Iron is an essential element to support the growth and survival of Trichomonas vaginalis. It plays a critical role in the host-parasite interaction, and modulates the expression of virulence factors in this protozoan. In this work, parasites grown in iron-rich and iron-depleted media were analyzed by (i) light and scanning electron microscopy and (ii) 2-DE and MS. Withdrawal of iron from the culture medium resulted in dramatic changes in both the morphology and in the proteome pattern of T. vaginalis. Trophozoites underwent transformation from ellipsoid or amoeboid forms to rounded cells, whose flagella and axostyle were internalized. Forty-five proteins differentially expressed in parasites cultivated in the absence of iron were identified. In iron-depleted parasites, enzymes involved in energetic metabolism, proteolysis and hydrogenosomal iron-sulfur (Fe-S) proteins were down-regulated or even suppressed. Among up-regulated proteins, six isoforms of actin were detected. In addition, phosphoenolpyruvate carboxykinase, putative lactate dehydrogenase, and putative adenosine triphosphatase were also up-regulated or were exclusively observed in gels related to iron-depleted parasites. Our data demonstrate that iron has a pivotal role in the regulation of the morphological transformation of T. vaginalis and modulates the expression of both Fe-S and non-Fe-S proteins in the parasite.

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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 P2X7 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 P2X7 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 P2X7 receptors are co-localized to the membrane of ATP-s cells and peritoneal macrophages.

The FMVI strain of Trichomonas vaginalis was freshly isolated from an asymptomatic patient, and its morphological properties and virulence in vitro compared with the well-established JT strain. The morphological variability of the parasites was assessed by differential interference microscopy and both scanning and transmission electron microscopy. The FMV1 strain presented nearly 20% amoeboid cells whereas the JT strain presented high percentages of ellipsoid but no amoeboid cells. The FMV1 morphotype population was unaltered after at least 1 year of subculturing. Electron microscopy revealed that this strain produced numerous pseudopod structures which mediated intimate contact and interdigitation among trophozoites. Dead FMV1 parasites were often phagocytosed by conspecific cells. We also compared the cytolytic capacity of these two populations against epithelial MDCK cells and its contact dependence. The FMV1 strain rapidly adhered to plastic or glass surfaces and to MDCK monolayers. This strain destroyed about 93% of the epithelial cells in 90 min whereas the cytolytic activity of the JT parasites was very much lower (about 41%). Parasite supernatants displayed no cytolytic activity, indicating contact-mediated lysis. The protozoan virulence in vitro did not correlate well with the clinical observations. The implications of these results are discussed.

Proteolytic activities of 5 strains of Leishmania (Viannia) braziliensis isolated from Brazilian and Colombian patients, presenting distinct clinical manifestations, were characterized and compared using whole-promastigote extracts and extracellular secretions. Zymographic assays concerning whole-cell extracts and supernatants resulted in the detection of high molecular weight bands, ranging from 50 to 125 kDa. Proteolytic activities from both whole-cell extracts and supernatants were optimal in a pH range 5.5 to 9.0 for all analysed strains. Such protease activities were inhibited when 10 mM 1,10-phenanthroline was assayed, strongly suggesting that the enzymes responsible for hydrolysis of the substrate belong to the metalloproteases class. Distinct profiles of metalloproteases were observed among the studied L. (V.) braziliensis strains. Differences among the microorganisms might be related to the geographical origin of the strains and/or to the clinical presentation.

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.

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In the present study, we identified and characterized the cysteine peptidase (CP) profiles of Trichomonas vaginalis isolates exhibiting high- and low-virulence phenotypes using a combination of two-dimensional SDS-PAGE (2DE), tandem mass spectrometry (MS/MS), and data mining. Seven of the eight CPs identified belong to Clan CA, family C1, cathepsin L-like CP, and one belongs to Clan CD, family C13, asparaginyl endopeptidase-like CP. Quantitative and qualitative differences in CP expression were detected between the isolates. BLAST analysis followed by CLUSTAL alignment of amino acid sequences of differentially expressed CPs showed identity or high homology to previously described CP cDNA clones CP1, CP3, CP4, and to a secreted CP fraction of 30 kDa involved in apoptosis of vaginal epithelial cells. One- and two-dimensional-substrate gel analyses revealed the differential CP profiles between the isolates, indicating that the combination of zymography with 2DE and MS/MS might be a powerful experimental approach to map and identify active peptidases in T. vaginalis. Toxicity exerted upon HeLa cells by high- and low-virulence isolates was 98.3% and 31%, respectively. Pretreatment of parasites with specific Clan CA papain-like CP inhibitor l-3-carboxy-2,3-trans-epoxypropionyl-leucylamido(4-guanidino)butane (E-64) drastically reduced the cytotoxic effect to 21.7% and 0.8%, respectively, suggesting that T. vaginalis papain-like CPs are the main factors involved in the cellular damage.