The patatin-like phospholipase PfPNPLA2 is involved in the mitochondrial degradation of phosphatidylglycerol during Plasmodium falciparum blood stage development.

Plasmodium falciparum is an Apicomplexa responsible for human malaria, a major disease causing more than ½ million deaths every year, against which there is no fully efficient vaccine. The current rapid emergence of drug resistances emphasizes the need to identify novel drug targets. Increasing evidences show that lipid synthesis and trafficking are essential for parasite survival and pathogenesis, and that these pathways represent potential points of attack. Large amounts of phospholipids are needed for the generation of membrane compartments for newly divided parasites in the host cell. Parasite membrane homeostasis is achieved by an essential combination of parasite de novo lipid synthesis/recycling and massive host lipid scavenging. Latest data suggest that the mobilization and channeling of lipid resources is key for asexual parasite survival within the host red blood cell, but the molecular actors allowing lipid acquisition are poorly characterized. Enzymes remodeling lipids such as phospholipases are likely involved in these mechanisms. P. falciparum possesses an unusually large set of phospholipases, whose functions are largely unknown. Here we focused on the putative patatin-like phospholipase PfPNPLA2, for which we generated an glmS-inducible knockdown line and investigated its role during blood stages malaria. Disruption of the mitochondrial PfPNPLA2 in the asexual blood stages affected mitochondrial morphology and further induced a significant defect in parasite replication and survival, in particular under low host lipid availability. Lipidomic analyses revealed that PfPNPLA2 specifically degrades the parasite membrane lipid phosphatidylglycerol to generate lysobisphosphatidic acid. PfPNPLA2 knockdown further resulted in an increased host lipid scavenging accumulating in the form of storage lipids and free fatty acids. These results suggest that PfPNPLA2 is involved in the recycling of parasite phosphatidylglycerol to sustain optimal intraerythrocytic development when the host resources are scarce. This work strengthens our understanding of the complex lipid homeostasis pathways to acquire lipids and allow asexual parasite survival.

The WD40-Protein PfWLP1 Ensures Stability of the PfCCp-Based Adhesion Protein Complex in Plasmodium falciparum Gametocytes.

Members of the WD40-repeat protein family can be found in all eukaryotic proteomes where they usually serve as interaction platforms for the assembly of large protein complexes and are therefore essential for the integrity of these complexes. In the malaria parasite Plasmodium falciparum, the WD40-repeat protein PfWLP1 has been shown to interact with members of distinct adhesion protein complexes in the asexual blood stages and gametocyte stages. In this study, we demonstrate that the presence of PfWLP1 is crucial for both the stability of these gametocyte-specific adhesion complexes as well as for gametocyte maturation and gametogenesis. Using reverse genetics, we generated a PfWLP1-knockdown parasite line for functional characterization of the protein. Knockdown of PfWLP1 resulted in a slight reduction of gametocyte numbers and significantly the impaired ability of the gametocytes to exflagellate. PfWLP1-knockdown further led to reduced protein levels of the Limulus coagulation factor C-like (LCCL)-domain proteins PfCCp1 and PfCCp2, which are key components of the adhesion complexes. These findings suggest that the interaction of PfWLP1 with members of the PfCCp-based adhesion complex ensures complex stability and thereby contributes to gametocyte viability and exflagellation.

Plasmodium falciparum S-Adenosylmethionine Synthetase Is Essential for Parasite Survival through a Complex Interaction Network with Cytoplasmic and Nuclear Proteins.

S-adenosylmethionine synthetase (SAMS) is a key enzyme for the synthesis of the lone methyl donor S-adenosyl methionine (SAM), which is involved in transmethylation reactions and hence required for cellular processes such as DNA, RNA, and histone methylation, but also polyamine biosynthesis and proteostasis. In the human malaria parasite Plasmodium falciparum, PfSAMS is encoded by a single gene and has been suggested to be crucial for malaria pathogenesis and transmission; however, to date, PfSAMS has not been fully characterized. To gain deeper insight into the function of PfSAMS, we generated a conditional gene knockdown (KD) using the glmS ribozyme system. We show that PfSAMS localizes to the cytoplasm and the nucleus of blood-stage parasites. PfSAMS-KD results in reduced histone methylation and leads to impaired intraerythrocytic growth and gametocyte development. To further determine the interaction network of PfSAMS, we performed a proximity-dependent biotin identification analysis. We identified a complex network of 1114 proteins involved in biological processes such as cell cycle control and DNA replication, or transcription, but also in phosphatidylcholine and polyamine biosynthesis and proteasome regulation. Our findings highlight the diverse roles of PfSAMS during intraerythrocytic growth and sexual stage development and emphasize that PfSAMS is a potential drug target.

A patatin-like phospholipase functions during gametocyte induction in the malaria parasite Plasmodium falciparum.

Patatin-like phospholipases (PNPLAs) are highly conserved enzymes of prokaryotic and eukaryotic organisms with major roles in lipid homeostasis. The genome of the malaria parasite Plasmodium falciparum encodes four putative PNPLAs with predicted functions during phospholipid degradation. We here investigated the role of one of the plasmodial PNPLAs, a putative PLA2 termed PNPLA1, during blood stage replication and gametocyte development. PNPLA1 is present in the asexual and sexual blood stages and here localizes to the cytoplasm. PNPLA1-deficiency due to gene disruption or conditional gene-knockdown had no effect on intraerythrocytic growth, gametocyte development and gametogenesis. However, parasites lacking PNPLA1 were impaired in gametocyte induction, while PNPLA1 overexpression promotes gametocyte formation. The loss of PNPLA1 further leads to transcriptional down-regulation of genes related to gametocytogenesis, including the gene encoding the sexual commitment regulator AP2-G. Additionally, lipidomics of PNPLA1-deficient asexual blood stage parasites revealed overall increased levels of major phospholipids, including phosphatidylcholine (PC), which is a substrate of PLA2 . PC synthesis is known to be pivotal for erythrocytic replication, while the reduced availability of PC precursors drives the parasite into gametocytogenesis; we thus hypothesize that the higher PC levels due to PNPLA1-deficiency prevent the blood stage parasites from entering the sexual pathway.

Phospholipases during membrane dynamics in malaria parasites.

Plasmodium parasites, the causative agents of malaria, display a well-regulated lipid metabolism required to ensure their survival in the human host as well as in the mosquito vector. The fine-tuning of lipid metabolic pathways is particularly important for the parasites during the rapid erythrocytic infection cycles, and thus enzymes involved in lipid metabolic processes represent prime targets for malaria chemotherapeutics. While plasmodial enzymes involved in lipid synthesis and acquisition have been studied in the past, to date not much is known about the roles of phospholipases for proliferation and transmission of the malaria parasite. These phospholipid-hydrolyzing esterases are crucial for membrane dynamics during host cell infection and egress by the parasite as well as for replication and cell signaling, and thus they are considered important virulence factors. In this review, we provide a comprehensive bioinformatic analysis of plasmodial phospholipases identified to date. We further summarize previous findings on the lipid metabolism of Plasmodium, highlight the roles of phospholipases during parasite life-cycle progression, and discuss the plasmodial phospholipases as potential targets for malaria therapy.

Role of the Endothelial Layer in the Deswelling Process of Organ-Cultured Human Corneas Before Transplantation.

PURPOSE: Before corneal transplant surgery, a deswelling process of organ-cultured corneas is required. This study compares the deswelling kinetics of corneas with an intact endothelial cell layer and disrupted or removed endothelium by measuring central corneal thickness (CCT) over time using anterior segment spectral domain optical coherence tomography. METHODS: Ten donor pairs were cultured in organ culture. The right and left corneas were alternately assigned to one of 2 deswelling groups. Deswelling in the first group [endothelial group (EG)] was induced using a medium with dextran 5%. Corneas of the second group [nonendothelial group (NEG)] were deprived of their endothelial cell layer by trypsinization and were then placed in the same deswelling medium. CCT (mean ± SD) was measured by anterior segment spectral domain optical coherence tomography before deswelling (0 hours) and after 1, 2, 3, 6, 12, 24, 48, 72, and 144 hours. Deswelling kinetics was analyzed through the nonlinear platform in SAS/JMP11 Pro. RESULTS: Before deswelling, CCT was 1071.0 µm (±129.6 µm) and 1133.8 µm (±124.3 µm) in the EG and NEG, respectively. Minimum corneal thickness was obtained after 24 hours in the EG (531.9 ± 47.5 µm) and 6 hours in the NEG (645 ± 81.2 µm). CCT was significantly (P < 0.01) higher in the NEG than EG after more than 6 hours. CONCLUSIONS: Corneal dehydration after organ culture seems to be a multifactorial process, which not only depends on osmotic effects of the deswelling compound but also requires the presence of an intact endothelial cell layer.

The Plasmodium falciparum blood stages acquire factor H family proteins to evade destruction by human complement.

The acquisition of regulatory proteins is a means of blood-borne pathogens to avoid destruction by the human complement. We recently showed that the gametes of the human malaria parasite Plasmodium falciparum bind factor H (FH) from the blood meal of the mosquito vector to assure successful sexual reproduction, which takes places in the mosquito midgut. While these findings provided a first glimpse of a complex mechanism used by Plasmodium to control the host immune attack, it is hitherto not known, how the pathogenic blood stages of the malaria parasite evade destruction by the human complement. We now show that the human complement system represents a severe threat for the replicating blood stages, particularly for the reinvading merozoites, with complement factor C3b accumulating on the surfaces of the intraerythrocytic schizonts as well as of free merozoites. C3b accumulation initiates terminal complement complex formation, in consequence resulting in blood stage lysis. To inactivate C3b, the parasites bind FH as well as related proteins FHL-1 and CFHR-1 to their surface, and FH binding is trypsin-resistant. Schizonts acquire FH via two contact sites, which involve CCP modules 5 and 20. Blockage of FH-mediated protection via anti-FH antibodies results in significantly impaired blood stage replication, pointing to the plasmodial complement evasion machinery as a promising malaria vaccine target.

Comparison of Gebauer SLc and Moria CBm Carriazo-Barraquer ALK Microkeratomes for Descemet's Stripping Automated Endothelial Keratoplasty Preparation.

PURPOSE: We compared the hand-guided Moria Carriazo-Barraquer (CBm) microkeratome with the fully automatic SLc microkeratome for Descemet's stripping automated endothelial keratoplasty (DSAEK)-lamella preparation and storage, vis-à-vis accuracy, endothelial cell loss (ECL), and lamellar surface roughness (LSR). METHODS: A total of 18 human corneas were dissected with both the 300 µm CBm multi-use (n = 9) and the 300 µm SLc (n = 9) single-use heads, after which they were incubated for 6 d in a 5% dextran medium. Before preparation (0 h) and 1, 24, and 144 h after dissection, ECL and corneal thickness (CT) were measured by ultrasound pachymetry (USP) and optical coherence tomography (OCT). LSR was assessed by scanning electron microscopy (SEM) and evaluated by three masked observers. RESULTS: Prior to cutting, CTs did not differ significantly between OCT or USP measurements, with a high correlation between the two modalities (r(2)= 0.94, p < 0.0001). One hour after preparation the anterior lamella showed a significantly higher dissection depth with the CBm (429.4 ± 21.8 µm) than the SLc (311.7 ± 54.8 µm, p = 0.0006), with the variance of the SLc system showing a trend towards higher values (p = 0.07). Anterior and posterior lamellae swelled significantly in the subsequent culture period. Both groups showed a significant ECL 1 h after preparation (p < 0.0001) with no significant difference between the systems (1 h: p = 0.44; CBm: - 9.4%, SLc: -11.7%), which stabilized over 144 h (144 h CBm: -13.9%, 144 h SLc: -10.3%). LSR did not differ significantly between both systems (p = 0.60). CONCLUSIONS: The SLc system agrees more with the designated cutting depth than the CBm. The dissection produced a comparable LSR and a ∼10% ECL independently of the system. Further incubation of the prepared lamellae led to a swelling, but no further ECL.