The Actinomyosin Motor Drives Malaria Parasite Red Blood Cell Invasion but Not Egress

Abstract

Apicomplexa are obligate intracellular parasites that actively invade, replicate within, and egress from host cells. The parasite actinomyosin-based molecular motor complex (often referred to as the glideosome) is considered an important mediator of parasite motility and virulence. Mature intracellular parasites often become motile just prior to egress from their host cells, and in some genera, this motility is important for successful egress as well as for subsequent invasion of new host cells. To determine whether actinomyosin-based motility is important in the red blood cell egress and invasion activities of the malaria parasite, we have used a conditional genetic approach to delete GAP45, a primary component of the glideosome, in asexual blood stages of Plasmodium falciparum Our results confirm the essential nature of GAP45 for invasion but show that P. falciparum does not require a functional motor complex to undergo egress from the red blood cell. Malarial egress therefore differs fundamentally from induced egress in the related apicomplexan Toxoplasma gondiiIMPORTANCE Clinical malaria results from cycles of replication of single-celled parasites of the genus Plasmodium in red blood cells. Intracellular parasite replication is followed by a highly regulated, protease-dependent process called egress, in which rupture of the bounding membranes allows explosive release of daughter merozoites which rapidly invade fresh red cells. A parasite actinomyosin-based molecular motor (the glideosome) has been proposed to provide the mechanical force to drive invasion. Studies of the related parasite Toxoplasma gondii have shown that induced egress requires parasite motility, mediated by a functional glideosome. However, whether the glideosome has a similar essential role in egress of malaria merozoites from red blood cells is unknown. Here, we show that although a functional glideosome is required for red blood cell invasion by Plasmodium falciparum merozoites, it is not required for egress. These findings place further emphasis on the key role of the protease cascade in malarial egress.

Keywords: GAP45; Plasmodium falciparum; egress; glideosome; invasion; malaria.

FIG 1

Efficient conditional silencing of GAP45 expression. (A) Schematic representation of the Cas9-based approach for conditional disruption of the GAP45 gene in the DiCre-expressing P. falciparum line B11 (a derivative of 1G5DC; see Materials and Methods). Green and red arrows represent the positions of hybridization of oligonucleotide primers used in panel B. Lollipops, translational stop codons. Red arrowheads, loxP sites. Light blue boxes, recodonized sequence. Red block, introduced HA3 epitope tag. (B) Diagnostic PCR confirming successful modification of the GAP45 locus in a GAP45:loxP P. falciparum clone and RAP-mediated excision of the floxed GAP45-HA3 transgene. Mock-treated parasites were treated with vehicle only (DMSO). (C) Western blots showing successful epitope tagging and rapamycin-inducible ablation of GAP45-HA3 expression. Note that HA3-tagged GAP45 migrates more slowly on SDS-PAGE gels than unmodified GAP45 due to its increased molecular mass. Expression of the unrelated parasite kinase CDPK1 was used as a loading control. (D) IFA showing the subcellular localization of GAP45-HA3 to the periphery of intracellular merozoites in mock-treated GAP45:loxP parasites and the loss of GAP45-HA3 upon RAP treatment. Over 99% of schizonts examined by IFA were GAP45 and HA negative following RAP treatment. Bar, 5 µm.

FIG 2

GAP45 is essential for proliferation of asexual blood-stage P. falciparum. (A) Growth curves showing replication of the GAP45-HA3:loxP parasite line following RAP or mock (DMSO) treatment. RAP-induced excision of the GAP45-HA3 locus produced parasites that were unable to replicate in vitro. Means from three replicates plotted. Error bars, standard deviations. (B) Schematic representation of the genetic complementation strategy used to introduce a second copy of the GAP45 gene and its promoter sequence into the Pfs47 locus of a GAP45-HA3:loxP parasite clone, generating the GAP45-HA3:loxPcomp line. Lollipops, translational stop codons. (C) Growth curves showing proliferation of the GAP45-HA3:loxPcomp parasite line following treatment with RAP or DMSO. The presence of the second GAP45 gene copy at the Pfs47 locus allowed the parasites to grow normally following RAP-mediated excision of the floxed GAP45-HA3 gene at the endogenous GAP45 locus. Means from three replicates plotted. Error bars, SD. (D) IFA showing continued expression of GAP45 from the modified Pfs47 locus following RAP-mediated silencing of GAP45-HA3 expression in GAP45-HA3:loxPcomp parasites. Bar, 5 µm.

FIG 3

ΔGAP45 parasites show defects in expression of the glideosome components MyoA and MTIP. (A) IFA showing the subcellular localization of GAP45-HA3, MTIP, MyoA, and GAP50 in segmented schizonts of ΔGAP45 (RAP) and mock-treated (DMSO) GAP45:loxP parasites. Loss of GAP45-HA3 also resulted in loss of detection of MTIP and MyoA at the IMC upon RAP treatment. Bars, 5 µm. (B) Western blots showing the decreased overall abundance of MTIP and MyoA proteins in the absence of GAP45. (C) TEM images showing similar merozoite and IMC morphologies in GAP45-HA3 and ΔGAP45 parasites. Merozoite plasma membrane (PM), merozoite IMC, and schizont PVM are indicated. Bar, 0.5 µm.

FIG 4

GAP45 is not required for egress. (A) First and final frames from a 40-min time-lapse video of ΔGAP45 schizonts undergoing egress. Schizonts from the first frame that subsequently rupture over the course of the video are circled in black (approximately 80% of the total population). Schizonts that do not rupture are circled in white. (B) Quantification of egress in mock-treated (DMSO) and RAP-treated (ΔGAP45) GAP45:loxP parasites. The top plot shows the proportion of schizonts that undergo egress in each 40-min video. The bottom plot shows the time taken for the schizonts in each video to egress. There were no significant differences between the RAP- and mock-treated populations in the efficiency or kinetics of egress. (C) Western blot showing similar levels of the P50 form of SERA5 (which results from egress-associated proteolytic processing of the P126 precursor [27, 58]) released into culture supernatants of DMSO- and RAP-treated GAP45:loxP schizonts following 45 min of egress.

FIG 5

GAP45 is required for invasion but not for microneme discharge. (A) Flow cytometry-based invasion assay showing failure of ΔGAP45 parasites to develop from schizonts (t = 0 h) through rings (t = 2 h) to trophozoites (t = 24 h). The inset image shows a merozoite, stained with anti-MSP1 antibody and an Alexa 488 secondary antibody (green) and Hoechst stain (blue), bound to an uninfected RBC stained with wheat germ agglutinin (WGA) conjugated to Alexa 647 (magenta). Bar, 5 µm. Comparable results were observed in five additional flow cytometry-based invasion assays. (B) Giemsa-stained thin films showing the presence of ring-stage parasites in the invaded mock-treated (DMSO) GAP45:loxP parasites and absence of rings in the RAP-treated (ΔGAP45) GAP45:loxP parasites. Bar, 5 µm. This was observed in more than 10 experiments. Quantification of rings by manual counting of Giemsa-stained films in one representative experiment is shown (right). (C) IFA showing relocalization of microneme protein AMA1 to the merozoite periphery in both mock-treated and ΔGAP45 parasites. Bar, 5 µm.