Pathogenesis of Plasmodium falciparum malaria: the roles of parasite adhesion and antigenic variation

Malaria results in up to 2.5 million deaths annually, with young children and pregnant women at greatest risk. The great majority of severe disease is caused by Plasmodium falciparum. A characteristic feature of infection with P. falciparum is the accumulation or sequestration of parasite-infected red blood cells (RBCs) in various organs, such as the brain, lung and placenta, and together with other factors is important in the pathogenesis of severe forms of malaria. Sequestration results from adhesive interactions between parasite-derived proteins expressed on the surface of infected RBCs and a number of host molecules on the surface of endothelial cells, placental cells and uninfected RBCs. Some receptors for parasite adhesion have been implicated in particular malaria syndromes, such as intercellular adhesion molecule 1 in cerebral malaria and chondroitin sulfate A and hyaluronic acid in placental infection. The principal parasite ligand and antigen on the RBC surface, P. falciparum erythrocyte membrane protein 1 encoded by a multigene family termed var, is clonally variant, enabling evasion of specific immune responses. An understanding of these host-parasite interactions in the context of clinical disease and immunity may reveal potential targets to prevent or treat severe forms of malaria. Received 25 June 2001; received after revision 22 August 2001; accepted 24 August 2001     

Membrane-shed vesicles from the parasite <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>: characterization and their association with cell interaction

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor–ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.     

Encapsulation ofPsorospermium haeckeli by the haemocytes ofAstacus leptodactylus

Summary Psorospermium haeckeli, a parasite of freshwater crayfish, has sometimes been observed encapsulated within the tissues ofAstacus leptodactylus. This observation does not agree with earlier reports. The lack of tolerance is discussed in relation to variations affecting either the parasite or its host.     

Enzymatic characterization of nine endoparasite species of small ermine moths (Yponomeutidae)

Summary Eight hymenopterous and 1 dipterous species, all endoparasitic in eggs, larvae, or pupae of small ermine moths (Yponomeuta) were investigated for their allozyme variation at 3–29 loci. The mean heterozygosity level of the hymenopterous species is one-third of that of the dipterous species. Zymogram patterns of the parasite larvae do not interfere with those of the host.     

Cryopreservation of parasites

Summary In this review, advances in cryopreservation of helminth parasites are reported. Our own studies demonstrate that metacestodes ofEchinococcus multilocularis can be maintained in a viable state for at least 1–2 years by appropriate deep-freezing and storage in liquid nitrogen. Infective larvae of the nematodeToxocara canis cryopreserved for 1 week in liquid nitrogen were maintained after thawing in vitro in a chemically defined medium for 35 weeks. Although motility of previously deep-frozen larvae was reduced they produced secretory/excretory antigens of similar immunodiagnostic quality as those from unfrozen larvae. Whereas infective larvae of several species of trichostrongylids can be easily cryopreserved, the infective larvae of the cattle lungworm,Dictyocaulus viviparus, and muscle larvae ofTrichinella spiralis are more sensitive to damage by subzero temperatures. Therefore, survival rates after cryopreservation are low, but improvement of the cooling schedules appears to be feasible. It is concluded that cryopreservation of certain stages of helminth and protozoan parasites is a useful technique for long-term storage of defined isolates, which can contribute considerably to reducing the number of experimental animals usually required for serial passages.     

Mechanisms of cellular invasion by intracellular parasites

Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world’s population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite–host cell interactions, forming the basis of the parasite’s cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.     

Development of malaria parasites in mosquitoes fed with ookinetes suspended in defined media

Information concerning the specific nutritional requirements of malarial parasites developing in the mosquito host has been difficult to obtain, owing primarily to the complex nature of the blood meal that accompanies the parasites and the lack of success in culturing the complete invertebrate cycle ofPlasmodium in vitro. The present report describes a blood-free system for infecting mosquitoes with ookinetes ofPlasmodium berghei and for allowing the latter to develop into infective sporozoites. Ookinetes cultured in vitro were separated from blood proteins, suspended in defined medium, and fed toAnopheles stephensi mosquitoes through a membrane. The mosquitoes were then maintained on the same defined medium plus 5% sucrose. Infectivity of the parasites was demonstrated 17–19 days later by intracardial inoculation of the macerated mosquitoes into hamsters. This system makes it possible to evaluate nutritional factors that affect parasite development in the mosquito host under controlled conditions.This project was supported, in part, by the Public Health Service research grant AI-18345 from the National Institute of Allergy and Infectious Diseases to Prof. K. Maramorosch, and the Charles and Johanna Busch Memorial Fund at Rutgers, The State University of New Jersey.     

Accessibility of host cell lineages to medaka stem cells depends on genetic background and irradiation of recipient embryos

Chimera formation is a powerful tool for analyzing pluripotency in vivo. It has been widely accepted that host cell lineages are generally accessible to embryonic stem (ES) cells with the actual contribution depending solely on the intrinsic pluripotency of transplanted donor cells. Here, we show in the fish medaka (Oryzias latipes) that the host accessibility to ES cell contribution exhibits dramatic differences. Specifically, of three albino host strains tested (i ¹ , i ³ and af), only strain i ¹ generated pigmented chimeras. Strikingly, this accessibility is completely lost in i ¹ but acquired in i ³ after host γ-irradiation. Host irradiation also differentially affected ES cell contribution to somatic organs and gonad. Therefore, the accessibility of various host cell lineages can vary considerably depending on host strains and cell lineages as well as on irradiation. Our findings underscore the importance of host genotypes for interpreting donor cell pluripotency and for improving ES-derived chimera production.     

The surface glycopeptidolipids of mycobacteria: structures and biological properties

One of the most important opportunistic pathogens associated with acquired immunodeficiency syndrome (AIDS) is the M. avium complex. M. avium infections are found in up to 70% of individuals in advanced stages of AIDS. It is apparent that M. avium can replicate in host macrophages and persist for long periods. This group of mycobacteria are distinguished by the presence of unique, highly antigenic, surface-located lipids known as the glycopeptidolipids (GPLs). The GPLs are the chemical basis of the 31 distinct serovars of the M. avium complex, and have also been identified in some other species. The M. avium lipids are immunosuppressive and can induce a variety of cytokines that affect general host responses. Despite extensive chemical characterization of the structures of these GPLs, much work is needed to elucidate the molecular mechanism involved in this complex glycosylation pathway and its genetic basis. The challenges for the future lie in explaining the roles of these copious products in the intracellular life and infectivity of mycobacteria. The intention of our review is to offer a concise account of the structures of the M. avium lipids, their putative roles in the host responses, bacterial physiology and pathogenesis, particularly in immunocompromised patients such as those infected with human immunodeficiency virus (HIV). Advances in chemical synthesis of the various haptenic oligosaccharides are also given to demonstrate how these have helped to define the immunogenic determinants. We believe that future research should involve the creation of conditional mutants defective in these lipids for both functional and biosynthesis studies which will complement biological assays using chemically defined or modified neoglycoconjugates. Received 7 May 2001; received after revision 28 June 2001; accepted 28 June 2001     

Inhibitory effect of halocyamine,an antimicrobial substance from ascidian hemocytes,on the growth of fish viruses and marine bacteria

Summary Halocyamine A, an antimicrobial substance isolated from hemocytes of the solitary ascidianHalocynthia roretzi, inhibited in vitro the growth of fish RNA viruses (infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus). Pretreatment of RNA virus with halocyamine A reduced the infectivity of the virus toward host cells. The growth of marine bacteria,Achromobacter aquamarinus andPseudomonas perfectomarinus, was also inhibited by halocyamine A but that ofAlteromonas putrefaciens andVibrio anguillarum was not. These results suggest that halocyamine may have a role in the defense mechanisms ofH. roretzi against marine viruses and bacteria.     

Comparative evaluation of 7 helminth antigens in the enzyme-linked immunosorbent assay (E.L.I.S.A.)

Summary 112 sera from Europeans with parasitologically proven helminthiasis were tested in the enzyme-linked immunosorbent assay (E.L.I.S.A.) against 6 crude extracts of various helminths (2 of adult worms:Dipetalonema viteae, Fasciola hepatica; 3 of eggs:Ascaris suum, Toxocara canis, Schistosoma mansoni; and ofEchinococcus granulosus scolices) and against bovine hydatid fluid. Each serum was tested simultaneously at a fixed dilution of 1:160 against all antigens. Extensive cross-reactions were observed, leading to the conclusion that non-purified helminth antigens, even in combination, are of limited value for reliable serodiagnosis in E.L.I.S.A.     

Evolution and ecological correlates of uniparental reproduction in freshwater snails

We review the spatial and temporal correlates of uniparental reproduction in freshwater snails as they pertain to the ecological hypotheses for the maintenance of biparental sex. The biogeographic evidence from two species (Potamopyrgus antipodarum andBulinus truncatus) presently supports the Red Queen hypothesis that biparental reproduction is selected as a way to reduce the risk to progeny of parasite attack. Uniparental reproduction in these species is associated with low levels of infection by parasites (castrating digenetic trematodes), suggesting that parthenogenesis or self-fertilization can replace cross-fertilization when the risk of infection is low. In addition, inB. truncatus, the opportunity for cross-fertilization coincides with the season in which parasite attack is highest. In a third species (Campeloma decisum), parthenogenetic reproduction is correlated with latitude and the presence of a non-castrating trematode that may prevent cross-fertilization; these patterns suggest that parthenogenesis has been selected as a mechanism to assure reproduction. Finally, we discuss the spotty taxonomic distribution of parthenogenetic species.     

Functional interaction between TRPC1 channel and connexin-43 protein: a novel pathway underlying S1P action on skeletal myogenesis

We recently demonstrated that skeletal muscle differentiation induced by sphingosine 1-phosphate (S1P) requires gap junctions and transient receptor potential canonical 1 (TRPC1) channels. Here, we searched for the signaling pathway linking the channel activity with Cx43 expression/function, investigating the involvement of the Ca²⁺-sensitive protease, m-calpain, and its targets in S1P-induced C2C12 myoblast differentiation. Gene silencing and pharmacological inhibition of TRPC1 significantly reduced Cx43 up-regulation and Cx43/cytoskeletal interaction elicited by S1P. TRPC1-dependent functions were also required for the transient increase of m-calpain activity/expression and the subsequent decrease of PKCα levels. Remarkably, Cx43 expression in S1P-treated myoblasts was reduced by m-calpain-siRNA and enhanced by pharmacological inhibition of classical PKCs, stressing the relevance for calpain/PKCα axis in Cx43 protein remodeling. The contribution of this pathway in myogenesis was also investigated. In conclusion, these findings provide novel mechanisms by which S1P regulates myoblast differentiation and offer interesting therapeutic options to improve skeletal muscle regeneration.     

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal “sterile α-motif” domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> chromosomal passenger complex is essential for the organization of a functional mitotic spindle: a prerequisite for productive endodyogeny

The phylum Apicomplexa encompasses deadly pathogens such as malaria and Cryptosporidium. Apicomplexa cell division is mechanistically divergent from that of their mammalian host, potentially representing an attractive source of drug targets. Depending on the species, apicomplexan parasites can modulate the output of cell division, producing two to thousands of daughter cells at once. The inherent flexibility of their cell division mechanisms allows these parasites to adapt to different niches, facilitating their dissemination. Toxoplasma gondii tachyzoites divide using a unique form of cell division called endodyogeny. This process involves a single round of DNA replication, closed nuclear mitosis, and assembly of two daughter cells within a mother. In higher Eukaryotes, the four-subunit chromosomal passenger complex (CPC) (Aurora kinase B (ARKB)/INCENP/Borealin/Survivin) promotes chromosome bi-orientation by detaching incorrect kinetochore–microtubule attachments, playing an essential role in controlling cell division fidelity. Herein, we report the characterization of the Toxoplasma CPC (Aurora kinase 1 (Ark1)/INCENP1/INCENP2). We show that the CPC exhibits dynamic localization in a cell cycle-dependent manner. TgArk1 interacts with both TgINCENPs, with TgINCENP2 being essential for its translocation to the nucleus. While TgINCENP1 appears to be dispensable, interfering with TgArk1 or TgINCENP2 results in pronounced division and growth defects. Significant anti-cancer drug development efforts have focused on targeting human ARKB. Parasite treatment with low doses of hesperadin, a known inhibitor of human ARKB at higher concentrations, phenocopies the TgArk1 and TgINCENP2 mutants. Overall, our study provides new insights into the mechanisms underpinning cell cycle control in Apicomplexa, and highlights TgArk1 as potential drug target.     

Babesia bovis: the effect of acute inflammation and isoantibody production in the detection of babesial antigens

Summary Immunoblots ofBabesia bovis antigen contain dominant antigens which react not only with antisera toB. bovis but with sera from naive calves recovering from an acute inflammatory reaction. It seems likely these antigens are from the host rather than the parasite.     

Gel-electrophoretic description of European populations ofTerellia virens (Loew) (Diptera,Tephritidae); implications for its use as an agent for the biological control ofCentaurea spp. (Asteraceae) in North America

Allozyme frequencies of 15 enzyme loci, 14 of which were polymorphic, were used to characterize sevenTerellia virens populations originating from three allopatrically distributedCentaurea species. The two populations whose origins were geographically furthest apart, from Israel (onC. iberica) and from Switzerland (onC. vallesiaca), showed relatively high values of genetic distance from the 5 populations sampled in Austria and Hungary (onC. maculosa) (Nei's D>0.07). The latter five displayed a high degree of genetic similarity. No diagnostic (fixed) allelic differences were observed between these three groups ofT. virens populations, but they could be well characterized by significant differences in allelic frequencies at 9 enzyme loci. Independently of this study, the populations from Switzerland (C. vallesiaca) and eastern Austria (C. maculosa) were selected as potential source populations for future introductions into North America for the biological control of introducedC. maculosa andC. diffusa. Based on the observed genetic differences and results from field experiments on the host specificity of these two potential source populations, it is argued that host specificity screening tests should be conducted separately for local (host plant) populations, as such populations might accept a different set of hosts. Biotype mismatch and the risk of spill-overs to native species could thus possibly be reduced.     

Mast cells enter a teleost's brain by Xth cranial nerve in response toDiplostomum phoxini (Trematoda)

Summary Experimental infection byDiplostomum phoxini of the brain of laboratory hatched and rearedPhoxinus phoxinus induces the migration along the Xth cranial nerve of periodic acid Schiff positive granular leucocytes (PAS-GLs). These differentiate and grow into cells that lie between the parasite and the neurones of the host. The transformed cells are associated with elevated levels of heparin and serotonin in the brain; little histamine was detected. The reactive cell is identified as a mast cell.This work was carried out with M.R.L. Johnston's guidance. Teresa Townshend is thanked for assistance with techniques to hatch and raiseP. phoxinus.     

Possible origin of zeaxanthin in the marine sponge,Reniera japonica

As part of a study to clarify the origins of biologically active substances in marine sponges, the carotenoids produced by two species of marine bacteria,Flexibacter sp. strain number DK30213 and DK30223, associated with the marine sponge,Reniera japonica, were investigated. Both bacteria were found to produce zeaxanthin [(3R, 3R)-dihydroxy-,-carotene] which is widely distributed in marine organisms. This carotenoid was also detected in the host sponge, suggesting the transport of zeaxanthin from the microorganisms to the host. As zeaxanthin plays the role of a quencher and scavenger for active species of oxygen, it is presumed that the sponge accumulates the bacterial product as a defense substance against the active oxygen species produced under irradiation by strong sunlight. It is thought that the bacteria are symbionts of the host sponge and act by obtaining the solid substrate and medium needed for settlement and growth from the host, and by producing and transmitting the biologically active substance to the host. Zeaxanthin-producing bacteria are also considered to have potential for practical uses by the aquacultural, pharmaceutical and food industries.