Identifying the essential genes ofMycobacterium aviumsubsp.hominissuiswith Tn-Seq using a rank-based filter procedure

Abstract

15Mycobacterium avium (Mav) is increasingly recognized as a significant cause of morbidity, particularly in 16 elderly patients or those with immune deficiency or underlying structural lung disease. Generally, Mav 17 infection is treated with 2-3 antimicrobial drugs for at least 12 months. Identification of genes essential 18 for Mav growth may yield novel strategies for improving curative therapy. We have generated 19 saturating genome-wide transposon mutant pools in a commonly used laboratory strain of 20Mycobacterium avium subsp. hominissuis (MAC109) and developed a computational technique for 21 classifying annotated genomic features as essential (ES), growth defect (GD), growth advantage (GA), or 22 no-effect (NE) based on the in vitro effect of disruption by transposon. We identified 270 features as ES 23 with 230 of these overlapping with ES features in Mycobacterium tuberculosis. These results may be 24 useful for identifying drug targets or for informing studies requiring genetic manipulation of 25Mycobacterium avium, which should seek to avoid disrupting ES features to ensure bacterial viability. 26 27 Importance:28 Mycobacterium avium subsp. hominissuis is an emerging cause of morbidity in vulnerable populations in 29 many countries. It is known to be particularly difficult to treat, often requiring years of antibiotic 30 therapy. In this study we report the genes of Mycobacterium avium subsp. hominissuis that are required 31 for the organism to grow in vitro. Our findings may help guide future research into identifying new drugs 32 to improve the treatment of this serious infection. 33 34

Genetic determinants of intrinsic antibiotic tolerance in Mycobacterium avium

“…We found 2 mutants that displayed hypertolerance to three of the four antibiotics tested (CLR, MOX, RFB): DFS55_10765 (annotated as a pyruvate kinase, rv1617 ) and DFS55_20040 (DUF1707 domain-containing protein, rv0966c ). Interestingly, Rv1617 deficiency is associated with a large growth defect in M. tuberculosis (34,35), but the same phenotype is not observed in DFS55_10765-deficient M. avium (35). This suggests that the metabolic impact of pyruvate kinase deficiency is remarkably different between MAC and M. tuberculosis .…”

“…DNA was extracted from regrown samples, as described previously (short-read sequencing protocol) (38). DNA was processed for Tn-Seq, as described previously (35). Libraries were sequenced (2×75bp) on an Illumina HiSeq 2500 by the Johns Hopkins GRCF High Throughput Sequencing Center.…”

“…avium (35). This suggests that the metabolic impact of pyruvate kinase deficiency is remarkably different between MAC and M. tuberculosis.…”

“…The setup for our genome-wide differential susceptibility screen is shown in Figure 1. We inoculated a 1-mL frozen aliquot of a previously generated diverse Himar1 transposon mutant pool (consisting of approximately 1.2 × 10 6 unique mutants) (35) into 300 mL of 7H9/30% OADC contained in a 1.3-L roller bottle. This was shaken at 37 o C for 24 hours to reduce bacterial clumping (220 rpm, 0.75 in [1.905 cm] orbit).…”

“…Transposon insertion sequencing (Tn-seq) is a powerful technique for determining bacterial genotype-phenotype relationships, particularly the requirement for specific bacterial genes for growth and/or survival under controlled stress conditions (30)(31)(32)(33). Modifications of this technique have been used to define essential genes for in vitro growth of M. tuberculosis (34) and M. avium (35). Xu et al screened a saturated transposon mutant library in the presence of partially inhibitory concentrations of various antibiotics with diverse mechanisms of action to identify genetic determinants of intrinsic antibiotic susceptibility in M. tuberculosis (36).…”