Infection, Genetics and Evolution 10 (2010) 586–590
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Short communication
LepBank: A Leptospira sequence repository and a portal for phylogenetic studies Marcus R. Eslaba˜o a, Odir A. Dellagostin a,*, Gustavo M. Cerqueira b,** a b
Centro de Biotecnologia, Universidade Federal de Pelotas, 96010900 Pelotas, RS, Brazil Centro de Biotecnologia, Instituto Butantan, 05503900 Sa˜o Paulo, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 December 2009 Received in revised form 20 February 2010 Accepted 26 February 2010 Available online 7 March 2010
Leptospirosis is a neglected infectious disease that constitutes a threat to both humans and animals. Comprehension about the epidemiological behavior and population dynamics of Leptospira may be helpful for the development of control measures. Thus, an effort was made to organize leptospiral sequences in a new and specific database. In addition, online bioinformatics tools were clustered in a web portal to facilitate sequences manipulation by scientists. LepBank (http://.lepbank.ufpel.edu.br) is a Leptospira sequences repository and a suite for systematics, which brings simplicity to leptospirosis research, integrating sophisticated online programs to a sequence database. We intend the database to be useful for the leptospirosis scientific community, providing standardized and high quality information and facilitating research into key aspects of the Leptospira taxonomy and phylogeny. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Leptospira Leptospirosis Bioinformatics Sequencing Genomics
1. Introduction Leptospirosis is a neglected infectious disease that represents a public health concern, mainly among developing countries (Bharti et al., 2003; Levett, 2001). This disease is caused by pathogenic spirochetes belonging to the Leptospira genus. Several animal species may be infected and serve as reservoirs, shedding bacteria in the urine. Humans are generally infected through contact with contaminated water and/or soil. In developed countries, leptospirosis represents an emerging disease due to outbreaks associated to occupational and recreational activities (Haake et al., 2002; Morgan et al., 2002; Ricaldi and Vinetz, 2006). However, the major public health burden of leptospirosis is found in developing countries, within poor rural environments and urban slum communities (Bharti et al., 2003; Levett, 2001; Maciel et al., 2008; Reis et al., 2008). Worldwide, more than 500,000 severe leptospirosis cases are notified annually, with a mortality rate up to 50% (McBride et al., 2005; WHO, 2003; Padilla Perez et al., 1998). In Brazil, the mean number of cases is approximately 2/100,000 inhabitants, per year, with a mortality rate ranging between 10 and 50% (Ko et al., 1999; McBride et al., 2005; Spichler et al., 2007; Vieira and Brauner, 2002).
** Corresponding author at: Centro de Biotecnologia, Laboratorio de Biotecnologia Molecular II, Instituto Butantan, Avenida Vital Brasil 1500, Butanta, 05503900 Sa˜o Paulo, SP, Brazil. Tel.: +55 11 3722 0019; fax: +55 11 3726 1505. * Corresponding author at: Centro de Biotecnologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil. Tel.: +55 53 3275 7587; fax: +55 53 3275 7350. E-mail addresses:
[email protected] (O.A. Dellagostin),
[email protected],
[email protected] (G.M. Cerqueira). 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.02.014
Leptospires are highly motile bacteria capable of active skin and mucosa penetration followed by rapid systemic dissemination, immediately after infection. Among susceptible hosts (humans and animals), the systemic infection produces, in diverse organs, severe manifestations including jaundice, renal failure and severe pulmonary hemorrhage. However, in reservoir-animals, such as rats or domestic mice, renal colonization is observed within the renal tubules (Bharti et al., 2003; Levett, 2001), without disease manifestation (Athanazio et al., 2008). Prophylaxis is mediated by the use of whole cell vaccines. Bacterins are widely available for animals, although only a few countries allow their commercialization for human use. These preparations may present a number of disadvantages such as low or no efficiency for broad range protection against all Leptospira serovars, the possibility of side effects and need for boosts. Thus, efforts have been done for more than a decade to overcome these drawbacks. Studies involving the use of the modern biotechnology have been conducted to evaluate the immunoprotective potential conferred by different vaccine candidates (Haake et al., 1999; Branger et al., 2001, 2005; Palaniappan et al., 2006; Chang et al., 2007; Seixas et al., 2007; Silva et al., 2007; Faisal et al., 2008, 2009; Yan et al., 2009). However, so far only a few targets revealed potential to constitute, in the future, a recombinant vaccine. The characterization of Leptospira isolates is a prerequisite for epidemiological studies. The Leptospira genus is divided in more than 270 serovars based on lipopolyssacharide (LPS) carbohydrate component heterogeneity. Moreover, DNA–DNA hybridization data demonstrated the existence of 20 Leptospira species (Adler ˜ a Moctezuma, 2010; Cerqueira and Picardeau, 2009). and de la Pen
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Sequencing is a worldwide available method, which produces easy to interpret and reliable data. Moreover, sequencing is progressively cheaper approach that may be employed as a routine for isolates classification. Housekeeping and outer membrane protein genes have been characterized and employed in the Leptospira species classification, i.e. the rrs (Cerqueira et al., 2010; Morey et al., 2006; Paster and Dewhirst, 2000; Paster et al., 1991), lipL41 (Haake et al., 2004), rpoB (La Scola et al., 2006), gyrB (Slack et al., 2006); S10-spc-a locus (Victoria et al., 2008) and ligB (Cerqueira et al., 2009a). The proposed taxonomic markers generated similar independent results in terms of strains clustering into branches representing species. In addition, studies involving the concatenation of some of the loci mentioned and others have enabled the generation of multilocus sequence typing (MLST) schemes (Ahmed et al., 2006; Thaipadungpanit et al., 2007; Victoria et al., 2008; Leon et al., 2010). This approach has been also applied to the identification of a dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand (Thaipadungpanit et al., 2007). A large number of Leptospira sequences were generated over almost 20 years of phylogenetic and taxonomic studies. All this information has been deposited in large databases such as GenBank (www.ncbi.nlm.nih.gov/Genbank/index.html) (Benson et al., 2009) and MLST.net (www.mlst.net) (Chan et al., 2001). While the former works as a sequence repository, the latter includes tools that enable the creation of MLST schemes. In addition, the IS Finder program (http://www-is.biotoul.fr/is.html) (Siguier et al., 2006) accepts sequences belonging specifically to insertion sequences and it is also capable of identifying such sequences into large genomic data. However, researchers in the field of Leptospira systematics claim for a specific database that includes sequences with taxonomic value, and which, at the same time, enables the interaction with important online tools for both taxonomists and phylogeneticists. Thus, we created the LepBank, a sequence repository and a web portal aiming to improve the systematics of Leptospira. 2. Development of LepBank The LepBank (Fig. 1A) was created on the top of a Firebird 2.1 SQL server, www.firebirdsql.org (Weinberg and Groff, 2002). Various tables were developed using the SQL to archive the sequence data along with other relevant deposit information. All the algorithms to retrieve, modify and deposit datasets (Fig. 1B) were written in Delphi language (www.embarcadero.com/products/delphi). Delphi enables the management of both user and sequence information, execution of exact and similarity searches, sequence alignment through MUSCLE and computation of phylogenetic trees by PhyML. Data retrieval by LepBank server is performed by the search engine using the keywords: search method (strain; serovar; serogroup; species; gene; author name); sequences or accession numbers (Fig. 1C). The search for either exact or similar sequences is possible through specific sections. The sequence similarity search was based on a score method; resembling that performed by BLAST (Morgulis et al., 2008). However, the LepBank search tool was newly developed to run in
Fig. 1. Screen dumps of LepBank user interface exemplifying its applications: the initial homepage displaying a simple menu (A); a typical submission form for sequences deposit (B); the results page retrieved by searching the word ‘‘Leptospira’’. Sequence selection for alignment is possible by ticking the left box besides each entry, and sending the request to MUSCLE server (short arrow) (C); the
alignment results and, at the bottom of the same frame, the link for phylogenetic tree creation by TreeDyn (long arrow) (D); and the resulting phylogenetic tree. Note that strains names in the tree may be easily modified through the link ‘‘Change leaf name’’, positioned among the options below the tree figure, and subsequent clicking over the name to be modified. Bootstrap values are displayed in the tree. The initial lowercase letter indicates the species name (ina–L. inadai, f–L. fainei, k–L. kirschneri, i–L. interrogans, n–L. noguchii, w–L. weilii, b–L. borgpetersenii and s–L. santarosai) and are followed by type strains names (E).
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the Windows platform. Briefly, it searches the largest match between the query and subject sequences. Then, the whole query sequence is aligned against the subject sequences found. For each nucleotide match the software attributes one point. At the end, a percentage identity is presented based on the score of the alignment of the query sequence against each subject sequence deposited in LepBank. The results are displayed from the most similar to the less similar (top to bottom). Results are shown in different pages; minimizing web page charging time. As an extension to the sequence search–retrieval process; the MUSCLE alignment tool (Edgar, 2004) was incorporated into LepBank for multiple alignment analysis. The alignment window is available at http://lepbank.ufpel.edu.br; but it runs from the MUSCLE server (www.ebi.ac.uk/muscle) (Fig. 1D). A phylogeny module was also created to use the alignment results and it incorporates two other online tools: PhyML (http://atgc.lirmm.fr/phyml/) (Guindon et al., 2009); a program responsible for calculating phylogenetic trees distances and bootstrap values; and TreeDyn (http://www.treedyn.org/) (Chevenet et al., 2006); an online software for phylogenetic trees construction based on the calculations of PhyML (Fig. 1E).
lepbank.ufpel.edu.br). All submitters should be registered into the database to ensure security and organizational requirements. LepBank is likely to be a very efficient online resource for taxonomists. Other databases containing leptospiral sequences are currently available, and they have made an important contribution to the field. A number of sequence-types of Leptospira spp. were previously deposited in MLST.net (http://leptospira.mlst.net). Now, LepBank is expected to perform constant sequence mining into MLST.net and GenBank. This approach is likely to support the identification and characterization of novel isolates worldwide. In addition, the alignment and phylogenetic analyses performed by MUSCLE and PhyML, respectively, through LepBank, may help to identify candidate loci for MLST analysis. Leptospiral sequences that allow discrimination up to the isolate level should be easily identified within the database, using the integrated bioinformatics tools. Gene sequences identified to contain discriminative power may be used in combination to manually constitute novel superloci for multilocus analysis, or add upon the existing MLST schemes. Thus, we have also made MUSCLE and PhyML accessible for dendrograms construction through the ‘‘Bioinformatics tools’’ link. 5. Discussion
3. Scheme, interface and functioning An electronic form is accessible under ‘‘Registration’’. General information and information about the registration (user name and password) can also be updated in ‘‘Update personal info’’ and ‘‘Login update’’, respectively. Using the ‘‘Sequence search’’ link, users are able to rapidly retrieve exact sequences that completely match with the query sequences, and by the use of ‘‘Similarity search’’ it is possible to obtain the results of either identical or similar sequences. The link ‘‘Sequence submission’’ is specific for new deposits and there are two options: ‘‘Single’’ for single sequences submission and ‘‘Multiple’’ for a batch submission in a single file, both in the FASTA format. Sequence updates or modifications can be done in ‘‘Sequence update’’ or by direct contact with the LepBank managers. Full postal and electronic addresses for correspondence are available at the LepBank home page, located in ‘‘Contact’’. Sequence submitters are invited to register before depositing one or multiple sequence(s). The scheme of the LepBank web server is presented in Fig. 1. The web server employs user-specific security for the sequence deposit process and no bugs were observed during the functioning of the database components. The 16S rRNA gene sequences from the type strains L. interrogans strain RGA, Leptospira kirschneri strain 3522C, Leptospira noguchii CZ214K, Leptospira borgpetersenii strain Veldrat Batavia 46, Leptospira weilii strain Celledoni. Leptospira santarosai strain LT821, Leptospira inadai strain 10 and Leptospira fainei strain But6 were selected in the LepBank results page, after search by the term ‘‘16S’’. The sequences were aligned by MUSCLE and then used to construct a phylogenetic tree. The method employed was the neighbor-joining and the model was p-distance. One thousand bootstrap replications were adopted to provide confidence in the nodes. The LepBank interface is user-friendly on account of frames, simple menus and tools operating. The database is also easily accessible by computers harboring Windows, Macintosh and Linux operational systems. LepBank was also tested and is fully functional using different web browsers. The user interface is divided into different sections, each one dedicated to a specific function. 4. Availability and utility LepBank is freely accessible to all users with non-profit interests and can be reached through its website (http://
There is a growing need to facilitate organization and utilization of sequence data through a web portal, which contains specific online tools for systematics. Such dataset is likely to help scientists to lead efforts towards comprehension of leptospirosis epidemiology and population dynamics. Rational vaccine design for leptospirosis is also believed to be supported by epidemiological analyses conducted with the help of LepBank. The leptospiral genomes sequenced so far demonstrated gene synteny for a number of ORFs, contributing to our knowledge about the genetic diversity of Leptospira (Bulach et al., 2006; Nascimento et al., 2004a, 2004b; Picardeau et al., 2008; Ren et al., 2003). To date, more than 270 serovars are recognized into Leptospira genus, but their complete discrimination by PCR-sequencing approaches have not been obtained (Cerqueira and Picardeau, 2009). Efforts have been done by the use of the Multilocus Sequence Typing (MLST) technique and considerable advances were achieved (Ahmed et al., 2006; Thaipadungpanit et al., 2007; Victoria et al., 2008). Yet, a recent work involving a superlocus containing the ligB (Cerqueira et al., 2009a), secY (Victoria et al., 2008), rpoB (La Scola et al., 2006) and lipL41 (Ahmed et al., 2006; Haake et al., 2004) genes sequences appeared to be the most promising for serovar discrimination based on PCR-sequencing methodology (unpublished). The difficulties to identify new taxonomic markers include the possibility of occurrence of evolutionary events such as horizontal gene transfer (HGT) (Haake et al., 2004; McBride et al., 2009), low extent of polymorphism of the target sequences (Morey et al., 2006) or the peculiar genetic distribution among serovars (Cerqueira et al., 2009a). Moreover, specific information for taxonomists appears to be diffuse. A database exists, which archives and organizes MLST data (Chan et al., 2001). We aim to collaborate with this important initiative, providing support to the identification of new and useful sequences for further epidemiological studies. Yet, two important resources to metagenomic analysis of Leptospira spp. deserve to be constantly accessed: LeptoList (http://bioinfo.hku.hk/LeptoList) (Fang et al., 2005) and MaGe-magnifying genomes (www.cns.fr/agc/mage) (Vallenet et al., 2006). Both projects correspond to high-quality sequence annotation and repositories, which provide bioinformatics tools for genome mining. Thus, the electronic information contained in LepBank will be objectively focused on Leptospira classification, while conserving the simplicity for easy manipulation and interpretation. LepBank may be also useful to gain insights about evolution and migration patterns of Leptospira populations.
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The LepBank database currently houses 2055 entries, covering 17 different genes (lipL32, lipL41, ompL1, 16S, ligA, ligB, ligC, gyrB, secY, rpoB, pntA, sucA, pfkB, tpiA, mreA, glmU, fadD), one intergenic region and the whole locus S10-spc-a. The sequences used to initially constitute and standardize LepBank were manually obtained from GenBank, and direct submissions are now encouraged. LepBank is available to the whole scientific community. 6. Conclusion LepBank is a sequence repository and a web portal constituted by widely known, recognized and easy to use online tools. The database design and content management is based on SQL and the information can be easily displayed using frames in any web browser. The database will be helpful to understand the systematics of Leptospira through taxonomic and phylogenetic analysis, by the use of free online bioinformatics tools incorporated into LepBank. We believe LepBank will be useful for scientists interested to study the epidemiological behavior of Leptospira spp. Acknowledgements The authors thank Robson G. Dossa and Moˆnica L. Vieira for their critical review and thoughtful advices on this manuscript. MRS is supported by CNPq, Brazilian Ministry of Science and Technology, Brazil. GMC is supported by FAPESP. References ˜ a Moctezuma, A., 2010. Leptospira and leptospirosis. Vet. MicroAdler, B., de la Pen biol. 140, 287–296. Ahmed, N., Devi, S.M., Valverde Mde, L., Vijayachari, P., Machang’u, R.S., Ellis, W.A., Hartskeerl, R.A., 2006. Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species. Ann. Clin. Microbiol. Antimicrob. 5, 28. Athanazio, D.A., Silva, E.F., Santos, C.S., Rocha, G.M., Vannier-Santos, M.A., McBride, A.J., Ko, A.I., Reis, M.G., 2008. Rattus norvegicus as a model for persistent renal colonization by pathogenic Leptospira interrogans. Acta Trop. 105, 176–180. Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Sayers, E.W., 2009. GenBank. Nucleic Acids Res. 37, D26–31. Bharti, A.R., Nally, J.E., Ricaldi, J.N., Matthias, M.A., Diaz, M.M., Lovett, M.A., Levett, P.N., Gilman, R.H., Willig, M.R., Gotuzzo, E., Vinetz, J.M., 2003. Leptospirosis: a zoonotic disease of global importance. Lancet Infect. Dis. 3, 757–771. Branger, C., Chatrenet, B., Gauvrit, A., Aviat, F., Aubert, A., Bach, J.M., Andre-Fontaine, G., 2005. Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1. Infect. Immun. 73, 4062–4069. Branger, C., Sonrier, C., Chatrenet, B., Klonjkowski, B., Ruvoen-Clouet, N., Aubert, A., Andre-Fontaine, G., Eloit, M., 2001. Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination. Infect. Immun. 69, 6831–6838. Bulach, D.M., Zuerner, R.L., Wilson, P., Seemann, T., McGrath, A., Cullen, P.A., Davis, J., Johnson, M., Kuczek, E., Alt, D.P., Peterson-Burch, B., Coppel, R.L., Rood, J.I., Davies, J.K., Adler, B., 2006. Genome reduction in Leptospira borgpetersenii reflects limited transmission potential. Proc. Natl. Acad. Sci. U.S.A. 103, 14560–14565. Cerqueira, G.M., McBride, A.J., Picardeau, M., Ribeiro, S.G., Moreira, A.N., Morel, V., Reis, M.G., Ko, A.I., Dellagostin, O.A., 2009a. Distribution of the leptospiral immunoglobulin-like (Lig) genes in pathogenic Leptospira spp. and application of ligB to typing leptospiral isolates. J. Med. Microbiol. 58, 1173–1181. Cerqueira, G.M., McBride, A.J.A., Queiroz, A., Pinto, L.S., Silva, E.F., Hartskeerl, R.A., Reis, M.G., Ko, A.I., Dellagostin, O.A., 2010. Monitoring Leptospira strain collections: the need for quality control. Am. J. Trop. Med. Hyg. 82, 83–87. Cerqueira, G.M., Picardeau, M., 2009. A century of Leptospira strain typing. Infect. Genet. Evol. 9, 760–768. Chan, M.S., Maiden, M.C., Spratt, B.G., 2001. Database-driven multi locus sequence typing (MLST) of bacterial pathogens. Bioinformatics 17, 1077–1083. Chang, Y.F., Chen, C.S., Palaniappan, R.U., He, H., McDonough, S.P., Barr, S.C., Yan, W., Faisal, S.M., Pan, M.J., Chang, C.F., 2007. Immunogenicity of the recombinant leptospiral putative outer membrane proteins as vaccine candidates. Vaccine 25, 8190–8197. ˜ uls, A.L., Jacq, B., Christen, R., 2006. TreeDyn: towards Chevenet, F., Brun, C., Ban dynamic graphics and annotations for analyses of trees. BMC Bioinform. 7, 439. Edgar, R.C., 2004. MUSCLE: a multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797.
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