Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth

Abstract

Mycobacterium kansasii (Mk) is a resilient opportunistic human pathogen that causes tuberculosis‐like chronic pulmonary disease and mortality stemming from comorbidities and treatment failure. The standard treatment of Mk infections requires costly, long‐term, multidrug courses with adverse side effects. The emergence of drug‐resistant isolates further complicates the already challenging drug therapy regimens and threatens to compromise the future control of Mk infections. Despite the increasingly recognized global burden of Mk infections, the biology of this opportunistic pathogen remains essentially unexplored. In particular, studies reporting gene function or generation of defined mutants are scarce. Moreover, no transposon (Tn) mutagenesis tool has been validated for use in Mk, a situation limiting the repertoire of genetic approaches available to accelerate the dissection of gene function and the generation of gene knockout mutants in this poorly characterized pathogen. In this study, we validated the functionality of a powerful Tn mutagenesis tool in Mk and used this tool in conjunction with a forward genetic screen to establish a previously unrecognized role of a conserved mycobacterial small RNA gene of unknown function in colony morphology features and biofilm formation. We also combined Tn mutagenesis with next‐generation sequencing to identify 12,071 Tn insertions that do not compromise viability in vitro. Finally, we demonstrated the susceptibility of the Galleria mellonella larva to Mk, setting the stage for further exploration of this simple and economical infection model system to the study of this pathogen.

Small RNA profiling inMycobacteriuminsights into stress adapt ability

“…Currently, many more regulatory sRNA have been identified in Mycobacterium using saturated transposon mutagenesis (Budell, et al 2020). More recent studies have evidenced the importance of sRNA in pathogenesis.…”

“…Initial analysis of the M. tb transcriptome by RNA-seq reveal the presence of at least 15 sRNAs (Westermann 2018). Currently, many more regulatory sRNA have been identified in Mycobacterium using saturated transposon mutagenesis (Budell, et al 2020). More recent studies have evidenced the importance of sRNA in pathogenesis.…”

Genetic Manipulation of Non-tuberculosis Mycobacteria

“…This is four times higher than the estimate for M. tuberculosis and such biases could result in an overestimation of the number of essential genes in forward genetic screens in this organism. Evaluation of ΦMycoMarT7 in Mycobacterium kansasii revealed that while temperature sensitivity was maintained, plaque formation in M. kansasii was 800-fold less efficient than for M. smegmatis ( Budell et al, 2020 ). Mapping of the insertion sites for the 14,700 M. kansasii mutants revealed that 82% of the insertions in the chromosome were in open reading frames (ORFs); a similar percentage (83%) was observed for the pMK12478 plasmid present in this strain.…”

“…Most studies in NTMs have used transposon mutagenesis to select for mutants with specific phenotypes. Examples include pigmentation variants in M. marinum ( Gao et al, 2003 ), M. kansasii mutants with altered colony morphology ( Budell et al, 2020 ) and M. avium mutants with altered antibiotic susceptibility ( Cangelosi et al, 2006 ) (see Table 2 for details). The Tn phoA transposon was developed to perform more targeted screens that identify mutants with insertions in membrane or secreted proteins ( Taylor et al, 1987 ).…”

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“…An initial analysis of the M. tb transcriptome by RNA-seq revealed the presence of at least 15 sRNAs (Westermann, 2018). Currently, many more regulatory sRNAs have been identified in Mycobacterium using saturated transposon mutagenesis (Budell et al, 2020). More recent studies have evidenced the importance of sRNA in pathogenesis.…”