Brazilian Journal of Medical and Biological Research (2004) 37: 459-478 interrogans genome features 459 ISSN 0100-879X

Genome features of serovar Copenhageni

A.L.T.O. Nascimento1, 1Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brasil S. Verjovski-Almeida2, 2Instituto de Química, 3Instituto de Biociências and M.A. Van Sluys3, 4Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil C.B. Monteiro-Vitorello5, 5Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, L.E.A. Camargo5, Piracicaba, SP, Brasil L.A. Digiampietri6, 6Laboratório de Bioinformática, Instituto de Computação, R.A. Harstkeerl7, Universidade Estadual de Campinas, Campinas, SP, Brasil P.L. Ho1, M.V. Marques4, 7Koninklijk Instituut voor de Tropen/Royal Tropical Institute (KIT), KIT Biomedical M.C. Oliveira3, Research, Amsterdam, The Netherlands J.C. Setubal6,10, 8The David Geffen School of Medicine at University of California, Los Angeles, D.A. Haake8,9 and CA, USA E.A.L. Martins1 9Division of Infectious Diseases, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA, USA 10Virginia Bioinformatics Institute and Department of Computer Science, Virginia Polytechnic Institute and State University Bioinformatics 1, Blacksburg, VA, USA

Abstract

Correspondence We report novel features of the genome sequence of Leptospira Key words A.L.T.O. Nascimento interrogans serovar Copenhageni, a highly invasive spirochete. Lep- • Leptospira interrogans Centro de Biotecnologia, tospira species colonize a significant proportion of rodent populations • Outer membrane proteins Instituto Butantan, worldwide and produce life-threatening infections in mammals. Ge- • Lipopolysaccharides Av. Vital Brazil, 1500 nomic sequence analysis reveals the presence of a competent transport • Transport 05503-900 São Paulo, SP • Regulatory systems Brasil system with 13 families of genes encoding for major transporters Fax: +55-11-3726-1505 including a three-member component efflux system compatible with E-mail: [email protected] the long-term survival of this organism. The leptospiral genome contains a broad array of genes encoding regulatory system, signal Research supported by FAPESP transduction and methyl-accepting chemotaxis proteins, reflecting the and CNPq. organism’s ability to respond to diverse environmental stimuli. The identification of a complete set of genes encoding the enzymes for the cobalamin biosynthetic pathway and the novel coding genes related to

Received 20 February, 2004 lipopolysaccharide biosynthesis should bring new light to the study of Accepted March 8, 2004 Leptospira physiology. Genes related to toxins, lipoproteins and several surface-exposed proteins may facilitate a better understanding of the Leptospira pathogenesis and may serve as potential candidates for vaccine.

Introduction lis. Leptospira consists of a genetically di- verse group of pathogenic and non-patho- Spirochetes are motile, helically shaped genic or saprophytic species (1). Leptospiro- which include the genera Leptospira, sis is a widespread zoonotic disease: trans- , and . Bor- mission to humans occurs through contact relia and Treponema are the causative agents with domestic or wild animal reservoirs or an of , and syphi- environment contaminated by their urine.

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Infection produces a wide spectrum of clini- features of the L. interrogans serovar Co- cal manifestations. The early phase of illness penhageni that should contribute to under- is characterized by fever, chills, headache, standing the molecular mechanisms of lep- and severe myalgias. The disease progresses tospiral physiology, pathogenesis and facili- in 5 to 15% of the clinical infections to tate the identification of candidates for broad- produce severe multisystem complications range vaccines. such as jaundice, renal failure and hemor- rhagic manifestations (2). In developed coun- Material and Methods tries, is associated with recre- ational activities (1) while in developing The sequenced strain, Fiocruz L1-130, countries it produces large urban epidemics was isolated as described by Nascimento et with mortality mainly during the rainy sea- al. (5). The sequencing strategy adopted fol- son (3). Leptospirosis also represents a ma- lows the basic outline of the Xylella genome jor economic problem producing abortions, project (6). Library construction, sequenc- stillbirths, infertility, failure to thrive, re- ing, assembly, and finishing were carried out duced milk production, and death in animals by the Agronomical and Environmental Ge- such as cows, pigs, sheep, goats, horses, and nomes consortium [http://aeg.lbi.ic.unicamp. dogs (1). Environmental control measures br] and by Instituto Butantan. The genome are difficult to implement because of the was assembled using phrap from shotgun long-term survival of pathogenic leptospires reads, cosmid reads and PCR-product se- in soil and water and the abundance of wild quences. Scaffolding was performed using and domestic animal reservoirs (1). Lep- domestic software. Finishing criteria are tospira are classified according to serovar based on consensus base phred quality of at status - more than 200 pathogenic serovars least 20 and consensus base covered by at have been identified. Structural heterogene- least one read sequence of each DNA strand ity in lipopolysaccharide (LPS) moieties ap- (6). The first base of the sequence was cho- pears to be the basis for the large degree of sen based on our hypothesis for the origin of antigenic variation observed among sero- the replication locus, which was in turn based vars (1). The development of vaccines has on the presence of certain genes and on GC- been pursued as a strategy for the prevention skew variation. Genome annotation and com- of leptospirosis. At present, vaccines are parative genomics were done as previously based on inactivated whole cell or mem- described (7). Detection of potentially sur- brane preparations of pathogenic leptospires face-exposed integral membrane proteins was which induce immune responses against lep- carried out as described by Nascimento et al. tospiral LPS (1). However, these vaccines (5). Sequences from 16S rDNA were manu- do not induce long-term protection against ally assembled using ESEE 3.2. Phyloge- infection and do not provide cross-protec- netic analyses were performed based on two tive immunity against heterologous leptospi- matrices (34 taxa and 1255 positions; 24 ral serovars. Protein antigens conserved taxa and 1375 positions) using the program among pathogenic serovars may contribute PAUP 4.0b8 (8). Divergence time was esti- to overcoming the limitations of the cur- mated based on 1445 positions of the 16S rently available vaccines. rRNA sequences. A constant rate of 1 to 2% The genome sequence of Leptospira per 50 million years was assumed (9). interrogans serovar Lai was recently pub- The sequences have been deposited lished (4) and comparative genome analysis in Genbank under accession numbers with L. interrogans serovar Copenhageni AE016823 (chromosome I) and AE016824 has been performed. We report here new (chromosome II).

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Results and Discussion supported monophyletic groups (Figure 2), one of them formed by the pathogenic strains Genome analysis (e.g., L. interrogans) and the other formed by the non-pathogenic strains (e.g., L. biflexa). The Leptospira genome consists of two At the base of the clade of the pathogenic circular chromosomes with a total of strains, L. inadai and L. fainei form a well- 4,627,366 base pairs (bp), chromosome I supported assemblage. Similar results were with 4,277,185 bp and chromosome II with obtained by Postic et al. (14) based on 16S 350,181 bp (5). Circular representations of rDNA analyses. In these analyses L. interro- both chromosomes are depicted in Figure 1. gans serovars formed a well-supported mono- The origin of replication of the large repli- phyletic cluster closely related to L. kirchneri con was identified between the dnaA and (Figure 2B). Considering a constant diver- dnaN genes, as in other bacterium genomes gence rate of 1 to 2% per 50 million years for (10). GC nucleotide skew (G - C/G + C) the 16S rDNA (9), separation time between analysis (11) confirmed the origin of replica- the two main assemblages (L. interrogans tion of the large replicon and indicated two versus L. biflexa) was estimated at 590 to putative sites for the small replicon. 295 million years. As previously described (12), rRNA genes L. interrogans belongs to a growing num- in L. interrogans are not organized into op- ber of multichromosomal prokaryotes, in- erons, as in most other bacteria, but are cluding Vibrio cholerae (15) and Ralstonia scattered over the chromosome I (Figure 1). solanacearum (16). The small replicon of L. L. interrogans serovar Copenhageni has one interrogans was previously suggested to be rrf gene, two rrl genes and two rrs genes a second chromosome based upon the local- coding for 5S, 23S and 16S rRNA, respec- ization of the metF gene which encodes an tively. As in other parasitic strains, L. inter- essential biosynthetic pathway enzyme, meth- rogans serovar Copenhageni has only one ylene tetrahydrofolate reductase (17). The rrf (5S) gene, which is located close to the genome sequence reveals that genes encod- origin replication region as described before ing enzymes for metabolic pathways, such for other strains of L. interrogans (12). Com- as glycolysis and the tricarboxylic acid cycle, paring the complete rrs (16S) sequences for as well as the enzymes for biosynthesis path- L. interrogans, serovars Copenhageni, Lai ways of amino acid and co-factor are also and Canicola the identity among the se- distributed between the two chromosomes. quences is of 99.9 to 100%. The rrf (5S) Sequence analysis of chromosome II shows sequence identity comparing Lai and Co- that an almost complete operon of genes penhageni is 100% and the rrl (23S) is 99%. coding for the protoheme biosynthesis path- Based on ribosomal genes, Copenhageni and way is present (hemAIBCENYH). Although Lai are closely related, as supported by the no homologue of the gene coding for uropor- whole genome comparison (5). phyrinogen III synthetase (hemD) was found, The are divided into three experimental evidence has shown that the major phylogenetic groups, or families: Spi- hemC gene is able to cope with hemD activ- rochaetaceae, which includes Borrelia and ity (18). Therefore, L. interrogans has the Treponema among others; Brachyspiraceae ability to synthesize protoheme de novo. In and Leptospiraceae, which contain two gen- addition, 13 genes clustered in chromosome era, Leptospira and Leptonema (13). Phylo- II coding for the cobalamin biosynthesis path- genetic analyses based on 16S rDNA se- way were identified (cobC, cobD, cbiP, cobP, quences, using Leptonema as an outgroup, cobB, cobO, cobM, cobJ, cbiG, cobI, cobL, show that Leptospira are split into two well- cobH, cobF) (Figure 3). Orthologues of

Braz J Med Biol Res 37(4) 2004 462 A.L.T.O. Nascimento et al. aller ively) color-coded by category role; circle 3: G + C content. Numbers serovar Copenhageni genome. The two large and small chromosomes L. interrogans Circular representation of the are 23S, 16S and 5S, respectively. Note: the two replicons not drawn to same scale. Chromosome II (CII) is 12 times sm rrf and Leptospira interrogans. rrs , rrl than chromosome I (CI). Figure 1. Circular maps of the two chromosomes in are presented. Circles 1 and 2 (from the outside in), all predicted protein-coding regions (forward reverse strand, respect on the outer circle are base pairs. rRNA genes,

Braz J Med Biol Res 37(4) 2004 Leptospira interrogans genome features 463 is used Leptonema GenBank accession numbers are in parenteses. , based on a matrix with 24 taxa and 1375 positions. B . Consensus phylogenetic distance tree constructed using 16S rDNA sequences. Numbers on the branches are bootstrap values (2000 Leptospira interrogans , Based on a matrix with 34 taxa and 1255 positions; A Figure 2. Phylogenetic analysis of as outgroup. replicates) for distance (top), parsimony (below the branch) and maximum likelihood (10 replicates, below branch, italics).

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cobGKN genes, known to be involved in the tains both uptake and efflux transport sys- cobalamin pathway (19) were not found. tems, and ATP hydrolysis energizes the trans- However, there are two predicted coding port. The porters of the ABC superfamily sequences inside this operon in chromosome consist of two integral membrane proteins II that could perform these steps. One has an (that determine specificity of the transported oxidoreductase NAD-binding domain solute) and two cytoplasmic ATP-hydrolyz- (LIC20133) and the other is a [2Fe-2S] ferre- ing proteins present as homodimers or heter- doxin involved in electron transfer odimers. The uptake systems (but not the (LIC20135). In addition, other genes present efflux systems) additionally possess extra- in the genome coding for reductases such as cytoplasmic solute-binding receptors (one LIC11145, LIC13354, LIC12391, and or more per system), which in Gram-nega- LIC10522 could also cope with these activi- tive bacteria are found in the periplasm. We ties. The presence of cysG in chromosome I, found 21 ABC efflux systems, including a gene that encodes a multifunctional pro- drug and heavy metal export and detoxifica- tein with methylase, oxidase and ferrochela- tion, lipoprotein-releasing and hemolysin tase activities (20), may also be a cobalt- export systems. The remaining 9 are ABC

inserting enzyme in the B12 pathway. Other uptake systems, including iron, sulfate, genes involved in this biosynthesis pathway nickel, phosphate, dipeptide, amino acid, were found in chromosome I (cysG/hemX/ and carbohydrate uptake. There is one F- cobA, cobT/cobU, cobS) (Figure 3). In fact, type ATP-synthase system (8 proteins), as experimental evidence has recently shown mentioned above, and 3 P-type cation-trans- that leptospires can grow in medium in the port ATPases (5 proteins). absence of B12 (Hartskeerl RA, unpublished We found 59 secondary electrochemical results). This is contrary to the previous potential-driven transporters (65 proteins) statement that L. interrogans is unable to including the largest family of secondary synthesize B12, and that is why it is an transporters, the resistance-nodulation-cell essential component of the EMJH semi-syn- division superfamily with 12 members, 5 of thetic medium (1,4). Thus, L. interrogans, which belong to the heavy metal efflux fam- unlike the spirochetes Borrelia burgdoferi ily (with 7 proteins) and 7 to the multiple and , have the complete drug efflux family (with 8 proteins). These repertoire of genes for de novo synthesis of secondary efflux transporters are energized protoheme and cobalamin. The functional by proton-motive force and show the widest link between the two replicons supports the substrate specificity among all known multi- view that the small replicon is in fact a drug pumps, ranging from most of the cur- second chromosome. rently used antibiotics, disinfectants, dyes, and detergents to simple solvents. The sec- Transport ond largest secondary transporter family is the major facilitator superfamily with 9 mem- Among the 220 Leptospira transport pro- bers including drug:cation antiporters (8 pro- teins, we found 148 proteins comprising 108 teins) and a glycerol-phosphate:inorganic different major primary and secondary trans- phosphate antiporter (1 protein). Additional porters (Figure 4), as defined by Paulsen et secondary transporters include three sodium- al. (21). There are 34 major primary ATP- solute symporters (one sodium-glucose co- driven transporters including 30 ABC-trans- transporter), three sodium-bile-acid sympor- porters (53 proteins), the largest protein fam- ters, and symporters for phosphate, di- ily in L. interrogans, as usually is the case for tripeptides, glutamate, amino acids, uracil, bacteria (22). The ABC superfamily con- sulfate as well as an ammonium:potassium

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Figure 3. The cobalamin biosynthetic pathway of Leptospira interrogans. Genetic organization of the cobalamin biosynthetic locus in the two chromosomes (CI and CII) of L. interrogans serovar Copenhageni.

Figure 4. Major transporters of Leptospira inter- rogans. Major primary transporters are driven by ATP hydrolysis and include ABC-transporters, P- type ion transporters and the ATP-synthase. All other transporters indicated are the major sec- ondary transporters, which are driven by the transmembrane electrochemical potential. RND = resistance-nodulation-cell division superfamily.

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antiporter, a glycerol-3-P:Pi antiporter, two which may have functions analogous to ani- drug-sodium antiporters and two polysac- mal ferritin, are also present and may pro- charide exporters. Secondary transporters vide both iron detoxification and storage, also include 7 members of the TonB family that would prevent free iron in Leptospira of auxiliary proteins for energizing the outer from driving oxidative reactions. membrane receptor-mediated active trans- port. Leptospira has many iron-transporting Regulatory systems and signal transduction proteins in addition to the cobalamin/iron periplasmic binding protein component of There are many genes encoding signal the ABC-transport system (LIC13403) men- transduction proteins in Leptospira, indicat- tioned above. Four members of the outer ing that this organism has developed a vast membrane receptor family have been identi- array of regulatory systems that enable it to fied, which are probably involved in the respond to environmental signals. This vari- transport of iron dicitrate (LIC10714), hemin ety of regulatory domains is found in non- (LIC10964), ferric hydroxamate (LIC11345) obligate parasitic bacteria, indicating a much and ferrienterobactin (LIC12998). In addi- greater need to interpret the signals from the tion, an orthologue of the ferrous iron up- environment in order to respond properly, take (FeoB) protein of E. coli was found while obligate parasites have a much lower (LIC11402). FeoB has been characterized as number and variety of domains in their sig- an Fe2+ uptake system and possesses an nal transduction proteins (25). There are 80 ATP/GTP binding motif at its N-terminal genes encoding components of the phospho- hydrophilic domain, therefore being prob- rylation-mediated signal transduction path- ably energized by ATP or GTP hydrolysis way: 29 histidine kinases (HK), 30 response (23). regulators (RR) and 18 hybrid kinase/regu- lators (HK/RR). These members of the two- Oxygen defense component systems present several domains organized into different arrangements (Fig- The Leptospira genome contains several ure 5). Nineteen of the histidine kinases are genes encoding enzymes with peroxidase located in the inner membrane, nine are cy- activities, such as catalase, glutathione per- toplasmic and one is probably found in the oxidase and thiol peroxidase. However, su- periplasm, as predicted by the PSORT pro- peroxide dismutase orthologues and two gram [http://psort.nibb.ac.jp/]. important regulons, SoxRS and OxyR, nor- On the other hand, only a third of the mally responsible for the defense against hybrid HK/RR are membrane-bound, sug- oxidative stress, are absent (5). It is possible gesting that these hybrid proteins are most that metalloporphyrins (24) could provide likely to be involved in phosphorylation cas- defense against oxidative damage, since L. cades, although some of them have the sen- interrogans is competent for porphyrin bio- sor PAS domain. The PAS domain has been synthesis and has metal uptake transport pro- reported to sense different environmental teins. Genes coding for co-migratory bacte- stimuli, like oxygen, redox state, nitrogen rioferritin (LIC20093 and LIC10732), thiol availability or light, and it may or may not be peroxidase (LIC12765) and peroxiredoxin associated with co-factors such as heme or (LIC11219) with alkyl hydroperoxide re- FAD (26). A two-component protein con- ductase (AhpC) and thiol-specific antioxi- taining a PAS sensor domain was found to dant activities, respectively, were also iden- be required for virulence of M. tuberculosis tified. Two predicted coding sequences for in mice, probably because it senses radical bacterioferritin (LIC11310 and LIC13209), oxygen intermediates generated by macro-

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Figure 5. Domain architecture of putative signal transduction proteins from Leptospira interrogans. The domain organization of histidine kinases, response regulators, hybrid histidine kinase/response regulators and other peptides probably associated with signal transduction is shown schematically. The number of peptides containing each domain organization is shown on the right, and the genes are found distributed over the large or small replicons, except for the seven peptides containing the PAS/GGDEF arrangement, which are encoded by genes located in tandem in the large replicon. AAA = ATPase; Cache = small ligand binding domain; EAL = phosphodiesterase; GAF = cGMP-binding domain; GGDEF = diguanylate cyclase; HK = histidine kinase domain; HTH = helix-turn-helix; PAS = sensor domain (may include a PAS/PAC arrangement); PP = phosphatase; RR = response regulator domain; SP = signal peptide; TM = transmembrane region.

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phage phagocytosis (27), and probably this include three related genes encoding puta- is also the case for Leptospira. tive DNA-binding proteins (LIC20104, The response regulators are the cytoplas- LIC20105 and LIC20178), which contain 6 mic effectors of the message, which become transmembrane domains in the amino-termi- functional after being phosphorylated at an nus and one helix-turn-helix domain of the aspartate residue by the cognate histidine AraC type at the carboxyl-terminus. These kinase. The RRs may possess a second effec- genes are present in chromosome II, and two tor domain (Figure 5), which will perform its of them are clustered. Orthologues with a C- ultimate function, such as the DNA-binding terminal DNA-2binding domain and a hy- helix-turn-helix domain that allows the RR drophobic N-terminal region were described to regulate transcription. Other noticeable in other bacteria, including Borrelia and Tre- domains found in L. interrogans RRs are the ponema (31), but their function is unknown. GGDEF and EAL motifs, which correspond to putative diguanylate cyclase and phos- Motility and chemotaxis phodiesterase domains, respectively, and a phosphatase domain similar to the mamma- The L. interrogans genome comprises a lian phosphatase 2C that may be involved in relatively large number of motility and che- the phosphorelay. motaxis genes. Enteric bacteria usually have Cyclic nucleotides are likely to have a about 50 genes coding for structural and major regulatory role in Leptospira. There functional proteins involved in motility (32). are 19 homologues of adenylate/guanylate A similar number of genes have been identi- cyclases, two specific phosphodiesterases fied for the spirochetes T. pallidum and B. and 7 cyclic nucleotide-binding proteins that burgdorferi (33,34). Apparently, the motil- probably have a regulatory function. There ity and chemotaxis apparatus of L. interro- are 12 genes encoding proteins containing gans is more complex since its genome con- the GGDEF motif, seven of which are orga- tains at least 79 putative motility-associated nized in tandem in chromosome I (LIC11131 genes. All genes are well conserved among to LIC11125), and they also have a PAS/ L. interrogans, T. pallidum and B. burgdor- PAC sensor domain in the amino-terminal feri and 42 genes were found to be common region, suggesting that they are the product to all three genera. However, the leptospiral of gene duplication. The diguanylate cyclase genome contains multiple copies of a num- activity of the GGDEF domain is required ber of motility-associated genes, accounting for a novel regulatory mechanism involving partly for the higher number. For example, bis-(2',5')-cyclic diguanylic acid (c-di-GMP) the genome of serovar Copenhageni con- as an allosteric activator (28). Two of the tains 5 flaB genes, 4 motB genes and 2 motA GGDEF-containing proteins also have the genes compared to 3 flaB genes and one Cache domain, which is involved in chemo- copy of both motA and motB in the T. pallidum taxis signal transduction (29), an important genome and one copy each of flaB, motA, feature for Leptospira. There are eight puta- and motB in the B. burgdorferi genome. In tive diguanylate phosphodiesterases contain- addition, the L. interrogans genome con- ing an EAL domain, with three of them being tains 11 putative genes encoding methyl- associated with an RR domain (Figure 5). L. accepting chemotaxis proteins, which is interrogans also presents in its genome three roughly twice as many as in T. pallidum and serine/threonine kinases and two hybrid HK/ B. burgdorferi. Forty-eight of the 79 motil- RR with a GAF domain, which is a binding ity-associated genes are positioned into 14 domain for cGMP (30). gene clusters varying in size from 2 to 8 Other interesting findings in the genome genes. Thus, like in T. pallidum and B. burg-

Braz J Med Biol Res 37(4) 2004 Leptospira interrogans genome features 469 dorferi, the majority of the structural and ity. These putative hemolysins were first functional motility genes are positioned in identified in the spirochete Brachyspira hyo- potential operons. However, the operons dysenteriae. Hemolytic and cytotoxic activi- probably underwent extensive rearrange- ties of the recombinant TlyA, TlyB and TlyC ments as they are generally smaller, often proteins, expressed in E. coli, were detected corresponding to only parts of the major (36). TlyB belongs to the family of Clp ATP- Treponema and Borrelia operons. For ex- dependent proteases (37) and there are 3 ample, the flgB operon in B. burgdorferi genes coding for structurally related proteins consisting of 26 genes (35) has been dis- (LIC10339, LIC11814 and LIC12017). Thus, rupted into 6 fragments dispersed at 5 posi- 5 genes of the tlyABC class (LIC10284, tions in the leptospiral genome. Differences LIC10339, LIC11814, LIC12017, and in number of genes and operon organization LIC13143) were identified in the L. interro- might be associated with the high flexibility gans genome. of pathogenic leptospires enabling them to LIC12134 codes for an HlpA-related pro- survive and adapt to a variety of environ- tein which was characterized as a hemolysin ments and hosts. in , a common oral bacterium associated with periodontitis (38). Hemolysins Another identified gene (LIC10325) is re- lated to the hlyX gene which codes for a The primary lesion caused by Leptospira predicted hemolysin previously identified in is the damage to the endothelium of small L. borgpetersenii serovar Hardjo (Acces- blood vessels, leading to hemorrhage and sion number AAF09252, unpublished re- localized ischemia in multiple organs. As a sults). LIC11352 should also be mentioned consequence, renal tubular necrosis, hepato- as the gene which codes for LipL32 or cellular damage, meningitis, and myositis HAP-1, a ubiquitous lipoprotein of patho- may occur in the infected host (1). Hemoly- genic Leptospira (see Lipoproteins section sins may play a fundamental role in this toxic below) with hemolytic activity (39). process. Several genes coding for predicted hemolysins were identified in the L. interro- Surface-exposed proteins gans genome. Some of them are structurally related to sphingomyelinases C (LIC10657, Pathogenic leptospires require several LIC12631, LIC12632, LIC11040, and types of surface-exposed proteins for the LIC13198). Although generically called purpose of colonization and survival in the sphingomyelinases, it is possible that these mammalian host. Surface-exposed proteins genes code for hydrolases that act not only may be categorized as nonspecific porins, on sphingomyelins but also on other specific channels for nutrient acquisition, sphingolipids. Erythrocytes may represent a efflux channels, lipoproteins, adhesins, S- target for these enzymes since they are rich layer glycoproteins, peripheral membrane in glycosphingolipids like the antigenic de- proteins, or surface-maintenance proteins. terminants of the ABO system. Interestingly, Aside from S-layer proteins and peripheral LIC12631 and LIC12632 are organized as membrane proteins, these surface-exposed an operon, which may suggest a concerted proteins would be expected to be integrated expression and action. into the outer membrane via transmembrane The other class of genes coding for regions or lipid modification. The leptospi- hemolysins, tlyABC, was identified. Although ral genome contains homologues of SecY they were assigned to the same TlyABC and other secretory proteins involved in ex- class, they do not present structural similar- porting proteins with signal peptides across

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the cytoplasmic membrane. The leptospiral acquisition of certain trace nutrients. For genome also contains both the standard sig- example, iron is essential for leptospiral nal peptidase and the lipoprotein signal pep- growth and is bound by a number of high- tidase. The standard signal peptidase is in- affinity binding proteins in mammals, which volved in the hydrolysis of signal peptides of restrict its availability. Bacteria scavenge transmembrane outer membrane proteins and trace nutrients, including iron, heme, and periplasmic proteins, releasing them from vitamin B12, from their environment utiliz- the cytoplasmic membrane. Lipoprotein sig- ing the cytoplasmic membrane protein TonB nal peptidase hydrolyzes the signal peptides and a series of “TonB-dependent” OMPs of lipoproteins prior to lipidation of the N- (Figure 6). After binding, transport of the terminal cysteine. nutrient across the outer membrane into the periplasm by this type of channel is an en- Transmembrane outer membrane proteins ergy-dependent step requiring interaction of the TonB-dependent OMP with TonB. The Analysis of the L. interrogans genome L. interrogans genome contains a TonB reveals 83 beta-sheet transmembrane outer orthologue (LIC10889) and a large number membrane proteins (OMPs) and all except of TonB-dependent OMPs: LIC20151, one (OmpL1, LIC10973) (40) were previ- LIC20214, LIC10998, LIC10964, LIC10714, ously unknown. An example of a newly LIC12374, LIC12898, LIC12998, LIC11694, identified leptospiral protein with a trans- LIC11345, LIC10882, LIC10881, and membrane beta-sheet structure is LIC10642 LIC10896. which is predicted to be a member of the The leptospiral genome also contains OMP superfamily. OMPs involved in efflux pathways (Figure Among these newly identified OMPs is a 6). The type I efflux system is represented family of predicted coding sequences by an orthologue of TolC (LIC13135), the (LIC20202, LIC10995, LIC11465, and outer membrane exporter of hemolysin and LIC11103) belonging to the superfamily of drugs, along with an orthologue of CusC alpha/beta hydrolases, which includes bac- (LIC11941), an outer membrane exporter of terial lipases. Another transmembrane OMP copper ion. A second type of efflux pathway is LIC11623, a member of a family of highly found in the leptospiral genome belongs to conserved proteins of Gram-negative bacte- the resistance-nodulation-cell division su- ria, including N. meningitides Omp85, which perfamily, a three-member complex that cata- is thought to be a chaperonin involved in the lyzes substrate efflux via an H+ antiport mech- assembly of OMPs in the outer membrane anism. The three-member complex consists (41). OmpL1 belongs to the class of nonspe- of an resistance-nodulation-cell division cific porins allowing passage of small mol- transporter, a membrane fusion protein, and ecules (<1000 Daltons) across the leptospi- an outer membrane factor involved in the ral outer membrane (40). Nonspecific porins export of proteins, carbohydrates, drugs or allow both influx of nutrients and efflux of toxic heavy metals (Figure 6). The leptospi- products of metabolism. Another example is ral orthologues are most closely related to LIC11458, which is predicted to be a mem- those of the Alcaligenes eutrophus cobalt/ ber of the porin superfamily. cadmium/zinc export system consisting of the resistance-nodulation-cell division Specific channels transporter CzcA (LIC12224, LIC15510 and LIC11938), CzcB (LIC12306 and Survival in the mammalian host environ- LIC11940), and CzcC (LIC12307 and ment by bacterial pathogens requires the LIC11941). In addition, L. interrogans has

Braz J Med Biol Res 37(4) 2004 Leptospira interrogans genome features 471 two orthologues of the cation efflux system LipL31 (LIC11456) and LipL71 (LIC11003) protein CzcD (LIC11714 and LIC13205), (46). A total of 184 predicted coding se- which are members of the cation diffusion quences in the L. interrogans genome were facilitator family. CzcD of Bacillus subtilis found to have a lipoprotein signal peptidase has been shown to catalyze divalent cation cleavage site (5). All proposed lipoprotein- (Zn2+ or Cd2+) efflux in exchange for the coding sequences conform to the rule of uptake of two monovalent cations (K+ and having an L, I, V, or F in the -3 and/or -4 H+) in an electroneutral process energized position relative to cysteine and most of by the transmembrane pH gradient (42). them have A, G, S, or N in the -1 position relative to cysteine (47). Lipoproteins Proteolytic functions can be assigned to some of the newly identified lipoproteins: Experimental evidence for fatty acid four lipoproteins are thermolysin homologues modification of leptospiral lipoproteins has (LIC10715, LIC13320, LIC13321, and been described for the outer membrane lipo- LIC13322), and one is a predicted metallo- proteins LipL32 (LIC11352) (43), LipL36 protease (LIC11834). Several lipoproteins (LIC13060) (44), and LipL41 (LIC12966) may be involved in hemolysis: two lipopro- (45). The cytoplasmic membrane also con- teins are sphingomyelinase homologues tains lipoproteins, as demonstrated for (LIC10657 and LIC12632), and one is a

Figure 6. Model of leptospiral membrane architecture. Leptospires have two membranes, an outer membrane (OM) and a cytoplasmic or inner membrane (IM). As in Gram-positive bacteria, the peptidoglycan (PG) cell wall is closely associated with the IM. The leptospiral surface is dominated by lipopolysaccharide (LPS) carbohydrate side chains. Subsurface proteins include the cytoplasmic protein, GroEL, and the periplasmic endoflagella (EF). The IM contains lipoproteins such as LipL31 and transmembrane proteins such as signal peptidase (SP) and ImpL63. The OM contains lipoproteins including LipL41 and LipL36, and transmembrane proteins including the porin, OmpL1. Genomic sequence analysis reveals several novel types of outer membrane proteins (OMPs), including TonB- dependent OMPs involved in nutrient acquisition. BtuB is an example of a TonB-dependent OMP involved in uptake of vitamin B12. The type I efflux system is represented by TolC, which forms a complex with ATP-binding cassette (ABC) transporters to export hemolysins and other cytoplasmic components. The leptospiral genome also contains genes involved in a three-component efflux system consisting of an outer membrane factor (OMF), membrane fusion protein (MFP), and an inner membrane transporter, in this case CzcA, which is involved in heavy metal detoxification.

Braz J Med Biol Res 37(4) 2004 472 A.L.T.O. Nascimento et al.

phospholipase D homologue (LIC11754). plasmic form, glpQ. The leptospiral genome Lipoprotein LIC10972 is predicted to be contains two homologues of this enzyme, located in the outer membrane and is a MauG LIC13182 and LIC10293. The former should homologue belonging to a family of cyto- be the cytosolic form because it lacks a chrome c peroxidases that may be involved signal peptide while the latter should be the in defense against hydrogen peroxide. Other exported form because it has an N-terminal lipoproteins with putative enzymatic func- signal peptide. In other spirochetes, GlpQ is tions are homologues of glucose dehydro- associated with the outer membrane (50). genase (LIC12294) and cholesterol oxidase (LIC13202). Cytoplasmic membrane proteins

S-layer and peripheral membrane proteins The leptospiral genome contains a num- ber of proteins that belong to a large family S-layers are thought to provide structural of prokaryotic proteins with homology to the integrity to the bacterial surface (48). Al- peptidoglycan-associated portion of E. coli though an S-layer has not been observed in OmpA (51). These proteins are predicted to pathogenic leptospires, the genome contains be either cytoplasmic membrane (LIC20250, at least two proteins with S-layer homology LIC10592, LIC13479, and LIC10050) or (LIC10868 and LIC12952). Experimental periplasmic proteins (LIC10537 and evidence is needed to determine whether LIC10191), rather than OMPs, which is con- these proteins are actually S-layer compo- sistent with experimental evidence that the nents. spirochetal cell wall is more closely associ- Like S-layer proteins, peripheral mem- ated with the cytoplasmic membrane than brane proteins are not integral membrane the outer membrane. An interesting protein proteins. LipL45 is processed to a peripheral family is the mechanosensitive ion channel membrane associated with the outer mem- (LIC20069 and LIC12671). Two members

brane, P31LipL45 (49). P31LipL45 expression is of this protein family of M. jannaschii have dramatically up-regulated in stationary phase been functionally characterized and both cultures, and for this reason may have a form mechanosensitive ion channels (52). membrane-stabilizing function. At this time Therefore, this family is likely to also en-

it is unclear whether P31LipL45 is surface- code mechanosensitive channel proteins in exposed. Interestingly, the leptospiral ge- L. interrogans, playing a physiological role nome contains a number of predicted coding in bacterial osmoregulation. sequences with homology to LipL45 (LIC20102, LIC20114, LIC13414, and Lipopolysaccharides LIC10123). Lipopolysaccharides distinguish the lep- Surface-maintenance proteins tospiral surface from those of the other inva- sive spirochetes. Changes in genes involved Bacteria are likely to have a variety of in the LPS biosynthesis apparatus are thought proteins involved in maintaining the surface to account for serovar diversity among lepto- structural integrity. One such protein, glyc- spires (53). It has been shown that the lep- erophosphoryl diester phosphodiesterase, is tospiral LPS resembles a typical LPS from a protein belonging to this category which Gram-negative bacteria, containing pentoses, has been found in all spirochetal genomes hexoses and heptoses. Although the pre- studied to date. E. coli has two forms of this dominant sugar is rhamnose, a large variety enzyme, a cytosolic form, ugpQ, and a peri- of other sugar residues are found (1). Anti-

Braz J Med Biol Res 37(4) 2004 Leptospira interrogans genome features 473 bodies raised against LPS from different Seventy-seven other genes, probably re- Leptospira strains during infections are re- lated to LPS biosynthesis, were identified lated to polysaccharide structure in terms of along the genome. All the genes related to its sugar composition, number, repetitive- lipid A biosynthesis described in E. coli ness, and ramification (1). In Leptospira, as were identified in the Leptospira genome in many other bacteria, at least part of the (lpxA, lpxC, lpxD, lpxB, lpxK). However, the genes coding for enzymes of the polysaccha- predominant fatty acids in lipid A of L. ride biosynthesis pathway are found clus- interrogans are dodecanoic and hexade- tered in a region of chromosomes named O- canoic acid instead of hydroxymyrystoyl (14 antigen gene cluster (rfb locus) (53). carbons), which is the signature of Gram- The complete genome analysis reveals a negative bacteria (1). Comparative analysis large portion of about 119 kb (genome posi- between LpxA of E. coli and P. aeruginosa tion 2.538.470-2.554.255), in which all the (55) showed that few structural differences 108 predicted coding sequences are tran- in this enzyme determine changes in the fatty scribed in the same orientation (Figure 7). acid chain size incorporated during lipid A Interestingly, this region is dense in genes biosynthesis. The lower endotoxicity of lep- related to LPS biosynthesis (Table 1) and tospiral LPS as compared to LPS from Gram- includes the O-antigen gene cluster previ- negative bacteria has been reported (56). It ously described in L. interrogans serovar was also reported that leptospiral LPS in- Copenhageni (53,54). In the first 14 kb of duces a TLR2-dependent cell activation, in- this region the predicted genes seem not to stead of LTR4, the receptor frequently in- be related to LPS biosynthesis, but to DNA volved in the LPS immune response (57). replication, and some genes code for riboso- The lower endotoxicity and the difference in mal proteins. In the other 105 kb of this the mechanism of cell immune response ac- region there are 94 predicted genes 56 of tivation are supposed to be related to differ- which are clearly related to LPS biosynthe- ences in the lipid A structure. sis. These predicted genes encode enzymes Genes encoding enzymes of Kdo biosyn- for nucleotide sugar biosynthesis, such as thesis (kdsA, kdsB) such as Kdo-transferase the enzymes for dTDP-rhamnose biosynthe- (waaA or kdtA), which catalyzes the binding sis (54) for CMP-N-acetylneuraminic acid of Kdo to lipid IVA, were identified. Al- and for perosamine synthase. In addition, though typical Kdo was found in Leptospira many genes coding for sugar transferases, LPS, it was reported to be substituted at sugar modifications and for the O-antigen different carbon positions (1). processing, Wzx-flippase and Wzy-poly- Four paralogues encoding MsbA, the pro- merase, involved in oligosaccharide export- tein that transfers the flippase of lipid A in ing and assembly of the LPS, were identi- fied. Some genes encoding proteins of the Table 1. Number of genes at the lipopolysaccha- lipid A biosynthesis (lpxD) and transport ride 119-kb locus. (msbA) are also found is this region. The comparison with the corresponding Predicted protein function Nucleotide sugar biosynthesis 22 region in the genome of L. interrogans Sugar transferase 17 serovar Lai (4) revealed only three distinct Sugar epimerase 10 predicted coding sequences: two extra cop- Lipid A and polysaccharide transport 4 Hypothetical 26 (17 conserved) ies of genes for aminotransferase (LIC12197 Aminoacyl tRNA synthase 3 and LIC12198) and the absence of a gene DNA metabolism 4 encoding galactoside O-acetyltransferase Ribosomal protein 3 Others 19 (LA1622).

Braz J Med Biol Res 37(4) 2004 474 A.L.T.O. Nascimento et al. serovar L. borgpetersenii genes related to rhamnose biosynthesis in rfb ate the homologous genes from , was first indicated as the F, E, A, B, D, C serovar Copenhageni genome with 108 predicted coding sequences transcribed in the same L. interrogans The 119-kb region of Leptospira interrogans. Copenhageni (54). Figure 7. Lipopolysaccharide region of L. interrogans Hardjobovis (53). The region containing the predicted coding sequences, indicated by letters orientation. Orange arrows indicate predicted genes encoding polysaccharide biosynthesis-related proteins. Shaded regions indic

Braz J Med Biol Res 37(4) 2004 Leptospira interrogans genome features 475

Table 2. Insertion sequence (IS) elements present in Leptospira interrogans serovar Copenhageni.

Element Family Copies Length (bp) Transposase Terminal inverted repeats

ISlin1 IS110 11 1,423 2 (126aa + 195) 5’end AAACTCAACAtctCGCTCTTTAcGAATCGC 3’end GCGATTCaTAAAGAGCGttgTGTTGAGTTT

IS1500 IS3 8 1,234 2 (282aa + 99aa) 5’end TAACCTaGtgACGaAttAttGGACACaTTTT 3’end AAAAgGTGTCCgtTttTaCGTagCcAGGTTA

IS1501 IS3 5 1,230 2 (132aa + 85aa) 5’end TGAagTAGTcACATAAAAGTgGACAgCcaTTT 3’end AAAaaGgTGTCtACTTTTATGTtACTAggTCA

the inner membrane from the cytoplasmic side ously described insertion sequence (IS) ele- to the periplasmic side, were identified. One of ments IS1500, IS1501 (58), reminiscent in- the msbA genes is located in the 119-kb region. sertions of IS1533 (59) and IS1502 (60) and Another msbA gene is located upstream of an IS recently identified that we designated lpxK, as reported in many other genomes (55). ISlin1 (5) (Table 2). IS1500, IS1501 and It will be interesting to compare the set of ISlin1 account for 24 insertions distributed genes coding for enzymes related to LPS bio- throughout chromosome I. Together, IS ele- synthesis identified in the Leptospira genome ments and transposases comprise 2% of the to the orthologues in other microorganisms, genome. These elements are related to major in order to correlate LPS structural differ- IS families such as IS110 and IS3 that are ences and endotoxic activity. defined by their conserved amino acid motif (DDE) in the transposase. So far all the Comparative genomics and insertion insertions were found in intergenic regions, sequences having no mutagenic effects on L. interro- gans genes. A three-way comparison between the L. The L. interrogans genome provides new interrogans genome and the genomes of B. insights into biosynthesis pathway, transport burgdorferi and T. pallidum yields the fol- families, environmental response, and patho- lowing results: 1167 (31%) of the genes in L. genesis. A broad array of regulatory system interrogans Copenhageni are found in B. proteins enable this organism to respond to burgdorferi and/or in T. pallidum, 666 (41%) environmental signals. A new group of genes of the genes in B. burgdorferi are found in involved in LPS biosynthesis may contribute the Copenhageni genome, and 589 (57%) of to the elucidation of serovar diversity among the genes in T. pallidum are found in the L. leptospires. Several categories of surface- interrogans genome. A total of 362 pre- exposed proteins required for colonization dicted genes were found to be shared by all and survival in the mammalian host were three spirochetes, 45 of which are hypotheti- identified. These proteins may have a role in cal (detailed list in our project website: http:/ mechanisms of leptospiral pathogenesis and /aeg.lbi.ic.unicamp.br/world/lic/). These re- protective immunity. Available vaccines are sults show that a thorough analysis of the serovar-specific and have low efficacy (1). genome can significantly contribute to the Surface-exposed proteins that are conserved understanding of spirochete biology. among pathogenic serovars may be used for Our analysis of the L. interrogans ge- vaccine development for the prevention of nome revealed the presence of the previ- leptospirosis.

Braz J Med Biol Res 37(4) 2004 476 A.L.T.O. Nascimento et al.

Acknowledgments

We are deeply indebted to Drs. I. Raw (Fundação Consortium of the network Organization for Nucleotide Butantan, São Paulo, SP, Brazil) for use of laboratory Sequencing and Analysis (ONSA) for the genome sequenc- facilities and valuable support. We thank Dr. Albert I. Ko ing data, and Dr. L.C.C. Leite (Instituto Butantan) for a (Fiocruz, Salvador, BA, Brazil) for the strain Fiocruz L1- critical reading of this manuscript. 130, the Agronomical and Environmental Genomes (AEG)

Linear representation of the Leptospira interrogans serovar Copenhageni chromosomes (CI and CII). Genes are colored according to their biological role. Arrows indicate the direction of transcription. Insert The pie representation indicates the distribution of the number of genes according to their biological role. The numbers below protein-producing genes correspond to gene identification numbers (IDs). L

Additional information is contained in the project website http://aeg.lbi.ic.unicamp.br/world/lic/

Braz J Med Biol Res 37(4) 2004 Leptospira interrogans genome features 477

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