Comparative Genomics Of Type â…¥ Secretion Systems In Klebsiella Peneumoniae

The bacterial species Klebsiella pneumoniae is an increasingly important multi-drug resistance opportunistic pathogen of man,with dramatic increasing in the levels of nosocomial infections. Eleven Klebsiella pneumoniae genomes have been completely sequenced, including K. pneumoniae HS11286, an epidemic ST11 carbapenemase-producing clinical isolate obtained from the sputum of a patient in Shanghai, China in 2011. Type â…¥ secretion systems are a class of sophisticated cell contact-dependent apparatuses involved in mediating antagonistic or synergistic communications between bacteria and/or bacteria and eukaryotes. These apparatuses have recently been found to be widely distributed among Gram-negative bacterial species, such as Pseudomonas aeruginosa and Vibrio cholerae. However, there is a paucity of research on the issue in K. pneumoniae. The research work in this study would be helpful to explore the association among T6 SS, Klebsiella pneumoniae pathogenesis and bacterial antagonism. In this study, the T6 SS gene clusters and the effectors were predicted and compared by using bioinformatics approaches in view of K. pneumoniae genomics analysis.The T6 SS gene clusters in K. pneumoniae genomes were firstly identified. Data on 906 T6 SSs were found in 498 bacterial strains via text mining and bioinformatics prediction, including（i） 64 experimental T6 SSs found in 57 bacterial strains, 92 validated T6 SS effectors and 25 immunity proteins as well as 127 associated regulation factors via literature mining and manual curation of over seven-hundred directly relevant references;（ii） predicted data on T6 SSs in light of the component protein sequence similarity. Considering core component sequence similarity and genetic arranagement, T6 SSs are divided into three types: i, ii and iii. SecReT6 is a PostgreSQL-based integrated database providing comprehensive information on type â…¥ secretion systems（T6SSs） in bacteria. SecReT6 offers a unique, readily explorable archive of known and putative T6 SSs, and cognate effectors found in bacteria. A broad range of T6 SS gene cluster detection and comparative analysis tools are readily accessible via SecReT6, which may aid identification of effectors and immunity proteins around the T6 SS core components. Two putative T6SS-encoding loci, coding for more than five core components in the variable regions of the K. pneumoniae HS11286 genome, were identified and comparatively analyzed. SecReT6 also provides the online tool ‘T6SS-HMMER’ to perform HMM-profile-based detection of T6 SS core components encoded by user-supplied DNA sequences. Twenty-six T6 SS gene clusters in 11 sequenced K. pneumoniae genomes were identified using ‘T6SS-HMMER’. Two putative T6SS-encoding loci（T6SS-1 and T6SS-2）, respectively coding for 12 and 9 core components in the genomic regions（KPHS₂2970..KPHS₂3190 and KPHS₃2450..KPHS₃2770） of the K. pneumoniae HS11286 genome, were identified and comparatively analyzed.Three approaches have been applied to predict K. pneumoniae T6 SS effectors（T6SE） in this study:（i） SecReT6 collated and archived candidate secreted effectors showing similarity with reported T6SEs;（ii） T6 SS comparative analyses revealing that variable genomic regions might carry genes coding for the novel T6 SE and cognate immunity protein;（iii） T6 SE prediction based on sequence features extraction using the support vector machine. KPHS₂3105 and KPHS₂3060-KPHS₂3090 of K. pneumoniae HS11286 were respectivly predicted as a putative T6 SS effector and four cognate immunity proteins.Lastly, we apply and improve the identification approaches of T6 SS gene cluster to the bacterial type III, IV secetion systems and mobile genetics elements（MGE） such as prophages, integrative and conjugative elements, integrons and insertion sequences and genomic islands. Across the full spectrum of the bacterial domain, risk genes influencing bacterial infection contains such secretion systems as well as a vast repertoire of antibiotic resistance, virulence-associated and other traits spreading via horizontal gene transfer of various mobile genetic elements. STeP is a web-based analytical toolkit that performs sequence homology searches for user-defined gene clusters against multiple bacterial genomes for in silico profiling of virulence and antibiotic resistance traits of pathogenic bacteria. STeP integrated VRprofile currently takes less than 10 minutes to annotate 5.3 Mb K. pneumoniae HS11286 chromosomal sequence. Identified T3SS/T4SS/T6SS/T7 SS and MGEs could be visually and comparatively analysed against collected known elements in the back-end MobilomeDB database. The three other individual tools with complementary functionality to VRprofile include:（i） ‘CDSeasy’ which takes less than 30 minutes to complete gene-finding and initial functional annotation of any assembled complete or draft bacterial genome sequence, or outputs a ready file available for processing by the other VRprofile tools;（ii） ‘CGCfinder’ which quickly performs simultaneous MultiGeneBlast–facilitated homology searches of a user-defined gene cluster against up to fifty genomes in the database; and（iii） ‘COGviewer’ which provides a HMMER/RPS-BLAST result for a specific set of co-location-encoded COG/Pfam features to take account of potential low sequence similarity and/or gene arrangements.In this research, T6 SS and T6 SE were investigated in completely sequenced bacterial genomes based upon K. pneumoniae analyses. The built T6 SS database and T6 SE prediction tools are helpful to the study on exploring the association among T6 SS, pathogenesis and inter-bacterial antagonism in K. pneumoniae and other Gram-negative pathogenic bacteria. The developed pathogenicity and drug-resistance gene cluster prediction tool would provide a rapid prediction of bacterial pathogenicity and drug-resistance at the genomic level.