Implicating bites from a leishmaniasis sand fly vector in the loss of tolerance in pemphigus.

A possible etiological link between the onset of endemic pemphigus in Tunisia and bites of Phlebotomus papatasi, the vector of zoonotic cutaneous leishmaniasis, has been previously suggested. We hypothesized that the immunodominant P. papatasi salivary protein PpSP32 binds to desmogleins 1 and 3 (Dsg1 and Dsg3), triggering loss of tolerance to these pemphigus target autoantigens. Here, we show using far-Western blot that the recombinant PpSP32 protein (rPpSP32) binds to epidermal proteins with a MW of approximately 170 kDa. Coimmunoprecipitation revealed the interaction of rPpSP32 with either Dsg1 or Dsg3. A specific interaction between PpSP32 and Dsg1 and Dsg3 was further demonstrated by ELISA assays. Finally, mice immunized with rPpSP32 twice per week exhibited significantly increased levels of anti-Dsg1 and -Dsg3 antibodies from day 75 to 120. Such antibodies were specific for Dsg1 and Dsg3 and were not the result of cross-reactivity to PpSP32. In this study, we demonstrated for the first time to our knowledge a specific binding between PpSP32 and Dsg1 and Dsg3, which might underlie the triggering of anti-Dsg antibodies in patients exposed to sand fly bites. We also confirmed the development of specific anti-Dsg1 and -Dsg3 antibodies in vivo after PpSP32 immunization in mice. Collectively, our results provide evidence that environmental factors, such as the exposure to P. papatasi bites, can trigger the development of autoimmune antibodies.

Enhanced patient serum immunoreactivity to recombinant Mycobacterium tuberculosis CFP32 produced in the yeast Pichia pastoris compared to Escherichia coli and its potential for serodiagnosis of tuberculosis.

CFP32 is a Mycobacterium tuberculosis complex-restricted secreted protein that was previously reported to be present in a majority of sputum samples from patients with active tuberculosis (TB) and to stimulate serum antibody production. CFP32 (originally annotated as Rv0577 and also known as TB27.3) was therefore considered a good candidate target antigen for the rapid serodiagnosis of TB. However, the maximal sensitivity of CFP32 serorecognition may have been limited in earlier studies because recombinant CFP32 (rCFP32) produced in Escherichia coli was used as the test antibody-capture antigen, a potential shortcoming stemming from differences in bacterial protein posttranslational modifications. To further investigate the serodiagnostic potential of rCFP32 synthesized in different heterologous hosts, we expressed rCFP32 in the yeast Pichia pastoris. Compared to E. coli rCFP32, yeast rCFP32 showed a higher capacity to capture polyclonal antisera in Western blot studies. Likewise, yeast rCFP32 was significantly better recognized by the sera from TB patients and healthy Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals, by enzyme-linked immunosorbent assay (ELISA), than E. coli rCFP32. In subsequent testing, the yeast rCFP32-based antibody-capture ELISA had a sensitivity of 85% and a specificity of 98% for the discrimination of active TB cases (n = 40) from BCG vaccinees (n = 39). The sensitivity was surprisingly high for a single-antigen TB serodiagnostic test compared to tests using E. coli-expressed antigens. Overall, the trans-production of rCFP32 in P. pastoris significantly improved the serologic detection of CFP32-specific antibodies in patient sera, thereby offering a new, possibly better, modality for producing antigens of diagnostic potential for use in the development of immunoassays for both TB and other infectious diseases.