Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins

doi:10.3389/fmicb.2016.00795

. 2016 Jun 7:7:795.

doi: 10.3389/fmicb.2016.00795. eCollection 2016.

Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins

Nomakorinte Gcebe¹, Anita Michel², Nicolaas C Gey van Pittius³, Victor Rutten⁴

Affiliations

¹ Tuberculosis Laboratory, Agricultural Research Council - Onderstepoort Veterinary InstituteOnderstepoort, South Africa; Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of PretoriaOnderstepoort, South Africa.
² Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria Onderstepoort, South Africa.
³ Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Centre of Excellence for Biomedical Tuberculosis Research, Stellenbosch University Tygerberg, South Africa.
⁴ Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of PretoriaOnderstepoort, South Africa; Division of Immunology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht UniversityUtrecht, Netherlands.

PMID: 27375559
PMCID: PMC4894912
DOI: 10.3389/fmicb.2016.00795

Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins

Nomakorinte Gcebe et al. Front Microbiol. 2016.

. 2016 Jun 7:7:795.

doi: 10.3389/fmicb.2016.00795. eCollection 2016.

Authors

Nomakorinte Gcebe¹, Anita Michel², Nicolaas C Gey van Pittius³, Victor Rutten⁴

Affiliations

¹ Tuberculosis Laboratory, Agricultural Research Council - Onderstepoort Veterinary InstituteOnderstepoort, South Africa; Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of PretoriaOnderstepoort, South Africa.
² Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria Onderstepoort, South Africa.
³ Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Centre of Excellence for Biomedical Tuberculosis Research, Stellenbosch University Tygerberg, South Africa.
⁴ Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of PretoriaOnderstepoort, South Africa; Division of Immunology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht UniversityUtrecht, Netherlands.

PMID: 27375559
PMCID: PMC4894912
DOI: 10.3389/fmicb.2016.00795

Abstract

The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis identified a total of 609 proteins in the six PPDs and 22 of these were identified as shared between PPD of M.bovis and one or more of the NTM PPDs. Previously characterized M tuberculosis/M. bovis homologous immunogenic proteins detected in one or more of the nonpathogenic NTM in this study included CFP-10 (detected in M. malmesburii sp. nov. PPD), GroES (detected in all NTM PPDs but M. malmesburii sp. nov.), DnaK (detected in all NTM PPDs), and GroEL (detected in all NTM PPDs). This study confirms reports that the ESX-1, ESX-3, and ESX-4 regions are ancestral regions and thus found in the genomes of most mycobacteria. Identification of NTM homologs of immunogenic proteins warrants further investigation of their ability to cause cross-reactive immune responses with MTBC antigens.

Keywords: Esx family; M. fortuitum; M. komanii sp. nov.; M. malmesburii sp. nov.; M. nonchromogenicum; PE/PPE; non-tuberculous mycobacteria.

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Figures

**Figure 1**
**Alignment of **M. bovis** and NTM predicted CDS regions to those of **M. tuberculosis** visualized using BRIG**. Immunogenic proteins of interest are highlighted in red (also see Table 1).

**Figure 2**
**Alignment of **M. fortuitum** predicted CDS regions to those of **M. smegmatis** MC² 155 (CP001663.1) using BRIG**. Immunogenic proteins of interest identified in M. smegmatis are highlighted in red (esxB: locus tag MSMEG_0065, product hypothetical protein; esxA: locus tagMSMEG_0066, product early secretory antigenic target, 6 kDa; dnaK: locus tag MSMEG_0709, product of chaperone protein DnaK; mpt63: locus tag MSMEG_0828, product immunogenic protein MPT63; mpt64: locus tagMSMEG_2331, product immunogenic protein MPB64/MPT64; mpt70: locus tag MSMEG_5196, product fasciclin domain-containing protein; canA: locus tag MSMEG_4985, product carbonic anhydrase; tpx: locus tagMSMEG_3479, product thiol peroxidase).

**Figure 3**
**Alignment of **M. nonchromogenicum** predicted CDS regions to those of **M. smegmatis** MC² 155 (CP001663.1) using BRIG**. Immunogenic proteins of interest identified in M. smegmatis are highlighted in red (esxB: locus tag MSMEG_0065, product hypothetical protein; esxA: locus tagMSMEG_0066, product early secretory antigenic target, 6 kDa; dnaK: locus tag MSMEG_0709, product of chaperone protein DnaK; mpt63: locus tag MSMEG_0828, product immunogenic protein MPT63; mpt64: locus tagMSMEG_2331, product immunogenic protein MPB64/MPT64; mpt70: locus tag MSMEG_5196, product fasciclin domain-containing protein; canA: locus tag MSMEG_4985, product carbonic anhydrase; tpx: locus tagMSMEG_3479, product thiol peroxidase).

**Figure 4**
**Alignment of **M. malmesburii** sp. nov. predicted CDS regions to those of **M. rhodesiae** NBB3 (CP003169.1) using BRIG**. Immunogenic proteins of interest identified in M. rhodesiae are highlighted in red (dnaK: locus tagMycrhN_1341, product chaperone protein DnaK; mpt70: locus tag MycrhN_3596, product secreted/surface protein with fasciclin-like repeats; canA_1: locus tag MycrhN_1479, product sulfate permease-like transporter, MFS superfamily; canA_2: locus tag MycrhN_2217, product carbonic anhydrase; canA_3: locus tagMycrhN_2307, product isoleucine patch superfamily enzyme, carbonic anhydrase/acetyltransferase; canA_4: locus tag MycrhN_3599, product isoleucine patch superfamily enzyme, carbonic anhydrase/acetyltransferase; canA_5: locus tag MycrhN_3776, product carbonic anhydrase. Carbonic anhydrase genes have been numbered according to genomic position).

**Figure 5**
**Alignment of **M. komanii** sp. nov. predicted CDS regions to those of M. sp. JLS (CP000580.1) using BRIG**. Immunogenic proteins of interest identified in M. sp. JLS are highlighted in red (esxB: locus tag Mjls_0060, product hypothetical protein; esxA: locus tag Mjls_0061, product 6 kDa early secretory antigenic targetEsaT6; hsp20: locus tag Mjls_1109, product heat shock protein Hsp20; Mjls_1343: locus tag Mjls_1343, product of polysaccharide biosynthesis protein; cfp6: locus tag Mjls_1885, product low molecular weight protein antigen 6; cfp2: locus tag Mjls_3145, product low molecular weight antigen; Mjls_5331: locus tag Mjls_5331, product lipoprotein antigen family protein; dnaK: locus tag Mjls_0449, product molecular chaperone DnaK;mpt64: locus tag Mjls_1842, product immunogenic protein MPB64/MPT64; mpt70_1: locus tag Mjls_2023, product beta-Ig-H3/fasciclin; mpt70_2: locus tag Mjls_2024, product beta-Ig-H3/fasciclin; mpt70_3: locus tagMjls_4307, product beta-Ig-H3/fasciclin; Mjls_1176: locus tag Mjls_1176, product peptidase M22, glycoprotease; gcp: locus tag Mjls_1178, product putative DNA-binding/iron metalloprotein/AP endonuclease; canA_1: locus tag Mjls_3936, product carbonic anhydrase; canA_2: locus tag Mjls_4306, product carbonic anhydrase;canA_3: locus tag Mjls_5131, product carbonic anhydrase; Mjls_0685: locus tag Mjls_0685, product alkyl hydro peroxide reductase/ Thiol specific antioxidant/ Mal allergen; Mjls_3070: locus tag Mjls_3070, product alkyl hydro peroxide reductase; Mjls_3370: locus tag Mjls_3370, product alkyl hydro peroxide reductase; Mjls_3657: locus tag Mjls_3657, product alkyl hydro peroxide reductase.

**Figure 6**
Schematic representation of the genomic arrangement of orthologs of genes present in three ESAT-6 gene cluster regions: ESX-1, ESX-3, and ESX-4 of **M. fortuitum, M. malmesburii** sp. nov., **M. nonchromogenicum** (as well as ESX-2), and **M. komanii** sp. nov., compared to **M. tuberculosis** and **M. smegmatis**. *M. tuberculosis* annotation was also used for the NTM orthologs (except for *M. smegmatis*). Protein coding sequences are represented by blocked arrows reflecting the relative lengths of the genes and the direction of transcription. Genes of *M. tuberculosis* are color coded according to the protein families they encode for (Gey van Pittius et al., 2006). The unannotated genes in NTM encode for hypothetical proteins.

**Figure 7**
**Alignment of EsxA aa sequences of **M. fortuitum** ATCC 6841 (M. fortuit), I MC²155 (M. smegmat), **M. bovis**, and **M. tuberculosis** (M. tubercu)**. ^*Represents identical sequences observed in all species, and >indicates presence of the same aa residue in at least one of the NTM species. Highly immunogenic epitopes of *M. bovis* as described by Vordermeier et al. (2000, 2003, 2007) are underlined.

**Figure 8**
Alignment of EsxB aa sequences of **M. fortuitum** ATCC 6841 (M. fortuit), **M. smegmatis** MC² 155 (M. smegmat), **M. malmesburii** sp. nov (M. malmesb), **M. komanii** sp. nov., **M. bovis**, and **M. tuberculosis** (M. tubercu). ^*Represents identical sequences observed in all species, and >indicates presence of the same aa residue in at least one of the NTM species. Highly immunogenic epitopes of *M. bovis* as described by Vordermeier et al. (2000, 2003, 2007) are underlined.

**Figure 9**
Alignment of EsxH aa sequences of **M. fortuitum** ATCC 6841 (M. fortuit), **M. smegmatis** MC²155 (M. smegmat), **M. malmesburii** sp. nov. (M. malmesb), **M. komanii** sp. nov., **M. nonchromogenicum** (M. nonchro), **M. bovis**, and **M. tuberculosis** (M. tubercu). ^*Represents identical sequences observed in all species, and >indicates presence of the same aa residue in at least one of the NTM species. Highly immunogenic epitopes of *M. tuberculosis* as described by Skjøt et al. (2002) are underlined.

**Figure 10**
Alignment of PPE68 aa sequences of **M. fortuitum** ATCC 6841 (M. fortuit), **M. smegmatis** MC² 155 (M. smegmat), **M. nonchromogenicum** (M. nonchro), **M. komanii** sp. nov., **M. bovis**, and **M. tuberculosis** (M. tubercu). ^*Indicates identical aa sequences in all species, and >indicates same aa residue in at least one of the NTM species and *M. bovis/M. tuberculosis*. Highly immunogenic epitope of *M. tuberculosis*, as described by Mustafa (2014) are Underlined.

**Figure 11**
**Venn diagrams illustrating overlap of proteins identified among (A) PPDA-PPD-B, PPD-F, (B) PPD-A, PPD-B, PPD-K, (C) PPD-A, PPD-B, PPD-M, (D) PPD-A, PPD-B, and PPD-N**.

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References

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1. Akhter Y., Ehebauer M. T., Mukhopadhyay S., Hasnain S. (2012). The PE/ PPE multigene family codes for virulence factors and is possible source of mycobacterial antigenic variation: perhaps more. Biochimie 94, 110–116. 10.1016/j.biochi.2011.09.026 - DOI - PubMed
1. Alikhan N. F., Petty N. K., Zakour N. L. B., Beatson S. A. (2011). BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 12:12. 10.1186/1471-2164-12-402 - DOI - PMC - PubMed
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[1] Adékambi T., Berger P., Raoult D., Drancourt M. (2006). rpoB gene sequence-based characterisation of emerging non - tuberculous mycobacteria with descriptions of Mycobacterium bolletii sp. nov., Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov. Int. J. Syst. Evol. Microbiol. 56, 133–143. 10.1099/ijs.0.63969-0 - DOI - PubMed

[2] Adékambi T., Berger P., Raoult D., Drancourt M. (2006). rpoB gene sequence-based characterisation of emerging non - tuberculous mycobacteria with descriptions of Mycobacterium bolletii sp. nov., Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov. Int. J. Syst. Evol. Microbiol. 56, 133–143. 10.1099/ijs.0.63969-0 - DOI - PubMed

[3] Adékambi T., Drancourt M. (2004). Dissection of the phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. Int. J. Syst. Evol. Microbiol. 54, 2095–2105. 10.1099/ijs.0.63094-0 - DOI - PubMed

[4] Adékambi T., Drancourt M. (2004). Dissection of the phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. Int. J. Syst. Evol. Microbiol. 54, 2095–2105. 10.1099/ijs.0.63094-0 - DOI - PubMed

[5] Akhter Y., Ehebauer M. T., Mukhopadhyay S., Hasnain S. (2012). The PE/ PPE multigene family codes for virulence factors and is possible source of mycobacterial antigenic variation: perhaps more. Biochimie 94, 110–116. 10.1016/j.biochi.2011.09.026 - DOI - PubMed

[6] Akhter Y., Ehebauer M. T., Mukhopadhyay S., Hasnain S. (2012). The PE/ PPE multigene family codes for virulence factors and is possible source of mycobacterial antigenic variation: perhaps more. Biochimie 94, 110–116. 10.1016/j.biochi.2011.09.026 - DOI - PubMed

[7] Alikhan N. F., Petty N. K., Zakour N. L. B., Beatson S. A. (2011). BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 12:12. 10.1186/1471-2164-12-402 - DOI - PMC - PubMed

[8] Alikhan N. F., Petty N. K., Zakour N. L. B., Beatson S. A. (2011). BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 12:12. 10.1186/1471-2164-12-402 - DOI - PMC - PubMed

[9] Amoudy H. A., Ebrahimi B. H., Mustafa A. S. (2014). Immune responses against Mycobacterium tuberculosis-specific proteins PE35 and CFP10 in mice immunized with recombinant Mycobacterium vaccae. Saudi Med. J. 35, 350–359. - PubMed

[10] Amoudy H. A., Ebrahimi B. H., Mustafa A. S. (2014). Immune responses against Mycobacterium tuberculosis-specific proteins PE35 and CFP10 in mice immunized with recombinant Mycobacterium vaccae. Saudi Med. J. 35, 350–359. - PubMed

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Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins

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Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins

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