Host cell traversal is important for progression of the malaria parasite through the dermis to the liver

The malaria sporozoite, the parasite stage transmitted by the mosquito, is delivered into the dermis and differentiates in the liver. Motile sporozoites can invade host cells by disrupting their plasma membrane and migrating through them (termed cell traversal), or by forming a parasite-cell junction and settling inside an intracellular vacuole (termed cell infection). Traversal of liver cells, observed for sporozoites in vivo, is thought to activate the sporozoite for infection of a final hepatocyte. Here, using Plasmodium berghei, we show that cell traversal is important in the host dermis for preventing sporozoite destruction by phagocytes and arrest by nonphagocytic cells. We also show that cell infection is a pathway that is masked, rather than activated, by cell traversal. We propose that the cell traversal activity of the sporozoite must be turned on for progression to the liver parenchyma, where it must be switched off for infection of a final hepatocyte.Inst Pasteur, Unite Biol & Genet Paludisme, F-75724 Paris 15, FranceUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Montpellier 2, CNRS, UMR 5539, F-34095 Montpellier 05, FranceMie Univ, Sch Med, Tsu, Mie 5140001, JapanUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilWeb of Scienc

Production of IFN- by CD4+ T cells in response to malaria antigens is IL-2 dependent

T-cell immune responses are critical for protection of the host and for disease pathogenesis during infection with Plasmodium species. We examined the regulation of CD4+ T-cell cytokine responses during infection with Plasmodium berghei ANKA (PbA). CD4+ T cells from PbA-infected mice produced IFN-γ, IL-4 and IL-10 in response to TCR stimulation at levels higher than those from uninfected mice. This altered cytokine response was dependent on parasitemia. To examine the specificity of the response, mice were adoptively transferred with CD4+ T cells from OT-II TCR transgenic mice and were infected with PbA expressing OVA. Unexpectedly, CD4+ T cells from the OT-II-transferred wild-type PbA-infected mice showed high levels of IFN-γ production after stimulation with OVA and the cells producing IFN-γ were not OT-II but were host CD4+ T cells. Further investigation revealed that host CD4+ T cells produced IFN-γ in response to IL-2 produced by activated OT-II cells. This IFN-γ response was completely inhibited by anti-CD25 mAbs, and this effect was not due to the block of the survival signals provided by IL-2. Furthermore, IFN-γ production by CD4+ T cells in response to PbA antigens was dependent on IL-2. These findings suggest the importance of IL-2 levels during infection with malaria parasites and indicate that CD4+ T cells can produce IFN-γ without TCR engagement via a bystander mechanism in response to IL-2 produced by other activated CD4+ T cells

Centromere Plasmid: A New Genetic Tool for the Study of Plasmodium falciparum

The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite

セクレトームに基づくマラリア肝臓ステージの宿主寄生虫間相互作用の解明

application/pdfマラリア原虫は宿主である肝細胞の機能を巧みに制御し自身の生存に有利な環境を構築していると考えられる。この制御を分子レベルで理解することはマラリア原虫の感染を阻止する新たな戦略につながる。とりわけ肝細胞内に輸送される原虫タンパク質はこの制御の中心になっている可能性が高く重要である。本研究では、肝細胞内に輸送される原虫タンパク質群（セクレトーム）を同定することを目的として、NGSを用いた遺伝子大量解析を実施した。Liver stage malaria parasites establish environments suitable for their survival by manipulating the host hepatocyte. To understand molecular mechanisms of this control may lead to development of strategies to prevent malaria infection of humans. Parasite proteins exported to the host hepatocyte would have a central role in this control. In this study we performed mass sequencing analysis in this stage in order to identify whole proteins exported to the host hepatocyte (secretome) .2013年度～2016年度科学研究費補助金(基盤研究(A))研究成果報告書2525302

ターゲト－ムによるマラリア原虫ライフサイクルの統一的理解

application/pdf本研究ではマラリア原虫の各転写因子の機能、結合配列、標的遺伝子等の解析を実施した。この成果により,１つの転写因子が遺伝子を直接かつ包括的に制御し各ステージの遺伝子発現レパートリーを決定するというマラリア原虫のユニークな転写制御機構を実証することに成功した。また、マラリア原虫のライフサイクルをターゲトームの連鎖として捉え、各ステージ間の遺伝子発現のダイナミックな連関に光を当てることに成功したIn this study, we analyzed the function of each Plasmodium transcription factor, identified its binding sequences, and determined target genes. Our results demonstrated the unique transcriptional regulation mechanism of Plasmodium parasites, in which a stage-specific transcription factor directly and comprehensively regulates genes and determines the gene expression repertoire at each stage. We have also revealed the dynamic linkage of gene expression through the lifecycle by viewing it as a chain of targetomes.2017年度～2019年度科学研究費補助金(基盤研究(A))研究成果報告書17H0154

PbAP2-FG2 and PbAP2R-2 function together as a transcriptional repressor complex essential for Plasmodium female development.

Gametocyte development is a critical step in the life cycle of Plasmodium. Despite the number of studies on gametocyte development that have been conducted, the molecular mechanisms regulating this process remain to be fully understood. This study investigates the functional roles of two female-specific transcriptional regulators, PbAP2-FG2 and PbAP2R-2, in P. berghei. Knockout of pbap2-fg2 or pbap2r-2 impairs female gametocyte development, resulting in developmental arrest during ookinete development. ChIP-seq analyses of these two factors indicated their colocalization on the genome, suggesting that they function as a complex. These analyses also revealed that their target genes contained a variety of genes, including both male and female-enriched genes. Moreover, differential expression analyses showed that these target genes were upregulated through the disruption of pbap2-fg2 or pbap2r-2, indicating that these two factors function as a transcriptional repressor complex in female gametocytes. Formation of a complex between PbAP2-FG2 and PbAP2R-2 was confirmed by RIME, a method that combines ChIP and MS analysis. In addition, the analysis identified a chromatin regulator PbMORC as an interaction partner of PbAP2-FG2. Comparative target analysis between PbAP2-FG2 and PbAP2-G demonstrated a significant overlap between their target genes, suggesting that repression of early gametocyte genes activated by PbAP2-G is one of the key roles for this female transcriptional repressor complex. Our results indicate that the PbAP2-FG2-PbAP2R-2 complex-mediated repression of the target genes supports the female differentiation from early gametocytes

Plasmodium pre-erythrocytic stages: what's new?

The pre-erythrocytic (PE) phase of malaria infection, which extends from injection of sporozoites into the skin to the release of the first generation of merozoites, has traditionally been the 'black box' of the Plasmodium life cycle. However, since the advent of parasite transfection technology 13 years ago, our understanding of the PE phase in cellular and molecular terms has dramatically improved. Here, we review and comment on the major developments in the field in the past five years. Progress has been made in many diverse areas, including identifying and characterizing new proteins of interest, imaging parasites in vivo, understanding better the cell biology of hepatocyte infection and developing new vaccines against PE stages of the parasite