The Multifaceted Role of Protein Phosphatase 1 in Plasmodium.

Protein phosphatase type 1 (PP1) forms a wide range of Ser/Thr-specific phosphatase holoenzymes which contain one catalytic subunit (PP1c), present in all eukaryotic cells, associated with variable subunits known as regulatory proteins. It has recently been shown that regulators take a leading role in the organization and the control of PP1 functions. Many studies have addressed the role of these regulators in diverse organisms, including humans, and investigated their link to diseases. In this review we summarize recent advances on the role of PP1c in Plasmodium, its interactome and regulators. As a proof of concept, peptides interfering with the regulator binding capacity of PP1c were shown to inhibit the growth of P. falciparum, suggesting their potential as drug precursors.

Plasmodium pseudo-Tyrosine Kinase-like binds PP1 and SERA5 and is exported to host erythrocytes.

Pseudokinases play key roles in many biological processes but they are poorly understood compared to active kinases. Eight putative pseudokinases have been predicted in Plasmodium species. We selected the unique pseudokinase belonging to tyrosine kinase like (TKL) family for detailed structural and functional analysis in P. falciparum and P. berghei. The primary structure of PfpTKL lacks residues critical for kinase activity, supporting its annotation as a pseudokinase. The recombinant pTKL pseudokinase domain was able to bind ATP, but lacked catalytic activity as predicted. The sterile alpha motif (SAM) and RVxF motifs of PfpTKL were found to interact with the P. falciparum proteins serine repeat antigen 5 (SERA5) and protein phosphatase type 1 (PP1) respectively, suggesting that pTKL has a scaffolding role. Furthermore, we found that PP1c activity in a heterologous model was modulated in an RVxF-dependent manner. During the trophozoite stages, PbpTKL was exported to infected erythrocytes where it formed complexes with proteins involved in cytoskeletal organization or host cell maturation and homeostasis. Finally, genetic analysis demonstrated that viable strains obtained by genomic deletion or knocking down PbpTKL did not affect the course of parasite intra-erythrocytic development or gametocyte emergence, indicating functional redundancy during these parasite stages.

Human IgG1 Responses to Surface Localised Schistosoma mansoni Ly6 Family Members Drop following Praziquantel Treatment.

BACKGROUND: The heptalaminate-covered, syncytial tegument is an important anatomical adaptation that enables schistosome parasites to maintain long-term, intravascular residence in definitive hosts. Investigation of the proteins present in this surface layer and the immune responses elicited by them during infection is crucial to our understanding of host/parasite interactions. Recent studies have revealed a number of novel tegumental surface proteins including three (SmCD59a, SmCD59b and Sm29) containing uPAR/Ly6 domains (renamed SmLy6A SmLy6B and SmLy6D in this study). While vaccination with SmLy6A (SmCD59a) and SmLy6D (Sm29) induces protective immunity in experimental models, human immunoglobulin responses to representative SmLy6 family members have yet to be thoroughly explored. METHODOLOGY/PRINCIPAL FINDINGS: Using a PSI-BLAST-based search, we present a comprehensive reanalysis of the Schistosoma mansoni Ly6 family (SmLy6A-K). Our examination extends the number of members to eleven (including three novel proteins) and provides strong evidence that the previously identified vaccine candidate Sm29 (renamed SmLy6D) is a unique double uPAR/Ly6 domain-containing representative. Presence of canonical cysteine residues, signal peptides and GPI-anchor sites strongly suggest that all SmLy6 proteins are cell surface-bound. To provide evidence that SmLy6 members are immunogenic in human populations, we report IgG1 (as well as IgG4 and IgE) responses against two surface-bound representatives (SmLy6A and SmLy6B) within a cohort of S. mansoni-infected Ugandan males before and after praziquantel treatment. While pre-treatment IgG1 prevalence for SmLy6A and SmLy6B differs amongst the studied population (7.4% and 25.3% of the cohort, respectively), these values are both higher than IgG1 prevalence (2.7%) for a sub-surface tegumental antigen, SmTAL1. Further, post-treatment IgG1 levels against surface-associated SmLy6A and SmLy6B significantly drop (p = 0.020 and p < 0.001, respectively) when compared to rising IgG1 levels against sub-surface SmTAL1. CONCLUSIONS/SIGNIFICANCE: Collectively, these results expand the number of SmLy6 proteins found within S. mansoni and specifically demonstrate that surface-associated SmLy6A and SmLy6B elicit immunological responses during infection in endemic communities.

Drug-induced exposure of Schistosoma mansoni antigens SmCD59a and SmKK7.

BACKGROUND: Schistosomiasis is a serious health problem especially in developing countries and affects more than 243 million people. Only few anthelmintic drugs are available up to now. A major obstacle for drug treatment is the different developmental stages and the varying host compartments during worm development. Anthelmintic drugs have been tested mainly on adult schistosomes or freshly transformed cercariae. Knowledge concerning the larval stages is lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used in vitro-grown schistosomula (aged between 2 to 14 days) to investigate drug effects of the three anthelmintics praziquantel, artemether, and oxamniquine. Further, we analyzed the antibody accessibility of two exemplary schistosome antigens SmCD59a and SmKK7, before and after drug treatment. Our results demonstrated that praziquantel applied at a concentration of 1 µM inhibited development of all life stages. Application of 10 µM praziquantel led to dramatic morphological changes of all schistosomula. Artemether at 1 and 10 µM had differential effects depending on whether it was applied to 2-day as compared to 7- and 14-day schistosomula. While 2-day schistosomula were not killed but inhibited from further development, severe morphological damage was seen in 7- and 14-day schistosomula. Oxamniquine (1 and 10 µM) led to severe morphological impairment in all life stages. Analyzing the accessibility of the antigens SmCD59a and SmKK7 before drug treatment showed no antibody binding on living intact schistosomula. However, when schistosomula were treated with anthelmintics, both antigens became exposed on the larvae. Oxamniquine turned out to be most effective in promoting antibody binding to all schistosomula stages. CONCLUSION: This study has revealed marked differences in anthelmintic drug effects against larvae. Drug treatment increases surface antigen presentation and renders larvae accessible to antibody attack.

Plasmodium falciparum encodes a conserved active inhibitor-2 for Protein Phosphatase type 1: perspectives for novel anti-plasmodial therapy.

BACKGROUND: It is clear that the coordinated and reciprocal actions of kinases and phosphatases are fundamental in the regulation of development and growth of the malaria parasite. Protein Phosphatase type 1 is a key enzyme playing diverse and essential roles in cell survival. Its dephosphorylation activity/specificity is governed by the interaction of its catalytic subunit (PP1c) with regulatory proteins. Among these, inhibitor-2 (I2) is one of the most evolutionarily ancient PP1 regulators. In vivo studies in various organisms revealed a defect in chromosome segregation and cell cycle progression when the function of I2 is blocked. RESULTS: In this report, we present evidence that Plasmodium falciparum, the causative agent of the most deadly form of malaria, expresses a structural homolog of mammalian I2, named PfI2. Biochemical, in vitro and in vivo studies revealed that PfI2 binds PP1 and inhibits its activity. We further showed that the motifs 12KTISW16 and 102HYNE105 are critical for PfI2 inhibitory activity. Functional studies using the Xenopus oocyte model revealed that PfI2 is able to overcome the G2/M cell cycle checkpoint by inducing germinal vesicle breakdown. Genetic manipulations in P. falciparum suggest an essential role of PfI2 as no viable mutants with a disrupted PfI2 gene were detectable. Additionally, peptides derived from PfI2 and competing with RVxF binding sites in PP1 exhibit anti-plasmodial activity against blood stage parasites in vitro. CONCLUSIONS: Taken together, our data suggest that the PfI2 protein could play a role in the regulation of the P. falciparum cell cycle through its PfPP1 phosphatase regulatory activity. Structure-activity studies of this regulator led to the identification of peptides with anti-plasmodial activity against blood stage parasites in vitro suggesting that PP1c-regulator interactions could be a novel means to control malaria.

Plasmodium falciparum dynein light chain 1 interacts with actin/myosin during blood stage development.

Dynein light chain 1 (LC1), a member of the leucine-rich repeat protein family, has been shown to be engaged in controlling flagellar motility in Chlamydomonas reinhardtii and Trypanosoma brucei via its interaction with the dynein gamma heavy chain. In Plasmodium falciparum, we have identified the LC1 ortholog, designated Pfdlc1. Negative attempts to disrupt the dlc1 gene by reverse genetic approaches in both P. falciparum and P. berghei suggest either its essentiality for parasite survival or the inaccessibility of its locus. Expression studies revealed high levels of DLC1 protein in late trophozoites and schizonts, pointing to an unexpected role of this protein in blood-stage parasites as they do not have flagella. Interactions studies and co-immunoprecipitation experiments revealed that PfDLC1 was able to bind to P. falciparum myosin A and actin 1. The PfDLC1 interacting domains present in P. falciparum myosin A and actin 1 were mapped to sequences containing SDIE and/or EEMKT motifs present in the upper 50-kDa segment of the myosin A head domain and in the subdomain IV of actin 1, respectively. Detection of PfDLC1 by fluorescence tagging and immunofluorescence staining using specific antibodies showed a cytoplasmic location similar to actin and immunofluorescence studies showed a co-localization of PfDLC1 and myosin A. Taken together, these findings suggest that PfDLC1 might play an important role in P. falciparum erythrocytic stages by its interaction with myosin A and actin 1, known to be essential for parasite development.

Analysis of immune response patterns in naïve and Plasmodium berghei-infected young rats following a ferroquine treatment.

The direct antimalarial activity of ferroquine (FQ, SSR97193), a chloroquine (CQ) derivative, is well established. To determine whether the FQ anti-parasite activity affects the host immune properties, we have investigated its effect on several immunological parameters in young rats infected with Plasmodium berghei and compared it with that of CQ. In uninfected young rats, treatment with either drug did not show any impairment in the cellular distribution of spleen cells in their response to mitogens and did not induce the production of IL-10 in vivo. After infection, rats treated with CQ or FQ showed no parasitemia and survived with no recrudescence, in comparison with placebo. Nevertheless, FQ cured young rats more rapidly than its parent drug. Analysis of cellular distribution including CD4+TCR+, CD8+TCR+, NK and NKT cells in blood and spleen and the production of specific antibodies did not reveal any alteration of these parameters in infected young rats treated either with CQ or FQ. However, we observed a persistence of CD4+CD25+T-cells in infected CQ-treated rats when compared with infected FQ-treated rats, very likely related to the delay of blood parasite clearance by CQ-treatment. Another significant difference is that the CQ treatment dramatically inhibited the lymphoproliferative response of young infected rats when compared with FQ. Collectively, the absence of any observable immunotoxic effects due to FQ in naïve and infected young rats, together with previous results indicating the susceptibility to FQ of all Plasmodium falciparum field isolates and CQ-resistant strains make it a promising drug for malarial treatment.

The age-related resistance of rats to Plasmodium berghei infection is associated with differential cellular and humoral immune responses.

In this study, we investigated how the age of rats would affect the course of infection of and the immune response to Plasmodium berghei. Both young (4-week-old) and adult rats (8-week-old) can be infected with P. berghei ANKA strain, with significantly higher levels of infected red blood cells in young rats. While 100% of young rats succumbed to infection, adult rats were able to clear blood parasites and no mortality was observed. Analysis of cellular distribution and circulating cytokines demonstrated the persistence of CD4+/CD25+ T cells and high expression of circulating interleukin-10 (IL-10) during the progression of infection in young-susceptible rats, whereas high levels of CD8+ T cells and natural killer T cells are detected in adult-resistant rats. Analysis of antibody isotypes showed that adult rats produced significantly higher levels of interferon-gamma (IFN-gamma)-dependent IgG2c antibodies than young rats during infection. Further evaluation of the role of IL-10, IFN-gamma and of immune cells showed that only the adoptive transfer of spleen cells from adult-resistant rats was able to convert susceptibility of young-susceptible rats to a resistant phenotype. These observations suggest that cell-mediated mechanisms are crucial for the control of a primary infection with P. berghei in young rats.