cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites

Rôles des petites protéines GTPase Rab11 dans l'interaction Hôtes/Parasites

PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Non-Inferiority of a Single Injection of Sodium Hyaluronate Plus Sorbitol to Hylan G-F20: A 6-Month Randomized Controlled Trial.

INTRODUCTION: Several viscosupplement treatments are available for patients suffering from painful osteoarthritis (OA) of the knee, but few comparative clinical trials have been conducted. The primary objective of the trial was to demonstrate the non-inferiority of Synolis VA (80 mg hyaluronic acid and 160 mg sorbitol) (Group HA1) to Synvisc-One (48 mg hylan GF-20) (Group HA2) at Day 168 in terms of pain relief efficacy in patients with knee OA (Kellgren and Lawrence radiological stage II or III) in whom oral treatment with analgesics, NSAIDs or weak opioids provided insufficient clinical responses or were poorly tolerated. METHODS: This was a prospective, multicentre, comparative, randomized, double-blinded trial comparing the two previously indicated viscosupplements, HA1 and HA2. The average VAS pain score (1-100) was 62.5 at baseline (Day 0). The patients were randomized into two parallel groups at Day 0 and followed until Day 168. They received one injection of either HA1 or HA2. The primary end point was the evolution of the Western Ontario and McMaster University (WOMAC) pain index at D168 in the groups. One of the secondary end points was the daily assessment of this index by the patient for 7 days following the injection and thereafter at Day 14. The other secondary end points were the WOMAC pain, stiffness, function and total scores assessed at Day 28, Day 84 and Day 168. At Day 168, efficacy and satisfaction were assessed by the evaluator and by the patient using a Likert scale (7 points). Moreover, the number of strict responders in each group was evaluated according to the The Osteoarthritis Research Society International (OARSI) Standing Committee for Clinical Trials Response Criteria Initiative and the Outcome Measures in Rheumatology (OMERACT) criteria (OMERACT-OARSI). The per protocol (PP) population was used for the primary analysis. RESULTS: A total of 202 patients were randomized. The patients were predominantly female (66%). The median age of the whole population was 65 years, and the median body mass index was 27.4 kg/m(2). No statistically significant differences between the two treatment groups were observed for any of the demographic criteria. At Day 168, 197 had had no protocol violations (94 in the HA1 group and 103 in the HA2 group). The WOMAC pain score decreased in the two groups: - 29.2 ± 24.1 (SD) in the HA1 group and - 31.6 ± 25.5 (SD) in the HA2 group, confirming the non-inferiority of Synolis VA (P = 0.57 for the difference between groups). Regarding the secondary end points, no significant difference was observed at Day 14, Day 28, Day 84 or Day 168 for all the outcomes except stiffness at Day 28 (P = in favour of treatment received in HA2). The rate of responders was comparable between the two groups: 79% for HA1 and 77% for HA2. Both products were well tolerated. Serious adverse events were reported by four patients in the HA1 group and 3 in the HA2 group. CONCLUSION: In this trial, we confirmed the non-inferiority of Synolis VA compared to Synvisc-One at Day 168 according to the WOMAC pain score. Safety was satisfying and comparable in the two groups. TRIAL REGISTRATION: 2017-A00034-49

Characterization of an A-kinase anchoring protein-like suggests an alternative way of PKA anchoring in Plasmodium falciparum

International audienceBackground: The asexual intra‑erythrocytic multiplication of the malaria parasite Plasmodium falciparum is regulated by various molecular mechanisms. In eukaryotic cells, protein kinases are known to play key roles in cell cycle regula‑ tion and signaling pathways. The activity of cAMP‑dependent protein kinase (PKA) depends on A‑kinase anchoring proteins (AKAPs) through protein interactions. While several components of the cAMP dependent pathway—includ‑ ing the PKA catalytic and regulatory subunits—have been characterized in P. falciparum, whether AKAPs are involved in this pathway remains unclear. Here, PfAKAL, an open reading frame of a potential AKAP‑like protein in the P. falcipa-rum genome was identified, and its protein partners and putative cellular functions characterized. Methods: The expression of PfAKAL throughout the erythrocytic cycle of the 3D7 strain was assessed by RT‑qPCR and the presence of the corresponding protein by immunofluorescence assays. In order to study physical interactions between PfAKAL and other proteins, pull down experiments were performed using a recombinant PfAKAL protein and parasite protein extracts, or with recombinant proteins. These interactions were also tested by combining bio‑ chemical and proteomic approaches. As phosphorylation could be involved in the regulation of protein complexes, both PfAKAL and Pf14‑3‑3I phosphorylation was studied using a radiolabel kinase activity assay. Finally, to identify a potential function of the protein, PfAKAL sequence was aligned and structurally modeled, revealing a conserved nucleotide‑binding pocket; confirmed by qualitative nucleotide binding experiments. Results: PfAKAL is the first AKAP‑like protein in P. falciparum to be identified, and shares 23 % sequence identity with the central domain of human AKAP18δ. PfAKAL is expressed in mature asexual stages, merozoites and gametocytes. In spite of homology to AKAP18, biochemical and immunochemical analyses demonstrated that PfAKAL does not interact directly with the P. falciparum PKA regulatory subunit (PfPKA‑R), but instead binds and colocalizes with Pf14‑ 3‑3I, which in turn interacts with PfPKA‑R. In vivo, these different interactions could be regulated by phosphorylation, as PfPKA‑R and Pf14‑3‑3I, but not PfAKAL, are phosphorylated in vitro by PKA. Interestingly, PfAKAL binds nucleotides such as AMP and cAMP, suggesting that this protein may be involved in the AMP‑activated protein kinase (AMPK) pathway, or associated with phosphodiesterase activities. Conclusion: PfAKAL is an atypical AKAP that shares common features with human AKAP18, such as nucleotides binding. The interaction of PfAKAL with PfPKA‑R could be indirectly mediated through a join interaction with Pf14‑ 3‑3I. Therefore, PfPKA localization could not depend on PfAKAL, but rather involves other partners..org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated

Plasmodium falciparum gametocyte-infected erythrocytes do not adhere to human primary erythroblasts

Abstract Plasmodium falciparum gametocytes, the sexual stages responsible for malaria parasite transmission, develop in the human bone marrow parenchyma in proximity to the erythroblastic islands. Yet, mechanisms underlying gametocytes interactions with these islands are unknown. Here, we have investigated whether gametocyte-infected erythrocytes (GIE) adhere to erythroid precursors, and whether a putative adhesion may be mediated by a mechanism similar to the adhesion of erythrocytes infected with P. falciparum asexual stages to uninfected erythrocytes. Cell-cell adhesion assays with human primary erythroblasts or erythroid cell lines revealed that immature GIE do not specifically adhere to erythroid precursors. To determine whether adhesion may be dependent on binding of STEVOR proteins to Glycophorin C on the surface of erythroid cells, we used clonal lines and transgenic parasites that overexpress specific STEVOR proteins known to bind to Glycophorin C in asexual stages. Our results indicate that GIE overexpressing STEVOR do not specifically adhere to erythroblasts, in agreement with our observation that the STEVOR adhesive domain is not exposed at the surface of GIE

Transcript expression analysis of <i>phist</i> and <i>resa-like</i> genes during <i>P</i>. <i>falciparum</i> intraerythrocytic stages.

<p>Gene-specific transcript levels for select <i>phist</i> genes <b>A</b>) and for the <i>resa-like</i> genes <b>B</b>) were analyzed by real time PCR using cDNA prepared from intraerythrocytic synchronized asexual and gametocyte <i>P</i>. <i>falciparum</i> cultures. Note that <i>resa</i> (<i>PFA0110w</i>) and <i>resa2</i> (<i>PF11_0509</i>) are highly expressed relative to the other <i>resa-like</i> genes. Transcript expression was normalized to the expression of the control gene <i>arginyl tRNA synthetase</i> (<i>PFL0900c</i>). Hours post-synchronization indicate time in hours after adding purified schizonts to fresh blood culture. In the schematic depiction of <i>phist</i> and <i>resa-like</i> genes, yellow represents the signal sequence, and red represents the PEXEL/HT motif. The life cycle stage percentage in the population for each time point is shown in <b>C</b>). The letter “G” indicates mature gametocytes.</p

The <i>Plasmodium</i> PHIST and RESA-Like Protein Families of Human and Rodent Malaria Parasites

<div><p>The <i>phist</i> gene family has members identified across the <i>Plasmodium</i> genus, defined by the presence of a domain of roughly 150 amino acids having conserved aromatic residues and an all alpha-helical structure. The family is highly amplified in <i>P</i>. <i>falciparum</i>, with 65 predicted genes in the genome of the 3D7 isolate. In contrast, in the rodent malaria parasite <i>P</i>. <i>berghei</i> 3 genes are identified, one of which is an apparent pseudogene. Transcripts of the <i>P</i>. <i>berghei phist</i> genes are predominant in schizonts, whereas in <i>P</i>. <i>falciparum</i> transcript profiles span different asexual blood stages and gametocytes. We pursued targeted disruption of <i>P</i>. <i>berghei phist</i> genes in order to characterize a simplistic model for the expanded <i>phist</i> gene repertoire in <i>P</i>. <i>falciparum</i>. Unsuccessful attempts to disrupt <i>P</i>. <i>berghei PBANKA_114540</i> suggest that this <i>phist</i> gene is essential, while knockout of <i>phist PBANKA_122900</i> shows an apparent normal progression and non-essential function throughout the life cycle. Epitope-tagging of <i>P</i>. <i>falciparum</i> and <i>P</i>. <i>berghei phist</i> genes confirmed protein export to the erythrocyte cytoplasm and localization with a punctate pattern. Three <i>P</i>. <i>berghei</i> PEXEL/HT-positive exported proteins exhibit at least partial co-localization, in support of a common vesicular compartment in the cytoplasm of erythrocytes infected with rodent malaria parasites.</p></div

Immunolocalization of <i>P</i>. <i>berghei</i> PHIST proteins PBANKA_114540, PBANKA_122900, and IBIS within infected erythrocytes.

<p><b>A</b>) PBANKA_114540 co-localizes with PBANKA_122900 within erythrocyte cytoplasmic vesicles. Co-localization was assayed using rabbit polyclonal anti-PBANKA_114540 followed by goat anti-rabbit IgG conjugated with Alexa 488 (green), followed by rabbit polyclonal anti-PBANKA_122900 followed by goat anti-rabbit Alexa 595 (red). Nuclei were stained with DAPI (blue). Control experiments using secondary antibodies were negative. <b>B</b>) Co-localization of mCherry-tagged IBIS protein PBANKA_136550 with the PHIST protein PBANKA_122900 in fixed erythrocytes, by staining with rabbit polyclonal anti-PBANKA_122900 followed by goat anti-rabbit IgG conjugated with Alexa 488. Parasite nuclei were stained with Hoechst (blue). Both proteins partially co-localize inside vesicles in the erythrocyte cytoplasm.</p