Antimicrobial treatment improves mycobacterial survival in nonpermissive growth conditions

Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis

Oleoyl Coenzyme A regulates interaction of transcriptional regulator RaaS (Rv1219c) with DNA in Mycobacteria

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria

Investigation of mycobacterial survival in non-permissive growth conditions

Tuberculosis (TB) remains a major public health issue worldwide. Global TB control efforts are significantly limited by the need of lengthy therapeutic regimens for its causative agent - Mycobacterium tuberculosis (Mtb). Mtb drug response largely depends on its growth rate and a physiological state. Non-replicating and slow-growing mycobacteria are generally more tolerant to drugs targeting cell wall biosynthesis. However, the current study shows that, under certain growth non-permissive conditions, cell wall targeting antimicrobials (ethambutol, isoniazid, cerulenin) can promote Mycobacterium bovis BCG survival; the survival-promoting effect was only observed in sealed non-shaking flasks and accompanied by the accumulation of an unknown volatile compound. This phenomenon was mediated by a transcriptional regulator that controls expression of several ATP dependent efflux pumps and was designated as RaaS (for regulator of antibiotic assisted survival). RaaS binding to DNA was found to be regulated by coenzyme A derivatives of fatty acids (oleoyl CoA and stearoyl CoA). Addition of oleic acid (a precursor of oleoyl CoA) completely abolished the survival promoting effect of ethambutol, confirming the biological importance of this regulatory mechanism. Further investigations were focused on one of the RaaS regulon member – rv3489 that encodes a conserved protein with unknown function. Its over-expression in Mycobacterium smegmatis and Mtb resulted in a growth defect in Sauton’s medium, while the gene deletion in Mtb had no effect on the growth in any media tested or during the macrophage infection. In a pilot animal experiment, the Δrv3489 mutant had a growth advantage in murine lungs at early phases of infection, but was attenuated at later stages. Pull down assay results suggested that Rv3489 interacts with GlgE - a maltosyltransferase that interconnects trehalose and glycogen biosynthesis pathways. These studies lead to a couple of clinically relevant conclusions: (i) dysregulation of efflux pumps by interfering DNA binding of RaaS can kill non-replicating bacteria, but (ii) incautious use of efflux pump inhibitors can promote their survival

Oleoyl coenzyme A regulates interaction of transcriptional regulator RaaS (Rv1219c) with DNA in mycobacteria

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria