Presence of 46 kDa gelatinase on the outer membrane of Leptospira.

Leptospira infection involves the adhesion of the bacteria followed by invasion of the host crossing the extracellular matrix barrier. In an effort to understand the molecular mechanism of this process, the possibility of occurrence of matrix degrading enzymes from Leptospira was investigated. Zymographic analysis showed that the outer membrane of Leptospires contains a gelatinase of average molecular size of 46 kDa. The gelatinase exhibited maximum activity at neutral pH and was inhibited by metal chelators such as EGTA, EDTA, and Orthophenanthroline and was activated by calcium, magnesium, zinc, and copper, suggesting that it is a membrane-associated neutral matrix metalloproteinase. Analysis of the production of the enzyme by various serovars showed that the pathogenic serovars expressed significant amount of this enzyme while nonpathogenic forms either did not express or showed only very low activity, suggesting that this enzyme may be associated with pathogenesis of leptospirosis.

A zymography analysis of proteinase activity present in Leptospira.

Leptospirosis is a major public health problem caused by spirochete Leptospira which is an extracellular pathogen. During infection and invasion, the bacteria cross the physical barriers and later it encounter with the host defence mechanism. These processes may involve proteolytic degradation of the host tissue biomatrix. In an effort to understand the production and nature of Leptospiral proteinases, investigations were carried out using zymograpic methods. The results showed that the leptospires degrades different kind of protein substances such as gelatin, casein, and albumin. Gelatin zymography reveals that different serovars contain multiple gelatinases in the molecular weight range from 240 to 32 kDa. Studies using inhibitors suggested that the Leptospiral proteinases include metalloproteinases, serine or cysteine proteinases. The temperature sensitivity suggests that some of these proteinases are stable even at high temperatures. The presence of multiple gelatinases in Leptospira serovars suggests a critical role for these enzymes in Leptospiral invasion and pathogenesis.

A diagnostically useful 200-kDa protein is secreted through the surface pores of the filarial parasite Setaria digitata.

The excretory/secretory (ES) materials from filarial parasites form an important tool for immunodiagnosis of filariasis. We have raised monoclonal antibodies against ES proteins isolated from the medium incubated with live adult bovine filarial parasite Setaria digitata. The hybridoma were cloned and characterised with respect to the individual proteins of the ES materials. A secretory glycoprotein with molecular weight 200-kDa (gp200) was purified, localised and characterised using the specific monoclonal antibodies raised against it. The immunolocalisation study clearly showed that the protein is secreted out through the pores on the surface of both male and female parasites. The gp200 on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed dissociation into 11- to 66-kDa 'ladder' polypeptides, and all of them could be identified with the same monoclonal antibody. The gp200 is normally shed along with the ES materials. The screening of human sera samples using the monoclonal antibodies give promising results which showed that gp200 could be a potent candidate protein for the immunodiagnosis of filariasis by the method of antigen detection.

Molecular modeling and in-silico engineering of Cardamom mosaic virus coat protein for the presentation of immunogenic epitopes of Leptospira LipL32.

Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be EP4 > EP3 > EP2 > EP6.