Abstract
BackgroundAntigenic variation by malaria parasites was first described in Plasmodium knowlesi, which infects humans and macaque monkeys, and subsequently in P. falciparum, the most virulent human parasite. The schizont-infected cell agglutination (SICA) variant proteins encoded by the SICAvar multigene family in P. knowlesi, and Erythrocyte Membrane Protein-1 (EMP-1) antigens encoded by the var multigene family in P. falciparum, are expressed at the surface of infected erythrocytes, are associated with virulence, and serve as determinants of naturally acquired immunity. A parental P. knowlesi clone, Pk1(A+), and a related progeny clone, Pk1(B+)1+, derived by an in vivo induced variant antigen switch, were defined by the expression of distinct SICA variant protein doublets of 210/190 and 205/200 kDa, respectively. Passage of SICA[+] infected erythrocytes through splenectomized rhesus monkeys results in the SICA[-] phenotype, defined by the lack of surface expression and agglutination with variant specific antisera.Principal FindingsWe have investigated SICAvar RNA and protein expression in Pk1(A+), Pk1(B+)1+, and SICA[-] parasites. The Pk1(A+) and Pk1(B+)1+ parasites express different distinct SICAvar transcript and protein repertoires. By comparison, SICA[-] parasites are characterized by a vast reduction in SICAvar RNA expression, the lack of full-length SICAvar transcript signals on northern blots, and correspondingly, the absence of any SICA protein detected by mass spectrometry.SignificanceSICA protein expression may be under transcriptional as well as post-transcriptional control, and we show for the first time that the spleen, an organ central to blood-stage immunity in malaria, exerts an influence on these processes. Furthermore, proteomics has enabled the first in-depth characterization of SICA[+] protein phenotypes and we show that the in vivo switch from Pk1(A+) to Pk1(B+)1+ parasites resulted in a complete change in SICA profiles. These results emphasize the importance of studying antigenic variation in the context of the host environment.
Highlights
Antigenic variation in malaria entails the sequential expression of different high molecular weight parasite-encoded variant proteins from a large multigene family on the surface of infected red blood cells and this process can result in immune evasion and facilitate disease, reviewed in 1–3
Specific antibodies to any region of the schizontinfected cell agglutination (SICA) proteins were not available at the time when SICA[-] parasites were first identified, and it has remained an open question whether these parasites do not make any protein or whether it is produced but not transported to the surface of the infected red blood cells (iRBCs) [9,21,22]
When tested by IFA, the pan-SICACyto antiserum was positive as expected on all SICA[+] trophozoiteinfected RBCs but negative on SICA[-] trophozoite-infected RBCs with only an occasional, spurious fluorescing iRBC observed in the blood smears
Summary
Antigenic variation in malaria entails the sequential expression of different high molecular weight parasite-encoded variant proteins from a large multigene family on the surface of infected red blood cells (iRBCs) and this process can result in immune evasion and facilitate disease, reviewed in 1–3. When passaged in pre-exposed/immunologically boosted rhesus macaques, a switch in variant antigen expression was observed; newly developed clones derived therefrom had unique iRBC surface antigenic properties [8,9] These clones were key to demonstrate the clonal nature of the antigenic variation and they enabled the experimental identification of the SICA variant antigens as high molecular weight (~200 kDa) parasite-encoded proteins that were extractable in SDS, and predicted to be inserted in the membrane of the RBC with exposure on the surface of the infected cells [8,9,10,11,12,13]. These results emphasize the importance of studying antigenic variation in the context of the host environment
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