Identification and characterization of Mycobacterium tuberculosis antigens in urine of patients with active pulmonary tuberculosis: an innovative and alternative approach of antigen discovery of useful microbial molecules

Abstract

Despite the clear need to control tuberculosis, the diagnosis and prevention of this serious disease are poorly developed and have remained fundamentally unchanged for more than 50 years. Here, we introduce an innovative approach to directly identify Mycobacterium tuberculosis antigens produced in vivo in humans with tuberculosis. We combined reversed phase high performance liquid chromatography and mass spectrometry and categorize four distinct M. tuberculosis proteins produced presumably in lung lesions and excreted in the urine of patients with pulmonary tuberculosis. The genes (MT_1721, MT_1694, MT_2462 and MT_3444) coding for these proteins were cloned and the recombinant molecules were produced in Escherichia coli. The proteins were recognized by immunoglobulin G antibodies from tuberculosis patients but not from non-diseased subjects. In addition, the recombinant proteins were recognized strongly by peripheral blood mononuclear cells from healthy purified protein derivative of tuberculin-positive individuals and to a lesser extent from patients with tuberculosis. These molecules are the only proteins reported to date that are derived directly from bodily fluids of tuberculosis patients, therefore are interesting candidate antigens for the development of vaccine and/or antigen detection assay for accurate diagnosis of active tuberculosis.

Fig. 1

Purification and characterization of the recombinant proteins coded for by MT_1721, MT_1694, MT_3444 and MT_2462. Recombinant proteins containing six Histidine tag amino terminal residues were expressed in Escherichia coli BL-21(DE3)/pLysS followed by purification by affinity chromatography using Ni-NTA agarose matrix. Purity was evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (4–20% gradient polyacrylamide gel) and Coomassie blue staining (a). Characterization was by Western blot analysis. Recombinant antigens (100 ng) were submitted to electrophoresis under reducing conditions in a 4–20% gradient gel and transferred to polyvinylidene fluoride membrane followed by probing with immunoglobulin G (1 μg/ml) obtained from pools of human sera from either patients with pulmonary tuberculosis (b) or normal control subjects (c). Reactivity was detected with peroxidase-labelled Staphylococcus aureus protein A and developed using a chemiluminescent reagent (ECL). Lane 1, rMT1721; lane 2, rMT1694; lane 3, rMT3444; lane 4, rMT2462. Numbers on the left are the molecular weights of the markers (MWM) in kDa.

Fig. 2

Recognition of purified Mycobacterium tuberculosis recombinant proteins by human peripheral blood mononuclear cells (PBMC). Proliferative responses and interferon (IFN)-γ production by PBMC from (a) tuberculosis patients (n = 16) and (b) healthy purified protein derivative of tuberculin (PPD)⁺ subjects (n = 59) were evaluated following stimulation with the indicated recombinant antigens (5 μg/ml) and PPD (2 μg/ml). Proliferation was measured by [³H]-thymidine incorporation and results are expressed as stimulation index (SI). IFN-γ was measured by sandwich enzyme-linked immunosorbent assay in the culture supernatants. PBMC obtained from PPD^– donors did not respond to any antigen (not shown). Dots represent individual donors. Dashed lines represent arbitrary cut-offs (SI ≥ 5; IFN-γ ≥ 50 pg/ml) which were defined using the results obtained with PBMC from PPD^– donors (not shown). Pairwise multiple comparison procedures (Dunn's method) were used to compare the SI as well the levels of IFN-γ obtained for each antigen within the tuberculosis patient group and within the PPD skin test-positive healthy volunteers. Within the tuberculosis patient groups, the differences in the median values among the responses to the recombinant antigens were not great enough to exclude the possibility that differences were due to random sampling variability (SI, P = 0·24; IFN-γ, P = 0·631). Among the PPD skin test-positive healthy controls, significance (P < 0·05) was observed for the following comparisons for SI rMT1721 × rMT1694, rMT1721 × rMT2462, rMT3444 × rMT1694 and rMT3444 × rMT2462. No significance (P > 0·05) was observed between the SI for rMT1721 × rMT3444 as well as between rMT1694 × rMT2462. Significance (P < 0·05) for IFN-γ production was as follows: rMT1721 × rMT1694, rMT1721 × rMT2462 and rMT3444 × rMT2462. No significance (P > 0·05) was observed between rMT1721 × rMT3444, rMT1694 × rMT2462 and between rMT3444 × rMT1694. Comparisons between the responses to recombinant antigens by PBMC from tuberculosis patients versus healthy PPD⁺ individuals were performed by Fisher's exact test. Significance was positive only for rMT1721 antigen in both assays (SI, P = 0·023; IFN-γ production, P = 0·0012).