Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum.

Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.

The 3-phosphoinositide-dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.

Rapid antimicrobial susceptibility testing in patients with bacteraemia due to Enterobacterales: an implementation study.

AIMS OF THE STUDY: The goal of this descriptive study was to assess the performance as well as the extent of the clinical impact of rapid automated antimicrobial susceptibility testing in patients with bacteraemia due to Enterobacterales. We also aimed to analyse how rapid automated antimicrobial susceptibility testing influences clinical decision-making. METHODS: This single-centre study conducted at the University Hospital of Zurich included data from all consecutive patients with Enterobacterales bacteraemia from November 2019 to October 2020. There was no control group. The primary outcome was the effect of rapid automated antimicrobial susceptibility testing on antibiotic therapy (no adjustment, escalation to a broader-spectrum antibiotic or de-escalation to a narrower-spectrum antibiotic). Rapid automated antimicrobial susceptibility testing results were further compared to susceptibility tests using European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard methods and erroneous results were noted. Additionally, we investigated turnaround times for rapid automated antimicrobial susceptibility testing and routine diagnostic testing. RESULTS: We analysed 106 patients with 116 episodes of bacteraemia due to Enterobacterales, with Escherichia coli and Klebsiella pneumoniae being the most frequent isolates. Almost 8% of pathogens were multidrug resistant. Rapid automated antimicrobial susceptibility testing showed category agreement in 98.4% of all interpretable cases. A significant reduction of more than 20 h in turnaround times could be achieved with rapid automated antimicrobial susceptibility testing compared to the routine diagnostic workflow. In the majority of cases, rapid automated antimicrobial susceptibility testing had no effect, given that the empirical therapy was already correct or circumstances did not allow for de-escalation. In 38.8% of cases, antimicrobial therapy was adjusted, whereas eight cases were de-escalated based on rapid automated antimicrobial susceptibility testing alone. CONCLUSIONS: Rapid automated antimicrobial susceptibility testing may be a valuable and safe way to accelerate diagnosis. In particular, time to suitable therapy can be shortened in cases of incorrect therapy. However, physicians are reluctant to de-escalate antibiotic therapy based on rapid automated antimicrobial susceptibility testing alone, limiting its impact in everyday clinics. To further explore the potential of rapid automated antimicrobial susceptibility testing, a stringent/compulsory antibiotic stewardship programme would be a valuable next step.

Revisiting the Effect of Pharmaceuticals on Transmission Stage Formation in the Malaria Parasite Plasmodium falciparum.

Malaria parasites rely on specialized stages, called gametocytes, to ensure human-to-human transmission. The formation of these sexual precursor cells is initiated by commitment of blood stage parasites to the sexual differentiation pathway. Plasmodium falciparum, the most virulent of six parasite species infecting humans, employs nutrient sensing to control the rate at which sexual commitment is initiated, and the presence of stress-inducing factors, including antimalarial drugs, has been linked to increased gametocyte production in vitro and in vivo. These observations suggest that therapeutic interventions may promote gametocytogenesis and malaria transmission. Here, we engineered a P. falciparum reporter line to quantify sexual commitment rates after exposure to antimalarials and other pharmaceuticals commonly prescribed in malaria-endemic regions. Our data reveal that some of the tested drugs indeed have the capacity to elevate sexual commitment rates in vitro. Importantly, however, these effects are only observed at drug concentrations that inhibit parasite survival and only rarely result in a net increase of gametocyte production. Using a drug-resistant parasite reporter line, we further show that the gametocytogenesis-promoting effect of drugs is linked to general stress responses rather than to compound-specific activities. Altogether, we did not observe evidence for mechanistic links between the regulation of sexual commitment and the activity of commonly used pharmaceuticals in vitro. Our data hence does not support scenarios in which currently applied therapeutic interventions would promote the spread of drug-resistant parasites or malaria transmission in general.

The catalytic subunit of Plasmodium falciparum casein kinase 2 is essential for gametocytogenesis.

Casein kinase 2 (CK2) is a pleiotropic kinase phosphorylating substrates in different cellular compartments in eukaryotes. In the malaria parasite Plasmodium falciparum, PfCK2 is vital for asexual proliferation of blood-stage parasites. Here, we applied CRISPR/Cas9-based gene editing to investigate the function of the PfCK2α catalytic subunit in gametocytes, the sexual forms of the parasite that are essential for malaria transmission. We show that PfCK2α localizes to the nucleus and cytoplasm in asexual and sexual parasites alike. Conditional knockdown of PfCK2α expression prevented the transition of stage IV into transmission-competent stage V gametocytes, whereas the conditional knockout of pfck2a completely blocked gametocyte maturation already at an earlier stage of sexual differentiation. In summary, our results demonstrate that PfCK2α is not only essential for asexual but also sexual development of P. falciparum blood-stage parasites and encourage studies exploring PfCK2α as a potential target for dual-active antimalarial drugs.

A 39-Amino-Acid C-Terminal Truncation of GDV1 Disrupts Sexual Commitment in Plasmodium falciparum.

Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasite's life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood-stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study, we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase knockout (KO) lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but that both lines harbor the same mutation that leads to a truncation in the extreme C terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild-type parasite lines replicates the observed phenotype. Using a GDV1 overexpression line, we show that the truncation in the GDV1 C terminus does not interfere with the nuclear import of GDV1 or its interaction with HP1 in vitro but appears to be important to sustain GDV1 protein levels and thereby sexual commitment.IMPORTANCE Transmission of malaria-causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the life cycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here, we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C terminus of the gametocyte development 1 protein (GDV1), which abrogates sexual development. The mutation destabilizes the protein but not its interaction with its cognate binding partner HP1. This suggests an important role for the GDV1 C terminus beyond trafficking and protein stability.

Blunted sFasL signalling exacerbates TNF-driven neutrophil necroptosis in critically ill COVID-19 patients.

OBJECTIVES: Critically ill coronavirus disease 2019 (COVID-19) patients are characterised by a severely dysregulated cytokine profile and elevated neutrophil counts, impacting disease severity. However, it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the regulated cell death (RCD) programme of neutrophils. METHODS: Regulated cell death phenotype of neutrophils isolated from critically ill COVID-19 patients or healthy donors and stimulated with COVID-19 or healthy plasma ex vivo was assessed by flow cytometry, time-lapse microscopy and cytokine multiplex analysis. Immunohistochemistry of COVID-19 patients and control biopsies were performed to assess the in situ neutrophil RCD phenotype. Plasma cytokine levels of COVID-19 patients and healthy donors were measured by multiplex analysis. Clinical parameters were correlated to cytokine levels of COVID-19 patients. RESULTS: COVID-19 plasma induced a necroptosis-sensitive neutrophil phenotype, characterised by cell lysis, elevated release of damage-associated molecular patterns (DAMPs), increased receptor-interacting serine/threonine-protein kinase (RIPK) 1 levels and mixed lineage kinase domain-like pseudokinase (MLKL) involvement. The occurrence of neutrophil necroptosis MLKL axis was further confirmed in COVID-19 thrombus and lung biopsies. Necroptosis was induced by the tumor necrosis factor receptor 1 (TNFRI)/TNF-α axis. Moreover, reduction of soluble Fas ligand (sFasL) levels in COVID-19 patients and hence decreased signalling to Fas directly increased RIPK1 levels, exacerbated TNF-driven necroptosis and correlated with disease severity, which was abolished in patients treated with glucocorticoids. CONCLUSION: Our results suggest a novel role for sFasL signalling in the TNF-α-induced RCD programme in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

PfMAP-2 is essential for male gametogenesis in the malaria parasite Plasmodium falciparum.

In malaria parasites, male gametogenesis is a proliferative stage essential for parasite transmission to the mosquito vector. It is a rapid process involving three rounds of genome replication alternating with closed endomitoses, and assembly of axonemes to produce eight flagellated motile microgametes. Studies in Plasmodium berghei have highlighted tight regulation of gametogenesis by a network of kinases. The P. berghei MAPK homologue PbMAP-2 is dispensable for asexual development but important at the induction of axoneme motility. However, in P. falciparum, causing the most severe form of human malaria, PfMAP-2 was suggested to be essential for asexual proliferation indicating distinct functions for MAP-2 in these two Plasmodium species. We here show that PfMAP-2 is dispensable for asexual growth but important for male gametogenesis in vitro. Similar to PbMAP-2, PfMAP-2 is required for initiating axonemal beating but not for prior DNA replication or axoneme formation. In addition, single and double null mutants of PfMAP-2 and the second P. falciparum MAPK homologue PfMAP-1 show no defect in asexual proliferation, sexual commitment or gametocytogenesis. Our results suggest that MAPK activity plays no major role in the biology of both asexual and sexual blood stage parasites up until the point of male gametogenesis.