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Plasmodium sporozoites, the transmission form of the malaria parasite, successively invade salivary glands in the mosquito vector and the liver in the mammalian host. Sporozoite capacity to invade host cells is mechanistically related to their ability to glide on solid substrates, both activities depending on the transmembrane protein TRAP. Here, we show that loss-of- function mutations in two adhesive modules of the TRAP ectodomain, an integrin-like A-domain and a thrombospondin type I repeat, specifically decrease sporozoite invasion of host cells but do not affect sporozoite gliding and adhesion to cells. Irrespective of the target cell, i.e. in mosquitoes, rodents and cultured human or hamster cells, sporozoites bearing mutations in one module are less invasive, while those bearing mutations in both modules are non-invasive. In Chinese hamster ovary cells, the TRAP modules interact with distinct cell receptors during sporozoite invasion, and thus act as independently active pass keys. As these modules are also present in other members of the TRAP family of proteins in Apicomplexa, they may account for the capacity of these parasites to enter many cell types of phylogenetically distant origins.

The sporozoite stage of the Plasmodium parasite is formed by budding from a multinucleate oocyst in the mosquito midgut. During their life, sporozoites must infect the salivary glands of the mosquito vector and the liver of the mammalian host; both events depend on the major sporozoite surface protein, the circumsporozoite protein (CS). We previously reported that Plasmodium berghei oocysts in which the CS gene is inactivated do not form sporozoites. Here, we analyzed the ultrastructure of P.berghei oocyst differentiation in the wild type, recombinants that do not produce or produce reduced amounts of CS, and corresponding complemented clones. The results indicate that CS is essential for establishing polarity in the oocyst. The amounts of CS protein correlate with the extent of development of the inner membranes and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites. This is a first example of a protein controlling both morphogenesis and infectivity of a parasite stage.

Cathepsin L is a member of the papain superfamily of cysteine proteases and, like many other proteases, it is synthesized as an inactive proenzyme. Its prosegment shows little homology to that of procathepsin B, whose structure, the first for a cysteine protease proenzyme, has been determined recently. We report here the 3-D structure of a mutant of human procathepsin L determined at 2.2 A resolution, describe the mode of binding employed by the prosegment and discuss the molecular basis for other possible roles of the prosegment. The N-terminal part of the prosegment is globular and contains three alpha-helices with a small hydrophobic core built around aromatic side chains. This domain packs against a loop on the enzyme's surface, with the aromatic side chain from the prosegment being located in the center of this loop and providing a large contact area. The C-terminal portion of the prosegment assumes an extended conformation and follows along the substrate binding cleft toward the N-terminus of the mature enzyme. The direction of the prosegment in the substrate binding cleft is opposite to that of substrates. The previously described role of the prosegment in the interactions with membranes is supported by the structure of its N-terminal domain. The fold of the prosegment and the mechanism by which it inhibits the enzymatic activity of procathepsin L is similar to that observed in procathepsin B despite differences in length and sequence, suggesting that this mode of inhibition is common to all enzymes from the papain superfamily.

Shigella species are enteropathogens that invade epithelial cells of the human colon. Entry into epithelial cells is triggered by the IpaB, IpaC and IpaD proteins which are translocated into the medium through the specific Mxi-Spa machinery. In vitro, Shigella cells secrete only a small fraction of the Ipa proteins, the majority of which remains in the cytoplasm. We show here that upon interaction with cultured epithelial cells or in the presence of fetal bovine serum, S.flexneri release pre-synthesized Ipa molecules from the cytoplasm into the environment. Evidence is presented that IpaB and IpaD are essential for both blocking secretion through the Mxi-Spa translocon in the absence of a secretion-inducing signal and controlling secretion of the Ipa proteins in the presence of a signal. Subcellular localization and analysis of the molecular interactions of the Ipa proteins indicate that IpaB and IpaD associate transiently in the bacterial envelope. We propose that IpaB and IpaD, by interacting in the secretion apparatus, modulate secretion.