Comprehensive essentiality analysis of the Mycobacterium kansasii genome by saturation transposon mutagenesis and deep sequencing

Abstract

Mycobacterium kansasii (Mk) is an opportunistic pathogen that is frequently isolated from urban water systems, posing a health risk to susceptible individuals. Despite its ability to cause tuberculosis-like pulmonary disease, very few studies have probed the genetics of this opportunistic pathogen. Here, we report a comprehensive essentiality analysis of the Mk genome. Deep sequencing of a high-density library of Mk Himar1 transposon mutants revealed that 86.8% of the chromosomal thymine-adenine (TA) dinucleotide target sites were permissive to insertion, leaving 13.2% TA sites unoccupied. Our analysis identified 394 of the 5,350 annotated open reading frames (ORFs) as essential. The majority of these essential ORFs (84.8%) share essential mutual orthologs with Mycobacterium tuberculosis (Mtb). A comparative genomics analysis identified 139 Mk essential ORFs that share essential orthologs in four other species of mycobacteria. Thirteen Mk essential ORFs share orthologs in all four species that were identified as being not essential, while only two Mk essential ORFs are absent in all species compared. We used the essentiality data and a comparative genomics analysis reported here to highlight differences in essentiality between candidate Mtb drug targets and the corresponding Mk orthologs. Our findings suggest that the Mk genome encodes redundant or additional pathways that may confound validation of potential Mtb drugs and drug target candidates against the opportunistic pathogen. Additionally, we identified 57 intergenic regions containing four or more consecutive unoccupied TA sites. A disproportionally large number of these regions were located upstream of pe/ppe genes. Finally, we present an essentiality and orthology analysis of the Mk pRAW-like plasmid, pMK1248. IMPORTANCE Mk is one of the most common nontuberculous mycobacterial pathogens associated with tuberculosis-like pulmonary disease. Drug resistance emergence is a threat to the control of Mk infections, which already requires long-term, multidrug courses. A comprehensive understanding of Mk biology is critical to facilitate the development of new and more efficacious therapeutics against Mk. We combined transposon-based mutagenesis with analysis of insertion site identification data to uncover genes and other genomic regions required for Mk growth. We also compared the gene essentiality data set of Mk to those available for several other mycobacteria. This analysis highlighted key similarities and differences in the biology of Mk compared to these other species. Altogether, the genome-wide essentiality information generated and the results of the cross-species comparative genomics analysis represent valuable resources to assist the process of identifying and prioritizing potential Mk drug target candidates and to guide future studies on Mk biology.

Keywords: Mycobacterium kansasii; TnSeq; antitubercular target candidate; comparative mycobacterial gene essentiality; gene essentiality; mycobacterial ESX secretion system; mycobacterial comparative genomics; mycobacterial drug targets; mycobacterial orthology analysis; nontuberculous mycobacteria; nucleoid-associated protein; pRAW-like plasmid; plasmid-encoded ESX system; transposon mutagenesis; tuberculosis; type VII secretion system.

Fig 1

Visualization of the M. kansasii chromosome showing insertion counts, orthology data, and various sequence features. Tracks from innermost: (1) chromosome backbone with ruler; (2) insertion count plot with the height of the black bars representing the number of raw insertion counts at each TA site (minimum = 0, maximum = 45,000); (3) GC skew (green represents richness of G over C, purple represents richness of C over G; (4) GC content (blue represents above average GC content, yellow represents below average GC content); (5) M. kansasii annotated genes (bright red); (6) M. tuberculosis orthologs of Mk open reading frames (ORFs) (dark red); (7) M. avium subsp. hominissuis orthologs (yellow) of Mk ORFs; (8) M. abscessus orthologs (green) of Mk ORFs; and (9) M. smegmatis orthologs (blue) of Mk ORFs.

Fig 2

Visualization of the M. kansasii plasmid pMK12478 showing insertion counts and various sequence features. Tracks are as described in the legend of Fig. 1. Insertion count plot minimum = 0, maximum = 37,000.

Fig 3

TA site insertion saturation. Each data set represents TnSeq results from a single-plated library. Gray bars show the overall saturation levels (chromosome + plasmid) of individual data sets. The solid lines represent the cumulative densities of the chromosome (circles) and plasmid (triangles) obtained by adding one data set at a time. Each data set was generated by harvesting and processing plated libraries as described in Materials and Methods. Briefly, colonies from a single library were scraped from the surface of 7H10 agar, and genomic DNA was extracted from the mutant pool. Genomic DNA was fragmented, fragment ends were repaired, A-tailed, and ligated to T-tailed adapters containing random nucleotide barcodes. PCR enrichment of fragments containing Tn-chromosome junctions using a Tn-specific primer and an adapter-specific primer was followed by the selection of fragments between 400 and 600 bp via gel electrophoresis and then a second hemi-nested PCR amplification using tailed primers to introduce Illumina-specific sequences. The resulting fragment libraries were sequenced on the Illumina platform, obtaining at least 30 million 100-bp paired-end reads per sample. Reads were processed and mapped onto the Mk chromosome and the Mk plasmid pMK12478 using the TPP tool in TRANSIT.

Fig 4

Venn diagrams illustrating in vitro essential ORFs of M. kansasii compared to other mycobacteria species. Mutual orthologs were identified using Gview server as described in Materials and Methods. (A) M. tuberculosis essential ORFs defined by Dejesus et al. (35). (B) M. avium subsp. hominissuis essential ORFs defined by Dragset et al. (34). (C) M. abscessus essential ORFs defined by Rifat et al. (33). (D) M. smegmatis essential ORFs defined by Dragset et al. (36). Venn diagrams were generated in R using the Eulerr package and edited in Adobe Illustrator.