Academic literature on the topic 'Leptospira recombinant protein'

ABSTRACT 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.

<strong>ABSTRACT</strong> Major immunodominant proteins of <em>Leptospira interrogans</em> Icterohaemorrahagiae were cloned, expressed in prokaryotic system was purified and confirmed by western blot. The protective efficacy of purified recombinant proteins were assayed synergistically with adjuvant in hamster models. Three combination were assessed for homologous protection (i.e, r<em>Lip</em>L41, r<em>Omp</em>L1, r<em>Hap</em>I and r<em>Sph</em>H; r<em>Lip</em>L41, r<em>Omp</em>L1, r<em>Lig</em>A and r<em>Lig</em>B; r<em>Lig</em>A, r<em>Lig</em>B, r<em>Hap</em>I and r<em>Sph</em>H) each protein at 25 &micro;g and 37.5 &micro;g concentration. Among the combinations tested combination of immunogloblulins (r<em>Lig</em>A and r<em>Lig</em>B) with hemolysins (r<em>Hap</em>I and r<em>Sph</em>H) confered 100% protection in both the concentration tested, that is evident through the elevated elicited antibody levels.&nbsp; The combination of outer membrane proteins and the immunoglobulins stands next which is comparable with the former combination.&nbsp; The blend of recombinant outermembrane proteins and hemolysins did effect protection which is lesser than the coalescence of immunoglobulins and hemolysins.&nbsp; Membrane fractions of <em>E. coli</em> harboring pET15b used as internal control showed neither homologous protection nor enhanced antibody levels used to compare the experimental proteins over hamster models.&nbsp; Placebo controls receiving PBS showed similar results as that of internal controls. <strong>Key words </strong>: <em>Leptospiral&nbsp; recombinant protein, recombinant proteins, outer membrane proteins, hemolysins, immunoglobulin like proteins, immune response, challenge studies, , homology modeling</em> <strong><em>REFERENCES:</em></strong> Alves, V.A., Gayotto, L.C., Brito, T.De., Santos, R.T., Wakamatsu, A., Vianna, M.R. and Sakata, E.E. (1992).&nbsp; <em>Leptospiral</em> antigens in the liver of experimentally infected guinea pig and their relation to the morphogenesis of liver damage. Exp. Toxicol. Pathol. 44(7): 425-434. Artiushin, S., Timoney, J.F., Nally, J. and Verma. A. (2004).&nbsp; Host inducibile immunogenic <em>Sphingo</em>myelinases like protein, Lk73.5, of <em>Leptospira</em> <em>interrogans</em>.&nbsp; Infect. Immun. (72): 742-749. Bartlett, J.M.S. and Stirling, D. (2004). DNA Extraction form Fungi, Yeast and Bacteria. PCR Protocols Second Edition, Methods in Molecular Biology, Humana Press, (226): 53-54. Bernheimer , A.W., and Bey, R.F. 1986. Copurification of <em>Leptospira</em> <em>interrogans</em> serovar pomona hemolysin and <em>Sphingo</em>myelinase C. Infect. Immun. 54(1): 262-264. Bolin CA, Cassells, J.A., Zuerner, R.L. and Trueba, G. (1991). Effect of vaccination with a monovalent <em>Leptospira</em> <em>interrogans</em>&nbsp; serovar hardjo type hardjo-bovis vaccine on type hardjo-bovis infection of cattle. Am. J. Vet. Res. (52): 1639-1643. Branger, C., Sonrier, C., Chatrenet, B., Klonjkowski, B. and Ruvoen-Clouet, N. (2001). Identification of the hemolysis-associated protein I as a cross-protective immunogen of <em>Leptospira</em> <em>interrogans</em> by adenovirus-mediated vaccination.&nbsp; Infect. Immun. (69): 6831-6838. Branger, C., Chatrenet, B., Gauvrit, A., Aviat, F. and Aubert, A. (2005). Protection against <em>Leptospira</em> <em>interrogans</em> sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein I.&nbsp; Infect. Immun. (73): 4062-4069. Carvalho, E., Barbosa, A.S,&nbsp; Gomez, R.M., Oliveira, M.L.S., Romero, E.C.,&nbsp; Goncales, A.P., Morais, Z.M., Vasconcellos, S.A. and Ho, P.L. (2010).&nbsp; Evaluation of the Expression and Protective Potential of <em>Leptospiral</em> <em>Sphingo</em>myelinases. Curr. Microbiol. (60): 134-142. Choy, H.A., Kelley, M.M., Chen, T.L., Moller,A.K., Matsunaga, J. and Haake, D.A. (2007).&nbsp; Physiological osmotic induction of <em>Leptospira</em> <em>interrogans</em> adhesion: <em>Lig</em>A and <em>Lig</em>B bind extracellular matrix proteins and fibrinogen. Infect. Immun. (75): 2441-2450. Coutinho, M.L., Choy, H.A., Kelley, M.M., Matsunaga, J., Babbitt, J.T., Lewis, M.S., Aleixo, J.G. and Haake, D.A. (2011).&nbsp; A <em>Lig</em>A three domain region protects hamsters from lethal infection by <em>Leptospira</em> <em>interrogans</em>.&nbsp; PLoS Negl. Trop. Dis. 5(12): e1422. Porika Raju and Estari Mamidala (2015). Anti-diabetic activity of compound isolated from Physalis angulata fruit extracts in alloxan induced diabetic rats. The Ame J Sci &amp; Med Res, 2015,1(1); Pages 1 -6. doi:10.17812/ajsmr2015.11.1. Croda, J., Ramos, J.G.R., Matsunaga, J., Queiroz, A., Homma, A., Riley, L.W., Haake, D.A., Reis, M.G. and Ko, A.I. (2007).&nbsp; <em>Leptospira</em> immunoglobulin like proteins as a serodiagnostic marker for acute Leptospirosis. J. Clin. Microbiol. 45(5): 1528-1534. Croda, J., Figueira, C.P., Wunder Jr., A.E., Santos, C.S., Reis, M.G., Ko, A.I. and Picardeau, M. (2008).&nbsp; Targeted Mutagenesis in pathogenic <em>Leptospira</em> species: Disruption of the <em>Lig</em>B gene does not affect virulence in animal models of leptospirosis.&nbsp; Infect. Immun. 76(12): 5826-5833. Faine, S. (1994).&nbsp; <em>Leptospira</em> and leptospirosis, 1st ed. CRC press, Boca Raton, Fla. Faucher, J.F, Hoen, B. and Estavoyer, J.M. (2004). The management of Leptospirosis. Expert&nbsp;Opin. Pharmacother. 5(4): 819-827.. Goldstein, S.F. and Charon, N.W. (1990).&nbsp; Multiple exposure photographic analysis of motile spirochetes. Proc Natl Acad Sci, (87): 4895-4899. Haake, D.A., Mazel, M.K., McCoy, A.M., Milward, F., Chao, G., Matsunga, J. and Wager, E.A., (1999). <em>Leptospiral</em> outer membrane proteins <em>Omp</em>L1 and <em>LipL41</em> exhibit synergistic immune protection. Infect Immun. (67): 6572-6582. Isogai, E., Isogai, H., Kurebayashi, Y. and Ito, N. (1986). Biological activities of <em>Leptospiral</em> lipopolysaccharide. Zentralbl. Bakteriol. Mikrobiol. Hyg. A. (261): 53-64. Koizumi, N. and Watanabe, H. (2004).&nbsp; Leptopsira immunoglobulin-like protein elicit protective immunity.&nbsp; Vaccine (22): 1545-1552. Lee, S.H., Sangduk, K., Seung, C.P. and Min, J.K. (2002).&nbsp; Cytotoxic Activities of <em>Leptospira</em> <em>interrogans</em>&nbsp; Hemolysin <em>Sph</em>H as a Pore-Forming Protein on Mammalian Cells. Infect. Immun. (70): 315-322. Levett, P.N. (2001).&nbsp; Leptospirosis. Clin. Microbiol. Rev. (14): 296-326. Lourdault, K., Wang, L.C., Vieira, A., Matsunaga, J., Melo, R., Lewis, M.S., Haake, D.A. and Solecki, M.G. (2014).&nbsp; Oral immunization with <em>Escherichia coli</em> expressing a lapidated form of <em>Lig</em>A protects hamsters against challenge with <em>Leptospira</em> <em>interrogans</em> serovar copenhageni.&nbsp; Infect. Immun. 82(2): 893-902. Lucas, D.S., Cullen, P.A., Lo, M., Srikram, A. and Sermswan, R.W. (2011).&nbsp; Recombinant <em>Lip</em>L32 and <em>Lig</em>A from <em>Leptospira</em> are unable to stimulate protective immunity against leptospirosis in the hamster model.&nbsp; Vaccine. (29): 3413-3418. Matsunaga, J., Barocchi, M.A., Croda, J., Young, T.A., Sanchez, Y., Siqueira, I., Bolin, C.A., Reis, M.G., Riley, L.W., Haake, D.A. and Ko, A.I. 2003.&nbsp; Pathogenic <em>Leptospira</em> species express surface exposed proteins belonging to the bacterial immunoglobulin superfamily.&nbsp; Mol. Microbiol. (49): 929-945. Matsunaga, J., Werneid, K., Zuerner, R.L., Frank, A. and Haake, D.A. (2006).&nbsp; <em>Lip</em>L46 is a novel surface exposed lipoprotein expressed during <em>Leptospiral</em> dissemination in the mammalian host. Microbiology. (152): 3777-3786. Merino, S., Rubires, X., Knoche, S. and Tomas, J.M. (1995).&nbsp; Emerging pathogens: <em>Aeromonas </em>spp. Int. J. Food. Microbiol. (28): 157-168. Natarajaseenivasan, K., Shanmughapriya, S., Velineni, S., Artiushin, S.C. and Timoney, J.F. (2011). Cloning, Expression, and Homology Modeling of <em>Gro</em>EL Protein from <em>Leptospira</em> <em>interrogans</em>&nbsp; Serovar <em>Autumnalis</em> Strain N2. Genomics Proteomics Bioinform. 9(4-5): 151. Palaniappan, R.U., Chang, Y.F., Jusuf, S.S.D., Artiushin, S., Timoney, J.F., McDonough, S.P., Barr, S.C., Divers, T.J., Simpson, K.W., McDonough, P.L. and Mohammed, H.O. (2002). Cloning and Molecular Characterization of an Immunogenic <em>Lig</em>A Protein of <em>Leptospira</em> <em>interrogans</em>. Infect. Immun. 70(11): 5924-5930. Palaniappan, R.U., Chang, Y.F., Hassan, F., McDonough, S.P., Pough, M., Barr, S.C., Simpson, K.W., Mohammed, H.O., Shin, S., McDonough, P.L., Zuerner, R.L., Qu, J. and Roe, B. 2004.&nbsp; Expression of <em>Leptospiral</em> immunoglobulin like protein by <em>Leptospira</em> <em>interrogans</em> and evaluation of its diagnostic potential in a kinetic ELISA. J. Med. Microbiol. (53): 975-984. Palaniappan, R.U., McDonough, S.P., Divers, T.J., Chen, C.S., Pan, M.J., Matsumoto, M. and Chang, Y.F. (2006).&nbsp; Immunoprotection of recombinant <em>Leptospiral</em> immunoglobulin like protein A against <em>Leptospira interrogans </em>serovar Pomona infection. Infect. Immun. (74(3)): 1745-1750. Parthiban, M., Senthil Kumar, S., Balachandran, C., Kumanan, K., Aarthi, K.S. and Nireesha, G. 2015.&nbsp; Comparison of Immunoprotection of <em>Leptospira</em> recombinant proteins with conventional vaccine in experimental animals. Ind. J. Exp. Biol. (53): 779-785. Patti, J.M., Allen, B.L., McGavin, M.J. and Hook, M. (1994).&nbsp; MSCRAMM-mediated adherence of microorganisms to host tissues. Ann. Rev. Microbiol. (48): 585-617. Picardeau, M.D., Bulach, D.M. and Bouchier., C. (2008).&nbsp; Genome sequence of the saprophyte <em>Leptospira</em> <em>biflexa</em> provides insightsinto the evolution of <em>Leptospira</em> and the pathogenesis of leptospirosis. PLoS ONE 3: 1607 Pinnie, M. and Haake, D.A. (2009).&nbsp; A comprehensive approach to identification of surface-exposed, outer&nbsp; membrane spanning proteins of <em>Leptospira</em> <em>interrogans</em>. PLoS One (4): e6071. Seixas, F.K., da Silva, E.F., Hartwig, D.D., Cerqueira, G.M., Amaral, M. (2007).&nbsp; Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of <em>Leptospira</em> <em>interrogans</em> protects hamster from challenge.&nbsp; Vaccine. 26: 88-95. Silva, E.F., Medeiros, M.A., McBride, A.J., Matsunaga, J., Esteves, G.S., Ramos, J.G., Santos, C.S., Croda, J., Homma, A. and Dellgostin, O.A. (2007).&nbsp; The terminal portion of <em>Leptospiral</em> immunoglobulin-like protein <em>Lig</em>A confers protective immunity against lethal infection in the hamster model of leptospirosis.&nbsp; Vaccine. 25(33): 6277-6286. Trowbridge, A.A., Green, J.B., Bonnet, J.D., Shohet, S.B., Ponnappa, B.D. and Mccombs, W.B. 1981. Hemolytic anemia associated with leptospirosis morphologic and lipid studies. Am. J. Clin. Pathol. (76): 493-498. Zuerner, R.L., Alt, D.P., Palmer, M.V., Thacker, T.C. and Olsen, S.C. (2011).&nbsp; A <em>Leptospira</em> <em>borgpetersenii</em> serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. Clin. Vaccine. Immunol. (18): 684-691.

ABSTRACT We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

Pathogenic leptospires can bind to receptors on mammalian cells such as cadherins and integrins. Leptospira effectively adheres to cells, overcomes host barriers and spreads into the bloodstream, reaching internal target organs such as the lungs, liver and kidneys. Several microorganisms produce proteins that act as ligands of integrins through the RGD motif. Here, we characterized a leptospiral RGD-containing protein encoded by the gene lic12254. In silico analysis of pathogenic, intermediate and saprophytic species showed that LIC12254 is highly conserved among pathogenic species, and is unique in presenting the RGD motif. The LIC12254-coding sequence is greatly expressed in the virulent Leptospira interrogans L1-130 strain compared with the culture-attenuated L. interrogans M20 strain. We also showed that the recombinant protein rLIC12254 binds to αVβ8 and α8 human integrins most likely via the RGD motif. These interactions are dose-dependent and saturable, a typical property of receptor–ligand interactions. The binding of the recombinant protein lacking this motif—rLIC12254 ΔRAA—to αVβ8 was almost totally abolished, while that with the α8 human integrin was decreased by 65%. Taken together, these results suggest that this putative outer membrane protein interacts with integrins via the RGD domain and may play a key role in leptospirosis pathogenesis.

&nbsp; <strong>ABSTRACT</strong> Carboxy terminal domains of leptospiral immunoglobulins of r<em>Lig</em>A and r<em>Lig</em>B were assessed its hemolytic, cytotoxicity effects invitro and its protective efficacy was studied in hamster models. Experimental recombinant proteins of <em>Leptospira interrogans</em> Icterohaemorrhagiae were individually cloned, expressed in prokaryotic system.&nbsp; Purified expressed proteins confirmed by western blot, assessed for its protective efficacy using hamster models.&nbsp; 100% and 93% protection on homologues challenge was observed with r<em>Lig</em>A and r<em>Lig</em>B with adjuvant respectively which is evident with increased levels of antibody levels of r<em>Lig</em>A and r<em>Lig</em>B with 98% and 95% specificity.&nbsp; Cytotoxicity was maximum with 14% in Daudi cells with with r<em>Lig</em>B and 7% and r<em>Lig</em>A.&nbsp; 17% and 4% hemolytic activity with r<em>Lig</em>A, and r<em>Lig</em>B protein and 11% and 10% in pH-7.0 respectively. <em>E. coli </em>(harboring pET15b) membrane fraction used as placebo neither exerted cytotoxicity nor hemolysis was used to compare the leptospiral proteins.&nbsp; Leptospiral immunoglobulins showed least cytotoxicity and hemolytic activity with increased homologues protection in experimental animals <strong>Keywords: </strong><em>Leptospira recombinant protein, Leptospiral immunoglobulins, immune response, challenge studies, cytotoxic and hemolytic activity.</em> <strong><em>REFERENCES</em></strong> Alves, V.A., Gayotto, L.C., Brito, T.De., Santos, R.T., Wakamatsu, A., Vianna, M.R., and Sakata. E.E (1992). <em>Leptospiral </em>antigens in the liver of experimentally infected guinea pig and their relation to the morphogenesis of liver damage. Exp. Toxicol.&nbsp; Pathol. (44): 425-434. Andre-Fontaine, G., Branger, C., Gray, A.W and Klaasen, H.L. (2003).&nbsp; Comparison of the efficiacy of three commercial bacterins in preventing canine leptospirosis.&nbsp; Vet. Rec. (153): 165-169. Bartlett, J.M.S. and Stirling, D. (2004).&nbsp; DNA Extraction form Fungi, Yeast and Bacteria.&nbsp; PCR Protocols Second Edition, Methods in Molecular Biology, Humana Press, (226): 53-54. Bernheimer, A.W. and Bey, R.F. (1986). Copurification of <em>Leptospira interrogans</em> serovar pomona hemolysin and sphingomyelinase C. Infect. Immun. (54): 262-264. Bulach, D.M., Kalambaheti, T., Dela pena, M.A. and Adler, B. (2000). Functional analysis of genus is the rfb locus of <em>Leptospira</em> <em>borgpetersenii</em> serovar hardjo subtype hardjobovis. Infect. Immun.&nbsp; (68): 3793-3798. Choy, A.H., Kelley, M.M., Chen, T.L., Moller, A.K., Matsunga, J. and Haake., D.A. (2007).&nbsp; Physiological osmotic induction of Leptospira interrogans Adhesion: <em>Lig</em>A and <em>Lig</em>B Bind Extra cellular Matrix Proteins and Fibrinogen. Infect. Immun. (75):&nbsp; 2441-2450. Croda, J., Ramos, J.G., Matsunga, J., Queiroz, A., Homma, A., Riley, L.W., Haake, D.A., Reis, M.G. and Ko, A.I. (2007).&nbsp; <em>Leptospira</em> Immunoglobulin-like proteins as a serodiagnositc marker for acute leptospirosis.&nbsp; J. Clin. Microbiol. (45): 1528-1534. Croda, J., Figueira C.P., Wunder Jr.,, A.E., Santos, C.S., Reis, M.G. and Ko, A.I. (2008).&nbsp; Targeted Mutagenesis in Pathogenic Leptospira species: Disruption of the <em>Lig</em>B gene does not affect virulence in animal models of Leptospirosis. Infect. Immun.&nbsp; (76(12)) : 5826-5833. Del Real, G., Segers, R.P.A.M., Van der Zejst, B.A.M. and Gaastra, W. (1989). Cloning of a hemolysin gene from <em>Leptospira interrogans</em> serovar hardjo. Infect. Immun. (57): 2588-2590. Goldstein, S.F. and Charon, N.W. (1990). Multiple exposure photographic analysis of motile spirochetes. Pro. Nat. Acad.&nbsp; Sci. (87): 4895-4899. Haake, D.A., Mazel, M.K., McCoy, A.M., Milward, F., Chao, G., Matsunga, J. and Wager, E.A. (1999). <em>Leptospiral</em> outer membrane proteins <em>Omp</em>L1 and <em>Lip</em>L41 exhibit synergistic immune protection. Infect. Immun. (67): 6572-6582. Hartwig, D.D., Oliveira, T.L., Seixas, F.K., Forster, K.M., Rizzi, C., Hartleben, C.P., McBride A.J.A. and Dellagostin, O.A. (2010). High yield expression of leptospirosis vaccine candidates <em>Lig</em>A and LipL32 in the methylotrophic yeast Pichia pastoris.&nbsp; Microb. cell Fact. (9): 98. Isogai, E., Isogai, H., Kurebayashi,Y. and Ito, N. (1986). Biological activities of <em>Leptospiral </em>lipopolysaccharide. Zentralbl Bakteriol Mikrobiol Hyg A. (261): 53- 64. Ito, T. and Yanagawa, R. (1987). Leptospiral attachment to extracellular matrix of mouse fibroblast (L929) cells. Vet. Microbiol. (15): 89-96. Lee, S.H., Sangduk, K., Seung, C.P. and Min, J.K. (2002). Cytotoxic Activities of <em>Leptospira interrogans</em> Hemolysin <em>Sph</em>H as a Pore-Forming Protein on Mammalian Cells. Infect. Immun. (70): 315-322. Levett, P. N. (2001). Leptospirosis. Clin. Microbiol. Rev. (14): 296-326. Matsunga, J., barocchi, M.A., Croda, J., Young, T.A., Sanches, Y., Siqueira, I., Bolin, C.A., Reis, M.G., Riley, L.W., Haake, D.A. and Ko, A.I. (2003). Pathogenic Leptospira species express surface exposed proteins belonging to the bacterial immunoglobulin superfamily. Mol. Microbiol. (49): 929-945. Matsunga, J., Sanchez, Y., Xu, X. and Haake, D.A (2005).&nbsp; Osmolarity, a key environmental signal controlling expression of leptospiral proteins <em>Lig</em>A and <em>Lig</em>B and the extracellular release of <em>Lig</em>A.&nbsp; Infect. Immun. (73): 70-78. Naiman, B.M., Blumerman, S., Alt, D., Bolin, C.A., Brown, R., Zuerner, R. and Baldwin, C.L. (2002). Evaluation of type 1 immune response in&nbsp; aive and vaccinated animals following challenge with <em>Leptospira</em> borgpetersenii serovar hardjo: involvement of WC1and CD4 T cells. Infect. Immun. (70): 6147-6157. Natarajaseenivasan, K., Shanmughapriya, S., Velineni, S., Artiushin, S.C. and Timoney, J.F. (2011). Cloning, Expression, and Homology Modeling of GroEL Protein from <em>Leptospira interrogans</em> Serovar Autumnalis Strain N2. Genomics Proteomics Bioinform. (9(4-5)): 151-157. Palaniappan, R.U., Chang, Y.F., Hassan, F., Jusuf, S.S., Artiushin, Timoney, F.,&nbsp; McDonough, S.P.,&nbsp; Barr, S.C.,&nbsp; Drivers T.J.,&nbsp; Simpson, K.W., McDounough, P. and Mohammed, H.O.&nbsp; (2002).&nbsp; Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA. J. Med. Microbiol. (53): 975-984. Palaniappan, R.U., Chang, Y.F., Hassan, F., McDouough S.P., Pough, M., Barr, S.C., Simpson, K.W., Mohammed, H.O., Shin, S., McDounough, P., Zuerner, R.L., Qu, J. and Roe, B. (2004).&nbsp; Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA. J. Med. Microbiol. (53): 975-984. Sateesh Pujari and Estari Mamidala (2015). Anti-diabetic activity of Physagulin-F isolated from Physalis angulata fruits. The Ame J Sci &amp; Med Res, 2015,1(1):53-60. doi:10.17812/ajsmr2015113. Palaniappan, R.U., McDouough S.P., Drivers T.J., Chen, C.S., Pan, M.J.,&nbsp; Matsumoto, M and Chang Y.F. (2006).&nbsp; Immunoprotection of recombinant leptospiral immunoglobulin-like protein A against Leptospira interrogans serovar Pomona infection. Infect. Immun. (74): 1745-1750. Patti, J.M., Allen, B.L., McGavin, M.J. and Hook, M. (1994). MSCRAMM-mediated adherence of microorganisms to host tissues.&nbsp; Ann. Rev. Microbiol. (48): 585-617. Pinkney, M., Beachey, E. and Kehoe, M. (1989). The thio-activated toxin streptolysin O does not require a thiol group for cytolytic activity. Infect. Immun. (57): 2553-2558. Raghavan, U., Palaniappan, M., Chang, Y.F., Jusuf, S.S.D., Artiushin, S., Timoney, J.F., McDonough, S.P., Barr, S.C., Divers T.J., Simpson, K.W., McDonough, P.L. and Mohammed H.O. (2002). Cloning and Molecular Characterization of an Immunogenic <em>Lig</em>A Protein of <em>Leptospira interrogans.</em> Infect. Immun. (70(11)): 5924-5930. Raghavan, U., Palaniappan, M., Mc Donough, S.P., Divers, T.J., Chen, C.S., Pan, M.J.,&nbsp; Matsumoto, M. and Chang, Y.F. (2006).&nbsp; Immunoprotection of Recombinant Leptospiral Immunoglobulin like Protein a against Leptospira interrogans serovar Pomona infection.&nbsp; Infect. Immun. (74): 1745-1750. Reed, L.J. and Muench, H. (1938).&nbsp; A simple method of estimating fifty percent end points Am. J. . Hyg. (27): 493. Senthilkumar, T.M.A., Subathra, M. and Ramadass, P. (2008). Latex agglutination test for the detection of canine leptospiral antibodies using recombinant <em>Omp</em>L1 antigen. Veterinarski Arhiv (78): 393-399. Zuerner, R,L, Alt, D.P, Palmer, M.V, Thacker, T.C. and Olsen, S.C. (2011). A <em>Leptospira borgpetersenii</em> serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. Clin.&nbsp; Vac. Immunol. (18): 684.

Leptospira is one of the most common zoonotic diseases in the tropics and subtropics. Humans are infected by exposure to Leptospira contained water or food sources. Leptospirosis usually breaks out after the flood and causes several consequences for people and economy. Leptospirosis disease, if not rapidly detected and treated promptly, it causes serious consequences such as acute hepatitis-kidneys, meningitis and bleeding, heart and nerve complications, and severe illness can lead to death. Therefore, quick and accurate detection of Leptospira pathogen plays a very important role in Leptospirosis disease treatment. Among antigens of Leptospira, a conserved domain of LigB antigen (Leptospiral immunoglobulin-like protein) was reported that is present in the most of pathogenic serovars of Leptospira, but not in the non-pathogenic Leptospira biflexa, thus this conserved domain was used for production of Leptospirosis detection kits as well as vaccine for Leptospirosis. In order to create a kit for Leptospirosis diagonostic, especially detect anti-Leptospira antibodies in Leptospira infected serum and plasma samples, about 1kb gene fragment encoding for conserved domain of LigB (about 36 kb in molecular weight) was used as the material for producing of LigB protein by DNA recombinant technology. In this study, we present the results for cloning, expressing a conserved domain of LigB antigen in E. coli cells and purifying protein by affinity chromatography collumn. The result indicates that recombinant LigB protein was successfully expressed in E. coli Rosetta 1 and purified by Hitrap chealating collumn. The LigB protein concentration after purification reached 60 mg/L medium with 98% purity. This purified protein will be used as the materials for creating Leptospirosis kit.

Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch’s molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen’s ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease.

In determining the efficacy of new vaccine candidates for leptospirosis, the primary end point is death and an important secondary end point is sterilizing immunity. However, evaluation of this end point is often hampered by the time-consuming demands and complexity of methods such as culture isolation (CI). In this study, we evaluated the use of an imprint (or touch preparation) method (IM) in detecting the presence of leptospires in tissues of hamsters infected with Leptospira interrogans serovar Copenhageni. In a dissemination study, compared to CI, the IM led to equal or improved detection of leptospires in kidney, liver, lung and blood samples collected post-infection and overall concordance was good (κ=0.61). Furthermore, in an evaluation of hamsters immunized with a recombinant leptospiral protein-based vaccine candidate and subsequently challenged, the agreement between the CI and IM was very good (κ=0.84). These findings indicate that the IM is a rapid method for the direct observation of Leptospira spp. that can be readily applied to evaluating infection in experimental animals and determining sterilizing immunity when screening potential vaccine candidates.

Leptospirosis is a febrile disease and the etiological agents are pathogenic bacteria of the genus Leptospira. The leptospiral virulence mechanisms are not fully understood and the application of genetic tools is still limited, despite advances in molecular biology techniques. The leptospiral recombinant protein LIC11711 has shown interaction with several host components, indicating a potential function in virulence. This study describes a system for heterologous expression of the L. interrogans gene lic11711 using the saprophyte L. biflexa serovar Patoc as a surrogate, aiming to investigate its possible activity in bacterial virulence. Heterologous expression of LIC11711 was performed using the pMaOri vector under regulation of the lipL32 promoter. The protein was found mainly on the leptospiral outer surface, confirming its location. The lipL32 promoter enhanced the expression of LIC11711 in L. biflexa compared to the pathogenic strain, indicating that this strategy may be used to overexpress low-copy proteins. The presence of LIC11711 enhanced the capacity of L. biflexa to adhere to laminin (Lam) and plasminogen (Plg)/plasmin (Pla) in vitro, suggesting the involvement of this protein in bacterial pathogenesis. We show for the first time that the expression of LIC11711 protein of L. interrogans confers a virulence-associated phenotype on L. biflexa, pointing out possible mechanisms used by pathogenic leptospires.

Introduction: Leptospirosis is a zoonotic disease caused by the spirochete of genus Leptospira with widespread distribution in tropical, subtropical and temperate zones. Leptospirosis is often confused with other febrile illnesses including jaundice, dengue, and malaria. Generally, the disease is often underdiagnosed or misdiagnosed. Though leptospirosis is curable with antibiotic treatment, the laboratory diagnosis of the disease is specialized and open to interpretation with multiple kits available to detect the different serological markers of Leptospira. Moreover, when leptospirosis is misdiagnosed, the disease can lead to multi-organ failure and may have fatal effects. There is a need for strategies to develop vaccines and prevent leptospirosis. In the present study, the immunogenic potential of leptospiral recombinant protein LipL21 (rLipL21) and its truncated form I-LipL21 (rI-LipL21) was evaluated.&#x0D; Methodology: The recombinant proteins were established in cyclophosphamide treated BALB/c mice model infected with L. interrogans serovar Autumnalis strain N2.&#x0D; Results: The vaccination study showed 66% and 83% survivability among mice immunized with rLipL21 and rI-LipL21 respectively and post-challenge with leptospiral strain N2 compared to control groups that showed 100% lethality. Additionally, a significant increase in antibody levels and cytokine levels (TNF-a, IFN-γ and IL-10) was observed evidencing a marked stimulation of both humoral and cell-mediated immune response in mice immunized with rLipL21/rI-LipL21 compared to whole cell leptospiral lysate (WCL).&#x0D; Conclusions: This study evidenced protective immunization against leptospirosis with rLipL21 and rI-LipL21 recombinant proteins and are potential candidates for the development of leptospiral vaccine.

As bactérias patogênicas do gênero Leptospira são o agente causador da leptospirose, uma doença de importância global. As leptospiras patogênicas causam infecção em um amplo espectro de animais e no homem. As leptospiras podem invadir o corpo humano através de abrasões na pele e mucosa. A invasividade bacteriana depende de várias etapas, tais como: aderência, invasão e disseminação através dos tecidos do hospedeiro. Recentemente, nosso grupo identificou proteínas de membrana externa que atuam como adesinas de leptospira e/ou receptores de componentes do plasma hospedeiro, o que poderia contribuir para a patogenicidade bacteriana. Assim, o presente projeto tem como objetivo avaliar as propriedades funcionais do gene LIC10920, identificado na sequência genômica de Leptospira interrogans sorovar Copenhageni, como uma proteína hipotética, predita de membrana externa. A sequência LIC10920 foi amplificada por PCR e clonada no vetor de expressão pAE. O plasmídeo pAE contendo o inserto foi introduzido em estirpes de E. coli para a expressão da proteína. A proteína recombinante rLIC10920 foi purificada por cromatografia de afinidade a níquel e sua integridade estrutural foi avaliada pela técnica de dicroísmo circular. Camundongos foram imunizados com a LIC10920 para a avaliação da sua imunogenicidade. A presença de IgG humano contra LIC10920, foi avaliada por ELISA, em amostras de soro de pacientes com leptospirose. Assim, como a sua ligação com componentes da matriz extracelular e plasma do hospedeiro. Animais imunizados apresentaram alto título de anticorpos contra LIC10920. Além disso, a proteína foi reconhecida por anticorpos presente em amostras de soro humano infectado. A proteína foi capaz de interagir com plasminogênio e laminina de maneira dose-dependente e saturável. Em ambas as interações, a participação das regiões imunogênicas se mostrou importante. rLIC10920 foi capaz de capturar o plasminogênio direto do soro humano também de maneira dose-dependente. Por fim, foi observado que o plasminogênio ligado a rLIC10920 pode ser convertido em plasmina. A proteína em estudo é expressa durante a infecção e podemos atribuir a função de adesina, com papel na patogênese da bactéria.<br>Pathogenic bacteria of genus Leptospira are the causative agent of leptospirosis, a disease of global importance. Pathogenic leptospires cause infection in a broad spectrum of animals and humans. Pathogenic leptospires can efficiently invade the human body through skin and mucosa and promptly spread into blood vessels, reaching target organs. Bacterial invasiveness depends on several steps, such as adherence, invasion and throughout host tissues. Recently, our group has identified outer membrane proteins that act as leptospiral adhesins and/or receptors of host plasma components, which could contribute for bacterial pathogenesis. This project aims to evaluate the functional properties of the gene LIC10920, identified in the genome sequence of Leptospira interrogans serovar Copenhageni, as a predicted outer membrane protein of unknown function. The LIC10920 sequence was amplified by PCR, cloned into the expression vector pAE. Plasmids containing cloned DNA were introduced in E. coli strains for protein expression. The recombinant protein was purified by the metal affinity chromatography and its structural integrity was assessed by circular dichroism spectroscopy. Mice were subcutaneously immunized with LIC10920 for immunogenicity evaluation. The presence of IgG against LIC10920 in confirmed leptospirosis human serum samples was evaluated by ELISA. Binding of protein with extracellular matrix or plasma components was also assessed. Sera from immunized animals show that the rLIC10920 protein is capable to stimulate antibody immune response in mice. In addition, the protein is recognized by antibodies in leptospirosis human serum samples. The recombinant protein was capable of binding plasminogen and laminin. Dose-dependent and saturable binding was observed when increasing concentrations of the rLIC10920 were allowed adhere to a fixed concentration of plasminogen or of laminin, fulfilling the receptor-ligand interactions. In both cases, the participation of the immunogenic regions occurs, but in the case of laminin, the dependence is greater with structured epitopes. It has been shown that plasminogen linked to rLIC10920 can be converted to plasmin in the presence of activator. The recombinant protein was able to capture the plasminogen directly from normal human serum in a dose-dependent manner, suggesting the involvement of native protein in host-pathogen interactions. The protein under study is expressed during the infection and due to its capacity of interaction with host components, we may anticipate its role in leptospiral pathogenesis.

A leptospirose é uma doença sistêmica, causada por bactérias patogênicas do gênero Leptospira. O desenvolvimento de novas estratégias para prevenir a doença é necessário. As pesquisas atuais têm interesse em identificar antígenos conservados que estão envolvidos nas interações patógeno-hospedeiro. Dois genes de L. interrogans foram selecionados, clonados, expressos e suas respectivas proteínas caracterizadas. Os genes foram amplificados por PCR e clonados no vetor de expressão PAE. As proteínas recombinantes foram purificadas por cromatografia de afinidade ao metal. A proteína rLIC10821 foi capaz de se ligar a laminina, plasminogênio e fibrinogênio. Ambas as proteínas foram localizadas na membrana externa de acordo com as três metodologias utilizadas: imunofluorescência, proteinase K, leptospira intacta. A proteína rLIC10821 que interagiu com o PLG foi capaz de gerar plasmina. Após a interação da proteína rLIC10821 com o fibrinogênio, foi possível identificar uma diminuição de 60% no coágulo de fibrina.<br>Leptospirosis is a systemic disease caused by pathogenic bacteria of genus Leptospira. The development of new strategies to prevent the disease is needed. Currently research has focused to identify conserved antigens related to the host-pathogen interaction. Two genes of L. interrogans were selected, cloned, expressed and its proteins characterized. The genes were amplified by PCR and cloned into expression vetor pAE. The recombinant proteins were purified by chromatography of metal affinity. The protein rLIC10821 were able to bind to laminin, plasminogen and fibrinogen. Both proteins were localized in the outer membrane according three methodologies: immunofluorescence, proteinase K, intact leptospira. The protein rLIC10821 interacted with PLG was able to generate plasmin. After the interaction of the protein rLIC10821 with fibrinogen, we could identify a decrease of 60% in the fibrin clot.

Leptospirose é uma zoonose mundial que acomete várias espécies de mamíferos, incluindo humanos, causada por espécies de bactérias patogênicas do gênero Leptospira. Possui um quadro de manifestação clínico muito variado, podendo apresentar desde sintomas comuns a outras doenças como febre, calafrios, cefaleia, dores musculares, enjoo, vômitos, diarreia e uma forma mais severa da infecção denominada síndrome de Weill. Seguindo a estratégia de genômica funcional, foram selecionados genes de Leptospira interrogans sorovar Copenhageni, LIC10377, LIC11122, LIC11184 e LIC12287, tendo como critério a predição de localização das proteínas na membrana. Os fragmentos referentes aos genes LIC11122 e LIC12287 foram clonados no vetor pGEM-T Easy e subclonados no vetor de expressão pAE. As proteínas avaliadas interagem com laminina e plasminogênio, de forma dose-dependentes e saturáveis, sugerindo atuam nos processos de patogênese da bactéria. A presença de um fator sigma na superfície celular desempenhando um papel secundário, sugere que a rLIC11122 pode ser uma proteína moonlight.<br>Leptospirosis is a worldwide zoonosis that affects several species of mammals, including humans, caused by species of pathogenic bacteria of the genus Leptospira. It has a very varied clinical manifestation board and may have symptoms from common tropical diseases such as fever, chills, headache, muscle aches, nausea, vomiting, diarrhea and a severe syndrome of infection known as Weill syndrome. Following the functional genomics strategy, genes were selected from Leptospira interrogans serovar Copenhageni, LIC10377, LIC11122, LIC11184 and LIC12287, with the criterion of the prediction location of proteins in the membrane. The fragments related to genes LIC11122 and LIC12287 were cloned into pGEM-T Easy vector and subcloned into the expression vector pAE. The evaluated proteins interact with laminin and plasminogen, dose-dependent and saturable manner, suggesting participation in bacterial pathogenesis processes. The presence of a sigma factor on the cell surface plays a secondary role, suggests that performs a moonlight rLIC11122 protein.

A leptospirose é a zoonose mais disseminada no mundo e uma das principais causas de perda econômica no agronegócio. O estudo de novos antígenos de superfície de Leptospira interrogans, é intrigante e pode fornecer conhecimento na interação inicial patógeno-hospedeiro. Os genes LIC13059 e LIC10879, escolhidos por bioinformática, com predição de localização na superfície celular, foram clonados e as proteínas recombinantes expressas em E. coli, para avaliar a interação com componentes do hospedeiro. Após purificação, as proteínas encontravam-se estruturadas e foram reconhecidas por soro de indivíduos infectados. As proteínas recombinantes interagem com plasminogênio, fibrinogênio e laminina. rLIC13059, nomeada Lsa25.6, quando ligada ao fibrinogênio é capaz de inibir a formação de coágulo de fibrina e rLIC10879, nomeada Lsa16, interage com e-caderina, sugerido envolvimento na cascata de coagulação e ligação com o hospedeiro, respectivamente. O plasminogênio ligado às proteínas é convertido em plasmina, o que poderia ajudar a penetração bacteriana no hospedeiro.<br>Leptospirosis is the most widespread zoonosis and also a major cause of economic loss in animal production worldwide. The study of new surface antigens of Leptospira interrogans is intriguing and may shed light into the initial pathogen-host interactions. We set out to study two novel coding sequences LIC13059 and LIC10879 predicted to be located at the cell surface. The genes were cloned and the recombinant proteins were expressed in E. coli. The purified recombinant proteins presented secondary structures, and interacted with plasminogen, fibrinogen and laminin human components. rLIC13059, named Lsa25.6, when bound to fibrinogen was capable of inhibiting the formation of fibrin clot, while rLIC10879, named Lsa16, interacted with e-cadherin, a mammalian cell receptor, suggesting participation in coagulation pathway and host-cell binding, respectively. The plasminogen captured by both recombinant proteins could be converted into plasmin, a mechanism that could help bacterial penetration in the host.

O sequenciamento da L. interrogans sorovar Copenhageni e as análises bioinformáticas permitiram a identificação de candidatos vacinais e fatores de virulência. Foram selecionados dois genes, LIC11834 e LIC12253, que foram submetidos a ensaios de presença do DNA genômico e RNA mensageiro em diferentes sorovares de Leptospira. Observamos que o gene LIC12253 foi o mais presente entre os sorovares testados. Os genes foram clonados em vetor de expressão pAE e expressos em E. coli BL21 SI. As proteínas recombinantes foram purificadas e submetidas a ensaio de dicroísmo circular, o qual confirmou que ambas as proteínas estavam estruturadas. Por meio de testes de imunogenicidade em camundongos, ambas as proteínas mostraram-se imunogênicas, apresentando altos títulos de anticorpos, porém não foram capazes de promover resposta imune celular. Em ensaios de localização das proteínas nativas podemos observar a presença destas proteínas na membrana externa de Leptospira. Ensaios de reatividade com soros de pacientes diagnosticados com leptospirose mostraram que há reconhecimento das proteínas por anticorpos presentes nesses soros, sugerindo que as proteínas são expressas durante a infecção. Em ensaios de adesão a componentes de matriz extracelular e componentes do soro e plasma humano, rLIC11834 apresentou ligação à laminina, sendo nomeada de Lsa33, além de ligação ao plasminogênio, ao C4bp e ao fibrinogênio de forma dose-dependente; rLIC12253 apresentou ligação à laminina, sendo chamada de Lsa25, e ao C4bp de forma dose-dependente. Ensaios de desafio demonstraram que as proteínas não apresentam proteção contra infecção letal em hamsters. Assim, acreditamos que estas proteínas multifuncionais possam interagir com proteínas do hospedeiro e ter participação na patogênese da doença.<br>The genomic sequencing and the advances of bioinformatics analysis allowed the identification of new vaccine candidates and new virulence factors. Therefore, two genes from L. interrogans serovar Copenhageni, LIC11834 and LIC12253 were selected and subjected to assays for the presence of genomic DNA and mRNA in different serovars of Leptospira. These assays found that the gene LIC12253 was the most present among the tested serovars. The genes were then cloned in the expression vector pAE and expressed in E. coli BL21 SI. The recombinant proteins were purified and subjected to circular dichroism, which confirmed that both proteins presented secondary structures. Immunogenicity tests in mice showed that both proteins are immunogenic, with high antibodies titers, but don\'t induce cellular immune response. Localization assays of the native proteins on the leptospiras demonstrated the presence of the proteins on the surface of Leptospira. Reactivity assays with sera of patients diagnosed with leptospirosis showed that the recombinant proteins could be recognized by their antibodies, suggesting that they are expressed during infection. Adhesion assays to the extracellular matrix components, human serum and plasma components, showed that the protein rLIC11834 binds to laminin and was called Lsa33. In addition, Lsa33 interacts to plasminogen, to C4bp and to fibrinogen in a dose-dependent manner. The protein rLIC12253 showed binding to laminina, named Lsa25, and to C4bp in a dose-dependent manner. Challenge assays showed that both recombinant proteins don\'t afford protection against lethal infection in hamsters. Thus, we believe that these multifunctional proteins may interact with host proteins and may play a role in leptospiral pathogenesis.

Leptospirose é uma zoonose altamente disseminada causada por espiroquetas patogênicas do gênero Leptospira. Nos centros urbanos, os roedores são os mais importantes reservatórios da doença. Desde que o controle dos roedores e medidas sanitárias não são facilmente implementadas, o desenvolvimento de uma vacina é necessário para o combate da leptospirose. Desta maneira, os genes LIC10258, LIC12880 e LIC12238 foram selecionados por análises de bioinformática e amplificados do DNA genômico de L. interrogans por metodologia de PCR. A avaliação da capacidade de adesão das proteínas recombinantes com componentes da matriz extracelular mostrou que rLIC10258 interage com a laminina e fibronectina plasmática. Todas as proteínas recombinantes foram capazes de ligar ao plasminogênio e gerar plasmina, mostrando atividade proteolítica específica, enquanto que apenas rLIC12238 foi capaz de ligar ao fibrinogênio. Os resultados obtidos sugerem que estas proteínas podem desempenhar algum papel na patogênese da doença.<br>Leptospirosis is worldwide zoonosis caused by pathogenic spirochaetes of the genus Leptospira. In the urban settings, rodents are the most important reservoirs. Since the control of the rodents and sanitation measures are not easily implemented, the development of vaccine is necessary to combat the leptospirosis. Thus, the genes sequences of LIC10258, LIC12880 and LIC12238 were selected by bioinformatics analysis and amplified by PCR methodology from genomic DNA of L. interrogans. Evaluation of the adhesion capacity of the recombinant proteins with extracellular matrix components showed that rLIC10258 interacts to laminin and plasma fibronectin. All recombinant proteins were capable to bind plasminogen and to generate plasmin, showing specific proteolytic activity, whereas that only rLIC12238 was capable to bind fibrinogen. The results obtained suggest that these proteins may play a role in leptospiral pathogenesis.

A leptospirose é uma doença sistêmica, causada por bactérias patogênicas do gênero Leptospira. O desenvolvimento de novas estratégias para prevenir a doença é necessário. Vacinas surgem como fortes candidatas para contornar o problema. As pesquisas atuais têm interesse em identificar antígenos conservados que estão envolvidos nas interações patógeno-hospedeiro.O presente projeto selecionou três proteínas hipotéticas de L. interrogans para serem caracterizadas quanto ao seu papel na patogênese e avaliadas quanto ao seu potencial protetor. Os genes foram amplificados por PCR e clonados no vetor de expressão PAE. As proteínas recombinantes foram purificadas por cromatografia de afinidade e foram reconhecidas por soro de indivíduos infectados. As proteínas LIC13479 e LIC10050 foram capazes de se ligar a laminina, plasminogênio e fibronectina plasmática. Em relação à LIC10537, dois fragmentos recombinantes foram gerados. Apenas o fragmento 2 foi capaz de interagir com PLG. As proteínas que interagiram com o PLG foram capazes de gerar plasmina As proteínas foram capazes de estimular uma resposta imune e LIC13479 e LIC10050 exerceram proteção parcial no modelo de leptospirose em hamsters.<br>Leptospirosis is a systemic disease caused by pathogenic bacteria of genus Leptospira. The development of new strategies to prevent the disease is needed. Vaccines emerge as strong candidates to fight the problem.Currently research has focused to identify conserved antigens This project selected three hypothetical proteins of L. interrogans. Thesecoding sequences were characterized for their possible role in pathogenesis and their potential to protect animals against challenge with virulent leptospires. Genes were amplified by PCR and cloned into the expression vector pAE. The recombinant proteins were purified by metal affinity chromatography and were recognized by confirmed human leptospirosis serum samples.LIC13479 and LIC10050 proteins were able to bind with laminin, plasminogen and plasma fibronectin. The coding sequence LIC10537 was cloned in two fragments. Fragment 2was able to interact with plasminogen. All proteins were able to generate active plasmin. The recombinant proteins were able of inducing an immune response. Evaluation of immunoprotection in leptospirosis hamster model followed by challenge with virulent bacteria showed that the recombinant proteins conferred partial protection.

O sequenciamento genômico da L. interrogans sorovar Copenhageni e ferramentas de bioinformática nos permitiram selecionar 15 genes que codificam proteínas hipotéticas conservadas preditas de superfície. A conservação foi confirmada por PCR em seis dos sorovares mais predominantes de L. interrogans. As proteínas recombinantes foram clonadas em vetor de expressão de E. coli em fusão com seis resíduos de histidina, que permite a purificação por cromatografia de afinidade a metal. Seis proteínas apresentaram reatividade com anticorpos presentes em soros de pacientes com leptospirose. Através de um ensaio de adesão por ELISA, identificamos uma nova adesina de leptospira, Lsa21, que interage fortemente com laminina, colágeno IV e fibronectina plasmática. Usando a metodologia de Western blotting, identificamos mais nove possíveis adesinas. Nossos ensaios de desafio demonstraram que as proteínas rLIC12730 conferiu proteção contra infecção letal de L. interrogans em hamsters. Nossos dados sugerem que seja um promissor candidato na prevenção da leptospirose.<br>The whole-genome sequences of L. interrogans serovar Copenhageni and the bioinformatic tools allow us to choose fifteen genes encoding for conserved hypothetical proteins predicted to be exported to the membrane. The chosen genes were amplified by PCR from six predominant pathogenic serovars, the DNA cloned in an E. coli vector, the recombinant proteins expressed in fusion with 6xHis-tag at N-terminus and purified by metal affinity chromatography. Six proteins were recognized by antibodies present in sera from human patients diagnosed with leptospirosis. By ELISA-attachment assay, we have identified a novel adhesion, named Lsa21, that binds strongly to laminin, collagen IV, and plasma fibronectin. By western blotting assay, we have further identified nine novel probable adhesions. The immunization/challenge assays showed that the recombinant protein rLIC12730 afforded protection against lethal leptospiral inoculation in hamsters. Our data suggest that it is a promising candidate for prevention of leptospirosis.

Leptospirose é uma zoonose mundial que causa grandes prejuízos econômicos e sociais. Os mecanismos de patogenicidade da leptospira ainda não estão totalmente elucidados. Nesse trabalho foi avaliado o papel de três proteínas hipotéticas de superfície na patogenia da leptospirose: LIC11009, LIC11360 e LIC11975. Os genes foram clonados a partir do DNA da L. interrogans sorovar Copenhageni e as proteínas recombinantes foram purificadas por cromatografia de afinidade. RT-qPCR mostrou a transcrição dos genes em leptospiras. As proteínas recombinantes reagiram com soro de humanos diagnosticados com leptospirose. rLIC11009 induziu somente resposta imune Th1 em camundongos, enquanto que rLIC11360 e rLIC11975 induziram resposta Th1 e Th2. As três proteínas recombinantes se ligaram à laminina e plasminogênio, enquanto rLIC11360 e rLIC11975 também se ligaram à fibronectina plasmática e fibrinogênio. Em adição, rLIC11360 se ligou ainda aos reguladores do sistema complemento fator H e C4BP. Os resultados sugerem que as proteínas estudadas podem auxiliar a leptospira a evadir e invadir o hospedeiro.<br>Leptospirosis is a worldwide zoonosis that causes great economic and social losses. The pathogenic mechanisms of leptospira are not yet fully elucidated. In this study we evaluated the role of three hypothetical surface proteins in the leptospiral pathogenesis: LIC11009, LIC11360 and LIC11975. Genes were cloned from DNA of L. interrogans serovar Copenhageni and the recombinant proteins were purified by affinity chromatography. RT - qPCR data have shown that the genes are fully transcribed in leptospires. Recombinant proteins reacted with sera from humans diagnosed with leptospirosis. rLIC11009 induced Th1 immune response in mice, whereas rLIC11360 and rLIC11975 promoted both Th1 and Th2. The three recombinant proteins interacted with laminin and plasminogen while, rLIC11360 and rLIC11975, also interacted with plasma fibronectin and fibrinogen. In addition, rLIC11360 interacted with the complement regulators factor H and C4BP. These results suggest that the proteins tested can help leptospires to evade and invade the host.

A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. No mundo, aproximadamente 500.000 casos são reportados a cada ano, com 10% de taxa de mortalidade. Atualmente, vacinas contra leptospirose são compostas por células inativadas e são ineficazes em diferentes aspectos. Após analise do genoma, os genes LIC11121, LIC11087, LIC11228 e LIC11084 foram escolhidos para caracterização da imunogenicidade de suas respectivas proteínas. Esses genes foram clonados no vetor de expressão pAE e as proteínas recombinantes foram purificadas. Os resultados sugerem que essas proteínas podem estar localizadas na membrana externa, são imunogênicas, possivelmente expressas durante a infecção e que podem ter envolvimento em mecanismos de evasão do sistema imune e de patogenicidade da bactéria. Além disso, em um de dois experimentos, a proteína rLIC11084 induziu imunidade protetora parcial em hamsters imunizados frente desafio letal.<br>Leptospirosis is a zoonotic disease caused by pathogenic bacteria of genus Leptospira. In the world, nearly 500,000 cases are reported each year, with 10% of mortality rate. Currently, vaccines against leptospirosis are composed by inactivated cells that are ineffective in many aspects. After genome analysis, the genes LIC11121, LIC11087, LIC11228 e LIC11084 were chosen for immunogenicity characterization of their respective proteins. These genes were cloned in the pAE expression vector and the proteins encoded by LIC11087, LIC11228 and LIC11084 were purified. The results suggest the localization of these proteins in the bacterial outer membrane, are immunogenic, are possibly expressed during infection and may have involvement in mechanisms of immune system evasion and pathogenicity. Moreover, in one of two experiments, the rLIC11084 protein induced partial protective immunity of immunized hamsters against lethal challenge.

Leptospirosis is a worldwide zoonotic disease. Pathogenesis of leptospirosis is not well understood. Identification of virulence factors of pathogenic Leptospira and their roles in pathogenesis is crucial. Based on available whole-genome sequences of Leptospira, two presumptive virulence genes which are homologous to the invasionA (invA) gene of Rickettsia prowazekii and the mammalian cell entry (mce) gene of Mycobacterium tuberculosis, have been identified. The function of these genes may involve in the attachment and invasion of eukaryotic cells. We proposed that these gene homologues should be conserved in pathogenic serovars of Leptospira. Thus, our objective of the study is to determine the presence and the conservation of each gene in various serovars of pathogenic Leptospira. Ten different pathogenic serovars and one non-pathogenic serovar (serovar Patoc) leptospires were used in this study. Polymerase chain reaction using primers designed to bind to the conserved regions of each gene were performed. The amplified PCR products of the invA gene homologue were abtained in seven pathogenic serovars whereas eight pathogenic serovars contained the homologue of mce gene. Neither gene homologue was amplified in the non-pathogenic serovar. The nucleotide sequences of the invA gene homologue of seven serovars were greater than 99% identity. The mce gene homologues of eight serovars were approximately 90 to 100% nucleotide identity resultin in more than 98% amino acid identity. Therefore, the gene homologues of invA and mce are found to be conserved in most pathogenic serovars of Leptospira with high-degree similarity at nucleotide and amino acid levels. These two homologues were absent in non-pathogenic serovar. However, convalescent sera of patients with leptospirosis could not detect recombinant proteins of InvA and Mce. In vivo expression of these genes requires further investigations.