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Genome-wide transposon mutagenesis in pathogenic Leptospira species.

ABSTRACT: Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.

SUBMITTER: Murray GL

PROVIDER: S-EPMC2632054 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Genome-wide transposon mutagenesis in pathogenic Leptospira species.

Murray Gerald L GL Morel Viviane V Cerqueira Gustavo M GM Croda Julio J Srikram Amporn A Henry Rebekah R Ko Albert I AI Dellagostin Odir A OA Bulach Dieter M DM Sermswan Rasana W RW Adler Ben B Picardeau Mathieu M

Infection and immunity 20081201 2

Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 d ...[more]

PMID: 19047402

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Project description:Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups--pathogens, non-pathogens, and intermediates--based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological significance might contribute to our understanding of biology and infectivity of pathogenic spirochetes.

Project description:Mycobacterium abscessus is an emerging opportunistic human pathogen that naturally resists most major classes of antibiotics, making infections difficult to treat. Thus far, little is known about M. abscessus physiology, pathogenesis, and drug resistance. Genome-wide analyses have comprehensively catalogued genes with essential functions in Mycobacterium tuberculosis and Mycobacterium avium subsp. hominissuis (here, M. avium) but not in M. abscessus. By optimizing transduction conditions, we achieved full saturation of TA insertion sites with Himar1 transposon mutagenesis in the M. abscessus ATCC 19977T genome, as confirmed by deep sequencing prior to essentiality analyses of annotated genes and other genomic features. The overall densities of inserted TA sites (85.7%), unoccupied TA sites (14.3%), and nonpermissive TA sites (8.1%) were similar to results in M. tuberculosis and M. avium. Of the 4,920 annotated genes, 326 were identified as essential, 269 (83%) of which have mutual homology with essential M. tuberculosis genes, while 39 (12%) are homologous to genes that are not essential in M. tuberculosis and M. avium, and 11 (3.4%) only have homologs in M. avium. Interestingly, 7 (2.1%) essential M. abscessus genes have no homologs in either M. tuberculosis or M. avium, two of which were found in phage-like elements. Most essential genes are involved in DNA replication, RNA transcription and translation, and posttranslational events to synthesize important macromolecules. Some essential genes may be involved in M. abscessus pathogenesis and antibiotics response, including certain essential tRNAs and new short open reading frames. Our findings will help to pave the way for better understanding of M. abscessus and benefit development of novel bactericidal drugs against M. abscessus. IMPORTANCE Limited knowledge regarding Mycobacterium abscessus pathogenesis and intrinsic resistance to most classes of antibiotics is a major obstacle to developing more effective strategies to prevent and mitigate disease. Using optimized procedures for Himar1 transposon mutagenesis and deep sequencing, we performed a comprehensive analysis to identify M. abscessus genetic elements essential for in vitro growth and compare them to similar data sets for M. tuberculosis and M. avium subsp. hominissuis. Most essential M. abscessus genes have mutual homology with essential M. tuberculosis genes, providing a foundation for leveraging available knowledge from M. tuberculosis to develop more effective drugs and other interventions against M. abscessus. A small number of essential genes unique to M. abscessus deserve further attention to gain insights into what makes M. abscessus different from other mycobacteria. The essential genes and other genomic features such as short open reading frames and noncoding RNA identified here will provide useful information for future study of M. abscessus pathogenicity and new drug development.

Project description:Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.

Dataset Information

Genome-wide transposon mutagenesis in pathogenic Leptospira species.

Publications

Genome-wide transposon mutagenesis in pathogenic Leptospira species.

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