Antigenic characterization and cytolocalization of P35, the major Mycoplasma penetrans antigen

Mycoplasma penetrans is a mycoplasma with unique morphology, recently ident ified in urine samples collected from HIV-infected patients. This mycoplasm a has been found to be statistically associated with HIV infection, and to be cytopathic in vitro. The dominant antigen recognized during natural and experimental infections is an abundant lipoprotein, P35, which, upon extrac tion, segregates in the Triton X-114 detergent phase. It is used as the bas is of M. penetrans-specific serological assays. Although mycoplasma lipopro teins, including M, penetrans P35, are the main antigens recognized by the host humoral immune response, very little is known about the nature of the epitopes involved. Immunoelectron microscopy revealed that all P35 is expos ed at the cell surface and is distributed all over the membrane. P35 linear B-epitopes were mapped by an ELISA approach based on a set of overlapping peptides covering the entire mature polypeptide. The immunoreactivity of th e peptides was first tested with sera from immunized animals. The dominant B-epitopes were found at the C- and N-terminal regions, in partial agreemen t with algorithmic predictions. Patient sera were evaluated with the same a ssay. Only some reacted with linear epitopes whereas others did not, indica ting the importance of P35 nonsequential epitopes. Statistical analysis of the results allowed the definition of a set of peptides which were clearly immunodominant. Finally, the P35-encoding gene was modified by in vitro mut agenesis to allow the production and purification of a recombinant protein (rP35 Delta 0) in Escherichia coil. The antigenicity of rP35 Delta 0 was te sted by Western blotting and compared to that of another recombinant produc t, rP35 Delta 3, a truncated P35 polypeptide. Although rP35 Delta 0 reacted with the M. penetrans-seropositive patient sera tested, rP35 Delta 3 was o nly immunoreactive with one of six sera. This result confirmed that P35-non sequential epitopes dominate during M. penetrans infection. Our results hav e important implications for the understanding of lipoprotein antigenicity during mycoplasma infections. In addition, the P35-derived immunodominant s ynthetic peptides defined in this study, as well as the purified rP35 Delta 0, provide the antigenic material for the necessary improvement of M. pene trans serological assays.