Direct manipulation of malaria parasites with optical tweezers reveals distinct functions of Plasmodium surface proteins.

Plasmodium sporozoite motility is essential for establishing malaria infections. It depends on initial adhesion to a substrate as well as the continuous turnover of discrete adhesion sites. Adhesion and motility are mediated by a dynamic actin cytoskeleton and surface proteins. The mode of adhesion formation and the integration of adhesion forces into fast and continuous forward locomotion remain largely unknown. Here, we use optical tweezers to directly trap individual parasites and probe adhesion formation. We find that sporozoites lacking the surface proteins TRAP and S6 display distinct defects in initial adhesion; trap(-) sporozoites adhere preferentially with their front end, while s6(-) sporozoites show no such preference. The cohesive strength of the initial adhesion site is differently affected by actin filament depolymerization at distinct adhesion sites along the parasite for trap(-) and s6(-) sporozoites. These spatial differences between TRAP and S6 in their functional interaction with actin filaments show that these proteins have nonredundant roles during adhesion and motility. We suggest that complex protein-protein interactions and signaling events govern the regulation of parasite gliding at different sites along the parasite. Investigating how these events are coordinated will be essential for our understanding of sporozoite gliding motility, which is crucial for malaria infection. Laser tweezers will be a valuable part of the toolset.

Key factors regulating Plasmodium berghei sporozoite survival and transformation revealed by an automated visual assay.

Malaria is transmitted to the host when Plasmodium sporozoites are injected by a mosquito vector. Sporozoites eventually enter hepatocytes, where they differentiate into liver-stage parasites. During the first hours after hepatocyte invasion, the crescent-shaped sporozoites transform into spherical intracellular exoerythrocytic parasites. This process, which precedes genome replication, can be mimicked in vitro in the absence of host cells. Here, we developed an automated method to follow transformation and cell death of sporozoites in vitro. This assay provides a rapid tool to test sporozoite survival and to screen for antiparasitic drugs. We found that extracellular bicarbonate and high temperature trigger transformation, whereas physiological serum albumin concentrations and media lacking bicarbonate delayed sporozoite death. Because bicarbonate also triggers ookinete transformation and exflagellation of gametocytes, we suggest that a common molecular mechanism regulates similar aspects of stage conversion in Plasmodium.

Multistep adhesion of Plasmodium sporozoites.

Adhesion of eukaryotic cells is a complex process during which interactions between extracellular ligands and cellular receptors on the plasma membrane modulate the organization of the cytoskeleton. Pathogens particularly rely often on adhesion to tissues or host cells in order to establish an infection. Here, we examined the adhesion of Plasmodium sporozoites, the motile form of the malaria parasite transmitted by the mosquito, to flat surfaces. Experiments using total internal reflection fluorescence microscopy and analysis of sporozoites under flow revealed a stepwise and developmentally regulated adhesion process. The sporozoite-specific transmembrane proteins TRAP and S6 were found to be important for initial adhesion. The structurally related protein TLP appears to play a specific role in adhesion under static conditions, as tlp(-) sporozoites move 4 times less efficiently than wild-type sporozoites. This likely reflects the decreased intradermal sporozoite movement of sporozoites lacking TLP. Further, these three sporozoite surface proteins also act in concert with actin filaments to organize efficient adhesion of the sporozoite prior to initiating motility and host cell invasion.

Plasmodium sporozoite motility is modulated by the turnover of discrete adhesion sites.

Sporozoites are the highly motile stages of the malaria parasite injected into the host's skin during a mosquito bite. In order to navigate inside of the host, sporozoites rely on actin-dependent gliding motility. Although the major components of the gliding machinery are known, the spatiotemporal dynamics of the proteins and the underlying mechanism powering forward locomotion remain unclear. Here, we show that sporozoite motility is characterized by a continuous sequence of stick-and-slip phases. Reflection interference contrast and traction force microscopy identified the repeated turnover of discrete adhesion sites as the underlying mechanism of this substrate-dependent type of motility. Transient forces correlated with the formation and rupture of distinct substrate contact sites and were dependent on actin dynamics. Further, we show that the essential sporozoite surface protein TRAP is critical for the regulated formation and rupture of adhesion sites but is dispensable for retrograde capping.

Automated classification of Plasmodium sporozoite movement patterns reveals a shift towards productive motility during salivary gland infection.

The invasive stages of malaria and other apicomplexan parasites use a unique motility machinery based on actin, myosin and a number of parasite-specific proteins to invade host cells and tissues. The crucial importance of this motility machinery at several stages of the life cycle of these parasites makes the individual components potential drug targets. The different stages of the malaria parasite exhibit strikingly diverse movement patterns, likely reflecting the varied needs to achieve successful invasion. Here, we describe a Tool for Automated Sporozoite Tracking (ToAST) that allows the rapid simultaneous analysis of several hundred motile Plasmodium sporozoites, the stage of the malaria parasite transmitted by the mosquito. ToAST reliably categorizes different modes of sporozoite movement and can be used for both tracking changes in movement patterns and comparing overall movement parameters, such as average speed or the persistence of sporozoites undergoing a certain type of movement. This allows the comparison of potentially small differences between distinct parasite populations and will enable screening of drug libraries to find inhibitors of sporozoite motility. Using ToAST, we find that isolated sporozoites change their movement patterns towards productive motility during the first week after infection of mosquito salivary glands.

Microneme protein 8--a new essential invasion factor in Toxoplasma gondii.

Apicomplexan parasites rely on sequential secretion of specialised secretory organelles for the invasion of the host cell. First, micronemes release their content upon contact with the host cell. Second, rhoptries are discharged, leading to the formation of a tight interaction (moving junction) with the host cell, through which the parasite invades. The functional characterisation of several micronemal proteins in Toxoplasma gondii suggests the occurrence of a stepwise process. Here, we show that the micronemal protein MIC8 of T. gondii is essential for the parasite to invade the host cell. When MIC8 is not present, a block in invasion is caused by the incapability of the parasite to form a moving junction with the host cell. We furthermore demonstrate that the cytosolic domain is crucial for the function of MIC8 and can not be functionally complemented by any other micronemal protein characterised so far, suggesting that MIC8 represents a novel, functionally distinct invasion factor in this apicomplexan parasite.

Aquaporin-1 channel function is positively regulated by protein kinase C.

Aquaporin-1 (AQP1) channels contribute to osmotically induced water transport in several organs including the kidney and serosal membranes such as the peritoneum and the pleura. In addition, AQP1 channels have been shown to conduct cationic currents upon stimulation by cyclic nucleotides. To date, the short term regulation of AQP1 function by other major intracellular signaling pathways has not been studied. In the present study, we therefore investigated the regulation of AQP1 by protein kinase C. AQP1 wild type channels were expressed in Xenopus oocytes. Water permeability was assessed by hypotonic challenges. Activation of protein kinase C (PKC) by 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced a marked increase of AQP1-dependent water permeability. This regulation was abolished in mutated AQP1 channels lacking both consensus PKC phosphorylation sites Thr(157) and Thr(239) (termed AQP1 DeltaPKC). AQP1 cationic currents measured with double-electrode voltage clamp were markedly increased after pharmacological activation of PKC by either OAG or phorbol 12-myristate 13-acetate. Deletion of either Thr(157) or Thr(239) caused a marked attenuation of PKC-dependent current increases, and deletion of both phosphorylation sites in AQP1 DeltaPKC channels abolished the effect. In vitro phosphorylation studies with synthesized peptides corresponding to amino acids 154-168 and 236-250 revealed that both Thr(157) and Thr(239) are phosphorylated by PKC. Upon stimulation by cyclic nucleotides, AQP1 wild type currents exhibited a strong activation. This regulation was not affected after deletion of PKC phosphorylation sites in AQP1 DeltaPKC channels. In conclusion, this is the first study to show that PKC positively regulates both water permeability and ionic conductance of AQP1 channels. This new pathway of AQP1 regulation is independent of the previously described cyclic nucleotide pathway and may contribute to the PKC stimulation of AQP1-modulated processes such as endothelial permeability, angiogenesis, and urine concentration.

A continuous enzyme assay and characterisation of fructosyl amine oxidase enzymes (EC 1.5.3).

Enzymatic reversal of the Maillard reaction is a growing area of research. Fructosyl amine oxidase enzymes (EC 1.5.3) have attracted recent attention through demonstration of their ability to deglycate Amadori products, low molecular weight intermediates formed during the early stage of the Maillard reaction. Although stopped assays have been described, a bottleneck in current studies is the lack of continuous kinetic assays. Here, we describe the development of a continuous, coupled enzyme assay and its successful application to determining optimal storage conditions and the steady-state kinetic parameters of an enzyme from this group, amadoriase I. A K(m)(app) of 11 microM and a K(cat)(app) of 3.5s(-1) were determined using this assay using fructosyl propylamine as a substrate, which differ from previous reports. This method was also used to test the activity of two site-directed mutants of amadoriase I, H357N and S370A, which were found to be catalytically inactive.