Who research agenda for hand hygiene in health care 2023–2030 : a Delphi consensus‑building study

Introduction: Hand hygiene (HH) is at the core of infection prevention and control programmes and one of the most efective interventions for reducing health care-associated infections (HAIs). Gaps exist in achieving optimal HH improvement.peer-reviewe

The N-terminus of EXP2 forms the membrane-associated pore of the protein exporting translocon PTEX in Plasmodium falciparum

In order to facilitate a number of processes including nutrient acquisition and immune evasion, malaria parasites extensively remodel their host erythrocyte. This remodelling is to a large extent accomplished through protein export, a crucial process mediated by the Plasmodium translocon for exported proteins (PTEX) translocon which is comprised of three core components, HSP101, PTEX150 and EXP2. EXP2 has been structurally and electrophysiologically shown to form the pore that spans the vacuole membrane enveloping the parasite. Here, we biochemically investigate the structure and function of EXP2. By differential alkylation we provide direct evidence that cysteines C113 and C140 form an intramolecular disulphide bond, while C201 is predominantly in a reduced state. We demonstrate that EXP2 possesses a protease resistant, membrane-associated, N-terminal region of ∼20 kDa that does not project into the infected erythrocyte cytosol; however, its C-terminus does project into the vacuole space. We show that a putative transmembrane peptide derived from the N-terminal region of EXP2 is haemolytic and in a polymer-based osmotic protection assay, we demonstrate that this peptide forms a discrete haemolytic pore. This work provides further biochemical insight into the role, function and cellular arrangement of EXP2 as the pore-forming component for protein translocation

Knockdown of the translocon protein EXP2 in Plasmodium falciparum reduces growth and protein export.

Malaria parasites remodel their host erythrocytes to gain nutrients and avoid the immune system. Host erythrocytes are modified by hundreds of effector proteins exported from the parasite into the host cell. Protein export is mediated by the PTEX translocon comprising five core components of which EXP2 is considered to form the putative pore that spans the vacuole membrane enveloping the parasite within its erythrocyte. To explore the function and importance of EXP2 for parasite survival in the asexual blood stage of Plasmodium falciparum we inducibly knocked down the expression of EXP2. Reduction in EXP2 expression strongly reduced parasite growth proportional to the degree of protein knockdown and tended to stall development about half way through the asexual cell cycle. Once the knockdown inducer was removed and EXP2 expression restored, parasite growth recovered dependent upon the length and degree of knockdown. To establish EXP2 function and hence the basis for growth reduction, the trafficking of an exported protein was monitored following EXP2 knockdown. This resulted in severe attenuation of protein export and is consistent with EXP2, and PTEX in general, being the conduit for export of proteins into the host compartment

Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150

The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex

Synthesis and antiplasmodial activity of 3-furyl and 3-thienylquinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives

The aim of this study was to identify new compounds active against Plasmodium falciparum based on our previous research carried out on 3-phenylquinoxaline- 2-carbonitrile 1,4-di-N-oxide derivatives. Twelve compounds were synthesized and evaluated for antimalarial activity. Eight of them showed an IC50 < 1 μM against the 3D7 strain. Derivative 1 demonstrated high potency (IC50= 0.63 μM) and good selectivity (SI=10.35), thereby becoming a new lead-compound

<i>Plasmodium falciparum</i> Transfected with Ultra Bright NanoLuc Luciferase Offers High Sensitivity Detection for the Screening of Growth and Cellular Trafficking Inhibitors

<div><p>Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in <i>Plasmodium falciparum</i> and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.</p></div

<i>Plasmodium falciparum</i> stably expressing Nluc.

<p>(A) The Nluc gene was cloned in the pEF vector for stable expression in <i>P. falciparum</i>. (B) Aliquots (100 µL) of cultures at 1% hematocrit and 5% parasitemia corresponding to 500,000 <i>P. falciparum</i> infected RBCs transfected with pEF-Nluc were mixed with 1 volume of Nano-Glo Luciferase Assay Reagent and reporter activity measured (1∶1). Nano-Glo Luciferase Assay Reagent was further diluted in 10-fold increments in Luciferase Cell Culture Lysis Reagent and used to determine reporter activity of the same culture. As a negative control, wild type parasites (wt) were mixed 1∶1 with Nano-Glo Luciferase Assay Reagent. (C) Parasites stably transfected with pEF-Nluc were diluted in 2-fold increments in RPMI + RBC maintaining 0.5% hematocrit. For each sample, 10 µL of the culture dilutions were mixed with 40 µL of Nano-Glo diluted 1∶400 in water and measured in the luminometer for 2 s with the gain adjusted 10% below saturation for the brightest sample. The solid line represents the mean RLU after linear regression. The dashed line represents the background +3 standard deviations. (D) Nluc expressing parasites were cultured in varying concentrations of chloroquine (CQ) and their growth determined by the LDH standard method or by measuring reporter activity using Nano-Glo Luciferase Assay Reagent at its standard dilution (1∶1) or diluted 1∶1000 as described in (C). Similarly, wild type parasites were transiently transfected with pPfNluc and their growth determined using Nano-Glo Luciferase Assay Reagent (Transient). IC₅₀ was calculated by non-linear regression and represents the mean of 3 experiments. (E) The IC₅₀s determined in D were plotted with 95% confidence intervals (CI).</p

Summary of the assay parameters from the growth assays.

1<p>refers to luminescence measured from transiently transfected parasites using the standard Nano-Glo dilution.</p><p>Summary of the assay parameters from the growth assays.</p

NanoLuc is targeted to the PV and to the RBC.

<p>Diagrams of gene constructs and the IFA images are shown on the left and right respectively. Nluc fused at its N-terminus to (A) the N-terminal region of an exported protein (PEXEL), (B) to a secretion signal peptide (SP) or (C) original (cytosolic). The gene encoding each fusion protein was cloned in the pEF vector. Transfected parasites were analysed by IFA using antibodies to detect Nluc and the PVM marker Exp2. DAPI was used for nuclear staining. Size bar = 5 µm.</p

Quantification of Nluc in cellular compartments of infected RBCs.

<p>(A) Schematic of the iRBC’s compartments where RBC represents the exported fraction that is released after Equinatoxin treatment. The PV compartment was then released following treatment with 0.01% saponin and finally, the Parasite fraction was lysed by Nano-Glo Luciferase Assay Reagent. (B) Trophozoite stage parasites transfected with either the original Nluc, secreted SP-Nluc or the exported PEXEL-Nluc fusions were fractionated as shown in (A) and luciferase activity measured. Nluc activities as a percentage of the total for each parasite line are shown and represent the mean of 3 experiments +/−SEM. Similar to (B), (C) Schizonts and (D) Ring stage parasites were also fractionated. Statistical significance (* p&lt;0.05) was determined by 2 way ANOVA test comparing the percentage of reporter activity of each sub-cellular fraction among the 3 cell lines.</p