A Metabolic Signal For Low Biotin Transcriptional Induction Mediated By Pyruvate Carboxylase In Mycobacterium Smegmatis And Rip1 In Mycobacterium Tuberculosis Response To In Vitro And In Vivo Stress

Abstract

Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. We executed a genetic screen in Mycobacterium smegmatis and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. pyc::tn fails to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of the repressor bioQ in pyc::tn reverted biotin auxotrophy, as did reconstituting the pathway through heterologous expression of BioB and provision of DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Pyc is the most rapidly depleted biotinylated protein after biotin withdrawal, indicating that it is a sensitive indicator of this state. Metabolomics revealed several dysregulated metabolites that could mediate BioQ derepression. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism. Rip1 is a site-2 protease required for full virulence in the mouse, but the causes of this phenotype have yet to be elucidated. We tested ?rip1 for defects in models of host-mediated stress and found that it is sensitive to nitric oxide (NO), hypoxia, and multiple classes of antibiotics. Further examination of the determinants of these sensitivities demonstrated differing requirements for Rip1 functions and substrates in each of these models. ?rip1 is not defective for induction of the DosR regulon, which links NO and hypoxia. Further, we reversed the ?rip1 sensitivity to antibiotics with addition of NaHS, but were unable to do so for NO or diamide. To determine the contribution of NO to the ?rip1 in vivo phenotype, we infected nos2-/- mice with ?rip1. ?rip1 regained much of its virulence in nos2-/- mice, but did not recapitulate WT fully, indicating a partial role for NO. These mice developed granulomatous lesions, which are atypical of the murine model. Histopathological examinations demonstrated that these lesions possess numerous traits found in human pathology. We suggest that infection of nos2-/- with attenuated strains such as ?rip1 may be a useful model for studying the granuloma.