Decaprenylphosphoryl-β-D-Ribose 2′-Epimerase, the Target of Benzothiazinones and Dinitrobenzamides, Is an Essential Enzyme in Mycobacterium smegmatis

BACKGROUND: The unique cell wall of bacteria of the suborder Corynebacterineae is essential for the growth and survival of significant human pathogens including Mycobacterium tuberculosis and Mycobacterium leprae. Drug resistance in mycobacteria is an increasingly common development, making identification of new antimicrobials a priority. Recent studies have revealed potent anti-mycobacterial compounds, the benzothiazinones and dinitrobenzamides, active against DprE1, a subunit of decaprenylphosphoribose 2' epimerase which forms decaprenylphosphoryl arabinose, the arabinose donor for mycobacterial cell wall biosynthesis. Despite the exploitation of Mycobacterium smegmatis in the identification of DprE1 as the target of these new antimicrobials and its use in the exploration of mechanisms of resistance, the essentiality of DprE1 in this species has never been examined. Indeed, direct experimental evidence of the essentiality of DprE1 has not been obtained in any species of mycobacterium. METHODOLOGY/PRINCIPAL FINDINGS: In this study we constructed a conditional gene knockout strain targeting the ortholog of dprE1 in M. smegmatis, MSMEG_6382. Disruption of the chromosomal copy of MSMEG_6382 was only possible in the presence of a plasmid-encoded copy of MSMEG_6382. Curing of this "rescue" plasmid from the bacterial population resulted in a cessation of growth, demonstrating gene essentiality. CONCLUSIONS/SIGNIFICANCE: This study provides the first direct experimental evidence for the essentiality of DprE1 in mycobacteria. The essentiality of DprE1 in M. smegmatis, combined with its conservation in all sequenced mycobacterial genomes, suggests that decaprenylphosphoryl arabinose synthesis is essential in all mycobacteria. Our findings indicate a lack of redundancy in decaprenylphosphoryl arabinose synthesis in M. smegmatis, despite the relatively large coding capacity of this species, and suggest that no alternative arabinose donors for cell wall biosynthesis exist. Overall, this study further validates DprE1 as a promising target for new anti-mycobacterial drugs

Identification of a Novel Gene Product That Promotes Survival of Mycobacterium smegmatis in Macrophages

BACKGROUND: Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs. METHODOLOGY/PRINCIPAL FINDINGS: We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate. CONCLUSIONS/SIGNIFICANCE: MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria

DprE1 alignment.

<p>Homologs from <i>M. leprae</i> (ML0109), <i>M. tuberculosis</i> (Rv3790) and <i>M. smegmatis</i> (MSMEG_6382) were aligned using CLUSTALW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016869#pone.0016869-Larkin1" target="_blank">[25]</a>. Residues that are completely conserved are reverse shaded while similar residues are indicated in grey. The putative FAD-binding domain is shown by a solid line. The conserved cysteine residue that is altered in BTZ-resistant mycobacteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016869#pone.0016869-Makarov2" target="_blank">[11]</a> is indicated by an asterix.</p

Genetic strategy for conditional disruption of <i>MSMEG_6382</i>.

<p><b>A</b>. The recombination plasmid pPKC72 contained a cloned copy of <i>MSMEG_6382</i> interrupted by a non-polar kanamycin resistance cassette (<i>MSMEG_6382</i>::<i>aphA3</i>), a gentamycin resistance marker (Gm^r), a temperature-sensitive replication origin for <i>M. smegmatis</i> (ori<i>Ms</i> (ts)), a replication origin for <i>E. coli</i> (ori<i>Ec</i>) and a counterselectable marker encoding sucrose sensitivity (<i>sacB</i>). The construct was introduced into <i>M. smegmatis</i> at the permissive temperature (30°C). Integration of the plasmid by a single crossover at the position indicated was detected by growing the cells at the non-permissive temperature (42°C) in the presence of kanamycin. <b>B</b>. Genetic map of the single crossover, showing key restriction sites and fragments. <b>C.</b> Culturing the single crossover strain containing a rescue plasmid encoding <i>MSMEG_6382</i> gave rise to a disrupted copy of <i>MSMEG_6382</i> in the chromosome, producing the conditional knockout (6382CKO). Since the disruption of <i>MSMEG_6382</i> coincided with the loss of the <i>sacB</i> gene, the conditional knockout strain could be selected on sucrose plates. Double lines indicate chromosomal DNA, single lines indicate plasmid DNA. Restriction fragments hybridising to the <i>MSMEG_6382-</i>specific probe are shown in kilobases (kb).</p

MSMEG_6382 is essential for growth of <i>M. smegmatis</i> on Middlebrook agar.

<p>The conditional knockout strain 6382CKO was cultured at 30°C on Middlebrook 7H10 agar containing Kn and Sm, then subcultured onto Middlebrook 7H10 agar containing Kn at 30°C and 42°C and examined for growth. Wild-type <i>M. smegmatis</i> mc²155 control strain cultured at 30°C (<b>A</b>) and 42°C (<b>B</b>) on Middlebrook 7H10 agar without antibiotics; 6382CKO strain cultured at 30°C (<b>C</b>) and 42°C (<b>D</b>) on Middlebrook 7H10 agar containing Kn.</p

Conditional disruption of <i>MSMEG_6382</i>.

<p>Southern blot of genomic DNA digested with <i>Hin</i>dIII/<i>Not</i>I/<i>Xba</i>I. Lane 1, DNA molecular weight DNA markers of the sizes indicated (kilobases, kb); lane 2, wild-type <i>M. smegmatis</i> mc²155; lane 3, single crossover strain used to derive the conditional knockout; lane 4, conditional knockout of <i>MSMEG_6382</i>, designated 6382CKO.</p

The Mycobacterial cell wall and pathway for DPA biosynthesis.

<p><b>A</b>. The mycobacterial cell wall is a multilayered structure containing many components unique to these and closely related bacteria, including phosphatidylinositol mannosides (PIM), lipoarabinomannans (LAM), trehalose monomycolates (TMM), trehalose dimycolates (TDM) mycolic acids and arabinogalactan. The biosynthetic pathways for these unique components are a rich source of potential drug targets. Arabinose sugars are shown in orange. <b>B</b>. Decaprenylphosphoryl arabinose (DPA) is formed by the epimerization of decaprenylphosphoryl ribose (DPR) by DprE1 (Rv3790) and DprE2 (Rv3791). DPA serves as an arabinose donor in cell wall biosynthesis, contributing to arabinogalactan and LAM assembly (orange arrows).</p

Identification of a membrane protein required for lipomannan maturation and lipoarabinomannan synthesis in corynebacterineae

Mycobacterium tuberculosis and related Corynebacterineae synthesize a family of lipomannans (LM) and lipoarabinomannans (LAM) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. Here we describe a new membrane protein, highly conserved in all Corynebacterineae, that is required for synthesis of full-length LM and LAM. Deletion of the Corynebacterium glutamicum NCgl2760 gene resulted in a complete loss of mature LM/LAM and the appearance of a truncated LM (t-LM). Complementation of the mutant with the NCgl2760 gene fully restored LM/LAM synthesis. Structural studies, including monosaccharide analysis, methylation linkage analysis, and mass spectrometry of native LM species, indicated that the ΔNCgl2760 t-LM comprised a series of short LM species (8–27 residues long) containing an α1–6-linked mannose backbone with greatly reduced α1–2-mannose side chains and no arabinose caps. The structure of the ΔNCgl2760 t-LM was similar to that of the t-LM produced by a C. glutamicum mutant lacking the mptA gene, encoding a membrane α1–6-mannosyltransferase involved in extending the α1–6-mannan backbone of LM intermediates. Interestingly, NCgl2760 lacks any motifs or homology to other proteins of known function. Attempts to delete the NCgl2760 orthologue in Mycobacterium smegmatis were unsuccessful, consistent with previous studies indicating that the M. tuberculosis orthologue, Rv0227c, is an essential gene. Together, these data suggest that NCgl2760/Rv0227c plays a critical role in the elongation of the mannan backbone of mycobacterial and corynebacterial LM, further highlighting the complexity of lipoglycan pathways of Corynebacterineae