Functional impact of a conservative missense mutation in the Mycobacterium bovis BCG Moreau heat shock response regulator hrcA gene

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BACKGROUND: The genus Mycobacterium includes important human pathogens such as M. tuberculosis, the etiological agent of human tuberculosis. A critical factor in its pathogenic success is its ability to adapt efficiently to various stress conditions, including heat shock. This response mitigates the accumulation of misfolded proteins by upregulating heat shock proteins, primarily chaperones, through complex regulatory networks, including transcriptional repressors such as HspR and HrcA. The vaccine strain M. bovis BCG Moreau genotypically differs from M. tuberculosis in several aspects, including a conservative missense mutation in hrcA, substituting valine with leucine at residue 67 near its DNA-binding domain. This genetic variation could impact stress adaptation and vaccine-associated traits through altered regulation of heat shock proteins. RESULTS: Structural modeling of BCG Moreau's HrcA revealed local rearrangements accommodating the larger leucine side chain, with maintained overall protein architecture. Electrophoretic mobility shift assays (EMSA) demonstrated reduced binding of Moreau's HrcA to CIRCE elements upstream of groES and groEL2 compared to the BCG Pasteur strain. RT-qPCR revealed unexpected patterns, with greater transcriptional induction of the groES operon and groEL2 in the Pasteur strain under heat shock conditions. Western blotting analysis corroborated these findings, showing increased GroEL2 levels in both intracellular and secreted fractions of BCG Pasteur. These observations suggest the involvement of multiple regulatory layers beyond direct HrcA-mediated repression. CONCLUSIONS: Our findings suggest that M. bovis BCG Moreau may employ distinct mechanisms to modulate its heat shock response, with the V67L mutation in HrcA potentially contributing to these differences. The lower baseline expression of immunogenic heat shock proteins in BCG Moreau, combined with its distinct regulatory response to stress, may explain its reported characteristics of reduced adverse reactions while maintaining protective immunity. These results provide new insights into strain-specific variations in heat shock protein regulation and their potential impact on vaccine performance.

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