Toxin-antitoxin systems in mycobacterial infections

With an increase in antibiotic therapy failure, there has been a surge of research to understand antibiotic persistence. The distinct subpopulation of cells that can survive antibiotic treatment are called “persisters”. The persister cells also account for group of cells that cause “persistent infection” by evading human immune system, for example Mycobacterium tuberculosis (Mtb) causing latent tuberculosis infection. The bacteria can use same mechanisms for both antibiotic persistence and immune evasion. Several studies have implicated toxin-antitoxin (TA) systems as an important player in cell persistence. TA systems are composed of two adjacent genes: one encoding a toxin that causes growth arrest, and second encoding a cognate antitoxin that inhibits the toxin activity. Upon stress, the toxin is activated through degradation of its corresponding antitoxin. Unlike exotoxins, toxins of TA systems are not secreted. Instead, they act upon the cell itself by inducing growth inhibition that can lead to a dormant state. The Mtb genome consists of an unusually high number of TA systems (>80) compared to other bacteria, with the Virulence-Associated Protein (Vap) BC family accounting for 50 of those TA systems. These VapC toxins are endoribonucleases that target unique single-stranded RNA sequences through recognition of a combination of sequence and structure determinants. To date, the RNA targets of all VapC toxins within Mtb cell have not been identified and the mechanism these toxins use to increase persistence is not understood. This study aims to understand the role of three Mtb VapC toxins: VapC2, VapC4, and VapC21. To do so, a specialized 5’ RNA-seq method was used to accurately detect toxin- cleaved RNAs. This method utilizes distinct 5’ end left by the toxin upon cleavage. This method also allows to uncover any ribosome stalling, if present. We identified that VapC2 and VapC21 solely targets initiator tRNA cleaving at a single site within the anticodon stem-loop. Depletion of initiator tRNA causes inhibition in translation and eventually growth arrest. This decrease in growth might be necessary for Mtb to transition from active growth to latency during infection. We also identified that VapC4 targets tRNACys at a single site within anticodon sequence, thus inactivating it. Depletion of the pool of tRNACys causes ribosome stalling at Cys codons in actively translated transcripts. This ribosome stalling also allowed us to uncover several unannotated Cys- containing ORFs. VapC4 mimics a state of Cys starvation, which then activated Cys- attenuation at sORFs to globally redirect metabolism towards the synthesis of free Cys. The resulting newly enriched pool of Cys feeds into the synthesis of mycothiol, the glutathione counterpart in this pathogen that is responsible for maintaining cellular redox homeostasis during oxidative stress, as well as into a circumscribed subset of cellular pathways that enable cells to defend against oxidative and copper stresses characteristically endured by Mtb within macrophages. This study also designed to understand the role of VapC toxin in Mycobacterium abscessus (Mab) infections. Mab infections are on the rise, and it is the most notoriously difficult to treat due to its extreme resistance to antibiotics and disinfectants. Mab may enlist TA system to increase antibiotic persistence. A total of 22 novel putative TA systems were identified in 128 Mab clinical strains available on NCBI. VapC5 toxin was one of the more abundant toxins present in these clinical strains. Unlike Mtb VapC toxins, VapC5 toxin cleaves multiple tRNAs at a single site within their anticodon resulting in reduction in translation. VapC5 also induces the expression of multiple genes that underlie persistence/intrinsic antibiotic resistance in Mab. Finally, we also demonstrated that VapC5 expression increased persister cell formation after treatment with two Mab antibiotics. Understanding how TA systems play a role in persister cell formation can help us design better antibiotics or use currently available antibiotics more effectively.Ph.D.Includes bibliographical reference