Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions.

Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM) of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D) environment mainly composed of Collagen I (COL I). This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited "freeze and run" migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular "home"-macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model.

Effects of platelet-activating factor on the interaction of Trypanosoma cruzi with Rhodnius prolixus.

We investigated the effects of platelet-activating factor (PAF) on the interaction of Trypanosoma cruzi with Rhodnius prolixus. The parasites (epimastigotes) were treated with PAF and/or WEB 2086 (PAF antagonist) for 1 h prior to the interaction experiments. PAF stimulated both in vivo and ex vivo interactions between T. cruzi and R. prolixus while WEB 2086 abrogated these effects. PAF-treated epimastigotes also showed an increase in surface negativity and in the amount of surface sialic acid. Neither of these effects was observed when the epimastigotes were treated with neuraminidase following PAF treatment. In the ex vivo interaction experiments, the number of epimastigotes bound to the midguts of the insects was reduced when the epimastigotes had been treated with neuraminidase. We conclude that PAF modulates the interaction of T. cruzi with R. prolixus by altering the amount of sialyl residues at the surface of the parasite.

Group B Streptococcus induces tyrosine phosphorylation of annexin V and glutathione S-transferase in human umbilical vein endothelial cells.

Group B Streptococcus (GBS), a human pathogen that causes infection and invasive diseases in newborns, pregnant women and immunocompromised adults, has been shown to invade human umbilical vein endothelial cells (HUVECs). The objective of this study was to investigate the molecular mechanisms underlying GBS-HUVEC interaction, focusing specifically on the responsiveness of host protein tyrosine kinase (PTK). We found that GBS serotypes III and V induced actin reorganization and formation of stress fibers into HUVECs. Since rearrangements of the actin cytoskeleton into eukaryotic cells are usually associated with the activation of PTK, we decided to follow the expression of this class of kinases in the course of the interaction. Unexpectedly, treatment of HUVECs with genistein greatly increased both cytoadherence and intracellular viability, for all GBS strains studied. GBS increased tyrosine phosphorylation of two proteins with an apparent molecular mass of 35 and 23 kDa in HUVECs as demonstrated by Western blot analysis with anti-phosphotyrosine antibodies. Mass spectra analysis identified these proteins as annexin V and glutathione S-transferase. Studies are in progress to identify the role of these two proteins on GBS-HUVEC interaction.

Acanthamoeba interaction with extracellular matrix glycoproteins: biological and biochemical characterization and role in cytotoxicity and invasiveness.

Acanthamoeba are free-living amoebae that are dispersed in most environments. Occasionally, Acanthamoeba cause serious human infections, such as keratitis and encephalitis. During the infection process, amoebic adhesion to, and degradation of, host cells and their extracellular matrix (ECM) appear to be important requirements. We examined the interaction of Acanthamoeba with the ECM, and related this event to host cell destruction and tissue invasion. Pathogenic Acanthamoeba culbertsoni differentially attached on the ECM glycoproteins laminin-1, collagen-I, and fibronectin, as compared with non-pathogenic Acanthamoeba astronyxis. Binding to collagen-I and laminin-1 induced A. culbertsoni to become flattened and elongated. Because attachment on laminin-1 was higher in A. culbertsoni, laminin-1 was chosen for further analysis. A 55-kDa laminin-binding protein was identified in pathogenic amoebae, but it was not found in non-pathogenic amoebae. No differential cytotoxicity against distinct cell types was observed between A. culbertsoni incubated with or without ECM. On the other hand, binding on collagen-I or matrigel scaffolds induced a differential effect where A. culbertsoni invaded collagen-I matrices more rapidly. These results indicate that ECM recognition, as an antecedent to tissue invasion, may be a trait characteristic of pathogenic Acanthamoeba.

Intra-strain clonal phenotypic variation of Tritrichomonas foetus is related to the cytotoxicity exerted by the parasite to cultured cells.

As observed in most of the investigated trichomonads, a strain of Tritrichomonas foetus includes different parasite subpopulations. Such population diversity might account for important properties such as the ability of the parasite to destroy host cells. The aim of this study was to characterize the cytotoxicity exerted by subpopulations (named as K1, K2, K3, K4 and K5) of an isolate of T. foetus on epithelial cultured cells. The five populations studied here destroyed epithelial monolayers at different rates (from 25% to 55%), even though the cytoadhesion level and whole-cell protease activity were closely related among them. We were also able to detect differences in contact-dependent and contact-independent cytotoxicity mechanisms among the five populations. An extracellular parasite protease had varying activity among the parasite populations. The intensity of contact-independent cytotoxicity was strictly related to the degree of enzyme activation, suggesting that such a protease might be involved in the cytotoxicity mediated by T. foetus.

Modulation of intercellular communication in macrophages: possible interactions between GAP junctions and P2 receptors.

Gap junctions are connexin-formed channels that play an important role in intercellular communication in most cell types. In the immune system, specifically in macrophages, the expression of connexins and the establishment of functional gap junctions are still controversial issues. Macrophages express P2X(7) receptors that, once activated by the binding of extracellular ATP, lead to the opening of transmembrane pores permeable to molecules of up to 900 Da. There is evidence suggesting an interplay between gap junctions and P2 receptors in different cell systems. Thus, we used ATP-sensitive and -insensitive J774.G8 macrophage cell lines to investigate this interplay. To study junctional communication in J774-macrophage-like cells, we assessed cell-to-cell communication by microinjecting Lucifer Yellow. Confluent cultures of ATP-sensitive J774 cells (ATP-s cells) are coupled, whereas ATP-insensitive J774 cells (ATP-i cells), derived by overexposing J774 cells to extracellular ATP until they do not display the phenomenon of ATP-induced permeabilization, are essentially uncoupled. Western-blot and reverse-transcription polymerase chain reaction assays revealed that ATP-s and ATP-i cells express connexin43 (Cx43), whereas only ATP-s cells express the P2X(7) receptor. Accordingly, ATP-i cells did not display any detectable ATP-induced current under whole-cell patch-clamp recordings. Using immunofluorescence microscopy, Cx43 reactivity was found at the cell surface and in regions of cell-cell contact of ATP-s cells, whereas, in ATP-i cells, Cx43 immunoreactivity was only present in cytosolic compartments. Using confocal microscopy, it is shown here that, in ATP-s cells as well as in peritoneal macrophages, Cx43 and P2X(7) receptors are co-localized to the membrane of ATP-s cells and peritoneal macrophages.

Sialic acid residues on astrocytes regulate neuritogenesis by controlling the assembly of laminin matrices.

In the developing nervous system migrating neurons and growing axons are guided by diffusible and/or substrate-bound cues, such as extracellular matrix-associated laminin. In a previous work we demonstrated that laminin molecules could self-assemble in two different manners, giving rise to matrices that could favor either neuritogenesis or proliferation of cortical precursor cells. We investigated whether the ability of astrocytes to promote neuritogenesis of co-cultivated neurons was modulated by the assembling mode of the laminin matrix secreted by them. We compared the morphologies and neuritogenic potentials of laminin deposited by in vitro-differentiated astrocytes obtained from embryonic or neonatal rat brain cortices. We showed that, while permissive astrocytes derived from embryonic brain produced a flat laminin matrix that remained associated to the cell surface, astrocytes derived from newborn brain secreted a laminin matrix resembling a fibrillar web that protruded from the cell plane. The average neurite lengths obtained for E16 neurons cultured on each astrocyte layer were 198+/-22 and 123+/-13 microm, respectively. Analyses of surface-associated electrostatic potentials revealed that embryonic astrocytes presented a pI of -2.8, while in newborn cells this value was -3.8. Removal of the sialic acid groups on the embryonic monolayer by neuraminidase treatment led to the immediate release of matrix-associated laminin. Interestingly, laminin reassembled 1 hour after neuraminidase removal converted to the features of the newborn matrix. Alternatively, treatment of astrocytes with the cholesterol-solubilizing detergent methyl-beta-cyclodextrin also resulted in release of the extracellular laminin. To test the hypothesis that sialic-acid-containing lipids localized at cholesterol-rich membrane domains could affect the process of laminin assembly, we devised a cell-free assay where laminin polymerization was carried out over artificial lipid films. Films of either a mixture of gangliosides or pure ganglioside GT1b induced formation of matrices of morpho-functional features similar to the matrices deposited by embryonic astrocytes. Conversely, films of phosphatidylcholine or ganglioside GM1 led to the formation of bulky laminin aggregates that lacked a defined structure. We propose that the expression of negative lipids on astrocytes can control the extracellular polymerization of laminin and, consequently, the permissivity to neuritogenesis of astrocytes during development.

Tritrichomonas foetus: the role played by iron during parasite interaction with epithelial cells.

The aim of this work was to investigate the role played by iron during interaction of Tritrichomonas foetus with cultured epithelial cells. We have observed that the growth rate of T. foetus is influenced by the amount of iron available into culture medium. When organisms maintained for 24h in iron-depleted medium were transferred to an iron-rich one, many protozoan cells exhibited a cytokinesis blockage. Parasites maintained in iron-depleted medium exhibited a significant increase in cytoadhesion when compared with both controls and parasites that had been cultured in medium in which iron was replaced. T. foetus collected from iron-depleted medium also exhibited a reduction in its ability to destroy epithelial cell monolayers and a reduction in the activity of several cysteine proteases. Taken together, the results presented here demonstrate that iron may be an extracellular signal, which seems to modulate the ability of T. foetus to interact with host epithelial cells.