Compromised base excision repair pathway in Mycobacterium tuberculosis imparts superior adaptability in the host.

Saba Naz,Umesh Varshney,Lakshya Veer Singh,Divya Arora,Dhiraj Kumar,Vinay Kumar Nandicoori,Sathya Narayanan Nagarajan,Yogendra Singh,Pradeep Kumar,Shruti Dabral,Helena Ingrid Boshoff

doi:10.1371/journal.ppat.1009452

Abstract

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is a significant public health concern, exacerbated by the emergence of drug-resistant TB. To combat the host's dynamic environment, Mtb encodes multiple DNA repair enzymes that play a critical role in maintaining genomic integrity. Mtb possesses a GC-rich genome, rendering it highly susceptible to cytosine deaminations, resulting in the occurrence of uracils in the DNA. UDGs encoded by ung and udgB initiate the repair; hence we investigated the biological impact of deleting UDGs in the adaptation of pathogen. We generated gene replacement mutants of uracil DNA glycosylases, individually (RvΔung, RvΔudgB) or together (RvΔdKO). The double KO mutant, RvΔdKO exhibited remarkably higher spontaneous mutation rate, in the presence of antibiotics. Interestingly, RvΔdKO showed higher survival rates in guinea pigs and accumulated large number of SNPs as revealed by whole-genome sequence analysis. Competition assays revealed the superior fitness of RvΔdKO over Rv, both in ex vivo and in vivo conditions. We propose that compromised DNA repair results in the accumulation of mutations, and a subset of these drives adaptation in the host. Importantly, this property allowed us to utilize RvΔdKO for the facile identification of drug targets.

Highlights

The bacterium responsible for causing tuberculosis (TB) disease -Mycobacterium tuberculosis -is among the most notorious human pathogens prevalent across the world
The ung and udgB genes at the native loci were replaced with hygromycin resistant or chloramphenicol resistant cassette with the help of a specialized transduction method to generate RvΔung and RvΔudgB, respectively (Fig 1A and 1C)
To generate the combinatorial mutant, the native udgB gene in the RvΔung was replaced with chloramphenicol resistant cassette to generate RvΔdKO (Fig 1E)

Summary

Introduction

The bacterium responsible for causing tuberculosis (TB) disease -Mycobacterium tuberculosis -is among the most notorious human pathogens prevalent across the world. While the success rate of treatment for drug-susceptible TB is ~85%, and only ~57% for MDR-TB cases. These numbers decline further for patients co-infected with HIV [2,3]. The acquisition of drug resistance in Mtb is not a simple mechanism. It is a conglomeration of genetic events that occur sequentially, described as a probable pre-resistance state that predisposes pathogen to eventual antibiotic resistance [4]. In 10–40% of clinical cases, drug resistance cannot be explained by mutations in the direct targets, suggesting hitherto unknown mechanisms employed by the bacilli [5]. Mutations in DNA repair/replication genes such as dnaQ, alkA, nth and recF have been identified in drug-resistant strains [9,10,11]

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Results

Conclusion