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Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

ABSTRACT: 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.

SUBMITTER: Wang Y

PROVIDER: S-EPMC4640553 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

Wang Yanzhuo Y Zhuang Xuran X Zhong Yi Y Zhang Cuicai C Zhang Yan Y Zeng Lingbing L Zhu Yongzhang Y He Ping P Dong Ke K Pal Utpal U Guo Xiaokui X Qin Jinhong J

PLoS neglected tropical diseases 20151110 11

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 Leptosp ...[more]

PMID: 26555137

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Project description:BACKGROUND:Leptospirosis is a neglected zoonosis affecting animals and humans caused by infection with Leptospira. The bacteria can survive outside of hosts for long periods of time in soil and water. While identification of Leptospira species from human cases and animal reservoirs are increasingly reported, little is known about the diversity of pathogenic Leptospira species in the environment and how surveillance of the environment might be used for monitoring and controlling disease. METHODS AND FINDINGS:Water samples (n = 104) were collected from the peri-domestic environment of 422 households from farms, rural villages, and urban slums participating in a broader study on the eco-epidemiology of leptospirosis in the Los Rios Region, Chile, between October 2010 and April 2012. The secY region of samples, previously detected as pathogenic Leptospira by PCR, was amplified and sequenced. Sequences were aligned using ClustalW in MEGA, and a minimum spanning tree was created in PHYLOViZ using the goeBURST algorithm to assess sequence similarity. Sequences from four clinical isolates, 17 rodents, and 20 reference strains were also included in the analysis. Overall, water samples contained L. interrogans, L. kirschneri, and L. weilii, with descending frequency. All species were found in each community type. The distribution of the species differed by the season in which the water samples were obtained. There was no evidence that community-level prevalence of Leptospira in dogs, rodents, or livestock influenced pathogen diversity in the water samples. CONCLUSIONS:This study reports the presence of pathogenic Leptospira in the peri-domestic environment of households in three community types and the differences in Leptospira diversity at the community level. Systematic environmental surveillance of Leptospira can be used for detecting changes in pathogen diversity and to identify and monitor contaminated areas where an increased risk of human infection exists.

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.

Project description:BackgroundLeptospirosis is a complex zoonotic disease mostly caused by a group of eight pathogenic species (L. interrogans, L. borgpetersenii, L. kirschneri, L. mayottensis, L. noguchii, L. santarosai, L. weilii, L. alexanderi), with a wide spectrum of animal reservoirs and patient outcomes. Leptospira interrogans is considered as the leading causative agent of leptospirosis worldwide and it is the most studied species. However, the genomic features and phylogeography of other Leptospira pathogenic species remain to be determined.Methodology/principal findingsHere we investigated the genome diversity of the main pathogenic Leptospira species based on a collection of 914 genomes from strains isolated around the world. Genome analyses revealed species-specific genome size and GC content, and an open pangenome in the pathogenic species, except for L. mayottensis. Taking advantage of a new set of genomes of L. santarosai strains isolated from patients in Costa Rica, we took a closer look at this species. L. santarosai strains are largely distributed in America, including the Caribbean islands, with over 96% of the available genomes originating from this continent. Phylogenetic analysis showed high genetic diversity within L. santarosai, and the clonal groups identified by cgMLST were strongly associated with geographical areas. Serotype identification based on serogrouping and/or analysis of the O-antigen biosynthesis gene loci further confirmed the great diversity of strains within the species.Conclusions/significanceIn conclusion, we report a comprehensive genome analysis of pathogenic Leptospira species with a focus on L. santarosai. Our study sheds new light onto the genomic diversity, evolutionary history, and epidemiology of leptospirosis in America and globally. Our findings also expand our knowledge of the genes driving O-antigen diversity. In addition, our work provides a framework for understanding the virulence and spread of L. santarosai and for improving its surveillance in both humans and animals.

Project description:BackgroundPrevious genomic analysis of pathogenic Leptospira has identified two circular chromosomes but no plasmid. This study aims to investigate potential extrachromosomal elements of L.interrogans serovar Canicola strain Gui44.MethodologyTwo novel plasmids, pGui1 and pGui2, were isolated from the pathogenic strain Gui44, using a modified alkaline lysis method. Southern blotting was performed to determine the presence and size of them. Then, 454 and Hiseq sequencing were applied to obtain and analyze the complete sequences of the two plasmids. Furthermore, real-time quantitative PCR and next-generation sequencing were used to compare relative copy numbers of the two plasmids with that of the chromosomes. Finally, after serial passages in vitro for more than 2 years, the strain Gui44 was subsequently re-sequenced to estimate stability of the two plasmids.Principal findingsThe larger plasmid, pGui1, 74,981 base pairs (bp) in length with GC content of 34.63%, possesses 62 open reading frames (ORFs). The smaller plasmid, pGui2, is 66,851 bp in length with GC content of 33.33%, and contains 63 ORFs. The replication initiation proteins encoded by pGui1 and pGui2 demonstrate significant sequence similarity with LA1839 (86% and 88%), a well-known replication protein in another pathogenic L.interrogans serovar Lai strain Lai, suggesting the ability for autonomous plasmid replication. Quantitative PCR and next-generation sequencing confirms a single copy of both plasmids and their stable presence in the strain Gui44 with in vitro serial passages after more than 2 years. Interestingly, the two plasmids both contain a significant number of novel genes (35 in pGui1 and 52 in pGui2).ConclusionsThis report confirms the presence of two separate circular plasmids in serovar Canicola strain Gui44 and provides a new understanding of genomic organization, adaptation, evolution and pathogenesis of Leptospira, which will aid in the development of in vivo genetic manipulation systems in pathogenic Leptospira species.

Dataset Information

Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

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Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

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