UC Berkeley

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that establishes a pulmonary infection in the lung, and a quarter of the world’s population is estimated to be latently infected with Mtb. The genetics behind Mtb’s ability to survive in its primary niche, the phagosome of macrophages, is still under investigation. In chapter one, I introduce the history of Mtb forward genetics and discuss how laboratory techniques for forward genetics in Mtb have progressed over time. Then, I discuss mechanisms that are important for Mtb survival in the macrophage, including resistance against oxidative stress, nitrosative stress, and acidic pH. This chapter also reviews how Mtb acquires and metabolizes nutrients, specifically carbon and nitrogen sources.

In the second chapter, I present how a prokaryotic organelle, an encapsulin nanocompartment, is important for defense against oxidative stress in Mtb. Encapsulin nanocompartments are a method of compartmentalization in bacteria and archaea that are comprised of a proteinaceous cell surrounding an enzymatic cargo protein. Much of the previous work on encapsulin nanocompartments has been characterizing their biochemical properties rather than elucidating the contribution of nanocompartments to bacterial physiology. We show that Mtb assembles encapsulin nanocompartments containing the peroxidase DyP, which can detoxify hydrogen peroxide. A mutant lacking the DyP nanocompartment in Mtb is attenuated at acidic pH in the presence of hydrogen peroxide, and the DyP nanocompartment mutant is susceptible to pyrazinamide in broth and a murine mouse model.

Next, in chapter 3, the investigation of encapsulin nanocompartments is expanded to nontuberculosis mycobacteria, including Mycobacterium smegmatis (M. smeg) and Mycobacterium abscessus (M. abs). M. smeg and M. abs are predicted to encode the DyP nanocompartment in addition to a nanocompartment encoding a cysteine desulfurase (CyD). Genetic deletion nanocompartments in M. smeg and M. abs are largely unable to recapitulate the phenotypes observed in Mtb. However, M. smeg nanocompartment mutants have unique colony morphologies that are not observed in Mtb. Chapter 3 also investigates remaining hypotheses about Mtb nanocompartments, including the ability of Mtb DyP to use lipid peroxides as a substrate. We also explore the possibility that KatG, a catalase peroxidase in Mtb, acts redundantly with Mtb DyP in infection models.

In chapter 4, random barcode-transposon site sequencing (RB-TnSeq), a technique for high-throughput forward genetics, is used to uncover the function of unknown genes in Mtb. RB-TnSeq is a pooled, transposon-mutant based method wherein each transposon contains a unique twenty nucleotide barcode, which bypasses steps of laborious protocols used for standard TnSeq screens. We exposed an RB-TnSeq library in Mtb to a chemical library of carbon sources, nitrogen sources, and stress compounds. From the genetic screen data for Mtb growth on carbon sources, we find that the Nuo complex, one of the three NADH dehydrogenases encoded by Mtb, is specifically required for growth on propionate and asparagine. Additionally, we explore the possibility of D- lactate to act as a carbon source for Mtb. Finally, we identify novel mutants that confer resistance to pretomanid, a recently approved tuberculosis antibiotic. Broadening our knowledge concerning Mtb genetics and how the bacterium survives in the face of immunological stressors in vivo is critical for furthering the development of novel bacterial therapeutics.