TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities

Abstract

Once considered a phenotypically monomorphic bacterium, there is a growing body of work demonstrating heterogeneity among Mycobacterium tuberculosis (Mtb) strains in clinically relevant characteristics, including virulence and response to antibiotics. However, the genetic and molecular basis for most phenotypic differences among Mtb strains remains unknown. To investigate the basis of strain variation in Mtb, we performed genome-wide transposon mutagenesis coupled with next-generation sequencing (TnSeq) for a panel of Mtb clinical isolates and the reference strain H37Rv to compare genetic requirements for in vitro growth across these strains. We developed an analytic approach to identify quantitative differences in genetic requirements between these genetically diverse strains, which vary in genomic structure and gene content. Using this methodology, we found differences between strains in their requirements for genes involved in fundamental cellular processes, including redox homeostasis and central carbon metabolism. Among the genes with differential requirements were katG, which encodes the activator of the first-line antitubercular agent isoniazid, and glcB, which encodes malate synthase, the target of a novel small-molecule inhibitor. Differences among strains in their requirement for katG and glcB predicted differences in their response to these antimicrobial agents. Importantly, these strain-specific differences in antibiotic response could not be predicted by genetic variants identified through whole genome sequencing or by gene expression analysis. Our results provide novel insight into the basis of variation among Mtb strains and demonstrate that TnSeq is a scalable method to predict clinically important phenotypic differences among Mtb strains.

Genome-wide assessment ofMycobacterium tuberculosisconditionally essential metabolic pathways

“…Recent advances in Next-Generation Sequencing (NGS)-based approaches have transformed our ability to examine gene functions in a genome-wide manner. Transposon insertion sequencing (Tn-seq) has been widely used to conduct fitness profiling of gene functions in many bacterial species, including M. tuberculosis (3-10). In addition to fitness profiling, a lack of representation of specific transposon insertions within a saturated transposon library has been used to identify essential genes in genome-wide screens (3, 9, 10).…”

“…The gene essentialities determined by Tn-seq studies are accessible through publicly available databases, such as Tuberculist (17), Biocyc (18), and OGEE (19). Most of these gene essentiality data were obtained from Tn-seq studies that were carried out using a defined growth medium that was supplemented with a limited number of nutrients (7-10, 13, 15). Under this growth condition, genes for numerous essential central metabolic pathways can be rendered dispensable through supplementation.…”

CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units

“…One of the earliest applications of this technique, dubbed TraSH, for transposon site hybridization 4 , was quickly adapted in subsequent essentiality screens that incorporated NGS for transposon detection and analysis [5][6][7] , offering increasing depth of coverage and confidence in essentiality calls. This next generation of Tn-Seq approaches has been applied to questions of conditional essentiality in infection models 8 and, more recently, to elucidate differential gene essentialities between clinical M. tuberculosis isolates 9 .…”

Comparative Tn-Seq reveals common daptomycin resistance determinants inStaphylococcus aureusdespite strain-dependent differences in essentiality of shared cell envelope genes

“…Tn-Seq experiments are typically conducted in a single strain that is considered representative, and it is assumed that results from that strain can be extrapolated to other members of the species. Recent studies have challenged this notion, demonstrating clear strain-dependent gene requirements in Mycobacterium tuberculosis, Streptococcus pneumoniae , and Pseudomonas aeruginosa [18, 26, 72].…”

“…With these limitations, it is difficult to assess the congruence of gene essentiality across S. aureus strains. A recent comparative Tn-Seq analysis of Mycobacterium tuberculosis showed strain-to-strain differences in gene essentiality, with implications for antibiotic targets and acquisition of resistance; this work highlighted the importance of carrying out functional genomics studies of important pathogens in multiple strains using the same methodology [18].…”