The Micronemal Plasmodium Proteins P36 and P52 Act in Concert to Establish the Replication-Permissive Compartment Within Infected Hepatocytes

Within the liver, Plasmodium sporozoites traverse cells searching for a “suitable” hepatocyte, invading these cells through a process that results in the formation of a parasitophorous vacuole (PV), within which the parasite undergoes intracellular replication as a liver stage. It was previously established that two members of the Plasmodium s48/45 protein family, P36 and P52, are essential for productive invasion of host hepatocytes by sporozoites as their simultaneous deletion results in growth-arrested parasites that lack a PV. Recent studies point toward a pathway of entry possibly involving the interaction of P36 with hepatocyte receptors EphA2, CD81, and SR-B1. However, the relationship between P36 and P52 during sporozoite invasion remains unknown. Here we show that parasites with a single P52 or P36 gene deletion each lack a PV after hepatocyte invasion, thereby pheno-copying the lack of a PV observed for the P52/P36 dual gene deletion parasite line. This indicates that both proteins are equally important in the establishment of a PV and act in the same pathway. We created a Plasmodium yoelii P36mCherry tagged parasite line that allowed us to visualize the subcellular localization of P36 and found that it partially co-localizes with P52 in the sporozoite secretory microneme organelles. Furthermore, through co-immunoprecipitation studies in vivo, we determined that P36 and P52 form a protein complex in sporozoites, indicating a concerted function for both proteins within the PV formation pathway. However, upon sporozoite stimulation, only P36 was released as a secreted protein while P52 was not. Our results support a model in which the putatively glycosylphosphatidylinositol (GPI)-anchored P52 may serve as a scaffold to facilitate the interaction of secreted P36 with the host cell during sporozoite invasion of hepatocytes

Stable allele frequency distribution of the polymorphic region of SURFIN4.2 in Plasmodium falciparum isolates from Thailand

Plasmodium falciparum SURFIN 4.2 (PFD1160w) is a polymorphic protein expressed on the surface of parasite-infected erythrocytes. Such molecules are expected to be under strong host immune pressure, thus we analyzed the nucleotide diversity of the N-terminal extracellular region of SURFIN 4.2 using P. falciparum isolates obtained from a malaria hypoendemic area of Thailand. The extracellular region of SURFIN 4.2 was divided into four regions based on the amino acid sequence conservation among SURFIN members and the level of polymorphism among SURFIN 4.2 sequences; N-terminal segment (Nter), a cysteine-rich domain (CRD), a variable region 1 (Var1), and a variable region 2 (Var2). Comparison between synonymous and non-synonymous substitutions, Tajima\u27s D test, and Fu and Li\u27s D* and F* tests detected signatures of positive selection on Var2 and to a lesser extent Var1, suggesting that these regions were likely under host immune pressure. Strong linkage disequilibrium was detected for nucleotide pairs separated by a distance of more than 1.5 kb, and 7 alleles among 19 alleles detected in 1988-1989 still circulated 14 years later, suggesting low recombination of the analyzed surf 4.2 sequence region in Thailand. The allele frequency distribution of polymorphic areas in Var2 did not differ between two groups collected in different time points, suggesting the allele frequency distribution of this region was stable for 14 years. The observed allele frequency distribution of SURFIN 4.2 Var2 may be fixed in Thai P. falciparum population as similar to the observation for P. falciparum merozoite surface protein 1, for which a stable allele frequency distribution was reported

PEXEL-independent trafficking of Plasmodium falciparum SURFIN4.2 to the parasite-infected red blood cell and Maurer\u27s clefts.

SURFIN(4.2) is a parasite-infected red blood cell (iRBC) surface associated protein of Plasmodium falciparum. To analyze the region responsible for the intracellular trafficking of SURFIN(4.2) to the iRBC and Maurer\u27s clefts, a panel of transgenic parasite lines expressing recombinant SURFIN(4.2) fused with green fluorescent protein was generated and evaluated for their localization. We found that the cytoplasmic region containing a tryptophan rich (WR) domain is not necessary for trafficking, whereas the transmembrane (TM) region was. Two PEXEL-like sequences were shown not to be responsible for the trafficking of SURFIN(4.2), demonstrating that the protein is trafficked in a PEXEL-independent manner. N-terminal replacement, deletion of the cysteine-rich domain or the variable region also did not prevent the protein from localizing at the iRBC or Maurer\u27s clefts. A recombinant SURFIN(4.2) protein possessing 50 amino acids upstream of the TM region, TM region itself and a part of the cytoplasmic region was shown to be trafficked into the iRBC and Maurer\u27s clefts, suggesting that there are no essential trafficking motifs in the SURFIN(4.2) extracellular region. A mini-SURFIN(4.2) protein containing WR domain was shown by Western blotting to be more abundantly detected in a Triton X-100-insoluble fraction, compared to the one without WR domain. We suggest that the cytoplasmic region containing the WR may be responsible for their difference in solubility

Observation of morphological changes of female osmiophilic bodies prior to Plasmodium gametocyte egress from erythrocytes

Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Gametocytes, which differentiate from asexual-stage parasites, are activated by environmental changes when ingested into the mosquito midgut, and are rapidly released from erythrocytes prior to fertilization. Secretory proteins localized to osmiophilic bodies (OBs), organelles unique to gametocytes, have been reported to be involved in female gametocyte egress. In this study, we investigate the dynamics of OBs in activated gametocytes of Plasmodium falciparum and Plasmodium yoelii using the female OB-specific marker protein, G377. After activation, female gametocyte OBs migrate to the parasite surface and fuse to form large vesicles beneath the parasite plasma membrane. At the marginal region of female gametocytes, fused vesicles secrete contents by exocytosis into the parasitophorous vacuole space, prior to parasite egress via the break-down of the erythrocyte membrane. This is the first detailed description of how proteins are transported through osmiophilic bodies

Single amino acid substitution in Plasmodium yoelii erythrocyte ligand determines its localization and controls parasite virulence

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host–pathogen interactions and malaria virulence

Phenotypic Dissection of a <i>Plasmodium</i>-Refractory Strain of Malaria Vector <i>Anopheles stephensi</i>: The Reduced Susceptibility to <i>P. berghei</i> and <i>P. yoelii</i>

<div><p>Anopheline mosquitoes are the major vectors of human malaria. Parasite-mosquito interactions are a critical aspect of disease transmission and a potential target for malaria control. Current investigations into parasite-mosquito interactions frequently assume that genetically resistant and susceptible mosquitoes exist in nature. Therefore, comparisons between the <i>Plasmodium</i> susceptibility profiles of different mosquito species may contribute to a better understanding of vectorial capacity. <i>Anopheles stephensi</i> is an important malaria vector in central and southern Asia and is widely used as a laboratory model of parasite transmission due to its high susceptibility to <i>Plasmodium</i> infection. In the present study, we identified a rodent malaria-refractory strain of <i>A. stephensi mysorensis</i> (Ehime) by comparative study of infection susceptibility. A very low number of oocysts develop in Ehime mosquitoes infected with <i>P. berghei</i> and <i>P. yoelii</i>, as determined by evaluation of developed oocysts on the basal lamina. A stage-specific study revealed that this reduced susceptibility was due to the impaired formation of ookinetes of both <i>Plasmodium</i> species in the midgut lumen and incomplete crossing of the midgut epithelium. There were no apparent abnormalities in the exflagellation of male parasites in the ingested blood or the maturation of oocysts after the rounding up of the ookinetes. Overall, these results suggest that invasive-stage parasites are eliminated in both the midgut lumen and epithelium in Ehime mosquitoes by strain-specific factors that remain unknown. The refractory strain newly identified in this report would be an excellent study system for investigations into novel parasite-mosquito interactions in the mosquito midgut.</p></div