Transcriptional Profiling of Homologous Recombination Pathway Genes in Mycobacterium bovis BCG Moreau

Abstract

Mycobacterium bovis BCG Moreau is the main Brazilian strain for vaccination against tuberculosis. It is considered an early strain, more like the original BCG, whereas BCG Pasteur, largely used as a reference, belongs to the late strain clade. BCG Moreau, contrary to Pasteur, is naturally deficient in homologous recombination (HR). In this work, using a UV exposure test, we aimed to detect differences in the survival of various BCG strains after DNA damage. Transcription of core and regulatory HR genes was further analyzed using RT-qPCR, aiming to identify the molecular agent responsible for this phenotype. We show that early strains share the Moreau low survival rate after UV exposure, whereas late strains mimic the Pasteur phenotype, indicating that this increase in HR efficiency is linked to the evolutionary clade history. Additionally, RT-qPCR shows that BCG Moreau has an overall lower level of these transcripts than Pasteur, indicating a correlation between this gene expression profile and HR efficiency. Further assays should be performed to fully identify the molecular mechanism that may explain this differential phenotype between early and late BCG strains.

Keywords: DNA damage response pathways; Mycobacterium bovis BCG; homologous recombination; ultraviolet radiation exposure survival.

Figure 1

Survival (%) profile of M. bovis BCG Moreau compared to BCG Pasteur. After UV exposure of BCG Moreau and Pasteur for different periods, viability was assessed using CFU, comparing irradiated to non-irradiated cells. In (A), data are shown in log10 values, whereas in (B) absolute survival rate values are plotted. In (B), Y-axis segmentation was performed to better assess differences detected between Moreau and Pasteur strains. p-value: *** < 0.001. Data represent the mean ± SD of three independent experiments.

Figure 2

UV exposure survival profile of M. bovis BCG strains representing the four different genomic groups After 30 s UV exposure, viability (% survival) was assessed using CFU, comparing irradiated to non-irradiated cells. p-values: * < 0.05. Data represent the mean ± SD of three independent experiments.

Figure 3

Transcriptional profiling of the core and regulatory HR genes in M. bovis BCG Moreau and Pasteur. Comparative RT-qPCR analysis with primers detecting core (recA, ruvC, ruvA, ruvB, adnA and adnB) and regulatory (recX, lexA, pafB and pafC) HR genes from BCG Moreau and Pasteur samples after 30 s UV exposure. sigA transcription was used as an internal normalization control. Fold change was calculated setting Pasteur values as 1.0. p-values: * < 0.05; ** < 0.01; *** < 0.001. Data represent the mean ± SD of three independent experiments.