The Autophagy Machinery in Human-Parasitic Protists; Diverse Functions for Universally Conserved Proteins.

Autophagy is a eukaryotic cellular machinery that is able to degrade large intracellular components, including organelles, and plays a pivotal role in cellular homeostasis. Target materials are enclosed by a double membrane vesicle called autophagosome, whose formation is coordinated by autophagy-related proteins (ATGs). Studies of yeast and Metazoa have identified approximately 40 ATGs. Genome projects for unicellular eukaryotes revealed that some ATGs are conserved in all eukaryotic supergroups but others have arisen or were lost during evolution in some specific lineages. In spite of an apparent reduction in the ATG molecular machinery found in parasitic protists, it has become clear that ATGs play an important role in stage differentiation or organelle maintenance, sometimes with an original function that is unrelated to canonical degradative autophagy. In this review, we aim to briefly summarize the current state of knowledge in parasitic protists, in the light of the latest important findings from more canonical model organisms. Determining the roles of ATGs and the diversity of their functions in various lineages is an important challenge for understanding the evolutionary background of autophagy.

Exploring insights of syntaxin superfamily proteins from Entamoeba histolytica: a prospective simulation, protein-protein interaction, and docking study.

Entamoeba histolytica (Eh), a parasitic protozoan and the causative agent of invasive Amoebiasis, invade the host tissue through an effective secretory pathway. There are several lines of evidence suggesting that amoebic trophozoite pore-forming complex amoebapore and a large class of proteases enzymes including rhomboid proteases, cysteine proteases, and metalloproteases are implicated in host tissue invasion. For successful delivery of these molecules/cargos, trophozoites heavily rely on sorting machinery from the endoplasmic reticulum, Golgi to plasma membrane. Although, sole secretion machinery in E. histolytica is not characterized yet. Therefore, here our aim is to understand the properties of key molecules N-ethylmaleimide-sensitive fusion protein attached to protein receptors (SNAREs) in E. histolytica. SNAREs proteins are an important component of the membrane-trafficking machinery and have been associated in a range of processes including vesicle tethering, fusion as well as specificity of vesicular transport in all eukaryotic cells. SNARE proteins are architecturally simple, categorized by the presence of one copy of a homologous coiled-coil forming motif. However, the structural information and protein-protein interaction study of Eh-associated syntaxin proteins are still not known. Here, we characterize the syntaxin 1 like molecule and VAMP from Eh through physiochemical profiling, modeling, atomistic simulation, protein-protein interaction, and docking approaches on the proteins containing SNARE and synaptobrevin domain. The modeled structures and the critical residues recognized through protein interaction and docking study may provide better structural and functional insights into these proteins and may aid in the development of newer diagnostic assays.

From epigenetic landscape to phenotypic fitness landscape: Evolutionary effect of pathogens on host traits.

The epigenetic landscape illustrates how cells differentiate through the control of gene regulatory networks. Numerous studies have investigated epigenetic gene regulation but there are limited studies on how the epigenetic landscape and the presence of pathogens influence the evolution of host traits. Here, we formulate a multistable decision-switch model involving several phenotypes with the antagonistic influence of parasitism. As expected, pathogens can drive dominant (common) phenotypes to become inferior through negative frequency-dependent selection. Furthermore, novel predictions of our model show that parasitism can steer the dynamics of phenotype specification from multistable equilibrium convergence to oscillations. This oscillatory behavior could explain pathogen-mediated epimutations and excessive phenotypic plasticity. The Red Queen dynamics also occur in certain parameter space of the model, which demonstrates winnerless cyclic phenotype-switching in hosts and in pathogens. The results of our simulations elucidate the association between the epigenetic and phenotypic fitness landscapes and how parasitism facilitates non-genetic phenotypic diversity.

Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major.

Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance.

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process.

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.

[Update on host cell invasion by Toxoplasma gondii].

This paper reviewed the importance of micronemes, dense granules, rhoptry and major surface proteins of Toxoplasma gondii, and of calcium during the host cell invasion as well as the role of T. gondii proteases in the folding and processing of these proteins.

Metabolic implications for the mechanism of mitochondrial endosymbiosis and human hereditary disorders.

The endosymbiosis of proto-mitochondrial prokaryotes (PMP) into proto-eukaryotic host-cells was a major advance in eukaryotic evolution. The nature of the initial relationship remains the subject of controversy. Various conceptual models have been proposed, but none has definitive support. We construct a model of inter-species interactions based upon well-established respiratory pathways, describing the respective energy gain of host-cell and PMP resulting from varying levels of cooperation. The model demonstrates conflicting evolutionary strategies ("Prisoner's Dilemmas") in the interspecies molecular transfers. Nevertheless, we show that coercion and iterated, multilevel selection on both species encourage endosymbiosis. Mutualism is favored if host-cells are significantly more effective than PMPs at gathering food. Otherwise, an unambiguous asymmetry between host-cell and PMP benefits implies that the initial relationship consisted of the host-cell deriving a reproductive advantage at the PMPs' expense-a cellular version of farming. Other initial relationships such as oxygen-detoxification mutualism and parasitism are not strongly supported by the model. We compare the model behavior with experiments on mutant human mitochondria and find the model predicts proliferation rates consistent with that data. We derive from the evolutionary dynamics counter-intuitive therapeutic targets for two human hereditary mitochondrial disorders that reflect the ongoing effect of short-term selection at the mitochondrial level.

Signaling during pathogen infection.

Over the millennia, pathogens have coevolved with their hosts and acquired the ability to intercept, disrupt, mimic, and usurp numerous signaling pathways of those hosts. The study of host/pathogen interactions thus not only teaches us about the intricate biology of these parasitic invaders but also provides interesting insights into basic cellular processes both at the level of the individual cell and more globally throughout the organism. Host/pathogen relationships also provide insights into the evolutionary forces that shape biological diversity. Here we review a few recent examples of how viruses, bacteria, and parasites manipulate tyrosine kinase-mediated and Rho guanosine triphosphatase-mediated signaling pathways of their hosts to achieve efficient entry, replication, and exit during their infectious cycles.

Macrophage nutriprive antimicrobial mechanisms.

In addition to oxidative and antibiotic mechanisms of antimicrobial activity, macrophages are able to deprive intracellular pathogens of required nutrients. Thus, microbial killing may not rely only in the toxic environment the microbe reaches but also may result from the scarcity of nutrients in the cellular compartment it occupies. Here, we analyze evidence for such nutriprive (from the latin privare, to deprive of nutrients), antimicrobial mechanisms. Although the direct analysis of nutrient availability is most often not feasible, indirect evidence of lack of nutrients in the microbial organelles has been inferred from the study of mutants, the analysis of gene expression, and the consequences of changing the intracellular location of the pathogen. We propose that according to the microbe and its survival strategy, different mechanisms to impede access to nutrients may be constitutively present or may be induced by cytokines and other pathways. Thus, membrane transporters may remove nutrients from vacuolar compartments, and enzymes may degrade some growth factors. A series of diverse compounds may sequester other molecules required for microbial growth, as exemplified by the action of iron chelators. Modulation of vesicular trafficking may prevent the fusion of certain vesicles containing nutrients with those containing the pathogen, counteracting the evasion strategies of the pathogen. The understanding of these mechanisms will certainly help in designing new therapeutic and prophylactic approaches to preventing infectious diseases.

[Highest level of division in classification of organisms. 2. Archaebacteria, eubacteria and eukaryotes]. / Vysshii uroven' deleniia v klassifikatsii organizmov. 2. Arkhebakterii, éubakterii i éukarioty.

In three-domain system of organic world archaebacteria are considered as the third form of life alongside with eubacteria and eukaryotes. The author gives brief characteristics of all three groups with special focus on such diagnostic attributes as: plasmatic membrane and cellular wall, flagella, protein transcription, replication, topoisomerases, transcription, translation, glycosylation, chaperons and chaperonins, proteasomes and exosomes, histones, ATP-ases. The three-domain system has been proposed by several scientists but principal ideas were put by C. Woese. The systematics according Woese should reflect contemporary level of our knowledge of organisms. In the historical plan it once had to refuse dividing the organic world into plants and animals but accept the division into prokaryotes and eukaryotes. The science however goes further and turns now to the new level of generalizations based on the molecular aspects of cellular structures and processes. From this point of view, both plants and animals are uniform. As to prokaryotes they appeared to be non-monolithic group because of essentially different transcriptional and translational mechanisms. Therefore the detachment of archaebacteria as an independent group was the important step in the development of systematics. At the same time the three-domain system of organisms is typological and requires correction according to data on phylogenetic relatedness of these groups.

Cell biology of the intracellular infection by Legionella pneumophila.

Legionella pneumophila has become a paradigm for facultative intracellular pathogens that modulate biogenesis of their phagosomes into replicative niches. The ability to alter host cell biology and tailor it into a hospitable host for intracellular proliferation is at the crux of the mechanism of pathogenesis of Legionnaires' disease.

[Formation and diversity of parasitophorous vacuoles in parasitic protozoa. The Coccidia (Sporozoa, Apicomplexa)]. / Puti formirovaniia parazitofornykh vakuolei i ikh raznoobrazie u paraziticheskikh prosteishikh. Koktsidii (Sporozoa, Apicomplexa).

Data on parasitophorous vacuole (PV) formation in host cells (HC) harbouring different intracellular protozoan parasites have been reviewed and critically analysed, with special reference to the main representatives of the Coccidia. The vacuole membrane (PVM) is the interface between host and parasite, playing a role in nutrient acquisition by the parasite from the HC. The PV phenomenon is regarded as a generalized HC response to the introduction of alien bodies (microorganisms), which eventually reflects the evolutionary established host-parasite relationships at cellular, subcellular and molecular levels. Special attention has been paid to the existing morpho-functional diversity of the PVs within the same genera and species of parasites, and even at different stages of the parasite life cycle. The PVM is generally considered to derive from the HC plasmalemma, whose biochemical composition undergoes significant changes as the intravacuolar parasite grows. The original HC proteins are selectively excluded from the PVM, while those of the parasite are incorporated. As the result, the changed PVM becomes not fusigenic for HC lysosomes. For Toxoplasma gondii and other cyst-forming coccidia (Isospora, Sarcocystis), a definite correlation has been noticed between the extent of rhoptry and dense granule secrets released by a zoite during HC internalization, on the one hand, and the pattern of the PV that forms, on the other one. In T. gondii, tachyzoites, known to discharge abundant secrets, commonly force the development of PVs limited with a single unit membrane and equipped with a tubulovesicular network in the lumen. Unlike, bradyzoites known to be deficient in secretory materials trigger the formation of PVs with a three-membrane lining composed of the changed invaginated plasmalemma in addition to two membranes of endoplasmic reticulum. The two different types of PV harbour, respectively, exoenteric and enteric stages of T. gondii, the latter being confined to the cat intestine only. Unlike, all endogenous stages of the classic intestinal coccidia (Eimeria spp.) develop within PVs limited with a single membrane, with some invaginations extending into the PV lumen. Unusual PV patterns are characteristic of the extracytoplasmic eimerian coccidia (Cryptosporidium, Epieimeria) and adeleid haemogreagarines (Karyolysus). In cyst-forming coccidia, the PVM is actively involved in tissue cyst wall formation, thus protecting the encysted parasites from recognition by the host immune system. All this strongly suggests that the PV is far from being an indifferent membraneous vesicle containing a parasite, but represents a metabolically active compartment in infected cells. Since all the coccidia are obligate intracellular parasites, the mode of their intimate interaction with the HC, largely accomplished via the PV and its membrane, is vital for their survival as biological species.

Lysosomes and the plasma membrane: trypanosomes reveal a secret relationship.

Studies of the cell invasion mechanism of the parasite Trypanosoma cruzi led to a series of novel findings, which revealed a previously unsuspected ability of conventional lysosomes to fuse with the plasma membrane. This regulated exocytic process, previously regarded mostly as a specialization of certain cell types, was recently shown to play an important role in the mechanism by which cells reseal their plasma membrane after injury.

Fosforilación en células eucarióticas: papel de fosfatasas y quinasas en la biología, patogenia y control de protozoosis tisulares y sanguíneas / Phosphorylation in eukaryotic cells: role of phosphatases and kinases in biology, pathogenesis and control of intracellular and bloodstream protozoa

Cells respond to environmental or cellular changes, rapidly switching protein activities from one state to another. In eukaryotes, a way to achieve these changes is through protein phosphorylation cycles, involving independent protein kinase and protein phosphatase activities. Current evidences show that phosphatases and kinases are also involved in the molecular basis of immune response and in diseases such as diabetes obesity and Alzheimer. In protozoan parasites like Trypanosoma and Leishmania, several kinases and phosphatases have been identified, many of them have been cloned but in several cases their biological role remains undetermined. In this review, the state-of-the art is summarized and the role of phosphatases and kinases in biological phenomena such as remodeling, invasion and pathogenic capacity of protozoan parasites is described. The real chance to use these components of signal transduction pathways as target for chemotherapeutic intervention is also discussed

The flagellated parasite Trichomonas vaginalis: new insights into cytopathogenicity mechanisms.

Our knowledge concerning cytopathogenicity of Trichomonas vaginalis has been enriched in the past by numerous findings. In this paper, we review the latest advances in the field and discuss the different mechanisms and molecules responsible for the parasite's virulence.

Asociación de patógenos bacterianos a células eucariotas: el caso de parasitismo intracelular de Brucella abortus / Asociation of bacterian pathogens in eucariotas cells

Brucella abortus es un parásito intracelular capaz de infectar una gran variedad de mamíferos incluyendo al hombre. Esta bacteria provoca su internalización en células epiteliales induciendo rearreglos locales del esqueleto celular. Una vez en el interior de la célula hospedero, Brucella reside inicialmente en un comportamiento temprano de la cascada de endocitosis/fagocitosis; sin embargo, rápidamente el patógeno se desliga desde el mecanismo de transporte intracelular y se asocia a la cascada de autofagocitosis. En los estadíos tardíos de la infección Brucella prolifera en el retículo endoplasmático de las células infectadas. Las brucelas poseen un sistema regulador de la transcripción de genes de virulencia formado por una proteína sensora de membrana y una proteína reguladora citoplasmática: este sistema de dos componentes permite a las bacterias adaptarse a los diferentes microambientes por los cuales transita durante el proceso de infección intracelular. Estas propiedades biológicas podrían favorecer el uso de Brucella abortus como modelo útil para el diseño de vacunas recombinantes. (Rev Cost Cienc Méd 1999; 20(1-2): 85-102) PALABRAS CLAVE: Brucella abortus, Parásito, Tráfico intracelular, Fagosoma, Autofagocitosis, Retículo endoplasmático, Sistema regulador, Lipopolisacárido, Péptidos catiónicos, Vacuna recombinante

[New views on the pathogenesis and diagnosis of toxoplasmosis]. / Neue Aspekte zur Pathogenese und Diagnostik der Toxoplasmose.

T. gondii is one of the most occurring human pathogenic parasites in Europe. While the majority of immunocompetent individuals with T. gondii infection do not present clinical symptoms, congenital toxoplasmosis and reactivation of a latent infection in immunocompromised patients (i. e. patients with AIDS) are of high clinical relevance. A classification of T. gondii isolates is not available so far, although it was possible to demonstrate strain differences by the use of several methods, i. e. by using monoclonal antibodies. T. gondii seems to be able to infect any mammalian cell. Host cell-derived as well as parasite-derived factors seem to be important for the contact between host cell and parasite and the subsequent internalization. Following invasion, T. gondii is located within a parasitophorous vacuole that does not fuse with lysosomes. The multiplication rate of these obligately intracellular growing parasites decreases during conversion from the tachyzoite stage to the bradyzoite stage. Finally, the bradyzoites-harbouring cysts persist for the lifetime of the host. Reconversion from bradyzoites to tachyzoites may occur in immunocompromised patients. Probably, IFN-gamma is involved in this process. In addition to serological methods, direct detection of T. gondii using PCR or demonstration of circulating antigens might be routinely used as diagnostical tools in the future. Determination of specific IgA antibodies, which can be evaluated using the immunoblot technique, seem to be important for early serological diagnosis. The use of recombinant antigens might be helpful in future diagnosis to circumvent discrepancies between serological test results which could have resulted from strain-specific differences.