Identification of a Haarlem genotype-specific single nucleotide polymorphism in the mgtC virulence gene of Mycobacterium tuberculosis

Abstract

MgtC is a virulence factor common to several intracellular pathogens, including Mycobacterium tuberculosis, that might have been acquired through horizontal gene transfer. In the present study, we investigated the polymorphism of mgtC in clinical isolates representative of the main epidemic groups of M. tuberculosis. MgtC appears to have a low polymorphism rate in M. tuberculosis that consists exclusively of nonsynonymous mutations. We identified a single nucleotide polymorphism (SNP) at mgtC codon 182 (mgtC182) specifically associated with the Haarlem genotype. A simple PCR assay, called the "on/off switch assay," using phosphorothioate-modified primers and Pfu polymerase allowed us to distinguish Haarlem from non-Haarlem strains based on the mgtC182 SNP. The amino acid change (H182R) associated with the mgtC182 SNP in Haarlem strains does not appear to procure a selective advantage. Our results offer a simple and rapid tool to distinguish between Haarlem and non-Haarlem strains. In addition, the on/off switch assay, which allows the detection of SNPs on chromosomal DNA and M. tuberculosis cultures, provides a novel approach for the screening of known SNPs in M. tuberculosis.

FIG. 1.

On/off switch PCR-based assay to analyze the mgtC¹⁸² polymorphism. (A) Illustration of the on/off switch assay. The technique uses two sets of primers with an identical forward primer (SNP-F) and variable phosphorothioate-modified “SNP-specific” reverse primers. The SNP-specific primers differ only at the 3′-end nucleotide that is complementary to the wild-type sequence (SNP-182G-R) or the mutant sequence (SNP-182A-R). Two PCRs are performed, one with each of the two SNP-specific primers. Amplification occurs only if the 3′ end of the SNP-specific primer matches the sequence of the template. In case of mismatching, the mismatched phosphorothioate-modified reverse primer is trapped within the exo-domain of the Pfu polymerase during proofreading, blocking DNA polymerization. (B) Analysis of the mgtC¹⁸² polymorphism by on/off switch assay. Lanes 1 to 4, optimization of conditions for the assay using chromosomal DNA from Haarlem (lanes 1 to 2, CIP 0443) or non-Haarlem (lanes 3 to 4, CIP 0357) strains; lanes 5 to 16, analysis of the mgtC¹⁸² polymorphism using cultures of Haarlem (lanes 5 to 6, strain 14; lanes 7 to 8, strain 45) or non-Haarlem (lanes 9 to 10, ITM 990020; lanes 11 to 12 ITM 981277; lanes 13 to 14, ITM 021038, lanes 15 to 16, ITM 993123) strains. The reverse modified primer is either SNP-182A-R (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or SNP-182G-R (lanes 2, 4, 6, 8, 10, 12, 14, and 16).

FIG. 2.

Comparison of the activity of MgtC wild-type and MgtC R182H variant from M. tuberculosis by complementation assay in serovar Typhimurium. Plasmids expressing MgtC wild-type from M. tuberculosis (pNM13) or the MgtC R182H variant (pNM70) were introduced into a serovar Typhimurium ΔmgtC strain. A plasmid without the mgtC gene (pNM11) was used as a negative control. The data are means from three different experiments. Asterisks indicate statistical significance (P < 0.05) compared to pNM11. No significant difference was found between pNM13 and pNM70 plasmids. (A) Complementation assay for growth in magnesium-deprived NCE medium. (B) Complementation assay for replication in J774 macrophages. The percentage of replication comparatively to a wild-type strain of Salmonella serovar Typhimurium is shown.