Comparative genomics for mycobacterial peptidoglycan remodelling enzymes reveals extensive genetic multiplicity

  • Edith Erika Machowski1,

    Affiliated with

    • Sibusiso Senzani1,

      Affiliated with

      • Christopher Ealand1 and

        Affiliated with

        • Bavesh Davandra Kana1Email author

          Affiliated with

          BMC Microbiology201414:75

          DOI: 10.1186/1471-2180-14-75

          Received: 27 December 2013

          Accepted: 12 March 2014

          Published: 24 March 2014

          Abstract

          Background

          Mycobacteria comprise diverse species including non-pathogenic, environmental organisms, animal disease agents and human pathogens, notably Mycobacterium tuberculosis. Considering that the mycobacterial cell wall constitutes a significant barrier to drug penetration, the aim of this study was to conduct a comparative genomics analysis of the repertoire of enzymes involved in peptidoglycan (PG) remodelling to determine the potential of exploiting this area of bacterial metabolism for the discovery of new drug targets.

          Results

          We conducted an in silico analysis of 19 mycobacterial species/clinical strains for the presence of genes encoding resuscitation promoting factors (Rpfs), penicillin binding proteins, endopeptidases, L,D-transpeptidases and N-acetylmuramoyl-L-alanine amidases. Our analysis reveals extensive genetic multiplicity, allowing for classification of mycobacterial species into three main categories, primarily based on their rpf gene complement. These include the M. tuberculosis Complex (MTBC), other pathogenic mycobacteria and environmental species. The complement of these genes within the MTBC and other mycobacterial pathogens is highly conserved. In contrast, environmental strains display significant genetic expansion in most of these gene families. Mycobacterium leprae retains more than one functional gene from each enzyme family, underscoring the importance of genetic multiplicity for PG remodelling. Notably, the highest degree of conservation is observed for N-acetylmuramoyl-L-alanine amidases suggesting that these enzymes are essential for growth and survival.

          Conclusion

          PG remodelling enzymes in a range of mycobacterial species are associated with extensive genetic multiplicity, suggesting functional diversification within these families of enzymes to allow organisms to adapt.

          Keywords

          Peptidoglycan Transglycosylases Amidases D,D-carboxypeptidases Transpeptidases Endopeptidases

          Background

          Bacteria inhabit every environment on earth with a resilience that is central to their survival and consequently, they continue to serve as a major source of human disease. A critical factor, which has been central to the success of these organisms, is the diversity entrenched within their cell walls, which serves as a major barrier to drug treatment. The mycobacterial cell wall is an incredibly complex structure, with multiple layers that collectively constitute a waxy, durable coat around the cell, which serves as the major permeability barrier to drug action [14]. Considering this, the cell wall and related components are attractive for the mining of new drug targets, and remain relatively unexploited for drug discovery in the case of certain bacterial pathogens [2, 5, 6]. Peptidoglycan (PG or the murein sacculus) is a rigid layer that constricts the cell membrane and the cell within, providing mechanical stability to counteract imbalances of cytoplasmic turgour pressure, and plays an important role in determining cell size and shape [710]. Mycobacteria possess a highly complex additional lipid rich outer membrane, with different constituents anchored either directly to the cell membrane or to the PG [6, 11]. Arabinoglactan (AG), a structure unique to actinomycetes, is bound externally to an N-acetyl muramic acid (NAM) moiety of the PG [3, 12]. In mycobacteria, a certain proportion of the muramic acid is N-glycolylated [13] through the activity of NamH, a UDP-N-acetylmuramic acid hydroxylase [14]. This modification results in altered tumour necrosis factor α production [15, 16] however, abrogation of NamH activity does not lead to decreased virulence in mice [16].

          This serves as an anchor for further lipid rich cell wall components, either by covalent attachment to the mycolic acid layer or through non-covalent interactions [trehalose dimycolate (TDM); phthiocerol dimycocerosate (PDIM); phenolic glycolipids (PGL)] [3, 11, 12]. PG consists of repeated alternating sugars N-acetyl glucosamine (NAG) and NA/GM (muramic acid with or without the glycolyl modification), which are linked to a pentapeptide side chain [79, 17], Figure  1. The crosslinking of these subunits lead to a lattice-like structure around the cell.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-14-75/MediaObjects/12866_2013_2461_Fig1_HTML.jpg
          Figure 1

          PG units and chemical bonds associated with remodelling enzyme activities. At the top and bottom of the figure are shown the NAG-NA/GM sugar backbone in anti-parallel orientation. The NAM residues are designated as NA/GM to correspond to the N-glycolylation of muramic acid in mycobacteria. Enzymatic activities are indicated by arrows: Rpfs [yellow], PBPs [orange], endopeptidases [pink], L,D-transpeptidases [green] and amidases [blue], which are related to the corresponding colours in Table  1. Amino acid residues in the stem peptide are shown in black text. Pentapeptide stems are attached to the Carbon at position 3 of the NAM ring. Transglycosylase activities of Rpfs and the Pon domain indicate their ß-1,4-glycosidic bond substrate. Synthetic enzyme activities are shown on the left, that is those that generate bonds cross-linking the pentapeptides on opposing stems, by Pon and Pbp proteins at positions 4,3 (L-Ala to meso-DAP) or Ldt proteins at positions 3,3 (meso-DAP to meso-DAP). The hydrolytic enzyme activities are shown to the right. These include the amidases, the RipA endopeptidases and the DD-CPase (DacB) acting on the pentapeptide stem (pre- or post-crosslinking).

          The PG in bacterial cell walls is an incredibly dynamic structure that requires constant expansion and remodelling during growth to accommodate the insertion of new PG subunits, secretion apparatus, flagellae etc. [9, 10]. During cell division, pre-septal PG synthesis and subsequent degradation of the septum is critical to daughter cell separation; consequently these processes are carefully regulated [7]. In this regard, there is a diversity of enzymes involved in cross-linking, degradation and remodelling of PG, which are illustrated in Figure  1. A ubiquitous feature in bacteria is the genetic multiplicity associated with these functions, which presumably contributes to the ability of different organisms to adapt under varying environmental conditions [7, 9, 10]. In the case of Mycobacterium tuberculosis, the causative agent of tuberculosis, there is a dire need for new drugs with novel modes of action. The increased prevalence of drug resistant strains has raised concerns regarding the sustainability of the current treatment regimen. To address this, several aspects of mycobacterial metabolism are being assessed for potential new drug targets [18]. The genetic redundancy associated with PG biosynthesis together with the reliance on robust bacterial growth to achieve significant drug target vulnerability, has hampered drug development initiatives that target the cell wall [19]. For other bacterial pathogens, PG has been successfully used as an antibiotic target in the past, as evidenced by the widespread use of β-lactam antibiotics among others, the biosynthesis and degradation of this macromolecule in mycobacteria is meritorious of further investigation.

          In this study, we undertake a comprehensive analysis of the genomic repertoire of PG remodelling enzymes in various pathogenic and environmental mycobacteria to determine the level of genetic multiplicity/redundancy and degree of conservation. We focus on those enzymes involved in cross-linking and remodelling of the PG in the periplasmic compartment, including: resuscitation promoting factors (Rpfs), penicillin binding proteins (PBPs), transpeptidases, endopeptidases, and N-acetylmuramoyl-L-alanine amidases. Our data reveal extensive genetic multiplicity for the 19 strains analysed in this study, which allowed grouping of strains into three families based on their complement of PG remodelling enzymes, including the MTBC, other pathogenic mycobacteria and non-pathogenic environmental organisms.

          Results and Discussion

          The comparative genomics analysis for PG remodelling enzymes in mycobacterial species obtained from this study is summarised in Table  1. We analysed 19 distinct species/strains: Six of these belong to the MTBC, six are classified as other pathogenic bacteria [three of which belong to the Mycobacterium avium complex (MAC)] and six environmental species including Mycobacterium smegmatis. Mycobacterium leprae is listed separately due to its substantially reduced genome which emerges as an outlier in the analysis.
          Table 1

          Genetic complement for PG remodelling enzymes in 19 mycobacterial species

           

          MTB complex

          Other mycobacterial pathogens

          Environmental mycobacterial species

           
           

          M. tuberculosis H37Rv

          M. tuberculosis H37Ra

          M. tuberculosis CDC1551

          M. africanum GM041182

          M. bovis AF2122/97

          M. bovis BCG Pasteur 1173P2

          M. avium 104

          M. avium subsp. paraTB K-10

          M. intracellulare ATCC 13950

          M. ulcerans AGY99

          M. marinum M

          M. abscessus ATCC19977

          M. smegmatis mc 2 155

          M. vanbaalenii PYR–1

          M. sp. MCS

          M. sp. KMS

          M. sp. JLS

          M. gilvum PYR–GCK

          M. leprae TN

          Resuscitation promoting factors

          rpfA

          Rv0867c

          MRA_0874

          MT0890

          MAF_08760

          Mb0891c

          BCG_0919c

          MAV_0996

          MAP0805c

          OCU_08710

          MUL_0283

          MMAR_4665

          MAB_0869c

          MSMEG_5700

          Mvan_5049

          Mmcs_4479

          Mkms_4566

          Mjls_4862

          Mflv_1703

          ML2151

          rpfB

          Rv1009

          MRA_1018

          MT1038

          MAF_10190

          Mb1036

          BCG_1066

          MAV_1147

          MAP0974

          OCU_10320

          MUL_4651

          MMAR_4479

          MAB_1130

          MSMEG_5439

          Mvan_4801

          Mmcs_4264 Mmcs_1712

          Mkms_4350 Mkms_1758

          Mjls_ 4643 Mjls_1689

          Mflv_1932

          ML0240

          rpfC

          Rv1884c

          MRA_1895

          MT1932

          MAF_19060

          Mb1916c

          BCG_1921c

          MAV_2818

          MAP1607c

          OCU_26380

          MUL_2975

          MMAR_2772

          MAB_4080

          ML2030

          rpfD

          Rv2389c

          MRA_2413

          MT2458

          MAF_24030

          Mb2410c

          BCG_2403c

          rpfE

          Rv2450c

          MRA_2476

          MT2526

          MAF_24670

          Mb2477c

          BCG_2470c

          MAV_1722

          MAP2273c

          OCU_18020

          MUL_3723

          MMAR_3776

          MAB_1597

          MSMEG_4643/MSMEG_4640

          Mvan_3962/Mvan_3961

          Mmcs_3564/Mmcs_3563

          Mkms_3637/Mkms_3636

          Mjls_3569/ Mjls_3568

          Mflv_2619/Mflv_2620

          Penicillin binding proteins

          ponA1

          Rv0050

          MRA_0053

          MT0056

          MAF_00500

          Mb0051

          BCG_0081

          MAV_0071

          MAP0064

          OCU_00670

          MUL_0068

          MMAR_0069

          MAB_4901c

          MSMEG_6900

          Mvan_6036

          Mmcs_5372

          Mkms_5461

          Mjls_5748

          Mflv_0871

          ML2688c

          ponA2

          Rv3682

          MRA_3717

          MT3784

          MAF_36900

          Mb3707

          BCG_3741

          MAV_0446

          MAP0392c

          OCU_03970

          MUL_4257

          MMAR_5171

          MAB_0408c

          MSMEG_6201 MSMEG_4384

          Mvan_5442 Mvan_1068

          Mmcs_4825 Mmcs_1483

          Mkms_4911 Mkms_1505

          Mjls_5212 Mjls_1480

          Mflv_1365 Mflv_5209

          ML2308

          pbpA

          Rv0016c

          MRA_0018

          MT0019

          MAF_00160

          Mb0016c

          BCG_0046c

          MAV_0020

          MAP0019c

          OCU_00180

          MUL_0020

          MMAR_0018

          MAB_0035c

          MSMEG_0031

          Mvan_0025

          Mmcs_0017

          Mkms_0025

          Mjls_0017

          Mflv_0810

          ML0018

          pbpB

          Rv2163c

          MRA_2178

          MT2221

          MAF_21760

          Mb2187c

          BCG_2180c

          MAV_2330

          MAP1903c

          OCU_22960

          MUL_3508

          MMAR_3200

          MAB_2000

          MSMEG_4233

          Mvan_3529

          Mmcs_3262

          Mkms_3324

          Mjls_3273

          Mflv_2982

          ML0908

          PBP-lipo

          Rv2864c

          MRA_2889

          MT2933

          MAF_28690

          Mb2889c

          BCG_2886c

          MAV_3723

          MAP2936c

          OCU_35570

          MUL_2089

          MMAR_1840

          MAB_3167c

          MSMEG_2584 MSMEG_6319

          Mvan_2266 Mvan_4630

          Mmcs_2047 Mmcs_4955

          Mkms_2093 Mkms_5043

          Mjls_2030 Mjls_5336

          Mflv_4076 Mflv_2080

          ML1577c

          dacB1

          Rv3330

          MRA_3372

          MT3433

          MAF_33460

          Mb3363

          BCG_3400

          MAV_4305

          MAP3448

          OCU_41630

          MUL_1445

          MMAR_1192

          MAB_3681

          MSMEG_1661

          Mvan_1562

          Mmcs_1216

          Mkms_1233

          Mjls_1243

          Mflv_4869

          ML0691

          dacB2

          Rv2911

          MRA_2936

          MT2979

          MAF_29150

          Mb2935

          BCG_2932

          MAV_3766

          MAP2979

          OCU_36070

          MUL_2045

          MMAR_1797

          MAB_3234

          MSMEG_2433/MSMEG_2432

          Mvan_2184/Mvan_2183

          Mmcs_1962/Mmcs_1961

          Mkms_2008/Mkms_2007

          Mjls_1942/Mjls_1941

          Mflv_4179/Mflv_4180

          Rv3627c

          Rv3627c

          MRA_3663

          MT3729

          MAF_36340

          Mb3651c

          BCG_3685c

          MAV_0529

          MAP0436

          OCU_04440

          MUL_4203

          MMAR_5127

          MAB_0519

          MSMEG_6113

          Mvan_5380

          Mmcs_4778

          Mkms_4864

          Mjls_5164

          Mflv_1409

          ML0211

          MSMEG_1900

          MAB_2019

          MSMEG_1900

          Mvan_4520

          Mmcs_0342

          Mkms_0352

          Mjls_0331

          Mflv_2177

          Endo-Peptidases

          Rv0024

          Rv0024

          MRA_0027

          MT0027

          MAF_00240

          Mb0024

          BCG_0054

          MAV_0042

          MAP0036

          OCU_00360

          MUL_0042

          MMAR_0043

          ripA

          Rv1477

          MRA_1487

          MT1524

          MAF_15000

          Mb1513

          BCG_1539

          MAV_3301

          MAP1203

          OCU_31420

          MUL_1486

          MMAR_2284

          MAB_2728c

          MSMEG_3145

          Mvan_3656 Mvan_2747

          Mmcs_1440 Mmcs_2451

          Mkms_5716 Mkms_1458 Mkms_2496

          Mjls_ 2488 Mjls_4564 Mjls_4520

          Mflv_5292 Mflv_0895 Mflv_2839 Mflv_3663

          ML1812

          ripB

          Rv1478

          MRA_1488

          MT1525

          MAF_15010

          Mb1514

          BCG_1540

          MAV_3300

          MAP1204

          OCU_31410

          MUL_1487

          MMAR_2285

          MAB_2727c

          MSMEG_3146

          Mvan_2748 Mvan_3652

          Mmcs_2452 Mmcs_1447

          Mkms_2497 Mkms_1465 Mkms_5687 Mkms_5720

          Mjls_ 2489 Mjls_ 4472 Mjls_ 4557 Mjls_ 4529

          Mflv_5324 Mflv_5288 Mflv_0902 Mflv_2843

          ML1811

          ripD

          Rv1566c

          MRA_1578

          MT1617

          MAF_15930

          Mb1593c

          BCG_1619c

          MAV_3208

          MAP1272c

          OCU_30430

          MUL_1557

          MMAR_2381

          (MAB_2474)

          (MSMEG_3477)

          (Mvan_2970)

          (Mmcs_2672)

          (Mkms_2717)

          (Mjls_2702)

          (Mflv_3253)

          ML1214

          Rv2190c

          Rv2190c

          MRA_2205

          MT2245

          MAF_22010

          Mb2213c

          BCG_2206c

          MAV_2304

          MAP1928c

          OCU_22720

          MUL_3545

          MMAR_3234

          MAB_1974

          MSMEG_4256

          Mvan_3552 Mvan_3713

          Mmcs_3287 Mmcs_1435

          Mkms_3349 Mkms_5661 Mkms_1453

          Mjls_3298 Mjls_4528 Mjls_4570

          Mflv_2959 Mflv_5385 Mflv_5350 Mflv_2808 Mflv_0888

          ML0885

          L,D-transpeptidases

          ldt Mt1

          Rv0116c

          MRA_0123

          MT0125

          MAF_01170

          Mb0120c

          BCG_0150c

          MAV_5194

          MAP3520c

          OCU_50160

          MUL_4806

          MMAR_0316

          MAB_3165c

          MSMEG_3528

          Mvan_3019

          Mmcs_2729

          Mkms_2773

          Mjls_2759

          Mflv_3298

          ML2664

          ldt Mt2

          Rv2518c

          MRA_2545

          MT2594

          MAF_25330

          Mb2547c

          BCG_2539c

          MAV_1661

          MAP2322c

          OCU_17500

          MUL_3804

          MMAR_3872

          MAB_1530

          MSMEG_4745

          Mvan_4102 Mvan_3651 Mvan_5854

          Mmcs_1448 Mmcs_3641

          Mkms_5721 Mkms_3714 Mkms_1466

          Mjls_3646 Mjls_4532 Mjls_4556

          Mflv_2542 Mflv_5287 Mflv_0904

          ML0426

          ldt Mt3

          Rv1433

          MRA_1442

          MT1477

          MAF_14550

          Mb1468

          BCG_1494

          MAV_4834

          MAP3812c

          OCU_47330

          MMAR_3552

          MAB_4775

          MSMEG_0674

          Mjls_4515

          Mflv_1397

          ML0569

          ldt Mt4

          Rv0192

          MRA_0200

          MT0202

          MAF_01930

          Mb0198

          BCG_0229

          MAV_4986

          MAP3634

          OCU_48990

          MUL_1085

          MMAR_0435

          MAB_4537c

          MSMEG_0233

          Mvan_3694 Mvan_0177

          Mmcs_0151

          Mkms_5680 Mkms_0160

          Mjls_4535 Mjls_0141

          Mflv_5330 Mflv_2824 Mflv_5369 Mflv_0479

          ldt Mt5

          Rv0483

          MRA_0490

          MT0501

          MAF_04870

          Mb0493

          BCG_0524

          MAV_4666

          MAP3976

          OCU_45320

          MUL_4553

          MMAR_0809

          MAB_4061c

          MSMEG_0929

          Mvan_0824

          Mmcs_0654

          Mkms_0667

          Mjls_0647

          Mflv_0089

          ML2446

          Amidases

          ami1

          Rv3717

          MRA_3754

          MT3820

          MAF_37260

          Mb3744

          BCG_3777

          MAV_0385

          MAP0318

          OCU_03450

          MUL_4308

          MMAR_5233

          MAB_0318c

          MSMEG_6281

          Mvan_5529

          Mmcs_4905

          Mkms_4994

          Mjls_5273

          Mflv_1286

          ML2331

          ami2

          Rv3915

          MRA_3954

          MT4034

          MAF_39300

          Mb3946

          BCG_0021

          MAV_5303

          MAP4341

          OCU_51370

          MUL_5068

          MMAR_5479

          MAB_4942

          MSMEG_6935

          Mvan_6069

          Mmcs_5404

          Mkms_5493

          Mjls_5780

          Mflv_0837

          ML2704

          ami3

          Rv3811

          MRA_3851

          MT3918

          MAF_38260

          Mb3841

          BCG_3873

          MAV_0206

          MAP0209c

          OCU_02160

          MUL_4995

          MMAR_5375

          MAB_0168c

          MSMEG_6406

          Mvan_5652

          Mmcs_5022

          Mkms_5110

          Mjls_5403

          Mflv_1157

          ami4

          Rv3594

          MRA_3633

          MT3700

          MAF_36070

          Mb3625

          BCG_3659

          MAB_4807

          MSMEG_5315

          Mvan_3376

          Mmcs_4180

          Mkms_4246

          Mjls_4402

          Mflv_3152

          The names of the various organisms analysed are shown in the columns and gene complement is given in the corresponding rows. Mycobacteria are grouped as M. tuberculosis Cluster (MTBC), other pathogens, environmental species and M. leprae. Genes are sorted by functional groups in rows. The listing of a gene is based on its presence by protein BLAST analysis, either at curated sites or directly at NCBI. For all genes the protein sequence, in FASTA format, was obtained and utilised for phylogeny. Annotations for M. africanum (MAF_) and M. intracellulare (OCU_) were obtained directly from NCBI. BLAST analysis was performed against individual strains at NCBI using M. tuberculosis H37Rv homologues as the query sequence. The cut off was taken at a coverage of >90% and an identity of >40%. MSMEG_1900 was identified at SmegmaList. In the case of ripD, parentheses indicate the 63C-terminal amino acid truncation. Further in-depth information, and confirmation of gene annotation, was obtained by assessment of phylogeny based on protein sequences, Additional file 1 Figure S1-S7. Font differences in the M. tuberculosis H37Rv column indicate genes that have been annotated as essential by two different TraSH analyses – indicated in bold (Sassetti et al. [20]) and/or italicised (Griffin et al. [21]) are those genes identified as essential or required for optimal growth.

          Resuscitation promoting factors (lytic transglycosylases)

          Of all the enzymes identified in this study, the Rpf family is the most extensively studied. This group of enzymes are of particular interest due to demonstrated importance for reactivation from dormancy and essentiality for growth in Micrococcus luteus[22, 23]. Whilst Mi. luteus encodes a single, essential rpf gene, mycobacteria encode a multiplicity of rpf homologues and those present in M. tuberculosis, designated as rpfA-rpfE, encode closely related proteins all of which retain the Rpf domain [2426], Figure  2. These have been the subject of intense study due to the potential role they may play in reactivation disease in individuals that harbour latent TB infection [25, 2731]. In this regard, the five rpf genes present in M. tuberculosis are collectively dispensable for growth but are differentially required for reactivation from an in vitro model of non-culturability [32, 33]. Furthermore, the Rpfs are combinatorially required to establish TB infection and for reactivation from chronic infection in mice [3235]. For additional information, the reader is referred to several extensive reviews on this topic [25, 27, 28, 3638].
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-14-75/MediaObjects/12866_2013_2461_Fig2_HTML.jpg
          Figure 2

          Alignment and domains of M. tuberculosis H37Rv PG remodelling enzymes. Domain architecture is based on output from InterScanPro. All enzymes depicted are the M. tuberculosis H37Rv homologues. Amino acid sequences are grouped according to their common domains, as indicated by their colors: Rpf domains [yellow], PBPs [orange], endopeptidases [pink], LD-transpeptidases [green] and amidases [blue]. PonA proteins are grouped with PBPs. PFAM domains are annotated as follows: PF06737 Transglycosylase-like domain, PF00905 PBP transpeptidases domain, PF00912 Transglycosylase domain, PF00768 D-alanyl-D-alanine Carboxypeptidase domain, PF02113 D-Ala-D-Ala carboxypeptidase 3 (S13) family domain, PF00877 NlpC/P60 family domain, PF03734 L,D-transpeptidase catalytic domain, PF01520 N-acetylmuramoyl-L-alanine amidase amidase_3 domain, PF01510 N-acetylmuramoyl-L-alanine amidase amidase_2 domain. N-terminal signal sequence or transmembrane domains are displayed as purple and pink, respectively. Additional domains annotated at PFAM are as follows (in grey): PonA2, PF03793, PASTA domain; PbpB, PF03717, PBP dimerization domain; PBP-lipo, PF05223, NTF2-like N-terminal transpeptidase; Ami2, PF01471, Peptidoglycan-binding like; RpfB, PF03990, Domain of unknown function DUF348; RpfB, PF07501, G5 domain. Rv3627c retains two tandem copies of the PF02113 D-Ala-D-Ala carboxypeptidase 3 (S13) family domain, one of which is contracted. Figure not to scale.

          Rpfs are classified as lytic transglycosylases (LTs) based on sequence conservation and three-dimensional protein structure [29, 3941]. LTs cleave the ß-1,4-glycosidic bonds between the NAG-NA/GM sugar subunits, Figure  1, and their activity is required for insertion of new PG units and expansion of the glycan backbone [9]. In mycobacteria RpfB contains a lysozyme-like, transglycosylase-like PFAM domain, and consequently this group of enzymes are predicted to cleave the glycan backbone of PG [3941]. Direct evidence for this is lacking and moreover, the mechanism through which Rpf-mediated cleavage of PG results in growth stimulation remains unknown. The repertoire of rpf genes is highly conserved in the MTBC; in contrast, other pathogenic mycobacteria lack rpfD, including M. leprae, Table  1. Based on the distribution of rpfC and rpfD, we categorize the 19 strains analysed in this study into the MTBC (which retains all five rpf homologues present in M. tuberculosis), other pathogenic mycobacteria (which lack rpfD) and environmental strains (which lack both rpfC and rpfD). This classification is supported by phylogenetics analysis which confirms these clusters and duplication/loss of genes, Additional file 1: Figure S1. Recently, it has been shown that the Rpfs can serve as potent antigens [42] and Rpf-directed host immune responses allow for detection of TB in latently infected individuals [43]. It is noteworthy that strains lacking different combinations of rpf genes confer significant protective efficacy when used as vaccine strains in mice [44]. Hence, any variation in rpf gene complement between pathogenic mycobacteria may have significant consequences for broadly protective effects of future Rpf-based vaccines.

          The environmental species retain three rpf genes [rpfA, rpfB (duplicated in Mycobacterium sp. JLS, Mycobacterium sp. KMS, Mycobacterium sp. MCS) and rpfE], Table  1 and Additional file 1: Figure S1. Although rpfC (Rv1884c in M. tuberculosis) homologues have been annotated as present in all mycobacteria [45], our analysis shows that the M. tuberculosis rpfC homologue is absent from environmental species. Artemis Comparison Tool (ACT) whole genome alignment reveals that the region encoding rpfC in M. tuberculosis is absent in M. smegmatis and all other environmental mycobacteria (data not shown). Thus, based on gene synteny, there is no direct rpfC homologue in these strains. However, there is a local duplication of rpfE in all the environmental strains (annotated as MSMEG_4643 in M. smegmatis), Table  1, Additional file 1: Figure S1. Consequently, we re-annotate MSMEG_4640 to rpfE2, as a homologue of MSMEG_4643, rather than a homologue of Rv1884c. As RpfE interacts with the Rpf Interacting Protein A (RipA) [46], there may be some functional consequence to the presence of multiple copies in M. smegmatis and other environmental bacteria.

          The restriction of rpfC and rpfD homologues to pathogenic and MTBC strains, along with the duplication of rpfB in some environmental species, raises interesting questions regarding the nature of growth stimulation in these organisms. These differences suggest that the latter require fewer secreted Rpfs and are more reliant on the membrane bound RpfB homologue. This could be related to the fact that environmental organisms are required to grow in diverse niches of varying size and complexity making them more dependent on localised growth stimulatory activity through a membrane bound Rpf rather than paracrine signalling from diffusible Rpfs produced by neighbouring organisms. It is noteworthy that of all five homologues in M. tuberculosis, deletion of rpfB individually or in combination with rpfA results in colony forming defects and prolonged time to reactivation from chronic infection in mice [21, 34, 35].

          The role of Rpfs in TB disease in humans remains enigmatic. It has been demonstrated that sputum from patients with active TB disease, before the initiation of treatment, is characterised by a population of dormant bacteria that require Rpfs for growth [47]. These data provide tantalizing preliminary evidence that Rpfs play an important role in determining bacterial population dynamics in TB infected patients and moreover are critical for disease transmission. Within the granulomatous environment, it may be preferable for the bacterial population as a whole to facilitate emergence of fitter clones which are able to exit from arrested growth. This could explain clonal emergence in clinical samples if few strains are able to expand sufficiently to cause tubercular lung disease.

          Penicillin binding proteins

          Penicillin Binding proteins (PBPs) are a large family of evolutionarily related cell wall associated enzymes, that bind β-lactam antibiotics [48, 49]. PBPs are classified according to their molecular weight as either high molecular mass (HMM) or low molecular mass (LMM) and are broken down into Class A, Class B and Class C [49]. In mycobacteria, Class A PBPs constitute bi-functional enzymes designated as ponA1 (PBP1, Rv0050, [50]); and ponA2 (PBP1A, Rv3682 [51]), Figure  2. They contain separate domains for transpeptidase and transglycosylase activities. Both these genes are present in all mycobacteria and, as previously reported for M. smegmatis and other environmental strains, there is a duplication of ponA2 which was annotated as ponA3[51], Table  1 and Additional file 1: Figure S2.

          Class B PBP proteins PbpA (pbpA; Rv0016c, [52]), PbpB (pbpB; Rv2163c, [53]) and PBP-lipo (Rv2864c, [49]) are predicted to contain only transpeptidase domains and possibly additional dimerisation domains, but lack transglycosylase activities, Figure  2. Both PbpA and PbpB (FtsI) are involved in progression to cell division in M. smegmatis where gene deletion or depletion manifests in altered cell morphology and antibiotic resistance profiles [52]. In this family of PBPs – as exemplified by ponA2 - there is a distal duplication of PBP-lipo in the environmental strains, Table  1 and Additional file 1: Figure S3. No experimental data on this are currently available, but the lipophilic domain is speculated to allow for cell wall association.

          D,D-carboxypeptidases (DD-CPases) are designated as Class C PBPs and are generally present in high abundance [54]. DD-CPases remove the D-Ala residue at position 5 of pentapeptides [8] and through this activity prevent cross linking of the stem peptide into 4 → 3 bridges, Figure  1. In mycobacteria, the dacB2-encoded DD-CPase is not affected by penicillin – though it does bind the antibiotic [55]. Inhibition of DacB through treatment with meropenem results in the accumulation of pentapeptides in M. tuberculosis[56]. In this context, DD-CPases have been implicated in regulating the amount of cross-linking that can occur within the PG sacculus [8]. Our analysis shows that M. tuberculosis H37Rv encodes three distinct DD-CPase homologues: dacB1 (Rv3330), dacB2 (Rv2911) and Rv3627c, Table  1, Figure  2 and Additional file 1: Figure S4. Rv3627c carries two PF02113 domains, one of which is contracted. In the environmental species there is a local duplication of the dacB2 (Rv2911) homologue, leading to consecutive numbering of the resulting duplicated genes for example, MSMEG_2432 and MSMEG_2433 in M. smegmatis. In addition, a distant DD-CPase homologue (annotated as MSMEG_1900 in M. smegmatis) was identified in the environmental strains, as well as in M. abscessus but not in the other pathogenic mycobacteria and MTBC, Table  1. Two additional loci - Rv0907 and Rv1367c – were identified in M. tuberculosis by in silico analysis through their predicted ß-lactamase domains and are grouped among Class C PBPs [49]. Analysis of these proteins revealed that they retain a β-lactamase binding domain (of the AmpH family) but further classification into the functional classes studied herein proved difficult. Consequently, we have not analysed these genes further.

          Endopeptidases

          Endopeptidases are enzymes that cleave within the stem peptides in PG. In this study, we focus on the Nlp/P60 class of endopeptidases, which cleave within the stem peptides between positions 2 and 3 as exemplified by RipA, Figure  1. RipA is an essential PG hydrolytic enzyme that synergistically interacts with RpfB and RpfE [46, 57] to form a complex that is able to degrade PG. The RipA-RpfB hydrolytic complex is negatively regulated by PonA2 [58] suggesting a dynamic interplay between PG hydrolases, one that would be significantly nuanced with the presence of multiple RipA and Rpf homologues. In this regard, our analysis reveals four endopeptidases in M. tuberculosis that display strong homology to ripA, Table  1, Figure  2, Additional file 1: Figure S5. With the exception of Mycobacterium abscessus and M. leprae, pathogenic mycobacteria retain all five of these homologues. Environmental strains display enhanced expansion of endopeptidases, with the exception of the ripD homologue (Rv1566c). The functional consequence of this remains unknown but it is noteworthy that these strains have also expanded their rpfE and rpfB gene repertoire, suggesting that the multiplicity in this case allows for a greater number of RipA-RpfB/E protein complexes, as well as for protein complexes with different subunit composition. Dysregulated expression of RipA leads to dramatic alterations in cellular morphology and growth [59] suggesting that careful regulation of this protein, both at the expression level as well as by post-translational level is essential. Genetic expansion of RipA homologues along with two copies of RpfB and RpfE, both of which interact with RipA implies a functional consequence of this expansion. In addition, strong regulation of these multiple copies would be required to prevent any detrimental effects on cell growth.

          RipB displays strong sequence homology RipA in M. tuberculosis (100% amino acid identity over 58% coverage) and similar domain organization [60], but lacks the N-terminal motif, Figure  2, that has been implicated in auto inhibition by blocking the active site in the three-dimensional crystal structure [61]. More recently, high resolution crystal structures of RipB and the C-terminal module of RipA (designated as RipAc) revealed striking differences in the structure of these proteins, specifically in the N-terminal fragments that cross the active site [60]. Both RipB and RipAc are able to bind high molecular weight PG and retain the ability to cleave PG with variable substrate specificity, which is not regulated by the presence of the N-terminal domain [60]. This suggests that the N-terminus does not regulate PG degrading activity and in this context, the physiological consequences of the reduced size of RipB and RipD, Figure  2, remain unknown. The high degree of conservation of RipB across all pathogenic mycobacteria including M. leprae, Table  1, Additional file 1: Figure S5 indicates that variable substrate specificity in PG hydrolases in essential for pathogenesis. The Mycobacterium marinum homologues of Rv1477 and Rv1478, iipA and iipB (MMAR_2284 and MMAR_2285 respectively), Table  1, Additional file 1: Figure S5, have been implicated in macrophage invasion, antibiotic susceptibility and cell division [62]. As with the other enzymes assessed in this study, environmental mycobacteria display greater genetic multiplicity for these homologues, Table  1.

          Structural analysis of RipD reveals alterations in the catalytic domain, consistent with the inability of this protein to hydrolyse PG [63]. Nevertheless the core domain of RipD is able to bind mycobacterial PG and this binding is negatively regulated by the C-terminal region [63]. However, RipD homologues in the environmental mycobacteria lack the 63C-terminal amino acids, Table  1 (shown in parentheses), possibly allowing for stronger binding of this enzyme to PG.

          Rv2190c encodes another NlpC/P60-type PG hydrolase in mycobacteria. Deletion of this gene in M. tuberculosis results in altered colony morphology, attenuated growth in vitro, defective PDIM production and reduced colonisation of mouse lungs in the murine model of TB infection [64]. Consistent with this, homologues of Rv2190c are found in all pathogenic mycobacteria, Table  1, with notable genetic expansion in some environmental species. In contrast, the Rv0024 is absent from environmental species, suggesting that it could be required for intracellular growth or some other component of the pathogenic process, Table  1, Additional file 1: Figure S5.

          L,D - Transpeptidases

          L,D-transpeptidases (Ldt) are a group of carbapenem sensitive enzymes in M. tuberculosis[56] that contribute to the formation of a 3 → 3 link between the two adjacent mDAP (mDap → mDap bridges) residues in PG, distinct from the classic 4 → 3 link (D-Ala → mDAP), Figure  1. M. abscessus[65] and M. tuberculosis[66] exhibit increased ratios of the 3 → 3 cross-link in stationary axenic culture, indicating that mycobacteria are capable of modulating their PG at the level of transpeptidation in response to growth stage and the availability of nutrients. Both LdtMt1 and LdtMt2 (Rv0116c and Rv2518c respectively) were experimentally shown to affect M. tuberculosis H37Rv morphology, growth characteristics and antibiotic susceptibility in vivo[67]. The crystal structure of LdtMt2 places the extramembrane domain 80–100 Å from the membrane surface and indicates that this enzyme is able to remodel PG within this spatial region of the PG sacculus [68]. More recently, it has been demonstrated that the combinatorial loss of both LdtMt1 and LdtMt2 in M. tuberculosis resulted in morphological defects and altered virulence in the murine model of TB infection [69]. A notable variability of L,D-transpeptidase genes is found in mycobacteria, Table  1, Figure  2 and Additional file 1: Figure S6. Five homologues are present in all but one pathogenic strain, while multiple homologues are evident in most environmental strains. The exception is ldt Mt3 (Rv1433), which is absent from the pathogen Mycobacterium ulcerans and from the environmental species Mycobacterium vanbaalenii, M sp. MCS and M. sp. KMS, yet its presence in M. leprae suggests functional importance. As with RipA, M. gilvum shows the greatest expansion of the ldt genes. Biochemical characterisation of all five M. tuberculosis H37Rv homologues, LdtMt1 - LdtMt5, confirms PG cross-linking and/or ß-lactam acylating enzyme activities in all of these enzymes [70]. This activity can be abolished by treatment with imipenem and cephalosporins, indicating that this group of enzymes holds great promise for TB drug development [70, 71]. Moreover, the functionality of all the Ldt homologues present in M. tuberculosis raises interesting questions with respect to the functional consequences of the expansion of this protein family in environmental strains, which may require greater flexibility in Ldt function.

          Amidases

          While endopeptidases and transpeptidases are responsible for cleavage within or between peptide stems, amidases act to remove the entire peptide stem from the glycan strands, cleaving between the NA/GM moiety and the L-Ala in the first position of the stem peptide, Figure  1. The amidases have been implicated in PG degradation, antibiotic resistance/tolerance and cell separation in Escherichia coli and other organisms, and can be organised into 2 main families containing either an amidase_2 or amidase_3 – type domain [8, 9, 72]. The amidases of E. coli (which retains 5 amidases designated AmiA, AmiB, AmiC, AmiD and AmpD) have specific substrate requirements governed by the structural confirmation of the NAM carbohydrate moiety. Knockout of these amidases results in chaining phenotypes, abnormal cell morphologies and/or increased susceptibility to certain antibiotics [7274]. Amidases have also been implicated in spore formation, germination and cell communication in Bacillus subtilis[75, 76]. The role of amidases in mycobacterial growth, virulence and resuscitation from dormancy is unknown and any impact of these on mycobacterial morphology and antibiotic resistance remains to be demonstrated. Analysis of the amidase gene complement in mycobacteria reveals the presence of four homologues in M. tuberculosis, two containing the amidase_2 domain (ami3; Rv3811 and ami4; Rv3594) and two the amidase_3 domain (ami1; Rv3717 and ami2; Rv3915), Table  1, Figure  2 and Additional file 1: Figure S7. The crystal structure of Rv3717 from M. tuberculosis confirms that this enzyme is able to bind and cleave muramyl dipeptide [77]. The amidase family distinguishes itself from all other enzyme families by absence of a homologue (ami4) from non-MTBC pathogens and its presence in the MTBC and environmental strains. M. leprae retains only the ami1 and ami2 genes – both containing the amidase_3 domain. This suggests that amidase_2 domain amidase activity is dispensable specifically in this species, but required for peptidoglycan remodelling in the other pathogenic mycobacteria.

          Mycobacterium leprae

          Very little is known about in vitro growth and division of M. leprae, as it can only be grown in animal models. From our analysis, it is apparent that M. leprae habours notable genetic redundancy for PG remodelling enzymes (Table  1) in contrast to its minimal gene set for other areas of metabolism [78]. Considering that PG subunits or precursors cannot be scavenged from the host, it is expected that pathogenic bacteria would retain complete pathways for biosynthesis and remodelling of PG. However, the presence in M. leprae of multiple homologues within each class of PG remodelling enzyme assessed in this study, suggests that some level of multiplicity is required to ensure substrate flexibility. Further work in this regard is difficult due to the limited tractability of M. leprae for in vitro manipulation.

          Conclusions

          Mycobacteria represent a wide range of species with a great variety of phenotypes. Exposure to stresses which they encounter at various stages of their life cycles demands the ability to adapt. Consistent with this, many mycobacteria encode a multiplicity of genes for numerous important pathways such as respiration and cofactor biosynthesis [79, 80], which allows for a more nuanced regulation of physiology. The analysis performed herein summarises the general distribution of PG remodelling genes in diverse strains and reveals an emerging trend towards gene multiplicity in environmental mycobacteria. There is great conservation within the MTBC and other pathogenic mycobacteria. Of all strains, M. gilvum displays the greatest degree of gene expansion, containing a total 44 PG remodelling genes, Table  1. This organism has not been studied extensively but may represent a potential model system for understanding how the genetic multiplicity for PG remodelling enzymes contributes to bacterial physiology. As expected M. leprae shows a reduction in the number of genes that encode the enzymes assessed in this study but still retains more than one representative of each functional class. This, together with the striking degree of conservation in some families of PG remodelling enzymes in pathogenic mycobacteria, suggests that PG biosynthesis, remodelling and possibly recycling are all potential vulnerable pathways for drug development. The extracellular nature of these enzymes provides an added advantage for drug screening since small molecules need not enter the cell for biological activity. Entry of compounds into mycobacterial cells remains the major confounding factor in current drug development initiatives. Moreover, the lack of human counterparts would ensure a high degree of specificity. In conclusion, the gene complements for PG remodelling revealed in this study most likely reflect the differential requirements of various mycobacteria for murein expansion/turnover during colonisation of and proliferation within host organisms or environmental niches.

          Methods

          The 19 mycobacterial strain sequences used in this study were all complete and either published [24, 78, 8190] or directly submitted to GenBank [91] (Additional file 2: Table S1). The following sites were utilized for analysis of the genomes (Additional file 2: Table S2): The comparative genomic profile for the enzymes of interest were initiated by homology searches of known M. tuberculosis H37Rv genes at TubercuList [92], GenoList [93] or TBDB [94]. Where necessary for further analysis direct BLAST analysis was performed at NCBI [95], utilising protein sequence for BLASTp or DNA sequence for BLASTn particularly for the analysis of Mycobacterium sp. JLS, M. africanum and M. intracellulare which are not or only partially annotated at TBDB. To confirm the absence of genes, protein sequence was used for tBLASTn analysis. Additional homologues that are absent from M. tuberculosis H37Rv were identified by advanced search at SmegmaList (Mycobrowser) [96]. Where information was required for sequence level analysis, the Sanger Artemis Comparison Tool (ACT) [97] was utilized on annotated sequences obtained from the Integrated Microbial Genomes (IMG) site at the DOE Joint Genome Institute [98]. Phylogeny was established from FASTA files from all genes in Table 1 at EMBL-EBI by ClustalO [99] alignment and ClustalW2 [100] analysis and visualized using FigTree V1.4 software (http://​tree.​bio.​ed.​ac.​uk/​software/​figtree). Functional annotation of each of the M. tuberculosis proteins was identified at InterScanPro [101], for PFAM domains [102], signal sequences (SignalP) [103] and membrane anchoring domains (TMHMM) [104].

          Declarations

          Acknowledgements

          This work was supported by grants from the South African National Research Foundation (NRF), the Medical Research Council, the Department of Science and Technology. BK was supported by an Early Career Scientist award from the Howard Hughes Medical Institute. C.S.E was supported by postdoctoral fellowships from the NRF and the Centre of Aids Programme Research in South Africa (CAPRISA).

          Authors’ Affiliations

          (1)
          DST/NRF Centre of Excellence for Biomedical TB Research, Faculty of Health Sciences, University of the Witwatersrand, National Health Laboratory Service

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