Unraveling the Elusive Rhoptry Exocytic Mechanism of Apicomplexa.

Apicomplexan parasites are unicellular eukaryotes that invade the cells in which they proliferate. The development of genetic tools in Toxoplasma, and then in Plasmodium, in the 1990s allowed the first description of the molecular machinery used for motility and invasion, revealing a crucial role for two different secretory organelles, micronemes and rhoptries. Rhoptry proteins are injected directly into the host cytoplasm not only to promote invasion but also to manipulate host functions. Nonetheless, the injection machinery has remained mysterious, a major conundrum in the field. Here we review recent progress in uncovering structural components and proteins implicated in rhoptry exocytosis and explain how revisiting early findings and considering the evolutionary origins of Apicomplexa contributed to some of these discoveries.

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata.

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (ß-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.

Is Trichomonas tenax a Parasite or a Commensal?

Trichomonas tenax is considered a commensal organism found under poor oral hygiene conditions. T. tenax presents morphological similarities with T. vaginalis, and there are doubts concerning whether this protist is a parasite and whether it is a genetic variant of T. vaginalis. This study aimed to investigate the capacity of T. tenax to cause mammalian cell damage and compare its cytotoxicity with that of T. vaginalis. Protozoan-host cell interaction assays were performed with Madin-Darby canine kidney, HeLa, and gum cells and 3D spheroids, which were examined by scanning electron and transmission electron microscopy. Cellular viability experiments were also performed. T. tenax attached and had different forms when interacting with mammalian cells and caused damage with time-dependent host-cell viability. We observed that T. tenax produced plasma membrane projections and phagocytosed portions of the mammalian cells. In addition, T. tenax caused membrane blebbing and apoptotic bodies in HeLa cells, thus inducing cell death. Spheroids were also used in interaction assays with T. tenax and they were damaged by these cells. This study shows that T. tenax fulfills the requisites of a parasite, causing damage to different mammalian cells and behaving similarly to T. vaginalis when in contact with target cells in vitro.

[The glideosome, a unique machinery that assists the Apicomplexa in gliding into host cells]. / Un complexe moléculaire unique à l'origine de la motilité et de l'invasion des Apicomplexes.

Protozoan parasites belonging to the phylum Apicomplexa are of considerable medical and veterinary significance. These obligate intracellular parasites use a unique form of locomotion to traverse biological barriers and actively invade in and egress from host cells. An actin-myosin-based complex named "glideosome" drives this unusual substrate-dependent motility, which is essential for the establishment of the infection. The mechanisms involved in motility, invasion and egress are conserved throughout the phylum. This article describes the current knowledge on the invasion process of two experimentally tractable apicomplexan parasites: Toxoplasma gondii and Plasmodium falciparum.

The Cinderella syndrome: why do malaria-infected cells burst at midnight?

An interesting quirk of many malaria infections is that all parasites within a host - millions of them - progress through their cell cycle synchronously. This surprising coordination has long been recognized, yet there is little understanding of what controls it or why it has evolved. Interestingly, the conventional explanation for coordinated development in other parasite species does not seem to apply here. We argue that for malaria parasites, a critical question has yet to be answered: is the coordination due to parasites bursting at the same time or at a particular time? We explicitly delineate these fundamentally different scenarios, possible underlying mechanistic explanations and evolutionary drivers, and discuss the existing corroborating data and key evidence needed to solve this evolutionary mystery.

Avaliação do papel das células T CD8+ e análise proteômica nas Biópsias de pacientes com Leishmaniose Cutânea Localizada infectados por L. braziliensis / Assessing the role of CD8 + T cells and proteomic analysis in biopsies from patients with cutaneous leishmaniasis infected by L. braziliensis Located

Células T CD8+ são essenCIaIS na defesa contra vírus, mas pouco se sabe sobre a sua participação na defesa do hospedeiro contra parasitas, como a Leishmania. Nesse trabalho, investigou-se a participação das células T CD8+ no processo inflamatório e na indução da morte do parasita, bem como os perfis proteômicos das lesões dos pacientes com leishmaniose cutânea localizada (LCL) infectados por L. braziliensis. Observa-se uma alta porcentagem de células T CD8+ nas lesões dos pacientes com LCL, como sugerido pela maior frequência de células T CD8+CD45RO+ e células T CD8+CLA + em comparação com CMSP do próprio indivíduo. Após re-estimulação com L. braziliensis, a maioria das células T CD8+ presentes na lesão expressam marcadores citolíticos, CD 107a e Granzima B. Além disso, a co-cultura de macrófagos infectados com linfócitos T CD8+ resultou na libertação de granzima B. A utilização do inibidor da granzima B, assim como Z-V AO, Fas:Fc ou anti­ IFN-y não teve nenhum efeito sobre a morte do parasita. Por outro lado, a co-cultura de macrófagos infectados com células T CD4+ diminuiu a taxa de infecção, efeito que foi completamente revertido ao se utilizar o anti-IFN-y, enfatizando o papel determinante de células T CD4+IFN-/ na morte da Leishmania. Além disso, observou-se um total de 150 proteínas diferencialmente expressas entre as lesões de pacientes com LCL e as amostras de pele normal. Destas, cinquenta e nove proteínas foram identificadas. Entre elas, 13 apresentaram uma maior ou menor expressão nas amostras dos pacientes em relação a pele normal. Vinte e sete proteínas foram encontradas somente nas lesões dos pacientes e 18 somente nas amostras de pele normal. Essas proteínas foram associadas com processos de regulação biológica; incluindo apoptose, ciclo celular e resposta imune. Para explorar as interações entre as proteínas identificadas e as proteínas e os genes que poderiam ser afetados por elas, redes e sub-redes de interações foram geradas. Após análises de imuno-histoquímica, a presença de caspas e 9, caspase-3 e granzima B foi validada no sítio da lesão. A expressão da granzima B nas lesões dos pacientes correlacionou-se positivamente com a expressão da caspase 9 e a porcentagem de células positivas para TUNEL. Observou-se também uma maior porcentagem de células postivas para TUNEL expressando granzima B nas biópsias dos pacientes que apresentam um processo mais intenso de necrose. Além disso, a presença de granzime B, caspase 9 e caspase-3 foram correlacionados positivamente com o tamanho da, lesão. Neste estudo, podemos concluir que as células T CD8+Granzima B+ estão envolvidas na patogênese da L. braziliensis, através da ativação da apoptose no sítio inflamatório, favorecendo a progressão do dano tecidual observado nos pacientes com LCL

Interferon-inducible effector mechanisms in cell-autonomous immunity.

Interferons (IFNs) induce the expression of hundreds of genes as part of an elaborate antimicrobial programme designed to combat infection in all nucleated cells - a process termed cell-autonomous immunity. As described in this Review, recent genomic and subgenomic analyses have begun to assign functional properties to novel IFN-inducible effector proteins that restrict bacteria, protozoa and viruses in different subcellular compartments and at different stages of the pathogen life cycle. Several newly described host defence factors also participate in canonical oxidative and autophagic pathways by spatially coordinating their activities to enhance microbial killing. Together, these IFN-induced effector networks help to confer vertebrate host resistance to a vast and complex microbial world.

Revisiting the Acanthamoeba species that form star-shaped cysts (genotypes T7, T8, T9, and T17): characterization of seven new Brazilian environmental isolates and phylogenetic inferences.

Free-living amoebae of the genus Acanthamoeba are the agents of both opportunistic and non-opportunistic infections and are frequently isolated from the environment. Of the 17 genotypes (T1-T17) identified thus far, 4 (T7, T8, T9, and T17) accommodate the rarely investigated species of morphological group I, those that form large, star-shaped cysts. We report the isolation and characterization of 7 new Brazilian environmental Acanthamoeba isolates, all assigned to group I. Phylogenetic analyses based on partial (~1200 bp) SSU rRNA gene sequences placed the new isolates in the robustly supported clade composed of the species of morphological group I. One of the Brazilian isolates is closely related to A. comandoni (genotype T9), while the other 6, together with 2 isolates recently assigned to genotype T17, form a homogeneous, well-supported group (2·0% sequence divergence) that likely represents a new Acanthamoeba species. Thermotolerance, osmotolerance, and cytophatic effects, features often associated with pathogenic potential, were also examined. The results indicated that all 7 Brazilian isolates grow at temperatures up to 40°C, and resist under hyperosmotic conditions. Additionally, media conditioned by each of the new Acanthamoeba isolates induced the disruption of SIRC and HeLa cell monolayers.

[Autophagy and pathogens: «Bon appétit Messieurs!¼]. / Autophagie et pathogènes: «Bon appétit Messieurs!¼

Autophagy is a highly conserved, self-degradative pathway for clearance and recycling of cytoplasmic contents. This ubiquitous cell intrinsic process can be used as a defence mechanism against intracellular pathogens. Indeed autophagy is increased upon pathogen detection, and experimental extinction in vitro and in vivo of this cellular process has been demonstrated as a crucial role to control intracellular pathogens. Co-evolution between host-cells and pathogens has selected numerous micoorganisms able to avoid or usurp autophagy to their own benefit. Understanding mechanisms underlying the anti-microbial properties of autophagy as well as those used by certain pathogens to escape this cellular process might be crucial to manipulate this cellular function in order to prevent or treat infectious diseases.

Innate recognition of intracellular pathogens: detection and activation of the first line of defense.

The innate immune system constitutes the first line of defense against infections and is also important for initiating the development of an adaptive immune response. The innate immune system recognizes microbial infection through germline-encoded pattern recognition receptors, which are responsible for decoding the microbial fingerprint and activating an appropriate response against the invading pathogen. In this review, we present and discuss current knowledge on how the innate immune system recognizes intracellular pathogens, activates intracellular signaling, induces gene expression, and orchestrates the microbicidal response against pathogens with a habitat within host cells.

How cells clean house.

Inside the cytoplasm of a living cell, organelles called autophagosomes continually engulf bits of cytoplasm, along with damaged cell parts and invading bacteria and viruses. The "sweepings" are carried to digestive organelles for breakup and recycling. The process is called autophagy. Cell biologists are learning about autophagy in great detail by tracing the protein signals that drive and control the process. A fuller understanding of autophagy is opening up new options for treating cancer, infectious disease, immune disorders and dementia, and it may one day even help to slow down aging.

The propeptide of cruzipain--a potent selective inhibitor of the trypanosomal enzymes cruzipain and brucipain, and of the human enzyme cathepsin F.

Papain-like cysteine proteases of pathogenic protozoa play important roles in parasite growth, differentiation and host cell invasion. The main cysteine proteases of Trypanosoma cruzi (cruzipain) and of Trypanosoma brucei (brucipain) are validated targets for the development of new chemotherapies. These proteases are synthesized as precursors and activated upon removal of the N-terminal prodomain. Here we report potent and selective inhibition of cruzipain and brucipain by the recombinant full-length prodomain of cruzipain. The propeptide did not inhibit human cathepsins S, K or B or papain at the tested concentrations, and moderately inhibited human cathepsin V. Human cathepsin F was very efficiently inhibited (K(i) of 32 pm), an interesting finding indicating that cruzipain propeptide is able to discriminate cathepsin F from other cathepsin L-like enzymes. Comparative structural modeling and analysis identified the interaction between the beta1p-alpha3p loop of the propeptide and the propeptide-binding loop of mature enzymes as a plausible cause of the observed inhibitory selectivity.

Pulling together: an integrated model of Toxoplasma cell invasion.

The protozoan Toxoplasma gondii invades a wide array of animal cells using an actin/myosin-based motor complex to drive active penetration. This broad specificity implies that the parasite has developed a means of using a widely expressed receptor, many different receptors, or perhaps a receptor produced by T. gondii itself. Recently, there has been an explosion in identification of the molecules involved, including those that comprise the 'moving junction' that slides over the parasite as it invades. The emerging model is that invasion comprises at least seven steps that progressively increase the parasite's grip on the host surface, form the moving junction and enlist the motor complex to drive entry. These recent findings have led to new hypotheses regarding the parasite's broad host-specificity.

Reengineering transfusion and cellular therapy processes hospitalwide: ensuring the safe utilization of blood products.

Efforts to make blood transfusion as safe as possible have focused on making the blood in the bag as disease-free as possible. The results have been dramatic, and the costs have been correspondingly high. Although blood services will have to continue to deal with emerging pathogens, efforts to reduce the transfusion of infectious agents presently posing a risk will require high incremental costs and result in only improvements of a small magnitude. The other aspect of safe blood transfusion, the actual transfusion process performed primarily in hospitals, has been accorded considerably less interest. We should turn our attention to enhancing overall blood safety by focusing on improving the process of blood transfusion. Errors involving patient, specimen, and blood product identification put transfused patients at risk, increasing the mortality risk for some. Solutions that could improve the transfusion process are discussed as a focus of this article.

A new cruzipain-mediated pathway of human cell invasion by Trypanosoma cruzi requires trypomastigote membranes.

The intracellular protozoan Trypanosoma cruzi causes Chagas' disease, a chronic illness associated with cardiomyopathy and digestive disorders. This pathogen invades mammalian cells by signaling them through multiple transduction pathways. We previously showed that cruzipain, the main cysteine protease of T. cruzi, promotes host cell invasion by activating kinin receptors. Here, we report a cruzipain-mediated invasion route that is not blocked by kinin receptor antagonists. By testing different strains of T. cruzi, we observed a correlation between the level of cruzipain secreted by trypomastigotes and the capacity of the pathogen to invade host cells. Consistent with a role for cruzipain, the cysteine protease inhibitor N-methylpiperazine-urea-Phe-homophenylalanine-vinylsulfone-benzene impaired the invasion of human smooth muscle cells by strains Dm28c and X10/6 but not by the G isolate. Cruzipain-rich supernatants of Dm28c trypomastigotes enhanced the infectivity of isolate G parasites twofold, an effect which was abolished by the cysteine protease inhibitor l-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane and by thapsigargin, a drug that induces depletion of the intracellular Ca(2+) stores. The enhancement due to Dm28 supernatants was abolished upon cruzipain immunodepletion, and the activity was restored by purified cruzipain. In contrast, supernatants from isolate G trypomastigotes (with low levels of cruzipain) or supernatants from Dm28c epimastigotes or purified cruzipain alone did not enhance parasite invasion, indicating that the protease is required but not sufficient to engage this invasion pathway. We provide evidence that activation of this pathway requires cruzipain-mediated processing of a trypomastigote molecule associated with parasite-shed membranes. Our results couple cruzipain to host cell invasion through a kinin-independent route and further suggest that high-level cruzipain expression may contribute to parasite infectivity.

Intracellular parasite invasion strategies.

Intracellular parasites use various strategies to invade cells and to subvert cellular signaling pathways and, thus, to gain a foothold against host defenses. Efficient cell entry, ability to exploit intracellular niches, and persistence make these parasites treacherous pathogens. Most intracellular parasites gain entry via host-mediated processes, but apicomplexans use a system of adhesion-based motility called "gliding" to actively penetrate host cells. Actin polymerization-dependent motility facilitates parasite migration across cellular barriers, enables dissemination within tissues, and powers invasion of host cells. Efficient invasion has brought widespread success to this group, which includes Toxoplasma, Plasmodium, and Cryptosporidium.