Imaging malaria sporozoites in the dermis of the mammalian host

The initial phase of malaria infection is the pre-erythrocytic phase, which begins when parasites are injected by the mosquito into the dermis and ends when parasites are released from hepatocytes into the blood. We present here a protocol for the in vivo imaging of GFP-expressing sporozoites in the dermis of rodents, using the combination of a high-speed spinning-disk confocal microscope and a high-speed charge-coupled device (CCD) camera permitting rapid in vivo acquisitions. the steps of this protocol indicate how to infect mice through the bite of infected Anopheles stephensi mosquitoes, record the sporozoites' fate in the mouse ear and to present the data as maximum-fluorescence-intensity projections, time-lapse representations and movie clips. This protocol permits investigating the various aspects of sporozoite behavior in a quantitative manner, such as motility in the matrix, cell traversal, crossing the endothelial barrier of both blood and lymphatic vessels and intravascular gliding. Applied to genetically modified parasites and/or mice, these imaging techniques should be useful for studying the cellular and molecular bases of Plasmodium sporozoite infection in vivo.Inst Pasteur, Unite Biol & Genet Paludisme, F-75724 Paris 15, FranceWeb of Scienc

Towards systematic identification of Plasmodium essential genes by transposon shuttle mutagenesis

After the deciphering of the genome sequences of several Plasmodium species, efforts must turn to elucidating gene function and identifying essential gene products. However, random approaches are lacking and gene targeting is inefficient in Plasmodium. Here, we established shuttle transposon mutagenesis in Plasmodium berghei. We constructed a mini-Tn5 derivative that can transpose into parasite genes cloned in Escherichia coli, providing an efficient means of generating knockout fragments. A 10(4)-fold increase in frequencies of double-crossover homologous recombination in the parasite using a new electroporation technology permits to reproducibly generate pools of distinct mutants after transfection with mini-Tn5-interrupted sequences. The procedure opens the way to the systematic identification of essential genes in Plasmodium

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

Comparison of the Bordetella pertussis and Bordetella parapertussis Isolates Circulating in Saint Petersburg between 1998 and 2000 with Russian Vaccine Strains

We analyzed the Bordetella pertussis and Bordetella parapertussis isolates circulating in Saint Petersburg that were collected between 1998 and 2000 and compared them with isolates collected 40 years ago and Russian vaccine strains. The analysis involved serotyping, pulsed-field gel electrophoresis of chromosomal DNA after digestion with XbaI and SpeI, and sequencing of the ptxS1 and prn genes, which encode the S1 subunit of the pertussis toxin and the major adhesin pertactin, respectively. The Russian isolates were classified in five of the six pulsed-field gel electrophoresis groups identified in other European countries. The B. pertussis isolates currently circulating in Saint Petersburg differed from the Russian whole-cell vaccine strains and the isolates collected in the prevaccine era. However, their repartition in the major pulsed-field gel electrophoresis groups was slightly different from that of isolates collected in countries that have had a high level of vaccine coverage for a long time, probably because the level of vaccine coverage in Saint Petersburg has increased only recently, after decreasing until the early 1990s. Most of the B. parapertussis isolates studied were similar to those circulating in France. However, some variants were observed, perhaps because B. parapertussis infections are more common in children in this area

Calcium dynamics of Plasmodium berghei sporozoite motility

International audienceCalcium is a key signalling molecule in apicomplexan parasites and plays an important role in diverse processes including gliding motility. Gliding is essential for the malaria parasite to migrate from the skin to the liver as well as to invade host tissues and cells. Here we investigated the dynamics of intracellular Ca(2+) in the motility of Plasmodium berghei sporozoites by live imaging and flow cytometry. We found that cytosolic levels of Ca(2+) increase when sporozoites are activated in suspension, which is sufficient to induce the secretion of integrin-like adhesins that are essential for gliding motility. By increasing intracellular Ca(2+) levels artificially with an ionophore, these adhesins are secreted onto the sporozoite surface, however, the parasite is not capable of gliding. A second level of Ca(2+) modulation was observed during attachment to and detachment from a solid substrate, leading to a further increase or a decrease in the cytoplasmic levels of Ca(2+) respectively. We also observed oscillations in the intracellular Ca(2+) level during gliding. Finally, an intracellular Ca(2+) chelator, an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), and an inhibitor of the inositol triphosphate (IP3) receptor blocked the rise in intracellular Ca(2+) , adhesin secretion, and motility of activated sporozoites, indicating that intracellular stores supply Ca(2+) during sporozoite gliding. Our study indicates that a rise in intracellular Ca(2+) is necessary but not sufficient to activate gliding, that Ca(2+) levels are modulated in several ways during motility, and that a PI-PLC/IP3 pathway regulates Ca(2+) release during the process of sporozoite locomotion

Apicomplexa -specific tRip facilitates import of exogenous tRNAs into malaria parasites

International audienc

Flowcytometric and ImageStream RNA-FISH Gene Expression, Quantification and Phenotypic Characterization of Blood Stages and Sporozoites From Human Malaria Species

International audienceWe adapted the RNA FISH Stellaris method to specifically detect the expression of Plasmodium genes by flow cytometry and ImageStream (Flow-FISH). This new method accurately quantified the erythrocytic forms of (1) Plasmodium falciparum and Plasmodium vivax and (2) the sexual stages of P vivax from patient isolates. ImageStream analysis of liver stage sporozoites using a combination of surface circumsporozoite protein (CSP), deoxyribonucleic acid, and 18S RNA labeling proved that the new Flow-FISH is suitable for gene expression studies of transmission stages. This powerful multiparametric single-cell method offers a platform of choice for both applied and fundamental research on the biology of malaria parasites

Role of host cell traversal by the malaria sporozoite during liver infection

International audienceMalaria infection starts when the sporozoite stage of the Plasmodium parasite is injected into the skin by a mosquito. Sporozoites are known to traverse host cells before finally invading a hepatocyte and multiplying into erythrocyte-infecting forms, but how sporozoites reach hepatocytes in the liver and the role of host cell traversal (CT) remain unclear. We report the first quantitative imaging study of sporozoite liver infection in rodents. We show that sporozoites can cross the liver sinusoidal barrier by multiple mechanisms, targeting Kupffer cells (KC) or endothelial cells and associated or not with the parasite CT activity. We also show that the primary role of CT is to inhibit sporozoite clearance by KC during locomotion inside the sinusoid lumen, before crossing the barrier. By being involved in multiple steps of the sporozoite journey from the skin to the final hepatocyte, the parasite proteins mediating host CT emerge as ideal antibody targets for vaccination against the parasite