A point mutation in cycA partially contributes to the D-cycloserine resistance trait of Mycobacterium bovis BCG vaccine strains

Abstract

In mycobacteria, CycA a D-serine, L- and D-alanine, and glycine transporter also functions in the uptake of D-cycloserine, an important second-line anti-tubercular drug. A single nucleotide polymorphism identified in the cycA gene of BCG was hypothesized to contribute to the increased resistance of Mycobacterium bovis bacillus Calmette-Guérin (BCG) to D-cycloserine compared to wild-type Mycobacterium tuberculosis or Mycobacterium bovis. Working along these lines, a merodiploid strain of BCG expressing Mycobacterium tuberculosis CycA was generated and found to exhibit increased susceptibility to D-cycloserine albeit not to the same extent as wild-type Mycobacterium tuberculosis or Mycobacterium bovis. In addition, recombinant Mycobacterium smegmatis strains expressing either Mycobacterium tuberculosis or Mycobacterium bovis CycA but not BCG CycA were rendered more susceptible to D-cycloserine. These findings support the notion that CycA-mediated uptake in BCG is impaired as a result of a single nucleotide polymorphism; however, the partial contribution of this impairment to D-cycloserine resistance suggests the involvement of additional genetic lesions in this phenotype.

Figure 1. Analyses of CycA amino acid sequences.

(A) Partial CycA amino acid sequence alignment of various mycobacteria (the conserved G122 residue is indicated by the arrow). (B) 2-dimensional topological representation of M. bovis CycA with the G122S mutation found in all BCG (circled) in the extracellular loop (boxed) between the 3^rd and 4^th trans-membrane helices from the amino (N) terminus.

Figure 2. Characterization of cycA merodiploid BCG and M. smegmatis strains.

(A) Sequence of the SNP containing region in cycA from BCG-Pasteur::I425 and BCG-Pasteur::pYUB412 clones from the 5′ end. (B) CFUs of BCG-Pasteur::I425 (white bars) and BCG-Pasteur::pYUB412 (black bars) 4 days after treatment with indicated concentrations of DCS. (C) CFUs of BCG::I425 (solid squares) and BCG::pYUB412 (solid diamonds) obtained over time upon exposure to 32 µg/mL DCS. (D) CFUs of M. smegmatis::I425 (white bars) and M. smegmatis::pYUB412 (black bars) 24 hours after treatment with indicated concentrations of DCS. (E) CFUs of M. smegmatis::I425 (solid squares) and M. smegmatis::pYUB412 (solid diamonds) obtained over time upon exposure to 200 µg/mL DCS. Data points and error bars are means ± standard deviations. Statistically significant differences are denoted by asterisks (*, P<0.05; **, P<0.01). Representative of 3 independent experiments performed in duplicate.

Figure 3. Susceptibilities of M. smegmatis strains overexpressing cycA alleles to DCS.

(A) CFUs of recombinant M. smegmatis strains after 24 hours of treatment with indicated concentrations of DCS. (C) CFUs of recombinant M. smegmatis strains obtained over time upon exposure 200 µg/mL DCS. M. smegmatis harbouring pMD31 (black bars), pMDcycA_Mtb (white bars), pMDcycA_Mbov (light-gray bars) and pMDcycA_BCG (dark-gray bars). Data points and error bars are means ± standard deviations. Statistically significant differences are denoted by asterisks (*, P<0.05). Representative of three independent experiments performed in duplicate.