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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 2009 Jan 14;47(4):1002–1011. doi: 10.1128/JCM.01313-08

Genomic Comparison of PE and PPE Genes in the Mycobacterium avium Complex▿ †

Nick Mackenzie 1, David C Alexander 2, Christine Y Turenne 2, Marcel A Behr 2, Jeroen M De Buck 1,*
PMCID: PMC2668356  PMID: 19144814

Abstract

The Mycobacterium avium complex (MAC) comprises genomically similar but phenotypically divergent bacteria that inhabit diverse environments and that cause disease in different hosts. In this study, a whole-genome approach was used to examine the polymorphic PE (Pro-Glu) and PPE (Pro-Pro-Glu) gene families, implicated in immunostimulation and virulence. The four major groups of MAC organisms were examined, including the newly sequenced type strains of M. intracellulare and M. avium subsp. avium, plus M. avium subsp. paratuberculosis and M. avium subsp. hominissuis, for the purpose of finding genetic differences that could be exploited to design diagnostic tests specific to these groups and that could help explain their divergence in pathogenesis and host specificity. Unique and missing PPE genes were found in all MAC members except M. avium subsp. avium. Only M. intracellulare had a unique PE gene. Apart from this, most PE and PPE sequences were conserved, with average nucleotide sequence identities of 99.1 and 98.1%, respectively, among the M. avium subspecies, but only 82.9 and 79.7% identities with the PE and PPE sequences of M. intracellulare, respectively. A detailed analysis of the amino acid sequences was performed between M. avium subsp. paratuberculosis and M. avium subsp. hominissuis. Most differences were detected in the PPE proteins, with amino acid substitutions and frame shifts leading to unique amino acid sequences. In conclusion, several unique PPE proteins were identified in MAC organisms next to numerous polymorphisms in both the PE and PPE gene families. These substantial differences could help explain the divergence in phenotypes within the MAC and could lead to diagnostic tests with better discriminatory abilities.

Mycobacterium avium and Mycobacterium intracellulare are collectively known as the Mycobacterium avium complex (MAC). For genotypic, phenotypic, and historical reasons, multiple subspecies of M. avium are recognized; these include Mycobacterium avium subsp. paratuberculosis, M. avium subsp. hominissuis, Mycobacterium avium subsp. avium, and M. avium subsp. silvaticum (50). All four M. avium subspecies and M. intracellulare possess a high degree of genetic similarity but are capable of infecting a diverse range of host species.

M. avium subsp. paratuberculosis is the causative organism of Johne's disease (paratuberculosis), a debilitating chronic enteritis in ruminants (49), and has been implicated in Crohn's disease in humans (18). M. avium subsp. avium and M. avium subsp. silvaticum are limited almost exclusively to avian species (53), in which they cause tuberculosis. M. avium subsp. hominissuis is a recent designation and was added to reflect the distinction of human and porcine isolates from bird-type strains when genotypic methods showed that M. avium isolates from humans in particular but also pigs rarely shared the genetic profiles of organisms found in birds (38, 53). M. avium subsp. hominissuis and M. intracellulare are ubiquitous, saprophytic mycobacteria commonly found in soil and water (12, 17, 28). Best known for causing disseminated infection in patients infected with human immunodeficiency virus, M. intracellulare and M. avium subsp. hominissuis are increasingly recognized as emerging pathogens of immunocompetent hosts (9, 26, 29) and as the etiologic agents of chronic pulmonary infections.

Despite their divergent phenotypes and the diseases that they cause, the genomes of M. avium subsp. hominissuis and M. avium subsp. paratuberculosis share greater than 97% nucleotide identity over large regions of their genomes (5). This genetic similarity between M. avium subsp. paratuberculosis and other members of the MAC confounds the diagnosis of M. avium subsp. paratuberculosis infections by serological and PCR-based tests. In the current serological tests used to diagnose M. avium subsp. paratuberculosis infection, such as enzyme-linked immunosorbent assay, the gamma interferon release assay, and the agarose gel immunodiffusion assay, many of the antigens used are not specific for M. avium subsp. paratuberculosis (31). Most tests use complex, ill-defined mixtures of proteins derived from whole-cell or fractionated extracts of M. avium subsp. paratuberculosis, which allows cross-reactivity with antibodies generated against other mycobacterial species (56). The tests could be improved by using multiple, specific, well-defined antigens which would increase the consistencies, specificities, and sensitivities of the tests. The selection of such M. avium subsp. paratuberculosis-specific protein antigens requires a thorough comparison of the protein-coding sequences of all MAC members to allow differentiation between these closely related organisms to address clinical and epidemiologic needs.

For the detection of M. avium subsp. paratuberculosis by PCR, a few specific genetic sequences have previously been identified, such as the insertion sequence IS900 (21), the F57 element (41), and the hspX gene (15); but the value of these sequences for use in diagnostic assays for M. avium subsp. paratuberculosis infection remains unclear because of concerns about their specificity (13, 16, 42) or a lack of rigorous evaluations with large samples of clinical isolates. In fact, false-positive PCR results have been reported for suspected paucibacillary M. avium subsp. paratuberculosis infections in patients with Crohn's disease (30). All of these facts demonstrate that both in veterinary settings in which M. avium subsp. paratuberculosis is an established pathogen and in human investigations studying the putative link between M. avium subsp. paratuberculosis and Crohn's disease, there is a need to identify true M. avium subsp. paratuberculosis-specific sequences to develop new diagnostic tests.

A recent comparative study of the genomes of different mycobacterial species has indicated that the major differences among these species are in the gene products constituting the cell wall and the polymorphic gene families encoding the PE and the PPE proteins (36), which are unique to mycobacteria. The names PE and PPE are derived from the motifs Pro-Glu and Pro-Pro-Glu, respectively, found in conserved domains near the N termini of these proteins. The PE and PPE gene families are highly expanded in the pathogenic members of this genus but show a conspicuous paucity in the nonpathogenic species. Although no precise function is known for any member of these families, members of the PE and PPE families have been linked to virulence (34, 43) or have at least been shown to influence interactions with other cells (7). Some PPE proteins are thought to be expressed on the cell surface (7, 14) and have been found to be immunodominant antigens (8).

The first aim of the present study was to identify all PE and PPE orthologues in the major groups of the MAC. Although M. avium subsp. silvaticum is not included in the MAC, isolates of this subspecies are largely indistinguishable from M. avium subsp. avium (53). The second aim was to identify PE and PPE orthologues that are missing in some members of the complex. The final aim was to report how unique PPE genes, insertions or deletions (indels), and frame shifts in orthologue genes result in unique protein fragments in M. avium subsp. paratuberculosis that could be exploited as M. avium subsp. paratuberculosis-specific targets in the development of more specific diagnostic tests.

MATERIALS AND METHODS

Source material.

A Roche 454 pyrosequencer was used for genome sequencing. The genome data for M. avium subsp. avium strain TMC 25291T was collected and kindly provided by Vivek Kapur. Genome data for Mycobacterium intracellulare strain ATCC 13950T were generated at the Genome Quebec Innovation Centre, McGill University, and are deposited in GenBank as genome project 27955.

The genome sequences of M. avium subsp. paratuberculosis K-10 and M. avium subsp. hominissuis 104 were previously determined in the laboratory of V. Kapur (33) and at The Institute for Genomic Research (www.tigr.org), respectively.

Identification of orthologous PE and PPE genes.

The BLAST algorithm was used to find orthologues of all M. avium subsp. paratuberculosis and M. avium subsp. hominissuis PE and PPE genes in the fully sequenced genome of M. avium subsp. avium and M. intracellulare and vice versa. The corresponding genes and proteins, the latter only between M. avium subsp. paratuberculosis and M. avium subsp. hominissuis, were aligned in a pairwise manner; and the identity and gap were calculated by using a Ktuple of 2 and gap penalties of 7 and 4, respectively, and the DNAMAN (version 5.2.9) program (Lynnon Bio-Soft, Quebec, Canada).

Orthologues in other mycobacterial subspecies were identified by the BLASTp algorithm in the nonredundant GenBank coding sequence translations, RefSeq Proteins, and SwissProt databases and were subsequently compared by pairwise alignment.

Gap closing.

Alignment of the PE and PPE genes of M. avium subsp. hominissuis, M. avium subsp. avium, and M. avium subsp. paratuberculosis by the Vector NTI program (Invitrogen) revealed gaps in the sequences of the PPE genes (MaaPPE2, MaaPPE30, MaaPPE24, MaaPPE20, MaaPPE17, MaaPPE14, MaaPPE10, MaaPPE9, MaaPPE31, MaaPPE32, MaaPPE36, MaaPPE38, MaaPPE39) and the PE genes (MaaPE5 and MaaPE9) of the newly sequenced M. avium subsp. avium genome. Primers were designed around these gaps and were used to amplify the affected sequences from the genomic DNA of M. avium subsp. avium strain TMC 25291T. PCRs were performed with the Roche Expand high-fidelity PCR system; and the reaction mixtures (50 μl) contained 2 μl template DNA, each deoxynucleoside triphosphate at a concentration of 200 μM, 20 pmol of each primer, 5 μl of the manufacturer's PCR buffer containing MgCl2 (final MgCl2 concentration, 1.5 mM), and 1.75 U of Taq polymerase. The PCR conditions were denaturation at 94°C for 5 min, followed by 30 cycles of PCR with denaturation at 94°C for 45 s, annealing at 55°C for 1 min, and extension at 72°C for 2 min. The final extension time was 10 min at 72°C.

Identification of unique PE and PPE genes and proteins.

PE or PPE genes were considered unique when they had less than 65% DNA identity with their closest orthologue in all of the other subspecies. PE and PPE genes were defined to be missing from a member of the MAC complex if no orthologue with an identity higher than 65% with the PE and PPE genes of any of the other members could be found. PE and PPE protein fragments were identified as divergent when frame shifts were discovered or when stretches of more than 80 amino acids (aa) were <30% identical to the homologous protein.

Amino acid sequence variations in the PE and PPE proteins.

The indels were identified in the DNA sequences, and the conservative and mismatched amino acid substitutions in the protein sequences were analyzed manually on the basis of the amino acid physicochemical grouping described by Lim (35).

Categorization of PPE proteins in sublineages.

The PPE proteins were categorized in sublineages on the basis of the presence of the sublineage-specific motifs and PPE domain structure, as described previously (54).

Phylogenetic analysis of PPE genes.

PPE orthologues that were present in all four members of the M. avium complex and that had limited gaps in DNA sequence alignment were selected to be used for phylogenetic analysis of the MAC. The sequences of 14 PPE genes (MapPPE29, MapPPE28, MapPPE23, MapPPE19, MapPPE18, MapPPE16, MapPPE13, MapPPE8, MapPPE7, MapPPE31, MapPPE32, MapPPE33, MapPPE34, MapPPE35, or orthologues) were combined and concatenated, generating 16,851-bp sequences, and were aligned and compared by use of the ClustalW program (www.ebi.ac.uk/clustalw).

Single-nucleotide polymorphisms (SNPs) in the orthologues of MapPPE23 and MapPPE24 in M. avium subsp. avium, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis, and M. intracellulare were analyzed in detail; and a phylogram for both genes was created by use of the ClustalW program.

Nucleotide sequence accession numbers.

All M. avium subsp. avium PE and PPE gene locus sequences were deposited in GenBank under accession numbers EU854954 to EU854962 and EU864963 to EU854996 (Tables 1 and 2), respectively. All M. intracellulare PE and PPE gene locus sequences were deposited in GenBank under accession numbers EU854997 to EU855007 and EU855008 to EU855045, respectively.

TABLE 1.

Similarity of DNA sequences of PPE gene family orthologues in the MAC

MAC PPE locus name M. tuberculosis orthologue Locus name in:
Sub- lineage % Identity between the following pairs by nucleotide sequence alignment of PPE gene orthologues:
M. avium subsp. paratuberculosis M. avium subsp. hominissuis M. avium subsp. avium M. intracellulare M. avium subsp. paratuberculosis-M. avium subsp. hominissuis M. avium subsp. paratuberculosis- M. avium subsp. avium M. avium subsp. paratuberculosis- M. intracellulare M. avium subsp. hominissuis- M. avium subsp. avium M. avium subsp. hominissuis-M. intracellulare M. avium subsp. avium-M. intracellulare
MACPPE1 PPE20 MAP0123 Mav_0118 MaaPPE1 MiPPE1 II 99.7 99.3 83.4 97.9 81.4 81.1
MACPPE1 PPE20 MAP0124 Mav_0118 MaaPPE1 MiPPE1 (II)* 98.3 95.5 74.7 97.9 81.4 81.1
MACPPE2 PPE69 MAP0158 Mav_0152 MaaPPE2 MiPPE2 III 99.2 99.3 76.7 100.0 80.3 76.5
MACPPE3 PPE65 MAP0442 Mav_0535 MaaPPE3 MiPPE3 IV 99.0 98.8 77.3 99.0 80.7 77.3
MACPPE4 Aa Mav_0790c I
MACPPE5 A MAP0966c MaaPPE5 MiPPE5 V 98.6 83.0 82.8
MACPPE6 PPE15 MAP1003c Mav_1179 MaaPPE6 MiPPE6 IV 99.4 99.6 84.4 99.5 84.7 84.5
MACPPE7 A MAP2601 Mav_1322c MaaPPE7 MiPPE7 IV 99.1 99.0 77.0 99.9 76.9 76.8
MACPPE8 A MAP2600 Mav_1324c MaaPPE8 MiPPE8 IV 99.3 99.3 80.3 100.0 79.7 79.7
MACPPE9 A MAP2595 Mav_1329c MaaPPE9 MiPPE9 II 99.0 99.1 82.6 100.0 81.3 82.4
MACPPE10 PPE18 MAP2575c Mav_1347 MaaPPE10 MiPPE10 IV 99.0 99.1 81.4 99.9 81.0 81.1
MACPPE11 A Mav_1998 MaaPPE11 MiPPE11 IV 73.1 73.1 90.3
MACPPE12 A Mav_2006 IV
MACPPE13 A MAP2136c Mav_2039 MaaPPE13 MiPPE13 IV 99.0 99.1 80.8 99.9 80.8 80.8
MACPPE14 A MAP1813c Mav_2429 MaaPPE14 MiPPE14 II 97.5 97.5 83.2 99.0 83.4 83.2
MACPPE15 A NT03MA1810 Mav_2514c MaaPPE15 II 98.3 98.1 98.6
MACPPE16 A MAP1675 Mav_2746c MaaPPE16 MiPPE16 (IV)* 100.0 98.8 89.2 100.0 76.7 89.6
MACPPE16 A MAP1676 Mav_2746c MaaPPE16 MiPPE16 IV 99.4 98.6 72.3 99.5 76.7 70.5
MACPPE17 PPE33 MAP1522 Mav_2905c MaaPPE17 MiPPE17 IV 98.9 98.6 86.0 98.9 85.3 85.3
MACPPE18 PPE29, PPE32 MAP1521 Mav_2906c MaaPPE18 MiPPE18 IV 98.9 99.0 88.4 99.4 88.7 89.0
MACPPE19 PPE30, PPE33 MAP1519 Mav_2909c MaaPPE19 MiPPE19 IV 99.2 99.4 87.5 99.2 87.8 87.5
MACPPE20 PPE32 MAP1518 Mav_2910c MaaPPE20 MiPPE20 IV 98.4 98.3 86.2 99.9 86.0 86.0
MACPPE21 PPE31 MAP1516 Mav_2913c MaaPPE21 IV 98.6 98.6 100.0
MACPPE22 PPE31 MAP1515 Mav_2914c MaaPPE22 IV 98.2 98.1 99.9
MACPPE23 PPE26 MAP1506 Mav_2924c MaaPPE23 MiPPE23 IV 97.8 98.9 72.7 98.3 71.6 72.0
MACPPE24 PPE25 MAP1505 Mav_2925c MaaPPE24 MiPPE24 IV 93.9 97.6 74.7 93.4 74.3 74.6
MACPPE24 PPE26 MAP1506 Mav_2926c MaaPPE23 MiPPE23 IV 98.9 98.9 72.7 100.0 72.1 72.0
MACPPE24 PPE25 MAP1505 Mav_2928c MaaPPE24 MiPPE24 IV 97.6 97.6 74.7 99.8 74.6 74.6
MACPPE27 A MAP1155 Mav_3353c MaaPPE27 IV 98.2 98.2 99.4
MACPPE28 A MAP1153 Mav_3355c MaaPPE28 MiPPE28 IV 98.8 98.7 76.9 99.7 76.6 76.5
MACPPE29 A MAP1152 Mav_3356c MaaPPE29 MiPPE29 IV 99.1 99.1 70.0 100.0 70.2 70.2
MACPPE30 A MAP1144c MaaPPE30 MiPPE30 II 99.1 76.1 75.7
MACPPE31 A MAP2927 Mav_3715 MaaPPE31 MiPPE31 IV 98.3 98.6 68.6 98.6 68.6 68.3
MACPPE32 PPE50 MAP3184 Mav_4014 MaaPPE32 MiPPE32 IV 98.9 99.5 81.9 99.4 82.0 82.1
MACPPE33 PPE51 MAP3185 Mav_4015 MaaPPE33 MiPPE33 IV 98.7 98.6 76.6 99.0 76.7 76.9
MACPPE34 A MAP3419c Mav_4273c MaaPPE34 MiPPE34 IV 99.5 99.3 77.8 99.5 77.9 78.0
MACPPE35 A MAP3420c Mav_4274c MaaPPE35 MiPPE35 IV 99.5 99.3 83.8 99.5 83.8 83.6
MACPPE36 A MAP3490 Mav_4349 MaaPPE36 MiPPE36 II 99.1 99.3 82.7 98.9 81.5 81.7
MACPPE37 PPE10 MAP3939c Mav_4704 MaaPPE37 MiPPE37 V 99.7 99.5 89.6 99.5 89.5 89.8
MACPPE38 PPE4 MAP3782 Mav_4867c MaaPPE38 MiPPE38 II 98.7 98.6 84.3 99.1 85.4 84.8
MACPPE39 PPE2 Mav_4872c MaaPPE39 II 99.6
MACPPE40 A MAP3765 Mav_4879c MaaPPE40 MiPPE40 II 74.9 74.4 77.1 98.7 83.3 82.9
MACPPE41 PPE3 MAP3725 II
MACPPE42 A MAP3737 II
MACPPE43 PPE65 MiPPE43 IV
MACPPE44 A MiPPE44 IV
MACPPE45 A MiPPE45 IV
MACPPE46 PPE32 MiPPE46 IV
MACPPE47 A MiPPE47 IV
MACPPE48 PPE31 MiPPE48 IV
MACPPE49 A MAP1152 Mav_3356c MiPPE49 IV 99.1 99.1 70.5 100.0 70.7 70.7
    Mean 98.1 98.2 79.6 98.6 79.6 80.0
    SD 4.1 4.0 5.5 4.4 5.6 6.1
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a

A, absent; *, truncated gene.

TABLE 2.

Similarity of DNA sequences of PE gene family orthologues in the MAC

MAC PE locus name M. tuberculosis orthologue Locus name in:
% Identity by nucleotide sequence alignment of PE gene orthologues from the following pairs:
M. avium subsp. paratuberculosis M. avium subsp. hominissuis M. avium subsp. avium M. intracellulare M. avium subsp. paratuberculosis-M. avium subsp. hominissuis M. avium subsp. paratuberculosis-M. avium subsp. avium M. avium subsp. paratuberculosis-M. intracellulare M. avium subsp. hominissuis-M. avium subsp. avium M. avium subsp. hominissuis-M. intracellulare M. avium subsp. avium-M. intracellulare
MACPE1 Rv1386 Map0122 Mav_0117 MaaPE1 MiPE1 99.4 99.0 81.2 98.4 80.9 80.9
MACPE2 Rv3893 Map0157 Mav_0151 MaaPE2 MiPE2 100.0 100.0 77.6 100.0 79.2 77.6
MACPE3 Rv3622 Map0441 Mav_0534 MaaPE3 MiPE3 98.0 97.7 81.3 97.0 81.0 82.3
MACPE4 Rv1040 Map1003c Mav_1179c MaaPE4 MiPE4 99.5 99.3 83.9 99.2 83.8 84.8
MACPE5 Rv1195 Map2576c Mav_1346 MaaPE5 MiPE5 99.5 99.5 83.6 100.0 83.6 83.6
MACPE6 Rv1788 Map1514 Mav_2915c MaaPE6 MiPE7 99.3 99.3 69.0 100.0 68.3 68.3
MACPE7 Rv1791 Map1507 Mav_2923 MaaPE7 MiPE7 97.9 98.7 92.0 99.5 89.3 92.7
MACPE8 Rv1791 Map1507 Mav_2923 MaaPE7 MiPE8 97.9 98.7 71.3 99.5 73.1 72.0
MACPE9 Map4144 Mav_4488 MaaPE9 MiPE9 99.7 99.3 68.8 98.9 84.6 86.1
MACPE10 Rv0287 Map3783 Mav_4866 MaaPE10 MiPE10 99.7 99.0 91.5 99.7 92.0 91.8
MACPE11 Rv0285 Map3781 Mav_4868c MaaPE11 MiPE11 98.4 99.0 88.0 98.7 89.6 88.7
MACPE12 MiPE12
Mean 99.1 99.1 81.7 99.1 83.2 83.7
Stdev 0.8 0.6 8.1 0.9 6.7 7.2
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RESULTS

Identification of orthologous and unique sequences.

A total of 28 PPE genes were conserved in all four members of the complex (Table 1). Although most orthologues were intact, pseudogenes, frame-shifted genes, or remaining fragments of former complete PPE genes (MapPPE1, MapPPE16) were also observed. Map0123 and Map0124 (MapPPE1) were identified as nonoverlapping partial orthologues of MahPPE1, caused by an early stop codon in Map0123.

Map1675 and Map1676 (MapPPE16) were identified as nonoverlapping partial orthologues of MahPPE16, caused by an early stop codon in Map1675.

Over the whole complex, 49 different PPE paralogues existed, with 36 M. avium subsp. paratuberculosis, 38 M. avium subsp. hominissuis, 36 M. avium subsp. avium, and 38 M. intracellulare paralogues being detected. Consequently, there were almost identical duplicate genes (namely, the repeating pairs MahPPE23-MahPPE25, MahPPE24-MahPPE26, MapPPE21-MapPPE22, MahPPE21-MahPPE22, and MaaPPE21-MaaPPE22) and subspecies-specific paralogues in M. avium subsp. paratuberculosis (MapPPE41, MapPPE42), M. avium subsp. hominissuis (MahPPE4, MahPPE12), and M. intracellulare (MiPPE43, MiPPE44, MiPPE45, MiPPE46, MiPPE47, MiPPE48). The homologies of these loci and their closest orthologues are given in Table 3, as are other mycobacterial species with which the closest orthologues have homology. Additionally, missing paralogues, defined as missing from one subspecies but present in at least two members of the complex, were found in M. avium subsp. paratuberculosis (n = 2), M. avium subsp. hominissuis (n = 2), and M. intracellulare (n = 6) (Table 4). This comparison has allowed the definition of new PPE locus names for the MAC.

TABLE 3.

Similarity in DNA sequence between PE and PPE genes that are unique to a member of the MAC and their closest orthologue in other members of the complex or other mycobacterial species

Gene Unique PPE genes and closest orthologuesa
% Nucleotide sequence identity between genes from the following pairs:
Locus of non-MAC mycobacterial spp. with which homology exists
M. avium subsp. paratuberculosis M. avium subsp. hominissuis M. avium subsp. avium M. intracellulare M. avium subsp. paratuberculosis-M. avium subsp. hominissuis M. avium subsp. paratuberculosis-M. avium subsp. avium M. avium subsp. paratuberculosis-M. intracellulare M. avium subsp. hominissui-M. avium subsp. avium M. avium subsp. hominissui- M. intracellulare M. avium subsp. avium-M. intracellulare
PPE PPE33 PPE4 PPE33 PPE18 53.8 53.7 50.2 Rv1548c
PPE20 PPE12 PPE20 PPE43 57.9 59.5 58.8 Mt1850
PPE41 PPE36 PPE36 PPE40 58.2 55.6 59.5 Mt0269
PPE42 PPE39 PPE39 PPE40 60.8 60.1 56.4 Mt0269
PPE6 PPE6 PPE6 PPE45 55.4 42.3 42.7 Mt1745
PPE18 PPE18 PPE18 PPE46 58.4 58.5 57.6 Mt1850
PPE31 PPE31 PPE31 PPE47 60.4 60.2 60.1 Mul_2096
PPE18 PPE18 PPE18 PPE44 58.4 58.4 58.5 Mt1851
PPE18 PPE18 PPE18 PPE43 58.3 58.3 58.1 Mt1850
PPE21 PPE21 PPE21 PPE48 61.9 61.9 61.9 Mul_0947
PE PE7 PE7 PE7 PE11 63.0 64.0 64.0 Rv1791
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a

Unique PE and PPE genes are indicated in boldface.

TABLE 4.

Similarity in DNA sequence between PE and PPE genes that are missing from members of the MAC and their closest orthologues in other members of the complex

Orthologues of the missing PPE genesa
% Nucleotide sequence identity between genes from the following pairs:
M. avium subsp. paratuberculosis M. avium subsp. hominissuis M. avium subsp. avium M. intracellulare M. avium subsp. paratuberculosis-M. avium subsp. hominissuis M. avium subsp. paratuberculosis-M. avium subsp. avium M. avium subsp. paratuberculosis-M. intracellulare M. avium subsp. hominissuis-M. avium subsp. avium M. avium subsp. hominissuis-M. intracellulare M. avium subsp. avium-M. intracellulare
PPE5 PPE26 PPE5 PPE5 58.3 55.6 52.7
PPE30 PPE39 PPE30 PPE30 59.4 59.6 61.8
PPE27 PPE27 PPE27 PPE47 59.6 59.9 59.8
PPE15 PPE15 PPE15 PPE36 55.6 54.6 54.3
PPE22 PPE11 PPE11 PPE11 57.0 56.2 54.2
PPE36 PPE39 PPE39 PPE36 61.8 65.3 62.0 62.7
PPE22 PPE22 PPE22 PPE48 61.1 60.6 60.5
PPE21 PPE21 PPE21 PP448 61.9 61.9 61.9
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a

The closest orthologues in other members of the complex are indicated in boldface.

Close orthologues were found for the remaining PPE genes and for all M. avium subsp. hominissuis and M. avium subsp. paratuberculosis PE genes (Tables 1 and 2), including orthologues for the PE and the PPE gene fragments of the natural PE-PPE fusion in Map1003, both of which are found in the annotated gene Mav_1179 in two different reading frames with an 8-bp overlap. By performing BLASTn searches of the M. avium subsp. paratuberculosis genome with M. avium subsp. hominissuis PE sequences, a previously nonrecognized M. avium subsp. paratuberculosis PE gene, MapPE10, was discovered.

Deletions averaging 170 bp in size (size range, 38 bp to 464 bp) in 14 M. avium subsp. avium PPE genes compared to the sequences of their orthologues in M. avium subsp. paratuberculosis and M. avium subsp. hominissuis were initially found after compilation of the contigs. The affected regions in the M. avium subsp. avium genome were amplified by PCR, and subsequent sequencing confirmed that the gaps were due to insufficient overlap of the contigs. Several small indels between M. avium subsp. paratuberculosis and M. avium subsp. hominissuis still existed, and these are extensively covered in the next section. The numerous indels in the M. intracellulare PE and PPE genes were not investigated further.

With respect to the PE genes, 12 different PE paralogues were found (Table 2). M. avium subsp. paratuberculosis, M. avium subsp. hominissuis, and M. avium subsp. avium each had orthologues for 10 PE genes. M. intracellulare was found to have two MapPE7 orthologues (MiPE7 and MiPE8), with MiPE7 also being the closest orthologue to MapPE6 (Table 4). Again, the new names for all PE genes in MAC are defined in Table 2.

Sequence similarity.

Among the M. avium subspecies, the level of DNA identity was high for all conserved PE and PPE genes, with average levels of identity of 99.1% ± 0.8% and 98.3% ± 4.1%, respectively, for M. avium subsp. paratuberculosis, M. avium subsp. hominissuis, and M. avium subsp. avium. The only outliers were MapPPE24 and its first orthologue, MahPPE25; MapPPE40 and MahPPE40; and MahPPE11 and MaaPPE11. The level of identity between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis was high for all PE proteins, with an average of 98.0% ± 2.8%, but there was greater variability in the level of identity for the PPE proteins, with an average of 90.2% ± 21.2%; the variability was mainly caused by frame shifts.

The average levels of nucleotide sequence identity of the M. intracellulare PE and PPE genes with the M. avium PE and PPE genes were 82.0% ± 7.3% and 79.7% ± 5.7%, respectively.

Detailed comparison of M. avium subsp. paratuberculosis and M. avium subsp. hominissuis PE and PPE proteins and genes.

Orthologues of M. avium subsp. avium and M. avium subsp. hominissuis were found to be very similar, with an average level of nucleotide sequence identity of 98.6%, such that only M. avium subsp. hominissuis and M. avium subsp. paratuberculosis were selected for use in a more detailed amino acid sequence comparison (see Tables S1 and S2 in the supplemental material). While BLAST searches determined high levels of nucleotide sequence identity between sequences coding for the PE and PPE proteins and the flanking regions in M. avium subsp. paratuberculosis and M. avium subsp. hominissuis, large amino acid sequence differences caused by one or numerous frame shifts were observed in some PE and PPE loci. Frame shifts have created some unique M. avium subsp. paratuberculosis protein fragments in MapPE6 (aa 195 to 314), MapPPE1 (aa 185 to 301), and MapPPE15 (aa 62 to 368). Additional frame shifts in M. avium subsp. hominissuis genes MapPPE2 (aa 153 to 396) and MapPPE13 (aa 314 to 410) create differences in protein sequences between M. avium subsp. paratuberculosis and M. avium subsp. hominissuis but are not M. avium subsp. paratuberculosis specific because those frame shifts do not occur in M. avium subsp. avium.

Categorization of sublineages.

Of the 36 M. avium subsp. paratuberculosis PPE proteins, 10 belonged to sublineage II, 1 to sublineage III, 23 to sublineage IV, and 2 to sublineage V. Of the 38 M. avium subsp. hominissuis PPE proteins, 8 belonged to sublineage II, 1 to sublineage III, 28 to sublineage IV, and 1 to sublineage V. Of the 36 M. avium subsp. avium PPE proteins, 9 belonged to sublineage II, 1 to sublineage III, 24 to sublineage IV, and 2 to sublineage V. Three unique M. avium subsp. hominissuis PPE proteins belonged to sublineage IV, while the other two belonged to sublineage II. One unique M. avium subsp. paratuberculosis PPE protein belonged to the sublineage IV, while the other four belonged to sublineage II.

SNP analysis of orthologues of duplicated PPE genes.

Some PPE genes have duplicate paralogues in one of the members of the complex. MahPPE24 and MahPPE26 in M. avium subsp. hominissuis are both orthologues of MapPPE24, just as MahPPE23 and MahPPE25 are both orthologues of the single gene MapPPE23. The paralogues MahPPE23 and MahPPE25 have 93.3% amino acid identity, while the paralogues MahPPE22 and MahPPE24 have 98.3% amino acid identity. The order of the genes in M. avium subsp. hominissuis suggests duplication of both neighboring genes together, although an extra gene, Mav_2927, is inserted between the MapPPE24 and MapPPE23 orthologues MahPPE26 and MahPPE25, respectively.

SNP analysis of these orthologues (Tables 5 and 6) within the complex indicates that M. avium subsp. avium and M. avium subsp. paratuberculosis share SNPs with different orthologues in M. avium subsp. hominissuis. This suggests that the duplication of the neighboring PPE proteins was present in an ancestor of these members of the complex (M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis) and that M. avium subsp. avium and M. avium subsp. paratuberculosis have retained different paralogues of the duplicate. This is also illustrated by the phylograms of the MapPPE23 and MapPPE24 orthologues (Fig. 1).

TABLE 5.

SNPs in the orthologues of MACPPE23 in the MAC indicating genetic relation between the members

Orthologue SNP at base pair:
12 15 16 150 293 328 344 411 483 452 675 789 864 944 945 946 1039 1080 1125
MiPPE23 C G G G A C G G G C G T G A
MahPPE25 C A A C A T G G G C G C C A C C
MaaPPE23 C A A C A T G G G C G C C A C C
MahPPE23 T G G C A T G G G C G C G A T C
MapPPE23 type II T G G T T G G T C T C T C G T G G T T
MapPPE23 type III T G G T A T A G C C C T C G T T
MapPPE23 type I T G G T A T G G C C C T C G T T
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TABLE 6.

SNPs in the orthologues of MACPPE24 in the MAC indicating genetic relation between the members

Orthologue SNP at base pair:
381 562 563 564 571 572 577 579 582 585 588 590 593 597
MiPPE24 C G C G G T G G G G C A C G
MahPPE26 T A G C A T C G A G T G C G
MaaPPE24 T A G C A T C G A G T G C G
MahPPE24 T G C T G C G A G C C A G A
MapPPE24 C G C T G C G A G C C A G A
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FIG. 1.

FIG. 1.

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Phylograms of the MapPPE23 and MapPPE24 orthologues in the MAC based on their nucleotide sequences, as calculated by the use of ClustalW (EMBL-EBI) software. The relative branch lengths are given for all the orthologues.

Phylogenetic analysis.

A phylogram was drawn from the distances calculated with the ClustalW program (Fig. 2) for the concatenated nucleotide sequences of orthologues of 15 PPE genes of M. avium subsp. avium, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis, and M. intracellulare. M. intracellulare is the more distant relative within the MAC. M. avium subsp. avium and M. avium subsp. hominissuis are the closest relatives.

FIG. 2.

FIG. 2.

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Phylogram of the concatenated nucleotide sequences of orthologues of 15 PPE genes of members of the MAC, as calculated by the use of ClustalW (EMBL-EBI) software. The relative branch lengths are given for all the orthologues.

DISCUSSION

Studies involving comparisons of complete microbial genomes can readily reveal commonalities in gene content and genome organization between closely related bacteria and the major differences that distinguish them. While it is clear that bioinformatics has its limits in predicting the influences of gene content and genome organization on virulence and immunity, an important first step in determining the basis for differences in pathogenicity is the documentation of the differences between closely related organisms. Because of the previously reported link between PE and PPE proteins and pathogenicity for organisms of the M. tuberculosis complex, this protein family from phenotypically different MAC organisms represented a reasonable candidate for comparative analysis.

Previously, the PE and PPE genes in the fully sequenced and annotated genomes of M. tuberculosis H37Rv and CDC1551 were analyzed by comparative genomics (54). Although the genome of M. avium subsp. paratuberculosis strain K-10 was also analyzed, complete sequences for additional MAC organisms were not yet available, and it was not possible to detail the divergence of the PE and PPE proteins within the MAC. The genomes of several MAC organisms, including the type strains of M. avium subsp. avium and M. intracellulare, have now been sequenced. This provided an opportunity to reexamine the relationships between and evolutionary history of the members of the subfamilies of the PE and PPE protein families, to identify subfamily-specific characteristics, and to determine the extent of PE and PPE sequence similarity and variation.

In the present study, new uniform PPE and PE locus names for all members of the MAC were proposed. These uniform orthologue names will simplify the reporting of the findings of future studies of these polymorphic gene families in MAC. The names of the MAC PPE genes were rooted on the M. avium subsp. hominissuis genome because the sequence of M. avium subsp. paratuberculosis strain K-10 has previously been described to have small and large genomic inversions and, thus, a gene order that differs from those in M. avium subsp. hominissuis, M. avium subsp. avium, and other M. avium subsp. paratuberculosis isolates (5, 57).

This is the first time that the sequences of multiple genes that are potentially associated with virulence were compared between four members of the MAC. Previously, single genes (20, 46, 48, 52) and insertion sequences (10, 23, 32) were targeted to characterize MAC isolates. The study of multiple related genomes also provides insight into the distribution and evolution of these genes. The unique M. avium subsp. paratuberculosis genes are located in a subspecies-specific large sequence polymorphism and were likely acquired via horizontal gene transfer. Similarly, the unique M. avium subsp. hominissuis genes are found in genomic regions conserved among a subset of M. avium subsp. hominissuis isolates (44, 57). The missing orthologues of MACPPE05 and MACPPE11 also correspond to earlier deletions identified in M. avium subsp. hominissuis and M. avium subsp. paratuberculosis, respectively (44).

The present study demonstrated on average 98.6% nucleotide sequence similarity between the M. avium subsp. avium and the M. avium subsp. hominissuis PPE genes. This is in agreement with the high average similarities of 99.1 and 98.1% between the conserved PE and PPE genes of M. avium subsp. hominissuis and M. avium subsp. paratuberculosis, respectively. Indeed, very few genetic differences were previously found when the genomes of M. avium subsp. paratuberculosis and M. avium subsp. hominissuis were compared, with greater than 97% nucleotide sequence identity over large genomic regions and 100% nucleotide sequence identity of the 16S rRNA genes from these two subspecies being identified (5). In another study, the comparison of genes orthologous between M. avium subsp. paratuberculosis and M. avium subsp. hominissuis has revealed 98 to 99% nucleotide sequence identity (33). However, the high degree of nucleotide sequence identity observed among the M. avium subspecies does not hold true for the complete MAC, with only an average of 79.7% similarity between M. intracellulare and the M. avium subspecies. A previously observed mean similarity between M. intracellulare and M. avium was 91% (51).

From an analysis of 48% of the genome, only 27 predicted coding sequences were found to be absent in M. avium subsp. avium and M. avium subsp. hominissuis (2). In another report, 39 genes of M. avium subsp. paratuberculosis were found to be unique to M. avium subsp. paratuberculosis and were thus missing from M. avium subsp. hominissuis (33). Other reports indicate that M. avium subsp. paratuberculosis coding sequences are absent from M. avium subsp. hominissuis (3, 40). The unique PPE proteins mentioned above are not included in these lists since they were all recognized as members of the polymorphic PPE protein family, and the various other differences between M. avium subsp. paratuberculosis and M. avium subsp. hominissuis PPE proteins at the protein level were also not previously described. They are, however, equally valuable for consideration as targets for new discriminative diagnostic tests.

The present analysis indicates that all of the PE proteins and most of the PPE proteins of M. avium subsp. paratuberculosis are highly homologous to the corresponding proteins of M. avium subsp. hominissuis. This is in agreement with a comparison of other proteins between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis by Bannantine et al. (5), who described levels of similarity that ranged from 94 to 100%. However, one PE protein and six PPE proteins with high levels of nucleotide sequence identity were found to be very different at the protein level due to frame shifts that led to unique protein fragments in the M. avium subsp. paratuberculosis and the M. avium subsp. hominissuis PPE proteins. Other studies have shown that unique sequences have considerable potential for use in the development of more specific and sensitive diagnostic assays for the detection of M. avium subsp. paratuberculosis infection by both molecular assay- and immunoassay-based approaches (2-4, 40). It is also possible that these different amino acid sequences have significant impacts on the protein functions. Even the single amino acid substitutions identified could potentially have an influence on the immunogenicity and virulence of the PE and PPE proteins (25).

While unique and missing genes were identified in the M. avium subsp. hominissuis, M. avium subsp. paratuberculosis, and M. intracellulare genomes, most genes in all four members of the complex also contained numerous SNPs. These SNPs can be used for subspecies differentiation. SNPs are frequently used in epidemiological and evolutionary studies to differentiate between closely related species, subspecies, and strains of bacteria without knowledge of what effect the SNP may have on gene function or protein activity (1, 19, 24, 27, 47).

A study by Turenne et al. (52), who used a PCR- and sequencing-based strategy to investigate the complete hsp65 gene in the MAC, identified 10 SNPs that differentiated M. avium subsp. hominissuis and M. avium subsp. paratuberculosis. Other studies (37) showed that SNPs are a major source of genotypic variation within the MAC, and as demonstrated by Semret et al. (45), SNPs can be used in conjunction with large sequence polymorphisms to identify possible evolutionary paths within the MAC.

The existence of unique PPE proteins as well as subtle variations in the PE and PPE gene families, such as SNPs, might be the cause of the differences in pathogenesis between mycobacterial species, as previously suggested by Marri et al. (36). For example, if these PE and PPE genes are expressed in vivo, they could potentially cause the differential responses of bovine macrophages to M. avium subsp. avium/M. avium subsp. hominissuis and M. avium subsp. paratuberculosis, as observed previously (55). This previous suggestion is supported by previous observations that a specific M. avium subsp. hominissuis PPE protein is associated with the ability to grow in macrophages and is involved in virulence in mice (34). Other SNPs, such as the ones in the rpoV and mma3 genes of M. bovis, have been shown to have a marked impact on virulence and cellular functions (6, 11), and SNPs in M. tuberculosis (6, 11) have been shown to be responsible for altered phenotypes. The identification of SNPs in genes that are linked to virulence is the first step in explaining the differences in virulence that are caused by these SNPs.

The findings of the present analysis suggest that the duplication of the neighboring PPE genes (MapPPE23 and MapPPE24) was present in an ancestor of these members of the complex (M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis) and that M. avium subsp. avium and M. avium subsp. paratuberculosis have retained different paralogues of the duplicate, while M. avium subsp. hominissuis still contains both duplicates. This corresponds to the findings described in an earlier report that M. avium subsp. hominissuis is a diverse group of organisms from which two pathogenic clones (M. avium subsp. paratuberculosis and M. avium subsp. avium) have evolved independently (51).

The present study is based on the full genomic sequences of single isolates of M. avium subsp. paratuberculosis, M. avium subsp. hominissuis, M. avium subsp. avium, and M. intracellulare and thus will not identify sequence variations between subtypes (e.g., the bovine, ovine, and intermediate type variants of M. avium subsp. paratuberculosis) within a subspecies. Recently, SNPs have been found in the PPE gene sequence of MapPPE23. The sequences of the bovine strains appeared to consistently differ at 8 nucleotides from the sequences of the intermediate type strains and 7 nucleotides from the sequences of the ovine type strains and the homologous genes in M. avium subsp. hominissuis (MahPPE23 and MahPPE25, respectively) (22).

On the basis of the DNA sequences of the PPE genes, M. avium subsp. avium seems to be more closely related to M. avium subsp. hominissuis than to M. avium subsp. paratuberculosis. However, M. avium subsp. avium shares two PPE proteins (MACPPE5 and MACPPE30 with M. avium subsp. paratuberculosis that are absent in M. avium subsp. hominissuis. This could also indicate a function for these gene products in host adaptation because both M. avium subsp. avium and M. avium subsp. paratuberculosis are believed to be adapted to ruminant and bird hosts, respectively (53), whereas M. avium subsp. hominissuis has a more ubiquitous host distribution.

Two M. avium subsp. hominissuis-specific genes (MACPPE4 and MACPPE11) were found in this study. The corresponding gene products could be used to identify immune responses against this M. avium subspecies, and misinterpretations due to cross-reactivity in current diagnostics for Johne's disease would thereby be avoided. This subspecies is a heterogeneous group of strains with at least six distinct hsp65 sequences (hsp65 sequevars) (52). This has previously led to the suggestion that human isolates simply reflect what is found in their environment (44). It will be intriguing to know whether the M. avium subsp. hominissuis-specific MAC PPE genes are present in all sequevars and all environmental isolates or whether those genes are unique to isolates causing disease in humans.

After the demonstration of immune responses against one PPE protein in M. avium subsp. paratuberculosis-infected cows (39), studies are now under way to heterologously express the unique PPE M. avium subsp. paratuberculosis genes as well as the PPE genes coding for unique protein fragments for the construction of a partial protein array to evaluate the humoral and cell-mediated immunostimulatory capabilities of these recently discovered unique proteins. The combination of genomic information, molecular tools, and immunological assays will thus provide key insights into the host immune response to M. avium subsp. paratuberculosis infection. Overall, the elucidation of all of the unique sequences as well as those that may be associated with the cell surface of M. avium subsp. paratuberculosis provides a strong foundation on which to develop the next generation of specific and sensitive diagnostic assays for M. avium subsp. paratuberculosis.

In conclusion, the existence of several unique PPE proteins in both M. avium subsp. hominissuis and M. avium subsp. paratuberculosis was demonstrated, as were differences created by frame shifts and indels in both the PE and the PPE gene families. These substantial differences could help explain the important differences in phenotypes between members of the MAC.

Supplementary Material

[Supplemental material]
supp_47_4_1002__index.html (1.4KB, html)

Acknowledgments

We thank Vivek Kapur from the Department of Veterinary and Biomedical Sciences at Pennsylvania State University for providing us with the genome sequence data for M. avium subsp. avium.

This work has been supported by the Margaret Gunn Endowment for Animal Research (to J.M.D.B.).

Footnotes

▿

Published ahead of print on 14 January 2009.

†

Supplemental material for this article may be found at http://jcm.asm.org/.

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