Functional characterization of a redundant Plasmodium TRAP family invasin, TRAP-like protein, by aldolase binding and a genetic complementation test

Abstract

Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum Apicomplexa are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although TLP is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of TLP, encoding a redundant TRAP family invasin, in the malaria parasite.

FIG. 1.

In vitro aldolase binding assay to identify TRAP family invasins. Enzyme-linked immunosorbent assay plates coated with His-tagged fusion proteins of either PbTRAP, two PbTRAP loss-of-function mutants lacking the charged residues (trap-acid) or the penultimate tryptophan (trap-w/a), PfCTRP, PfTLP, a PfTLP loss-of-function mutant lacking the penultimate tryptophan (tlp-w/a), or PfEBA175 were incubated with a biotinylated GST-P. yoelii aldolase fusion protein. Bound aldolase was quantified in an avidin-substrate assay.

FIG. 2.

Schematic diagram of the Plasmodium TLP. (A) Representation of the primary structure of P. falciparum TLP (PFF0800w) and P. berghei TLP (AY484471). Displayed are the extracellular TSR, followed by an A domain, the transmembrane span (TM), and the CTD. (B) Comparison of the TSRs in selected TSR-containing proteins. Shown are the conserved tryptophans, the central dicysteine motif, and the cluster of positive residues. In addition to the TRAP family members TLP and TRAP, TSRs of P. berghei circumsporozoite protein (PbCSP), T. gondii micronemal protein 2 (MIC2), and human thrombospondin (thrombosp.) are shown. (C) Comparison of MIDAS motifs in the A-domain-containing proteins TLP, TRAP, and the integrin CD11b. Invariant residues of the MIDAS are highlighted in bold. (D) TLP has a TRAP family-like cytoplasmic domain (CTD). An amino acid alignment of the putative CTDs of P. falciparum and P. berghei TLP, P. berghei TRAP, P. falciparum CTRP, and T. gondii MIC2 are shown. The strictly conserved juxtamembrane tyrosine and penultimate tryptophan residues are shown in black, the carboxy-terminal acidic residues in gray.

FIG. 3.

Functional identification of TRAP family members in Plasmodium. (A) Schematic representation of the TRAP tail swap experiments. Shown are the cytoplasmic tails of P. berghei TRAP, a negative control with a large deletion of the major portion of the tail (Δtail), a positive control containing the tail of the P. falciparum TRAP ortholog (TRAP-tail), and the replacement of the TRAP tail with the corresponding region of PbTLP (TLP-tail), PfCTRP (CTRP-tail), PfEBA 175 (EBA175-tail), or a mutant version of PfEBA175 that contains an additional penultimate tryptophan (EBA175L/W-tail). Carboxy-terminal negatively charged residues are shown in bold, and the penultimate tryptophan is boxed. (B) Generation of the TRAP tail mutations by insertional replacement. The WT TRAP genomic locus is targeted with a SpeI-linearized insertion plasmid containing a 5′ truncation of the TRAP open reading frame, the corresponding tail swaps, the 3′ untranslated region of DHFR/TS, and the dhfr/ts positive selectable marker. Upon a single-crossover event, the region of homology is duplicated, resulting in a 5′ copy with the functional TRAP swap mutant and a nonfunctional 3′ mutant that lacks the promoter and the start codon. Integration-specific test primer combinations are indicated by arrows, and expected fragments are shown as lines. (C) The successful integration event in the resistant parasite population is confirmed by insertion-specific primer combinations.

FIG. 4.

Western blot analysis of TRAP tail swap parasites. Midgut sporozoite extracts from 100,000 WT or mutant sporozoites were separated on a 10% SDS gel and probed with polyclonal anti-PbTRAP-repeat serum (α-TRAP) and monoclonal anti-PbCSP antibodies.

FIG. 5.

Expression of TLP in invasive stages. (A) RT-PCR from poly(A)⁺ RNA of gradient-purified schizonts and salivary gland sporozoites of P. berghei using primers specific for PbTLP and, as a positive control, P. berghei merozoite capping protein 1 (PbMCP1). RT, reverse transcriptase; SPZ., sporozoites; BS, blood stages. (B) Quantitative real-time RT-PCR of P. yoelii MSP1 (white boxes) and PyTLP (gray boxes) using RNA from synchronized ring stages (Rings), early trophozoites (8 h) (Early T.), midstage trophozoites (12 h) (Mid T.), late trophozoites (16 h) (Late T.), and schizont (Schiz.) as a template. Change in gene expression levels is shown as mean values (± standard deviations) of stage-specific signal divided by the mean signal of the pooled RNA samples (pool). (C) RT-PCR from poly(A)⁺ RNA of synchronized P. falciparum blood stages (ring stages, trophozoites, and schizonts) with two pairs of gene-specific primers for PfTLP and primers for PfAMA1 (apical membrane antigen 1), PfMSP7 (merozoite surface protein 7), and PfACT1 (actin 1) as controls.

FIG. 6.

Targeted disruption of PbTLP. (A) Replacement strategy to generate tlp⁻ parasites. The WT TLP genomic locus is targeted with a KpnI/SacII-linearized replacement plasmid (pREP) containing 5′ and 3′ untranslated regions adjacent to the TLP open reading frame and the dhfr/ts positive selectable marker. Upon a double-crossover event, the open reading frame is replaced by the selectable marker. Replacement-specific test and WT primer combinations are indicated by arrows and expected fragments as lines. (B) Replacement-specific PCR analysis. The successful replacement event is verified by primer combination (test 1 and test 2) that can amplify only a signal from the REP locus. Absence of the WT signal from tlp⁻ parasites confirms the purity of the clonal population. (C) Absence of TLP transcripts in tlp⁻ parasites. cDNA from WT or tlp⁻ late-blood-stage parasites was amplified in the presence (+) or absence (−) of reverse transcriptase (RT) with two TLP-specific primer combinations (WT1 and WT2). As loading controls, RT-PCRs with myosin A (MyoA)- and merozoite surface protein 1 (MSP1)-specific primers were added. gDNA, wild-type genomic DNA.