Proteomic and Genetic Analyses Demonstrate that Plasmodium berghei Blood Stages Export a Large and Diverse Repertoire of Proteins

Abstract

Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.

In Vivo Function of PTEX88 in Malaria Parasite Sequestration and Virulence

“…The signature PEXEL/VTS motif is strictly conserved among different malaria parasite species. In addition, a growing list of PEXEL/VTS-negative exported proteins (PNEP) is being recognized in P. falciparum and other malaria parasite species (9,10). A corresponding Plasmodium translocon of exported proteins (PTEX) (11) is present in all mammalian Plasmodium parasites.…”

Murine Model for Preclinical Studies of Var2CSA-Mediated Pathology Associated with Malaria in Pregnancy

“…The extracellular region of var2CSA is fused to the exported protein EMAP1 (erythrocyte membrane-associated protein 1) of P. berghei that is localized at the surface membrane of P. berghei IEs (19). We found that PM pathology is more severe in mice infected with P. berghei-VAR than in mice infected with wild-type P. berghei.…”

“…A transgenic 1037cl1 strain that expresses C-terminally var2csa-tagged EMAP1 (gene model PBANKA_0836800; emap1::var2csa; line 1653cl3) was generated as previously reported for C-terminally mCherry-tagged EMAP1 (19). The synthetic 3D7-DBL1X-6ε gene (var2csa; PF3D7_1200600) (20) was first subcloned into PCR2.1 Topo (Invitrogen, Netherlands), and the C-terminal region of emap1 from pL1534 (BamHI/SpeI) was N-terminally fused to the 3D7-DBL1X-6ε gene (NdeI/SpeI).…”

“…Recently, we applied a combination of proteomic, genomic, and reverse-genetic approaches to identify proteins that are exported into the cytoplasm of infected erythrocytes of the rodent model parasite P. berghei (19). Two exported proteins that are transported to the IE surface were identified and were named EMAPs.…”

In Silico Characterization of Plasmodium falciparum and P. yoelii Translocon and Exoribonuclease II (RNase II) Identified in the Merozoite Rhoptry Proteome

“…At present, the full scope of proteins exported across the PVM is unknown. Proteins utilizing the Plasmodium exported element (PEXEL) and PEXEL negative exported proteins (PNEP) have been identified [41][42][43]. Exported proteins are trafficked to the Maurer's clefts in the erythrocyte cytoplasm in P. falciparum infected erythrocytes.…”