ABSTRACT
Background: Culture filtrate proteins (CFPs) of Mycobacterium tuberculosis are potential vaccine candidates. Objective: The aim was to study the influence of iron levels on CFPs and assess the immuno-protective potential of defined antigenic fractions from high (8 μg Fe/mL) and low iron (0.02 μg Fe / mL) cultures of M. tuberculosis. Materials and Methods: The CFPs of M. tuberculosis from high (CFP-high) and low (CFP-low) iron conditions were first compared to identify iron-regulated proteins and then fractionated to obtain ten antigen pools (CF-Ags H1- H5 and L1-L5) that were used to assess the immune response of TB patients and normal healthy controls. Results: Iron limitation resulted in the up-regulation of two novel iron-regulated low-molecular-weight proteins Irp-1 (in CF-Ag L4) and Irp-2 (in CF-Ag L5) and repression of two ESAT proteins (identified with monoclonal antibody HYB 76.8). The median stimulation indices (SIs) against most of the CF-Ags were high in pulmonary TB patients. The CF-Ags L1 and L2 showed statistically significant SI (P values of 0.0027 and 0.0029 respectively); the % case recognition was high with these antigens as well as with L4 ( P = 0.0275). IFN-γ in response to these CF-Ags was significantly high in the endemic normals; maximal expression was seen with CF-Ag L5 (median value of 233 pg mL -1 ) that was higher than the corresponding H5 (140 pg mL -1 ) and H3 and L3 (205 and 206 pg mL -1 respectively). Conclusions: CF-Ags L5, H3 and L3 showed immuno-protective potential in this geographical location.
Subject(s)
Adult , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/immunology , Bacterial Proteins/biosynthesis , Bacterial Proteins/immunology , Female , Humans , Iron/metabolism , Male , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/metabolism , T-Lymphocytes/immunology , Tuberculosis/immunology , Tuberculosis/microbiologyABSTRACT
PURPOSE: Leptospirosis is a zoonotic disease with humans getting the infection either from rodent hosts or from domestic animals. Urine contaminated environment is the common source of infection. This is an under-reported disease in Andhra Pradesh. We report a retrospective hospital-based study on 55 patients with suspected leptospirosis. METHODS: A total of 55 serum samples were collected from patients with suspected leptospirosis and subjected to serological testing by LeptoTek Dri-dot, microscopic agglutination test (MAT) and IgM enzyme-linked immunosorbent assay (ELISA). Identification of the predominant infecting serotype was done using a panel of 12 serovars. RESULTS: MAT analysis of all the 55 samples identified all cases to be positive. The predominant serogroup was Icterohaemorrhagiae (68%) followed by Australis (22%), Autumnalis (8%) and Javanica (2%). LeptoTek Dri-dot showed a sensitivity of 96% as compared to MAT. IgM ELISA done on 32 samples showed a sensitivity of 86.7% compared to MAT. CONCLUSIONS: MAT helped to identify Icterohemorrhagiae as the predominant serovar in this study. Despite the small number of samples analyzed, the data obtained establishes a need for a prospective study in this region.
Subject(s)
Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Hemagglutination Tests , Hospitals , Humans , Immunoglobulin M/immunology , India , Leptospira/growth & development , Leptospirosis/blood , Retrospective Studies , Serologic TestsSubject(s)
Bacterial Proteins/chemistry , Base Sequence , Carrier Proteins/chemistry , Hemeproteins/chemistry , Humans , Iron/metabolism , Leptospira/classification , Leptospira interrogans , Leptospirosis/microbiology , Models, Molecular , Molecular Sequence Data , Siderophores/metabolism , VirulenceABSTRACT
BACKGROUND: Iron deficiency has been shown to induce the expression of siderophores and their receptors, the iron-regulated membrane proteins in a number of bacterial systems. In this study, the response of Leptospira biflexa serovar Patoc strain Patoc I to conditions of iron deprivation was assessed and the expression of siderophores and iron-regulated proteins is reported. MATERIALS AND METHODS: Two methods were used for establishing conditions of iron deprivation. One method consisted of addition of the iron chelators ethylenediamine-N, N'-diacetic acid (EDDA) and ethylenediamine di-o-hydroxyphenylacetic acid (EDDHPA) and the second method involved the addition of iron at 0.02 microg Fe/mL. Alternatively, iron sufficient conditions were achieved by omitting the chelators in the former method and adding 4 microg Fe/mL of the medium in the latter protocol. Triton X-114 extraction of the cells was done to isolate the proteins in the outer membrane (detergent phase), periplasmic space (aqueous phase) and the protoplasmic cylinder (cell pellet). The proteins were subjected to SDS-PAGE for analysis. RESULTS: In the presence of the iron-chelators, four iron-regulated proteins (IRPs) of apparent molecular masses of 82, 64, 60 and 33 kDa were expressed. The 82-kDa protein was seen only in the aqueous phase, while the other three proteins were seen in both the aqueous and detergent fractions. These proteins were not identified in organisms grown in the absence of the iron chelators. The 64, 60 and the 33 kDa proteins were also demonstrated in organisms grown in media with 0.02 microg Fe/mL. In addition, a 24 kDa protein was found to be down-regulated at this concentration of iron as compared to the high level of expression in organisms grown with 4 microg Fe/mL. The blue CAS agar plates with top agar containing 0.02microg Fe/mL showed a colour change to orange-red. CONCLUSION: The expression of siderophores and iron-regulated proteins under conditions of iron deprivation was demonstrated in the non-pathogenic L.biflexa serovar Patoc.