighted in red are present in or have been previously shown to be duplicated from ESAT-6 (esx) gene cluster region 5 [1] and genes highlighted in yellow are members of the PGRS subfamily of the PE family. Arrows indicate orthologues of genes identified to be present within the M. smegmatis genome sequence. Five sublineages (including the PE_PGRS subfamily) are indicated by Roman numerals.

Figure 6

Phylogenetic reconstruction of the evolutionary relationships between the members of the PPE protein family. The phylogenetic tree was constructed from the phylogenetic analyses done on the 180 aa N-terminal domains of the PPE proteins. The tree was rooted to the outgroup, Rv3873 (PPE68), shown to be the first PPE insertion into the ESAT-6 (esx) gene clusters (region 1). The gene highlighted in purple is present in ESAT-6 (esx) gene cluster region 1, genes highlighted in green are present in or have been previously shown to be duplicated from ESAT-6 (esx) gene cluster region 3 [1], the gene highlighted in blue is present in ESAT-6 (esx) gene cluster region 2, genes highlighted in red are present in or have been previously shown to be duplicated from ESAT-6 (esx) gene cluster region 5 [1] and genes highlighted in yellow are members of the MPTR subfamily of the PPE family. Arrows indicate orthologues of genes present within the M. smegmatis genome sequence. Five sublineages (including the PPE-PPW, PPE-SVP and PPE-MPTR subfamilies) are indicated by Roman numerals.

Figure 7

Orthologues of M. tuberculosis PE genes present in the genomes of M. smegmatis, M. sp. KMS, M. avium paratuberculosis, M. leprae, M. ulcerans and M. marinum. PE genes identified in the genomes of M. smegmatis (highlighted in blue), M. sp. KMS (highlighted in grey), M. avium paratuberculosis (highlighted in purple), M. leprae (highlighted in yellow), M. ulcerans (highlighted in teal) and M. marinum (highlighted in green) are superimposed on the phylogenetic tree generated for the PE gene family in M. tuberculosis H37Rv (see Figure 5). M. avium paratuberculosis-, M. leprae-, M. ulcerans- and M. marinum-specific genes are omitted. M. flavescens, M. vanbaalenii, M. sp. MCS and M. sp. JLS PE genes show a similar distribution to M. smegmatis and are thus not indicated on the figure.

Figure 8

Orthologues of M. tuberculosis PPE genes present in the genomes of M. smegmatis, M. sp. KMS, M. avium paratuberculosis, M. leprae, M. ulcerans and M. marinum. PPE genes identified in the genomes of M. smegmatis (highlighted in blue), M. sp. KMS (highlighted in grey), M. avium paratuberculosis (highlighted in purple), M. leprae (highlighted in yellow), M. ulcerans (highlighted in teal), and M. marinum (highlighted in green) are superimposed on the phylogenetic tree generated for the PPE gene family in M. tuberculosis H37Rv (see Figure 6). M. avium paratuberculosis-, M. leprae-, M. ulcerans- and M. marinum-specific genes are omitted. M. flavescens, M. vanbaalenii, M. sp. MCS and M. sp. JLS PPE genes show a similar distribution to M. smegmatis and are thus not indicated on the figure.

Figure 9

Sequence alignments of orthologues of Rv0442c (PPE10) present in M. ulcerans, M. marinum, M. tuberculosis, M. bovis, M. microti, M. avium paratuberculosis and M. avium. Complete sequence alignment with orthologues from the genomes of all available species, showing conserved N-terminal regions and absence of MPTR region after base pair 180 in the M. avium complex. C-terminal region of Rv0442c protein is indicated, showing intergenic DNA region and start of the 3' flanking protein Rv0441c, proving that absence of the MPTR region is not due to wrong annotation of sequenced genomes. Homologous regions of the N-terminal part of Rv0441c are shaded in grey.

Figure 10

Southern hybridization analyses of the genomic DNA of 37 different species of the genus Mycobacterium. Summary of Southern blot results obtained with mycosin 5 (column 1) and PPE-MPTR probes (column 2 and 3) in comparison to previously-published results using MPTR and PGRS sequences, respectively (column 4 and 5), as indicated. Presence of hybridization signal is indicated by the word "Yes", while absence of signal is indicated by "No". The sign "-" indicates that hybridization was not performed in this species. Mycobacterial species are separated into fast- and slow-growing species (see Figure 3). * MPTR and PGRS hybridization results were obtained from previously-published studies by Hermans et al. [17], Ross et al. [75] and Poulet et al. [15]. ** data obtained from whole genome sequence information – see Table 1. *** negative results for Rv1917c and Rv1753c in M. marinum and M. ulcerans is in agreement with the genome sequencing data which indicated the absence of both of these genes within the genomes of this species. **** although previously published data indicated a failure of the MPTR repeat sequence to hybridize to the genomic DNA of these species, M. marinum- and M. ulcerans-specific PPE-MPTR genes have been identified in the current study through genome sequencing data.

Figure 11

Reconstruction of the evolutionary history of the PE and PPE gene families of the genus Mycobacterium. Schematic representation of the suggested evolutionary history of the PE and PPE gene families. The results of this study indicated that these genes were initially inserted into the ESAT-6 (esx) gene cluster region 1 after the duplication of the cluster, and have subsequently been duplicated along with the ESAT-6 regions. The expansion of the PE and PPE gene families have occurred in unison with the expansion of the ESAT-6 (esx) gene family, throughout the evolution of the genus. Members of the genus Mycobacterium investigated in this study, have diverged at the positions indicated. After each main duplication event involving a complete ESAT-6 (esx) gene cluster region, a number of secondary subduplications of the PE and PPE genes (in some cases associated with a copy of the ESAT-6 and CFP-10 genes, occurred from the newly duplicated ESAT-6 (esx) gene cluster region. The highly duplicated PE_PGRS and PPE-MPTR subfamilies originated after the divergence of the M. avium complex and M. leprae, respectively. Both families were present before the divergence of M. marinum/M. ulcerans and the M. tuberculosis complex.