Perforin-like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes.

Egress of malaria parasites from the host cell requires the concerted rupture of its enveloping membranes. Hence, we investigated the role of the plasmodial perforin-like protein PPLP2 in the egress of Plasmodium falciparum from erythrocytes. PPLP2 is expressed in blood stage schizonts and mature gametocytes. The protein localizes in vesicular structures, which in activated gametocytes discharge PPLP2 in a calcium-dependent manner. PPLP2 comprises a MACPF domain and recombinant PPLP2 has haemolytic activities towards erythrocytes. PPLP2-deficient [PPLP2(-)] merozoites show normal egress dynamics during the erythrocytic replication cycle, but activated PPLP2(-) gametocytes were unable to leave erythrocytes and stayed trapped within these cells. While the parasitophorous vacuole membrane ruptured normally, the activated PPLP2(-) gametocytes were unable to permeabilize the erythrocyte membrane and to release the erythrocyte cytoplasm. In consequence, transmission of PPLP2(-) parasites to the Anopheles vector was reduced. Pore-forming equinatoxin II rescued both PPLP2(-) gametocyte exflagellation and parasite transmission. The pore sealant Tetronic 90R4, on the other hand, caused trapping of activated wild-type gametocytes within the enveloping erythrocytes, thus mimicking the PPLP2(-) loss-of-function phenotype. We propose that the haemolytic activity of PPLP2 is essential for gametocyte egress due to permeabilization of the erythrocyte membrane and depletion of the erythrocyte cytoplasm.

A perforin-like protein mediates disruption of the erythrocyte membrane during egress of Plasmodium berghei male gametocytes.

Successful gametogenesis of the malaria parasite depends on egress of the gametocytes from the erythrocytes within which they developed. Egress entails rupture of both the parasitophorous vacuole membrane and the erythrocyte plasma membrane, and precedes the formation of the motile flagellated male gametes in a process called exflagellation. We show here that egress of the male gametocyte depends on the function of a perforin-like protein, PPLP2. A mutant of Plasmodium berghei lacking PPLP2 displayed abnormal exflagellation; instead of each male gametocyte forming eight flagellated gametes, it produced gametocytes with only one, shared thicker flagellum. Using immunofluorescence and transmission electron microscopy analysis, and phenotype rescue with saponin or a pore-forming toxin, we conclude that rupture of the erythrocyte membrane is blocked in the mutant. The parasitophorous vacuole membrane, on the other hand, is ruptured normally. Some mutant parasites are still able to develop in the mosquito, possibly because the vigorous motility of the flagellated gametes eventually leads to escape from the persisting erythrocyte membrane. This is the first example of a perforin-like protein in Plasmodium parasites having a role in egress from the host cell and the first parasite protein shown to be specifically required for erythrocyte membrane disruption during egress.

Changes in the transcriptome of the malaria parasite Plasmodium falciparum during the initial phase of transmission from the human to the mosquito.

BACKGROUND: The transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by dormant sexual precursor cells, the gametocytes, which become activated in the mosquito midgut. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they play a crucial role in spreading the tropical disease. The human-to-mosquito transmission triggers important molecular changes in the gametocytes, which initiate gametogenesis and prepare the parasite for life-cycle progression in the insect vector. RESULTS: To better understand gene regulations during the initial phase of malaria parasite transmission, we focused on the transcriptome changes that occur within the first half hour of parasite development in the mosquito. Comparison of mRNA levels of P. falciparum gametocytes before and 30 min following activation using suppression subtractive hybridization (SSH) identified 126 genes, which changed in expression during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or inner membrane complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were cell surface-associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence.For a subset of 27 genes, transcript changes during gametogenesis were studied in detail by real-time RT-PCR. Of these, 22 genes were expressed in gametocytes, and for 15 genes transcript expression in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of seven genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito. For selected genes, a regulated expression during gametogenesis was confirmed on the protein level, using quantitative confocal microscopy. CONCLUSIONS: The obtained transcriptome data demonstrate the regulations of gene expression immediately following malaria parasite transmission to the mosquito. Our findings support the identification of proteins important for sexual reproduction and further development of the mosquito midgut stages and provide insights into the genetic basis of the rapid adaption of Plasmodium to the insect vector.

Molecular mechanisms of host cell egress by malaria parasites.

Egress is a crucial step for malaria parasites to progress from one host cell to another. The rapid transition between host cells is mediated by the invasive merozoite stages. Merozoite egress from the enveloping cell includes the rupture of two membranes, the membrane of the parasitophorous vacuole and the host cell membrane. Egress from the host cell is also of importance for the intraerythrocytic gametocytes in order to undergo gametogenesis following their transmission to the mosquito during the blood meal. An increasing number of studies have aimed to identify the molecules involved in host cell egress by malaria parasites and decipher the sequence of membrane rupture. Recent work has acknowledged the crucial roles of plasmodial and host-derived proteases in membrane rupture and has indicated the involvement of secretory vesicles in priming the enveloping membranes for egress. This review highlights recent insight into the mechanisms of host cell egress by Plasmodium parasites. We will discuss the mode of egress of intrahepatic and intraerythrocytic parasites and their measures to evade the host immune system during this process.

Facing COVID-19: Early Recognition and Triage Tool for Medical Treatment Facilities With Limited Resources.

INTRODUCTION: Coronavirus Disease 2019 (COVID-19) is spreading all over the world. Health systems around the globe have to deal with decreased capabilities and exhausted resources because of the surge of patients. The need to identify COVID-19 patients to achieve a timely opportunity to treat and isolate them is an ongoing challenge for health care professionals everywhere. A lack of testing capabilities forces clinicians to make the crucial initial decision on the basis of clinical findings and routine diagnostic laboratory test. This article reviews the current literature and presents a new adapted protocol for diagnosing and triaging COVID-19 patients. A special emphasis lies on the stepwise approach guiding the medical provider to a triage decision that is suitable for the individual patient and the situation of the local medical treatment facility. MATERIALS AND METHODS: On March 30, 2020, a PubMed based literature research on COVID-19 following the preferred reporting items for systematic reviews and meta-analyses guidelines was performed. A diagnostic and triage tool for COVID-19 was designed based on the major findings in the reviewed literature. RESULTS: After a selection process, focusing on the topics "epidemiology," "clinical characteristics," and "diagnostic tools," 119 out of a total amount of 1,241 publications were selected to get an overview of the growing evidence. CONCLUSIONS: The designed Early Recognition and Triage Tool enables the medical provider to use the applicable modules of the protocol for capabilities of the local setting to get the most appropriate diagnostic and triage done. The tool should give guidance for the initial approach until specific testing for the COVID-19 virus is available.

Triage Decisions in the Context of COVID-19: Old Burden, New Challenge-The Structured Approach for Intensive Care Unit Triage (SAINT) Protocol.

INTRODUCTION: The medical treatment facilities (MTF) represent the equivalent of the healthcare system in the home countries, but they face the limitations of an outpost at the end of the supply chain. The capabilities are limited, and the necessary effort to extend the treatment capacity is tremendous. Algorithms based on scientific evidence or at least profound medical expertise are a tool to facilitate the decision-making process in triage under difficult circumstances. The aim of this article is to present a protocol that regards the specific entities military MTF abroad have to deal with in context of the COVID-19 pandemic. MATERIAL AND METHODS: To prepare our own health system within the Resolute Support mission, an interdisciplinary team of consultants and nurses located at the multinational role 2E, Camp Marmal, Mazar-e-Sharif, Afghanistan, individually reviewed medical databases and the current literature concerning triage on intensive care units. The identified literature was evaluated by all authors. In the next step, an adapted flow chart for triage on intensive care unit in MTF abroad was set up on the basis of existing triage tools found in the reviewed literature. RESULTS: The authors created the "Structured Approach for Intensive Care Unit Triage (SAINT)" protocol. It is an approach that fits to the specific entities (e.g., limited medical resources in the whole system, limited intensive care unit capabilities for long-term stay or organ replacement therapy, etc.) that determine the framework of the special military health system abroad. CONCLUSIONS: The presented triage protocol may be a tool for medical personnel to facilitate the difficult task of triaging. It provides guidance along patient-centered criteria like individual medical, ethical, and legal issues while taking into account the available resources. Future studies are needed to investigate the effectiveness of the SAINT protocol.

Perforin-like protein PPLP4 is crucial for mosquito midgut infection by Plasmodium falciparum.

The genomes of Plasmodium parasites encode for five perforin-like proteins, PPLP1-5, and four of them have previously been demonstrated to be involved in disruption of host cell barriers. We now show that the fifth perforin, PPLP4, is crucial for infection of the mosquito vector by Plasmodium falciparum parasites. PPLP4 is expressed in the blood and mosquito midgut stages in granular structures. In gametocytes, PPLP4 expression is specific to the female gender, while ookinetes show a PPLP4 localization at the apical pole. Gene disruption of pplp4 results in no phenotypical change during blood stage replication, gametocyte development or gametogenesis, while mosquitoes fed with PPLP4-deficient gametocytes display a severe reduction in oocyst numbers, and an accumulation of ookinetes in the mosquito midguts was observed. In conclusion, we propose an essential role for PPLP4 in infection of the mosquito midgut, presumably by mediating ookinete traversal through the midgut epithelium.

Inhibition of the SR protein-phosphorylating CLK kinases of Plasmodium falciparum impairs blood stage replication and malaria transmission.

Cyclin-dependent kinase-like kinases (CLKs) are dual specificity protein kinases that phosphorylate Serine/Arginine-rich (SR) proteins involved in pre-mRNA processing. Four CLKs, termed PfCLK-1-4, can be identified in the human malaria parasite Plasmodium falciparum, which show homology with the yeast SR protein kinase Sky1p. The four PfCLKs are present in the nucleus and cytoplasm of the asexual blood stages and of gametocytes, sexual precursor cells crucial for malaria parasite transmission from humans to mosquitoes. We identified three plasmodial SR proteins, PfSRSF12, PfSFRS4 and PfSF-1, which are predominantly present in the nucleus of blood stage trophozoites, PfSRSF12 and PfSF-1 are further detectable in the nucleus of gametocytes. We found that recombinantly expressed SR proteins comprising the Arginine/Serine (RS)-rich domains were phosphorylated by the four PfCLKs in in vitro kinase assays, while a recombinant PfSF-1 peptide lacking the RS-rich domain was not phosphorylated. Since it was hitherto not possible to knock-out the pfclk genes by conventional gene disruption, we aimed at chemical knock-outs for phenotype analysis. We identified five human CLK inhibitors, belonging to the oxo-ß-carbolines and aminopyrimidines, as well as the antiseptic chlorhexidine as PfCLK-targeting compounds. The six inhibitors block P. falciparum blood stage replication in the low micromolar to nanomolar range by preventing the trophozoite-to-schizont transformation. In addition, the inhibitors impair gametocyte maturation and gametogenesis in in vitro assays. The combined data show that the four PfCLKs are involved in phosphorylation of SR proteins with essential functions for the blood and sexual stages of the malaria parasite, thus pointing to the kinases as promising targets for antimalarial and transmission blocking drugs.