Toll-like receptor 4, Toll-like receptor 7 and Toll-like receptor 9 agonists enhance immune responses against blood-stage Plasmodium chabaudi infection in BALB/c mice.

BACKGROUND: Toll-like receptor (TLR) signals play vital roles during the blood-stage of malaria infections. However, the roles of TLR agonists in the regulation of immune responses and the development of protective immunity to malaria remain poorly understood. METHOD: BALB/c mice were pre-treated with TLR4, TLR7 and TLR9 agonists, followed by infection with Plasmodium chabaudi. After infection, splenic dendritic cells (DCs), Th1 cells and programmed death-1 (PD-1) expressed on Th1 cells, as well as regulatory T cells (Tregs) were analyzed by flow cytometry. The levels of IFN-γ, TNF-α, TGF-ß and IL-10 in splenocytes and IgG1 and IgG2a in serum were measured by ELISA. RESULT: Administration of TLR4, TLR7 and TLR9 agonists prior to infection improved disease outcomes. All TLR agonists promoted DC activation, and the proportions of Th1 cells increased. In TLR4, TLR7 and TLR9 agonist treated groups the levels of pro-inflammatory cytokines IFN-γ and TNF-α were elevated, and IgG1 and IgG2a serum levels were also significantly increased. TLR4, TLR7 and TLR9 agonists diminished the activation of Tregs and down-regulated the anti-inflammatory cytokines TGF-ß and IL-10. Finally, PD-1 expressed on Th1 cells were decreased in TLR4, TLR7 and TLR9 agonist treated groups compared with control groups. CONCLUSION: TLR4, TLR7 and TLR9 agonists activated DC-mediated innate immune responses and adaptive immune response, which against the blood-stage of Plasmodium and might be applied to malaria protection and treatment.

Plasmodium berghei serine/threonine protein phosphatase PP5 plays a critical role in male gamete fertility.

Sexual development in malaria parasites involves multiple signal transduction pathways mediated by reversible protein phosphorylation. Here, we functionally characterised a protein phosphatase, Ser/Thr protein phosphatase 5 (PbPP5), during sexual development of the rodent malaria parasite Plasmodium berghei. The recombinant protein phosphatase domain displayed obvious protein phosphatase activity and was sensitive to PP1/PP2A inhibitors including cantharidic acid (IC50 = 122.2 nM), cantharidin (IC50 = 74.3 nM), endothall (IC50 = 365.5 nM) and okadaic acid (IC50 = 1.3 nM). PbPP5 was expressed in both blood stages and ookinetes with more prominent expression during sexual development. PbPP5 was localised in the cytoplasm of the parasite and highly concentrated beneath the parasite plasma membrane in free merozoites and ookinetes. Targeted deletion of the pbpp5 gene had no influence on asexual blood-stage parasite multiplication or the survival curve of the infected hosts. However, male gamete formation and fertility were severely affected, resulting in almost complete blockade of ookinete conversion and oocyst development in the Δpbpp5 lines. This sexual development defect was rescued by crossing Δpbpp5 with the female defective Δpbs47 parasite line, but not with the male defective Δpbs48/45 line, thus confirming the essential function of the pbpp5 gene in male gamete fertility. Furthermore, the aforementioned PP1/PP2A inhibitors all had inhibitory effects on exflagellation of male gametocytes and ookinete conversion. In particular, endothall, a selective inhibitor of PP2A, completely blocked exflagellation and ookinete conversion at ∼548.3 nM. This study elucidated an essential function of PbPP5 during male gamete development and fertility.

Genetic Variations Associated with Drug Resistance Markers in Asymptomatic Plasmodium falciparum Infections in Myanmar.

The emergence and spread of drug resistance is a problem hindering malaria elimination in Southeast Asia. In this study, genetic variations in drug resistance markers of Plasmodium falciparum were determined in parasites from asymptomatic populations located in three geographically dispersed townships of Myanmar by PCR and sequencing. Mutations in dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps), chloroquine resistance transporter (pfcrt), multidrug resistance protein 1 (pfmdr1), multidrug resistance-associated protein 1 (pfmrp1), and Kelch protein 13 (k13) were present in 92.3%, 97.6%, 84.0%, 98.8%, and 68.3% of the parasites, respectively. The pfcrt K76T, pfmdr1 N86Y, pfmdr1 I185K, and pfmrp1 I876V mutations were present in 82.7%, 2.5%, 87.5%, and 59.8% isolates, respectively. The most prevalent haplotypes for pfdhfr, pfdhps, pfcrt and pfmdr1 were 51I/59R/108N/164L, 436A/437G/540E/581A, 74I/75E/76T/220S/271E/326N/356T/371I, and 86N/130E/184Y/185K/1225V, respectively. In addition, 57 isolates had three different point mutations (K191T, F446I, and P574L) and three types of N-terminal insertions (N, NN, NNN) in the k13 gene. In total, 43 distinct haplotypes potentially associated with multidrug resistance were identified. These findings demonstrate a high prevalence of multidrug-resistant P. falciparum in asymptomatic infections from diverse townships in Myanmar, emphasizing the importance of targeting asymptomatic infections to prevent the spread of drug-resistant P.falciparum.