Plasmodiumfalciparum infection induces dynamic changes in the erythrocyte phospho-proteome.

The phosphorylation status of red blood cell proteins is strongly altered during the infection by the malaria parasite Plasmodium falciparum. We identify the key phosphorylation events that occur in the erythrocyte membrane and cytoskeleton during infection, by a comparative analysis of global phospho-proteome screens between infected (obtained at schizont stage) and uninfected RBCs. The meta-analysis of reported mass spectrometry studies revealed a novel compendium of 495 phosphorylation sites in 182 human proteins with regulatory roles in red cell morphology and stability, with about 25% of these sites specific to infected cells. A phosphorylation motif analysis detected 7 unique motifs that were largely mapped to kinase consensus sequences of casein kinase II and of protein kinase A/protein kinase C. This analysis highlighted prominent roles for PKA/PKC involving 78 phosphorylation sites. We then compared the phosphorylation status of PKA (PKC) specific sites in adducin, dematin, Band 3 and GLUT-1 in uninfected RBC stimulated or not by cAMP to their phosphorylation status in iRBC. We showed cAMP-induced phosphorylation of adducin S59 by immunoblotting and we were able to demonstrate parasite-induced phosphorylation for adducin S726, Band 3 and GLUT-1, corroborating the protein phosphorylation status in our erythrocyte phosphorylation site compendium.

Trypanosoma cruzi trypomastigotes induce cytoskeleton modifications during HeLa cell invasion

It has been recently shown that Trypanosoma cruzi trypomastigotes subvert a constitutive membrane repair mechanism to invade HeLa cells. Using a membrane extraction protocol and high-resolution microscopy, the HeLa cytoskeleton and T. cruzi parasites were imaged during the invasion process after 15 min and 45 min. Parasites were initially found under cells and were later observed in the cytoplasm. At later stages, parasite-driven protrusions with parallel filaments were observed, with trypomastigotes at their tips. We conclude that T. cruzi trypomastigotes induce deformations of the cortical actin cytoskeleton shortly after invasion, leading to the formation of pseudopod-like structures.

Dematin, a component of the erythrocyte membrane skeleton, is internalized by the malaria parasite and associates with Plasmodium 14-3-3.

The malaria parasite invades the terminally differentiated erythrocytes, where it grows and multiplies surrounded by a parasitophorous vacuole. Plasmodium blood stages translocate newly synthesized proteins outside the parasitophorous vacuole and direct them to various erythrocyte compartments, including the cytoskeleton and the plasma membrane. Here, we show that the remodeling of the host cell directed by the parasite also includes the recruitment of dematin, an actin-binding protein of the erythrocyte membrane skeleton and its repositioning to the parasite. Internalized dematin was found associated with Plasmodium 14-3-3, which belongs to a family of conserved multitask molecules. We also show that, in vitro, the dematin-14-3-3 interaction is strictly dependent on phosphorylation of dematin at Ser(124) and Ser(333), belonging to two 14-3-3 putative binding motifs. This study is the first report showing that a component of the erythrocyte spectrin-based membrane skeleton is recruited by the malaria parasite following erythrocyte infection.

[Change of cytoskeleton and variance of Ca2+ in cultured cells during the invasion of Toxoplasma gondii].

OBJECTIVE: To explore the change of cytoskeleton and the variance of Ca2+ in cultured cells during the invasion of Toxoplasma gondii. METHODS: Tachyzoites suspensions were gathered by routine method and used to infect phagocytic cells (J774A.1) and non-phagocytic cells (HUVEC). The ability of T. gondii invading into the cells and the influence of cytoskeleton inhibitor, colchicine and cytochalasin D, were observed by microscopy. The rearrangement of cytoskeleton of cells was observed by fluoromicroscopy. By using laser scanning confocal microscope, the variance of Ca2+ in J774A.1 and HUVEC was detected. RESULTS: Ca2+ increased greatly in J774A.1 during the invasion of T. gondii (P<0.01) and PLC inhibitor, U73122, could block the increase of Ca2+ (P>0.05). The microfilaments of J774A.1 were agglomerated during the invasion of T. gondii. Cytoskeleton inhibitor, cytochalasin D (P<0.01) and colchicine (P< 0.05) significantly reduced the infection rate of J774A.1 cells. No considerable change of Ca2+ in HUVEC was found (P>0.05) during the invasion and cytoskeleton was not changed. Cytochalasin D and colchicine showed little effect on the infection rate of HUVEC. CONCLUSION: The concentration of Ca2+ increases greatly and cytoskeleton (mainly the microfilament) has been rearranged in phagocytic cell during the invasion of T. gondii, while both of them show no significant change in non-phagocytic cell.

Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis.

Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule beta (RELMbeta) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmbeta and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmbeta).

Host cell tropism underlies species restriction of human and bovine Cryptosporidium parvum genotypes.

It has been recognized recently that human cryptosporidiosis is usually caused by Cryptosporidium parvum genotype I ("human" C. parvum), which is not found in animals. Compared to C. parvum genotype II, little is known of the biology of invasion of the human-restricted C. parvum genotype I. The aims of the present study were (i) to explore and compare with genotype II the pathogenesis of C. parvum genotype I infection by using an established in vitro model of infection and (ii) to examine the possibility that host-specific cell tropism determines species restriction among C. parvum genotypes by using a novel ex vivo small intestinal primary cell model of infection. Oocysts of C. parvum genotypes I and II were used to infect HCT-8 cells and primary intestinal epithelial cells in vitro. Primary cells were harvested from human endoscopic small-bowel biopsies and from bovine duodenum postmortem. C. parvum genotype I infected HCT-8 cells with lower efficiency than C. parvum genotype II. Actin colocalization at the host parasite interface and reduction in levels of invasion after treatment with microfilament inhibitors (cytochalasin B and cytochalasin D) were observed for both genotypes. C. parvum genotype II invaded primary intestinal epithelial cells, regardless of the species of origin. In contrast, C. parvum genotype I invaded only human small-bowel cells. The pathogenesis of C. parvum genotype I differs from C. parvum genotype II. C parvum genotype I does not enter primary bovine intestinal cells, suggesting that the species restriction of this genotype is due to host tissue tropism of the infecting isolate.

Cryptosporidium parvum induces host cell actin accumulation at the host-parasite interface.

Cryptosporidium parvum is an intracellular protozoan parasite that causes a severe diarrheal illness in humans and animals. Previous ultrastructural studies have shown that Cryptosporidium resides in a unique intracellular compartment in the apical region of the host cell. The mechanisms by which Cryptosporidium invades host intestinal epithelial cells and establishes this compartment are poorly understood. The parasite is separated from the host cell by a unique electron-dense structure of unknown composition. We have used indirect immunofluorescence microscopy and confocal laser scanning microscopy to characterize this structure. These studies indicate that host filamentous actin is assembled into a plaque-like structure at the host-parasite interface during parasite invasion and persists during parasite development. The actin-binding protein alpha-actinin is also present in this plaque early in parasite development but is lost as the parasite matures. Other actin-associated proteins, including vinculin, talin, and ezrin, are not present. We have found no evidence of tyrosine phosphorylation within this structure. Molecules known to link actin filaments to membrane were also examined, including alpha-catenin, beta-catenin, plakoglobin, and zyxin, but none was identified at the host-parasite junction. Thus, Cryptosporidium induces rearrangement of the host cell cytoskeleton and incorporates host cell actin and alpha-actinin into a host-parasite junctional complex.

Non-detergent sulphobetaines enhance the recovery of membrane and/or cytoskeleton-associated proteins and active proteases from erythrocytes infected by Plasmodium falciparum.

A better understanding of the causative agent's biology and the definition of new targets for the development of drugs and/or specific immune responses is necessary to face the spred of drug-resistant malaria in developing countries and the absence of an efficient vaccine against this most important infectious disease. Non-detergent sulphobetaines enhance the recovery and isoelectric focussing of active Plasmodium falciparum proteases, cytoskeleton-associated proteins and Maurer's cleft-associated proteins. This is a significant advantage for the purification of such proteins and might help pinpoint their role for red blood cell rupture and merozoite release.

Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite.

Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. We demonstrate that invasion is critically dependent on actin filaments in the parasite, but not the host cell. Invasion into cytochalasin D (CD)-resistant host cells was blocked by CD, while parasite mutants invaded wild-type host cells in the presence of drug. CD resistance in Toxoplasma was mediated by a point mutation in the single-copy actin gene ACT1. Transfection of the mutant act1 allele into wild-type Toxoplasma conferred motility and invasion in the presence of CD. We conclude that host cell invasion by Toxoplasma, and likely by related Apicomplexans, is actively powered by an actin-based contractile system in the parasite.

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.

Changes in the cytoplasmic elements of cultured cells infected with Eimeria vermiformis sporozoites.

Epithelial-type (PK-15) and fibroblast-type (MDBK) mammalian cell cultures were inoculated with purified Eimeria vermiformis sporozoites. Matched samples from 0 to 93 h after inoculation (HAI) were processed for electron microscopy; half of the sample preparations were extracted with non-ionic detergent prior to fixation. Specimens were examined by both transmission and scanning electron microscopy. Numerous sporozoites were attached to the cultured cells from 2 to 93 HAI, usually near the cell periphery. Some host cell microvilli extended up and appeared attached to the sporozoites. Sporozoites fixed during the penetration process were markedly constricted at the site of entry; however, no noticeable changes occurred in the host cell membrane or surface microvilli during sporozoite invasion or in sporozoite-infected cells. In cells extracted with 1% Triton X-100, the host cytoskeleton was progressively reorganized about the parasites but changes were limited to the immediate area of the sporozoite. Around resident sporozoites, the cytoskeleton became less dense but also more ordered, which contrasted with adjacent cell areas. Cytoskeletal elements passed both over and under the parasites. The appearance of the cytoskeleton suggested that the host cell formed a loose, basket-like net of cytoskeletal elements about the parasite.

Interaction between pathogenic amebas and fibronectin: substrate degradation and changes in cytoskeleton organization.

Invasion of human tissues by the parasitic protozoan Entamoeba histolytica is a multistep process involving, as a first step, the recognition of surface molecules on target tissues by the amebas or trophozoites. This initial contact is followed by the release of proteolytic and other activities that lyse target cells and degrade the extracellular matrix. In other parasitic diseases, as well as in certain cancers, the interaction of invasive organisms or cells with fibronectin (FN) through specific receptors has been shown to be the initial step in target cell recognition. Interaction with FN triggers the release of proteolytic activities necessary for the effector cell migration and invasion. Here, we describe the specific interaction of Entamoeba histolytica trophozoites with FN, and identify a 37-kD membrane peptide as the putative receptor for FN. The interaction between the parasite and FN leads to a response reaction that includes the secretion of proteases that degrade the bound FN and the rearrangement of amebic actin into "adhesion plates" at sites of contact with FN-coated surfaces. The kinetics of the interaction was determined by measuring the binding of soluble 125I-FN to the trophozoites and visualization of the bound protein using specific antibodies. Degradation of FN was measured by gel electrophoresis and the release of radioactivity into the incubation medium. Focal degradation of FN was visualized as black spots under the trophozoites at contact sites with fluorescent FN. We conclude that the interaction of E. histolytica with FN occurs through a specific surface receptor. The interaction promotes amebic cytoskeleton changes and release of proteases from the parasite. The binding and degradation of extracellular matrix components may facilitate the migration and penetration of amebas into tissues, causing the lesions seen in human hosts.