Plasmodium falciparum infection induces dynamic changes in the erythrocyte phospho-proteome.

The phosphorylation status of red blood cell proteins is strongly altered during the infection by the malaria parasite Plasmodium falciparum. We identify the key phosphorylation events that occur in the erythrocyte membrane and cytoskeleton during infection, by a comparative analysis of global phospho-proteome screens between infected (obtained at schizont stage) and uninfected RBCs. The meta-analysis of reported mass spectrometry studies revealed a novel compendium of 495 phosphorylation sites in 182 human proteins with regulatory roles in red cell morphology and stability, with about 25% of these sites specific to infected cells. A phosphorylation motif analysis detected 7 unique motifs that were largely mapped to kinase consensus sequences of casein kinase II and of protein kinase A/protein kinase C. This analysis highlighted prominent roles for PKA/PKC involving 78 phosphorylation sites. We then compared the phosphorylation status of PKA (PKC) specific sites in adducin, dematin, Band 3 and GLUT-1 in uninfected RBC stimulated or not by cAMP to their phosphorylation status in iRBC. We showed cAMP-induced phosphorylation of adducin S59 by immunoblotting and we were able to demonstrate parasite-induced phosphorylation for adducin S726, Band 3 and GLUT-1, corroborating the protein phosphorylation status in our erythrocyte phosphorylation site compendium

Theileria parasites subvert E2F signaling to stimulate leukocyte proliferation

Intracellular pathogens have evolved intricate mechanisms to subvert host cell signaling pathways and ensure their own propagation. A lineage of the protozoan parasite genus Theileria infects bovine leukocytes and induces their uncontrolled proliferation causing a leukemia-like disease. Given the importance of E2F transcription factors in mammalian cell cycle regulation, we investigated the role of E2F signaling in Theileria-induced host cell proliferation. Using comparative genomics and surface plasmon resonance, we identified parasite-derived peptides that have the sequence-specific ability to increase E2F signaling by binding E2F negative regulator Retinoblastoma-1 (RB). Using these peptides as a tool to probe host E2F signaling, we show that the disruption of RB complexes ex vivo leads to activation of E2F-driven transcription and increased leukocyte proliferation in an infection-dependent manner. This result is consistent with existing models and, together, they support a critical role of E2F signaling for Theileria-induced host cell proliferation, and its potential direct manipulation by one or more parasite proteins

The H89 cAMP-dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes

In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor.Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria

TGF-b2 induction regulates invasiveness of theileria-transformed leukocytes and disease susceptibility

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence

A bovine lymphosarcoma cell line infected with theileria annulata exhibits an irreversible reconfiguration of host cell gene expression

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner

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

EhMAPK, the Mitogen-Activated Protein Kinase from Entamoeba histolytica Is Associated with Cell Survival

Mitogen Activated Protein Kinases (MAPKs) are a class of serine/threonine kinases that regulate a number of different cellular activities including cell proliferation, differentiation, survival and even death. The pathogen Entamoeba histolytica possess a single homologue of a typical MAPK gene (EhMAPK) whose identification was previously reported by us but its functional implications remained unexplored. EhMAPK, the only mitogen-activated protein kinase from the parasitic protist Entamoeba histolytica with Threonine-X-Tyrosine (TXY) phosphorylation motif was cloned, expressed in E. coli and functionally characterized under different stress conditions. The expression profile of EhMAPK at the protein and mRNA level remained similar among untreated, heat shocked and hydrogen peroxide-treated samples in all cases of dose and time. But a significant difference was obtained in the phosphorylation status of the protein in response to different stresses. Heat shock at 43°C or 0.5 mM H2O2 treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H2O2 treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H2O2 treatment reduced parasite viability significantly but heat shock and 0.5 mM H2O2 treatment failed to adversely affect E. histolytica viability. Therefore, a distinct possibility that activation of EhMAPK is associated with stress survival in E. histolytica is seen. Our study also gives a glimpse of the regulatory mechanism of the protein under in vivo conditions. Since the parasite genome lacks any typical homologue of mammalian MEK, the dual specificity kinases which are the upstream activators of MAPK, indications of the existence of some alternate regulatory mechanisms of the EhMAPK activity is perceived. These may include the autophosphorylation activity of the protein itself in combination with some upstream phosphatases which are not yet identified

Distribution of a Knockdown Resistance Mutation (L1014S) in Anopheles gambiae s.s. and Anopheles arabiensis in Western and Southern Kenya

In Kenya, insecticide-treated mosquito nets (ITNs) distributed to pregnant women and children under 5 years old through various programs have resulted in a significant reduction in malaria deaths. All of the World Health Organization-recommended insecticides for mosquito nets are pyrethroids, and vector mosquito resistance to these insecticides is one of the major obstacles to an effective malaria control program. Anopheles gambiae s.s. and Anopheles arabiensis are major malaria vectors that are widely distributed in Kenya. Two point mutations in the voltage-gated sodium channel (L1014F and L1014S) are associated with knockdown resistance (kdr) to DDT and pyrethroids in An. gambiae s.s. While the same point mutations have been reported to be rare in An. arabiensis, some evidence of metabolic resistance has been reported in this species. In order to determine the distribution of the point mutation L1014S in An. gambiae s.s. and An. arabiensis in southern and western Kenya, we collected larvae and screened for the mutation by DNA sequencing. We found high allelic and homozygous frequencies of the L1014S mutation in An. gambiae s.s. The L1014S mutation was also widely distributed in An. arabiensis, although the allelic frequency was lower than in An. gambiae s.s. The same intron sequence (length: 57 base) found in both species indicated that the mutation was introgressed by hybridization. The allelic frequency of L1014S was higher in both species in western regions, demonstrating the strong selection pressure imposed by long-lasting insecticide-treated nets (LLITN)/ITN on the An. gambiae s.s. and An. arabiensis populations in those areas. The present contribution of the L1014S mutation to pyrethroid resistance in An. arabiensis may be negligible. However, the homozygous frequency could increase with continuing selection pressure due to expanded LLITN coverage in the future

Effect of Acute Plasmodium falciparum Malaria on Reactivation and Shedding of the Eight Human Herpes Viruses

Human herpes viruses (HHVs) are widely distributed pathogens. In immuno-competent individuals their clinical outcomes are generally benign but in immuno-compromised hosts, primary infection or extensive viral reactivation can lead to critical diseases. Plasmodium falciparum malaria profoundly affects the host immune system. In this retrospective study, we evaluated the direct effect of acute P. falciparum infection on reactivation and shedding of all known human herpes viruses (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8). We monitored their presence by real time PCR in plasma and saliva of Ugandan children with malaria at the day of admission to the hospital (day-0) and 14 days later (after treatment), or in children with mild infections unrelated to malaria. For each child screened in this study, at least one type of HHV was detected in the saliva. HHV-7 and HHV-6 were detected in more than 70% of the samples and CMV in approximately half. HSV-1, HSV-2, VZV and HHV-8 were detected at lower frequency. During salivary shedding the highest mean viral load was observed for HSV-1 followed by EBV, HHV-7, HHV-6, CMV and HHV-8. After anti-malarial treatment the salivary HSV-1 levels were profoundly diminished or totally cleared. Similarly, four children with malaria had high levels of circulating EBV at day-0, levels that were cleared after anti-malarial treatment confirming the association between P. falciparum infection and EBV reactivation. This study shows that acute P. falciparum infection can contribute to EBV reactivation in the blood and HSV-1 reactivation in the oral cavity. Taken together our results call for further studies investigating the potential clinical implications of HHVs reactivation in children suffering from malaria

Cost of Mating and Insemination Capacity of a Genetically Modified Mosquito Aedes aegypti OX513A Compared to Its Wild Type Counterpart

The idea of implementing genetics-based insect control strategies modelled on the traditional SIT is becoming increasingly popular. In this paper we compare a genetically modified line of Aedes aegypti carrying a tetracycline repressible, lethal positive feedback system (OX513A) with its wild type counterpart with respect to their insemination capacities and the cost of courtship and mating. Genetically modified males inseminated just over half as many females as the wild type males during their lifetime. Providing days of rest from mating had no significant effect on the total number of females inseminated by males of either line, but it did increase their longevity. Producing sperm had a low cost in terms of energy investment; the cost of transferring this sperm to a receptive female was much higher. Continued mating attempts with refractory females suggest that males could not identify refractory females before investing substantial energy in courtship. Although over a lifetime OX513A males inseminated fewer females, the number of females inseminated over the first three days, was similar between males of the two lines, suggesting that the identified cost of RIDL may have little impact on the outcome of SIT-based control programmes with frequent releases of the genetically modified males