Towards a more precise serological diagnosis of human tegumentary leishmaniasis using Leishmania recombinant proteins.

BACKGROUND: Exposure to Leishmania induces a humoral immune response that can be used as a marker of parasite exposure. METHODOLOGY/PRINCIPAL FINDINGS: Herein, ELISA was used to screen sera from patients with Tegumentary Leishmaniasis (TL) against different L. infantum-chagasi-derived recombinant proteins (rHSP70, rH2A, rH2B, rH3, rH4 and rKMP11). Among the recombinant proteins, rHSP70 and rH2A showed the best reactivity against human sera obtained from endemic areas of TL. Receiver-Operator Characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for serodiagnosis of TL. ROC curves confirmed the superior performance of rHSP70 and rH2A, in comparison to the other tested recombinant proteins. Additionally, we evaluated the specificity of the response to rHSP70 and rH2A by testing sera obtained from patients with Chagas' disease, Tuberculosis, Leprosy or Systemic Lupus Erythematosus. In this case, rHSP70 displayed an increased ability to discriminate diseases, in comparison to SLA. CONCLUSION: Our results raise possibility of using rHSP70 for the serodiagnosis of TL.

Induction of cross-priming of naive CD8+ T lymphocytes by recombinant bacillus Calmette-Guerin that secretes heat shock protein 70-major membrane protein-II fusion protein.

Because Mycobacterium bovis bacillus Calmette-Guérin (BCG) unconvincingly activates human naive CD8(+) T cells, a rBCG (BCG-70M) that secretes a fusion protein comprising BCG-derived heat shock protein (HSP)70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed to potentiate the ability of activating naive CD8(+) T cells through dendritic cells (DC). BCG-70M secreted HSP70-MMP-II fusion protein in vitro, which stimulated DC to produce IL-12p70 through TLR2. BCG-70M-infected DC activated not only memory and naive CD8(+) T cells, but also CD4(+) T cells of both types to produce IFN-gamma. The activation of these naive T cells by BCG-70M was dependent on the MHC and CD86 molecules on BCG-70M-infected DC, and was significantly inhibited by pretreatment of DC with chloroquine. Both brefeldin A and lactacystin significantly inhibited the activation of naive CD8(+) T cells by BCG-70M through DC. Thus, the CD8(+) T cell activation may be induced by cross-presentation of Ags through a TAP- and proteosome-dependent cytosolic pathway. When naive CD8(+) T cells were stimulated by BCG-70M-infected DC in the presence of naive CD4(+) T cells, CD62L(low)CD8(+) T cells and perforin-producing CD8(+) T cells were efficiently produced. MMP-II-reactive CD4(+) and CD8(+) memory T cells were efficiently produced in C57BL/6 mice by infection with BCG-70M. These results indicate that BCG-70M activated DC, CD4(+) T cells, and CD8(+) T cells, and the combination of HSP70 and MMP-II may be useful for inducing better T cell activation.

Phosphorylation of Mycobacterium leprae heat-shock 70 protein at threonine 175 alters its substrate binding characteristics.

We have examined the functional properties including autophosphorylation of the Mycobacterium leprae Hsp70 homologue. Recombinant M. leprae Hsp70 had pH optima for its adenosine triphosphatase and autophosphorylating activities which were near pH 8 and 6, respectively. Both these activities were inhibited by reduced and alkylated bovine pancreatic trypsin inhibitor, but not other tested substrates. Autophosphorylation was augmented by up to 25 mM Ca2+. Using site-directed mutagenesis to construct two Thr-->Ala mutants at positions 175 and 193, and phosphoamino acid analysis, it was shown that Thr175 was the dominant threonine residue autophosphorylated in M. leprae Hsp70. Phosphorylation led to an increased affinity for a model polypeptide substrate, reduced and alkylated bovine albumin. These properties are compared with those of the DnaK protein of Escherichia coli.

The immune response to mycobacterial 70-kDa heat shock proteins frequently involves autoreactive T cells and is quantitatively disregulated in multiple sclerosis.

Heat shock proteins (HSP) are the most conserved molecules known to date that may also function as immune targets during infection. Hence, theoretically there is a high chance of cross-reactive responses to epitopes shared by host and microbe HSP. If not properly regulated, these responses may contribute to the pathogenesis of autoimmune disease. To determine if immune responses to HSP could contribute to the pathogenesis of multiple sclerosis, we raised T lymphocyte lines specific for the purified protein derivative of Mycobacterium tuberculosis (PPD) from patients with multiple sclerosis, patients with tuberculosis and from healthy individuals. These lines were then screened for their proliferative response to a M. tuberculosis 70-kDa heat shock protein (M.tb.HSP70). The relative frequency of the recognition of highly conserved sequences of M.tb.HSP70 compared to variable ones was also assessed by mapping experiments on those PPD specific T lymphocyte lines which also recognized the mycobacterial 70-kDa heat shock protein. In patients with multiple sclerosis, we observed a significantly higher estimated frequency of PPD-specific T lines responding to M.tb.HSP70 compared to healthy individuals and patients with tuberculosis. Furthermore, mapping experiments using recombinant proteins representing mycobacterial and human HSP70 sequences and a panel of synthetic peptides encompassing the whole sequence of Mycobacterium leprae HSP70, showed that the response to conserved epitopes of HSP70 is a frequent event in each of the three conditions studied, often leading to the cross-recognition of microbial and human sequences. These findings implicate the 70-kDa heat shock proteins as potential autoantigens in multiple sclerosis.