PDF(Pubmed)
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.
摘要：
kansasii分枝杆菌（Mk）是一种机会性病原体，经常从城市供水系统中分离出来，对易感人群构成健康风险。尽管它能引起结核病样肺病，很少有研究探讨这种机会病原体的遗传学。这里,我们报告了对Mk基因组的全面必要性分析。对MkHimar1转座子突变体的高密度文库进行深度测序显示，86.8％的染色体胸腺嘧啶-腺嘌呤（TA）二核苷酸靶位点允许插入，留下13.2%的TA网站空置。我们的分析确定了5350个注释开放阅读框(ORF)中的394个是必需的。这些必需的ORF中的大多数(84.8%)与结核分枝杆菌(Mtb)共享必需的相互直向同源物。比较基因组学分析确定了139Mk必需ORF，它们在其他四种分枝杆菌中共享必需直向同源物。在所有四个被确定为非必需的物种中，有13个Mk必需ORF共享直系同源物，而在所有物种中，只有两个Mk必需ORF是不存在的。我们使用此处报道的必要性数据和比较基因组学分析来强调候选Mtb药物靶标和相应Mk直系同源物之间的必要性差异。我们的发现表明，Mk基因组编码冗余或额外的途径，这些途径可能会混淆潜在的Mtb药物和针对机会性病原体的药物靶标候选物的验证。此外,我们确定了57个基因间区域,其中包含4个或4个以上连续未占据的TA位点.大量的这些区域位于pe/ppe基因的上游。最后,我们提出了MkpRAW样质粒的必要性和正交性分析，pMK1248.重要性Mk是与结核样肺病相关的最常见的非结核分枝杆菌病原体之一。耐药性的出现威胁到Mk感染的控制,这已经需要长期的，多种药物课程。对Mk生物学的全面了解对于促进开发针对Mk的新的和更有效的疗法至关重要。我们将基于转座子的诱变与插入位点鉴定数据分析相结合，以发现Mk生长所需的基因和其他基因组区域。我们还将Mk的基因重要性数据集与其他几种分枝杆菌的数据集进行了比较。该分析强调了与其他物种相比，Mk生物学的关键相似性和差异。总之,产生的全基因组重要性信息和跨物种比较基因组学分析的结果代表了宝贵的资源,可帮助识别和优先考虑潜在的Mk药物靶标候选物,并指导未来的Mk生物学研究.
