Hemin-binding proteins in pathogenic Leptospira : diagonastic potential of Hbpa and the identification of second hemin-buinding protein HbpB in Leptospira interrogans serovar Manilae

Abstract

Leptospirosis is an emerging zoonotic disease that is of concern globally and in India. The spirochetal pathogen belonging to the genus Leptospira causes high morbidity and mortality in animals and humans. Due to favoured transmission in monsoon seasons, this disease often breaks out as epidemics seasonally. The disease is usually self-limiting in humans but can progress to the severe fatal form known as Weil’s disease. The disease is usually self-limiting and the majority of patients recover without any adverse effects. The symptoms associated with the disease include severe body pain, headache, fever that is often mistaken for the common flu. If left untreated, the disease can progress to Weil’s disease, in which damage is seen in different organs, particularly liver, kidney and the lungs. Hemorrhagic manifestations and tissue damage is often fatal. Proper and timely diagnosis is the major lacuna in the control of this disease and hence it is often under-reported as it is clinically similar to flu, dengue and malaria. The lack of rapid, economic and easy-to-perform tests are lacking for the diagnosis of leptospirosis. The serological microscopic agglutination test (MAT) is used as the ‘gold standard’; however, due to several disadvantages it cannot be performed in routine diagnostic laboratories. Hence, there is a need to develop a simple serological test for the detection of antibodies against leptospiral-specific antigenic determinants. Understanding host- pathogen interactions result in the identification of novel molecules that can be used as diagnostic and vaccine candidates. Acquisition of iron by pathogenic Leptospira spp. was first reported by Sritharan and her group who demonstrated direct acquisition of iron by pathogenic L. interrogans serovar Lai by the hemin-binding protein HbpA, an 81 kDa TonB- dependant outer membrane protein up-regulated upon iron limitation. HbpA is present in pathogenic serovars and is absent in saprophytic leptospires. This, coupled to the in vivo expression of the protein made HbpA an ideal candidate for diagnosis. PCR amplification of hbpA from the genomic DNA of several clinical isolates identified all serovars belonging to L. interrogans spp. As the focus of this study was to develop an ELISA-based test for the serodiagnosis of leptospirosis, the diagnostic potential of HbpA was evaluated in xv both bovine and human serum samples by ELISA. Serum samples from patients with systemic leptospirosis were screened by MAT, HbpA-ELISA and the commercial PanBio ELISA. The performance of HbpA-ELISA was superior to PanBio ELISA and it was observed that ELISA-based screening identified larger number of samples compared to MAT. The latter is a specific test for leptospirosis but, due to its serovar specificity can give false negative if the respective serovar was no included in the reference panel for screening. This was reflected strongly in patients with leptospiral uveitis, 50% of whom tested MAT-negative, with the two ELISA tests identifying the majority of the cases with confirmed clinical symptoms of the disease. The mean anti-HbpA antibody titres in both the groups were significantly higher in patients when compared with controls (P<0.05) that included subjects with non-leptospiral uveitis, cataract and endemic healthy normal subjects. HbpA-ELISA showed higher sensitivity (93%) and specificity (58%) over PanBio ELISA. Screening of bovine serum samples by HbpA-ELISA was compared with MAT and ELISA based LilL41 and LipL32, two potential diagnostic antigens, specific for pathogenic serovars. The test showed a κ value of 0.920 for HbpA- ELISA vs LipL41-ELISA and 0.887 for HpbA-ELISA vs LipL32-ELISA indicating good agreement of HbpA with the two leptospiral antigens. All the three antigen- based ELISA tests showed 100% sensitivity with MAT; the specificity, however was low (~ 63%), for reasons of not including a larger panel of serovars. Since no epidemiological data is available in India on the prevailing serovars in a specific geographical region, a random panel of serovars had to be chosen. Thus, HbpA- ELISA was effective in the testing of both human and bovine serum samples and can possibly replace the cumbersome MAT. Early detection of the disease is of paramount importance in the development of control measures. The organisms, upon entry into the mammalian host remain in circulation for about a week. During this period, culture is possible but due to the fastidious nature of the pathogen, it has not been easy to culture from blood samples. Since antigen detection is more feasible, we developed monoclonal antibodies against HbpA. Four monoclones 1F5.1C, 2E7.1C, 7C2.1C and 10E3.1C, belonging to IgG2b sub-class expressed high titers of anti-HbpA antibodies, with negligible cross-reactivity with other bacterial pathogens. MAbs xvi 7C2.1C and 10E3.1C were specific for the C-terminal region of HbpA protein as determined by western-blot analysis and indirect ELISA. These MAbs, when used as primary antibody in sandwich ELISA were able to detect 0.48 ng of rHbpA. The essentiality of hbpA was studied in a hbpA mutant strain of L. interrogans serovar Manilae. The hbpA mutant strain M601, generated by random transposon mutagenesis was a kind gift by Adler and his group. In the presence of hemin as the sole source of iron, the mutant strain was unable to grow and showed no increase in cell number for almost 6-7 days after inoculation. Growth, though seen after 7 days was much lower compared to the wild-type (WT) strain. RNA transcripts of the two strains grown under high (10 ug Fe / mL), low (200 μm EDDA) and hemin (1 μg in medium pre-incubated with 200 μm EDDA) showed changes in the levels of several iron-regulated genes, previous reported by microarray analysis. In this study, an interesting observation was the six-fold increase in the level of LA3149, annotated as encoding a Ton-dependant outer membrane protein, possibly a hemin-receptor. Since this was indicative of a second hemin acquisition system, the gene was clones and expressed. The recombinant protein, proved to bind hemin-agarose beads was called as hemin- binding protein B (HbpB). Homology modeling with Phyre program using ferripyoverdine (Fpv) receptor from Psedomonas aeruginosa as template showed HbpB to fold into the characteristic β-barrel and the globular plug domain. The presence of the N-terminal TonB box, presence of the FRAP-NNHL motif with a conserved histidine residue located between the two motifs (aa 550) confirmed it was a TonB-dependant, hemin-binding protein. In conclusion, the potential of HbpA as a diagnostic antigen and the identification of HbpB as a second hemin-binding protein in the absence of HbpA are the major achievements from this study