Genomic Polymorphisms in Toxin-Antitoxin Systems and Identification of Novel Phylo-SNPs and Polymorphisms Associated with Drug Resistance/Susceptibility in Clinical Isolates of Mycobacterium tuberculosis from Mumbai, India

Abstract

27Toxin-antitoxin (TA) modules are one of the prominent determinants that triggers a persistent 28 state aiding Mycobacterium tuberculosis evasion to host generated stresses. The 79 29 characterized and putative TA systems described in M. tuberculosis are dominated by the 30 VapBC, MazEF, HigAB, RelBE and ParDE TA families, largely involved in persistence and 31 cell arrest. Hence, there is a need to maintain and conserve the TA loci in the chromosome of 32 the pathogen. It is essential to study the genomic differences of the TA systems in clinical 33 isolates along with its association to drug susceptibility patterns and lineage. In the current 34 study, the TA loci and their promoter sequences were analysed from the whole genome 35 sequence data of 74 clinical isolates. Mykrobe Predictor was used for lineage identification 36 and drug resistance predictions in the clinical isolates. Polymorphisms associated with 79.8% 37(63/79) TA systems were observed across 72 clinical isolates. Among the TA systems, the 38 isolates had a varying number of polymorphisms localised primarily in the toxin genes 39 (58.7%), antitoxin genes (40.7%) and chaperones (0.6%), due to Single Nucleotide 40 Polymorphism (SNP) resulting in transition (67.3%), transversion or frameshift mutations. 41 Our analysis suggests the presence of novel Phylo-SNPs by establishing high confidence 42 association of specific lineages to polymorphisms in the TA systems. Notably, association of 43 polymorphisms in Rv1838c-1839c (VapBC13), Rv3358-3357 (YefM/YoeB) and Rv0240-44 0239 (VapBC24) to Delhi/Central Asia lineage. The polymorphic loci of the 3 TA systems is 45 localised in the antitoxin gene of the Delhi/Central Asia strains, with a resultant silent 46 mutation. The assessment of correlation between TA polymorphisms and the drug resistance 47 Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis, with an annual estimate 52 of 10.4 million new cases globally, imposes a major burden of mortality and morbidity on the 53 human population [1]. The long duration of treatment and a large reservoir of people with 54 latent infection are major obstacles in controlling spread of the disease. Furthermore, 55 multidrug resistance is a deterrent for effective treatment of TB. Besides acquired drug 56 resistance, M. tuberculosis exhibits modified virulence, transmissibility and pathogenicity 57 [2]. The organism may employ endogenous mechanisms of stress evasion due to persistence 58 of the bacilli in the infected individual. The 'persister' state is a dormant state wherein the 59 bacterial cells are recalcitrant to the unfavourable conditions. The persistent cells revert back 60 to its proliferative growth on removal of the environmental stress factor such as nutrient 61 starvation or antibiotic presence. The persister cells are therefore not antibiotic resistant 62 mutants, but are highly antibiotic tolerant cells [3]. An important determinant of persistence 63 is the presence of multiple toxin-antitoxin (TA) systems [4]. 64 The TA syste...