Analyses of rat Pneumocystis carinii antigens recognized by human and rat antibodies by using western immunoblotting

Abstract

The major Pneumocystis carinii antigens inducing antibody responses in infected hosts were identified by Western immunoblotting techniques. The biochemical nature of these antigens was also elucidated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by protein staining revealed a major component with a molecular weight (MW) of greater than 205,000. This major component disappeared and a new major protein staining component of approximately 110,00 to 116,000 MW appeared when electrophoresis was done in the presence of ,3-mercaptoethanol. Periodic acid-Schiff staining revealed that this major component contains carbohydrate moieties. A major component in the 55,000to 60,000-MW region was visible with periodic acid-Schiff stain, but not with a protein stain, after electrophoresis in the presence of 1mercaptoethanol. The majority of sera tested from humans with diagnosed pneumocystosis and from rats allowed to recover from steroid-induced pneumocystosis reacted strongly with 110,000to 116,000-, and 55,000to 60,000-MW components. These sera often, but not always, detected antigens with MWs of approximately 170,000, 125,000, and 30,000 to 32,000. The data suggest that the antigenic composition of P. carinii is relatively complex and that rat and human P. carinii probably share antigenic determinants. Competitive studies between infection-derived human and rat antisera for the major rat P. carinii components revealed competition; rat antisera appeared to recognize a greater range of antigenic epitopes than did human antisera. Protease treatment of the antigenic components that had been immobilized on nitrocellulose paper destroyed their antigenic reactivity with rat antibody. Treatment with sodium periodate decreased reactivity of this 110,000to 116,000-MW component and completely destroyed the reactivity of the 55,000to 60,000-MW component with rat antibody. Pneumocystis carinii is a ubiquitous organism infecting many mammalian species, including man (14). Serological surveys indicate that detectable antibodies to P. carinii occur in 40 to 75% of the healthy adult population (9, 12). These data suggest that P. carinlii often occurs as a subclinical infection (14). Severe P. car-inii pneumonitis (PCP) similar to that seen in humans can be induced in rats by giving corticosteroids and a low-protein diet (17). In rats and humans, antibody titers against P. carinii usually rise upon

Ultrastructural localization of monoclonal antibodies against Pneumocystis carinii

“…We found that the epitopes recognized by SMoAb had a similar ultrastructural localization in organisms of human and rat origin, whereas the epitopes recognized by DMoAb were present only in organisms of human origin. This is consistent with previous observations suggesting the existence of both strainspecific and shared antigens in PC from different hosts (Gigliotti et al 1986;Graves et al 1986b;Kovacs et al 1989;Lundgren et a/. 1991;Gigliotti 1992).…”

“…Several research groups have produced monoclonal antibodies against PC, both for diagnostic and research purposes (Gigliotti et al 1986;Gigliotti et al 1988; Graves et al 1986a;Linder et al 1987; Magee et al 1991;Gigliotti 1992). Studies using these antibodies have shown that PC isolates from different hosts are antigenically dissimilar, although they share some common antigens (Graves et al 1986b;Kovacs et al 1989;Lundgren et al 199 1 ;Bauer et al 1993;Gigliotti et al 1993a). There is evidence of host speciesspecific genotypic variations (Sinclair et al 1991;Edlind et al 1992).…”

“…Many immunological studies have focused on the immunogenic 95 to 140 kDa glycoprotein termed gp 120 or gpA (Gigliotti et a/. 1986; Graves et al 1986b;Gigliotti 1992;Haidaris et a/. 1992;.…”

Translocation of surface antigen genes to a unique telomeric expression site in Pneumocystis carinii

“…Regardless of the host species from which they are obtained, P. c. carinii are covered by a large (90-120 kDa) glycoprotein called either major surface glycoprotein (MSG) Kovacs et al, 1988;Linke et al, 1989;Wada et al, 1993;Sunkin et al, 1994) or gpA (Haidaris et al, 1992;Wright et al, 1994). MSG is thought to play a crucial role in host-pathogen interactions because it is recognized by serum antibodies and T cells from exposed hosts (Graves et al, 1986;Gigliotti et al, 1988;Kovacs et al, 1988;Linke et al, 1989;Nakamura et al, 1989;Fisher et al, 1991;Lundgren et al, 1992;Smulian et al, 1993a;Garbe and Stringer, 1994), and binds to fibronectin (Pottratz and Martin, 1990), Surfactant Protein A (Zimmerman et al, 1992), and other host proteins (Ezekowitz et al, 1991;Limper et al, 1991;1993;Neese et al, 1994). Each P. c. carinii cell has the potential to express numerous different MSG isoforms because the genome of P. c. carinii encodes about 100 different MSG genes, distributed among all of the 15 discernible chromosomes (Kovacs et al, 1993;Wada et al, 1993;Sunkin et al, 1994).…”

Purification and characterization of a major human Pneumocystis carinii surface antigen.

“…Crossed affinoimmunoelectrophoresis is a technique that can be used to examine the possibility of carbohydrate-dependent microheterogeneity (7). With the human and the rat P carinii surface antigens being different both in their glycosylation and antigenicity (6,10,14,25), we undertook this study to further characterize the glycosylation of the major human Z? carinii glycoprotein Gp 95.…”