Characterizing CaeA-mediated rifampin tolerance in MTB

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ABSTRACT Tuberculosis (TB) is one of the top ten causes of death worldwide. Only two new classes of TB drugs (diarylquinolines and nitroimidazoles) have been introduced into clinical practice since the introduction of the current multidrug regimen in the 1970s. The lack of success in identifying novel drug targets suggests that the reinvestigation of mechanisms of action and resistance of currently available drugs may be a more fruitful approach. Rifampin, which targets the mycobacterial RNA polymerase, is one of the key sterilizing drugs permitting the modern “short-course” 6-month regimen. The lengthy duration of TB treatment required to prevent relapse reflects the ability of Mycobacterium tuberculosis (Mtb) to persist in the host in an altered physiological state characterized by reversible tolerance to first-line anti-tubercular drugs. Several mechanisms have been reported to contribute to Mtb antibiotic tolerance, including the induction of drug efflux pumps and reduced penetration of drugs into necrotic tuberculous lesions and across the waxy mycobacterial cell wall. Currently, there is significant interest in maximizing exposures to rifampin in order to eliminate Mtb “persisters”, however the mechanisms of tolerance to rifampin and other anti-tubercular drugs remain largely undefined. In order to address this significant knowledge gap, we conducted a high-throughput transposon (Tn) mutant screen to identify Mtb genes required for tolerance to rifampin in nutrient-rich broth and in physiologically relevant stress conditions. Mutants containing a Tn insertion in the rv2224c/caeA gene were 294-fold more susceptible to rifampin than was wild-type Mtb. This differential antibiotic susceptibility phenotype was confirmed using a targeted caeA deletion mutant (ΔcaeA) and its complemented strain. We also found that caeA deficiency is associated with increased rifampin accumulation and increased permeability to the polar molecule ethidium bromide in Mtb. Previous studies have shown that CaeA, a cell wall-associated carboxylesterase and serine protease, is critical for Mtb virulence. Disruption of caeA led to prolonged survival of infected mice and highly reduced lung pathology. Further supporting the potential utility of targeting CaeA in TB treatment, deficiency of this protein has been associated with increased Mtb susceptibility to the first-line drug ethambutol and the second-line drug streptomycin. Using a combination of targeted and unbiased approaches, the current proposal will test the hypothesis that CaeA mediates Mtb tolerance to rifampin by modifying Mtb cell wall composition, thereby reducing drug permeability. In proof-of-concept studies using a recently generated inducible CaeA degradation strain, we also will evaluate the treatment-shortening potential of targeting CaeA as adjunctive TB therapy in a clinically relevant animal model. Our findings are expected to have far-reaching implications for understanding antibiotic tolerance in Mtb and other persistent bacterial infections.