Redox defenses and evasion of reactive oxygen species mediated host immunity in Mycobacterium tuberculosis

Project Summary Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), caused 1.4 million deaths in 2017, more than any other pathogen. TB treatment remains difficult, and an effective vaccine has eluded research efforts for the past century.

A major barrier to ending the TB pandemic is a limited understanding of effective host immune responses and Mtb?s immune evasion strategies. Much in vitro work has been done to characterize Mtb infection of macrophages, its predominant cellular niche.

Whereas reactive oxygen species (ROS) are an important antimicrobial defense against diverse pathogens, Mtb is resistant to ROS.

We recently identified CpsA as a secreted virulence factor that inhibits NADPH oxidase recruitment to the Mtb-containing phagosome, thereby protecting Mtb from an oxidative burst. ROS is also an essential signal for LC3-associated phagocytosis (LAP), a noncanonical form of autophagy. Indeed, the ?cpsA mutant is rescued in mice defective in the NADPH oxidase and LAP.

Interestingly, in mice the ?cpsA mutant is severely attenuated during the first two weeks of infection and recovers substantially by 6 weeks, suggesting that CpsA is most important during the innate phase of infection before the activation of adaptive immunity. This phenotype coincides with a shift in cell types that are infected and the inflammatory response.

Therefore, we hypothesize that CpsA specifically protects Mtb against ROS in the cell types infected and inflammatory environment of the innate immune phase.

We will test our hypothesis by characterizing the ?cpsA mutant within different myeloid cells in vivo using flow-assisted cell sorting (FACS) and in mice that are deficient in alveolar macrophages, neutrophils, or monocyte-derived macrophages, as well as in mice that fail to mount an adaptive immune response against Mtb or that have cell type specific defects in LAP.

KatG is a catalase-peroxidase that is also important in ROS defense in Mtb. We will test whether CpsA and KatG cooperate in virulence by charactering a ?cpsA ?katG double mutant.

We hypothesize that the ?cpsA ?katG mutant will be more attenuated than either single mutant due to disinhibited ROS production by the host and reduced ROS detoxifying activity by Mtb. KatG activates the first-line drug isoniazid (INH), and mutations in katG confer INH resistance. CpsA, therefore, may permit transmission of INH- resistant katG mutants by protecting them against ROS.

Investigating the roles of ROS and the diverse myeloid cells involved in Mtb infection will impact strategies for host-directed therapies, targeting drug-resistant bacilli, and novel vaccine design.

This proposal is the topic of Steven Grigsby?s PhD thesis in Molecular Microbiology & Microbial Pathogenesis in the Medical Scientist Training Program at Washington University School of Medicine (WUSM).

The strength at WUSM in microbial pathogenesis and immunology makes it a perfect fit for the studies proposed by Steven Grigsby.

He has all of the necessary resources, input from a group of outstanding scientists, and a robust training plan, which will support his career development as an independent physician-scientist.