Mycobacterium avium subsp. hominissuis (MAH) is increasingly recognized as a significant cause of morbidity, particularly in elderly patients or those with immune deficiency or underlying lung impairment. Disease due to MAH is particularly difficult to treat, often requiring years of antibiotic therapy. Identification of genes essential for MAH growth may lead to novel strategies for improving curative therapy. Here we have generated saturating genome-wide transposon mutant pools in a strain of MAH (MAC109) and developed a novel computational technique for classifying annotated genomic features based on the in vitro effect of transposon mutagenesis. Our findings may help guide future genetic and biochemical studies of MAH pathogenesis and aid in the identification of new drugs to improve the treatment of these serious infections. The genus Mycobacterium contains a variety of difficult-to-treat pathogens frequently associated with pulmonary disease. One of these pathogens, Mycobacterium avium subsp. hominissuis (MAH), is an opportunistic pathogen associated with significant morbidity in the elderly and in patients with underlying lung disease 1,2 , as well as increased mortality in patients with AIDS 3. Similar to other mycobacteria, MAH is often difficult to treat effectively with existing antibiotic combinations. Current antibiotic regimens require a median of 5 months to convert the sputum to a culture-negative state 4 , with current guidelines recommending treatment for at least 1 year after sputum culture conversion 5. Furthermore, a large fraction of patients fail to convert after 1 year of therapy 4. Patients could greatly benefit from more potent and abbreviated therapies. Transposon sequencing (e.g., TraDIS 6 , Tn-Seq. 7 , INseq. 8) has been used extensively to profile haploid genomes and identify gene disruptions that affect bacterial growth under various conditions. Of potential interest in drug development are those drug targets which profoundly disrupt growth on nutrient-rich media (i.e., "essential" genes). In the current study, we have successfully generated genome-wide transposon mutant pools in MAH strain 109 (MAC109). This strain, which was originally isolated from the blood of an AIDS patient, has been characterized extensively in previous studies 9-13 and is known to infect mice and macrophages 11. We have utilized the transposon mutant pools we generated to identify genes critical for MAH growth in vitro with the goal of informing future research in MAH pathogenesis and drug development. In order to make gene essentiality predictions, we developed a new statistical approach for calling genes based on ranking the read counts from each mutant and applied this to new Tn-Seq data. We report our predictions of the essential genes of MAH and compare these with the predicted set of essential genes in the closely related human pathogen, Mycobacterium tuberculosis (Mtb). Results Constructing genome-wide transposon mutant pools in Mycobacterium avium subsp. hominissuis. To identify a suitable strain of ...
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
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