The interaction between the amoeba Balamuthia mandrillaris and extracellular matrix glycoproteins in vitro.

Balamuthia mandrillaris, a soil amoeba, is the causative agent of Balamuthia granulomatous amoebic encephalitis, a life-threatening brain infection. This amoeba is acquired from contaminated soil and may enter the host through cutaneous lesions or through nasal passages, migrating to the lungs or brain. During invasion, B. mandrillaris has access to components of the extracellular matrix (ECM) of the host. Therefore, we investigated the interaction of B. mandrillaris with 3 ECM glycoproteins (collagen-I, fibronectin and laminin-1) that are encountered in host connective tissues and at the basal lamina. Using optical microscopy, amoeba association on ECM-coated surfaces was examined. Binding of amoebae on laminin was greater than that on collagen or fibronectin. Laminin-adhered B. mandrillaris exhibited elongated and spread forms, distinctive from those observed for amoebae on a plastic surface. Collagen and fibronectin-adhered B. mandrillaris presented elongated shapes with cellular expansions. Binding to collagen, fibronectin, or laminin was inhibited when amoebae were pre-treated with sialic acid. Treatment with galactose resulted in diminished binding of amoebae on laminin, while mannose increased binding in all coating conditions tested. Dependence of divalent cations on amoeba binding was demonstrated for laminin-amoeba interaction. Collectively, the results indicate that B. mandrillaris recognizes specific glycoproteins of the mammalian extracellular matrix.

Trichomonas vaginalis virulence against epithelial cells and morphological variability: the comparison between a well-established strain and a fresh isolate.

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.

How laminin-1 can be recognized by the protozoan parasite Tritrichomonas foetus: possible role played by the extracellular matrix glycoprotein in both cytoadhesion and cytotoxicity exerted by the parasite.

The isoform 1 of the extracellular matrix glycoprotein Laminin is known to be an important ligand for some parasitic protozoa including Trichomonas vaginalis. The bovine parasite Tritrichomonas foetus seems to display a similar recognition process to laminin-1, as some amino acid sequences found in the LNS module of laminin-1 can also be recognized by this parasite. Which of the laminin-1 residing adhesion sequences are recognized by T. foetus, and the role played by such a protein-cell recognition process in both cytoadhesion and cytotoxicity exerted by the parasite are the subjects briefly reviewed and discussed here.

A preliminary investigation on the chemical composition of the cell surface of five enteropathogenic Escherichia coli serotypes.

The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100 degree C for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction.

Inhibition of polyamine synthesis arrests trichomonad growth and induces destruction of hydrogenosomes.

Trichomonad parasites such as Tritrichomonas foetus produce large amounts of putrescine (1,4-diaminobutane), which is transported out of the cell via an antiport mechanism which results in the uptake of a molecule of spermine. The importance of putrescine to the survival of the parasite and its role in the biology of T. foetus was investigated by use of the putrescine analogue 1, 4-diamino-2-butanone (DAB). Growth of T. foetus in vitro was significantly inhibited by 20 mM DAB, which was reversed by the addition of exogenous 40 mM putrescine. High-performance liquid chromatography analysis of 20 mM DAB-treated T. foetus revealed that putrescine, spermidine, and spermine levels were reduced by 89, 52, and 43%, respectively, compared to those in control cells. The DAB treatment induced several ultrastructural alterations, which were primarily observed in the redox organelles termed hydrogenosomes. These organelles were progressively degraded, giving rise to large vesicles that displayed material immunoreactive with an antibody to beta-succinyl-coenzyme A synthetase, a hydrogenosomal enzyme. A protective role for polyamines as stabilizing agents in the trichomonad hydrogenosomal membrane is proposed.

Basic surface properties of mononuclear cells from Didelphis marsupialis.

The electrostatic surface charge and surface tension of mononuclear cells/monocytes obtained from young and adult marsupials (Didelphis marsupialis) were investigated by using cationized ferritin and colloidal iron hydroxyde, whole cell electrophoresis, and measurements of contact angles. Anionic sites were found distributed throughout the entire investigated cell surfaces. The results revealed that the anionic character of the cells is given by electrostatic charges corresponding to -18.8 mV (cells from young animals) and -29.3 mV (cells from adult animals). The surface electrostatic charge decreased from 10 to 65.2% after treatment of the cells with each one of trypsin, neuraminidase and phospholipase C. The hydrophobic nature of the mononuclear cell surfaces studied by using the contact angle method revealed that both young and adult cells possess cell surfaces of high hidrofilicity since the angles formed with drops of saline water were 42.5 degrees and 40.8 degrees, respectively. Treatment of the cells with trypsin or neuraminidase rendered their surfaces more hydrophobic, suggesting that sialic acid-containing glycoproteins are responsible for most of the hydrophilicity observed in the mononuclear cell surfaces from D. marsupialis.

Cell surface alterations induced by methylene blue and direct electric current in Escherichia coli.

Cell surface properties, including hydrophobicity, zeta potential, carbohydrate and fatty acid components, were altered on treatment of E. coli K12 with methylene blue (MB) and direct electric current (DC). The treatment of fimbriated E. coli cells with MB greatly increased the agglutination of yeast cells when compared to untreated bacteria. However, this increased agglutination was markedly reduced when the bacteria were treated with MB plus DC. These results suggest that MB modifies cell surface components in the absence of light and these alterations are more pronounced when cells are treated simultaneously with MB and DC.

Surface charge and surface carbohydrates of Cyptobia salmositica virulent and avirulent forms and of C. bullocki (Kinetoplastida: Cryptobiidae).

The surface charge and surface carbohydrate residues of the virulent (freshly isolated from the fish blood) and avirulent forms (from culture) of Cryptobia salmositica and one strain of C. bullocki were studied. Measurements of the zeta potential of parasites showed that C. bullocki and the virulent form of C. salmositica had a net negative surface charge of about -15 mV, whereas the attenuated form of C. salmositica showed a surface charge of -7.9 mV. Enzymatic treatments of parasites with neuraminidase, trypsin, or phospholipase C indicated the presence of sialic acid residues, phosphate groups, and protein glycoconjugates as components of the Cryptobia surface that accounted for their surface charge. Residues of alpha-D-man, alpha- and beta-D-gal, alpha-D-galNAc, alpha-L-fuc, and D-glcNAc could be detected on the surface of all parasites by specific fluorescein isothiocyanate (FITC)- and colloidal gold-labeled lectins. The cell surface of the avirulent form of C. salmositica showed the strongest reactivity to almost all lectins tested. A remarkable binding pattern of lectins in the anterior region of parasites was observed.

Changes in the surface carbohydrate composition and exposure of anionic groups caused by beta-lactam antibiotics in streptococci.

Upon the addition of beta-lactam antibiotics at concentrations that caused a 50% reduction in the dry weight, beta-haemolytic streptococci produced increased amount of rhamnose, though the hexosamine content remained unchanged. These sugars are components of C-carbohydrate. Sialic acid content also increased in group B streptococcal surfaces and penicillin treatment generated new accessible surface sialic acid residues.

Ultrastructural aspects of the interaction between Escherichia coli and cultured epithelial cells.

Microorganisms from two Escherichia coli serotypes were allowed to interact with cultured epithelial cells and the interaction process was followed from 30 to 360 min. Destruction of the microvilli on the epithelial monolayers as well as of the cells themselves was observed only with 0111:H2 serotype.

The nature of anionic sites and the endocytic pathway in heart muscle cells.

The distribution and internalization of anionic sites in heart muscle cells (HMC) were studied by direct measurements of their zeta potential (ZP) and by ultrastructural cytochemistry. Our data showed that HMC are negatively charged and that their anionic sites are distributed over the entire sarcolemma. Treatments with neuraminidase and trypsin altered the ZP value and also reduced binding of cationized ferritin (CF) to the sarcolemma. Sialic acid was shown to be an important component on the surface of HMC, since its removal reduced the cell surface negative charge by 25%. Phospholipase C did not significantly change the surface charge, nor did it alter HMC reactivity to CF particles when compared with control cells. Endocytosis of anionic sites was investigated using two different protocols that allow follow-up of this dynamic process. Incubation of HMC with cationized ferritin particles at 37 degrees C induced a redistribution of ligand-bound anionic sites, followed by their internalization or detachment. The clustering of anionic sites on the surface of HMC indicates that these cells are characterized by a high level of membrane fluidity. CF particles were localized inside early and late endosomes, lysosomes, and also in ferritin-enriched vesicles near the sarcolemma. An endocytic pathway for anionic sites in HMC is discussed.

Sialic acid content and surface hydrophobicity of group B streptococci.

The sialic acid content and the cell-surface hydrophobicity index of 40 group B streptococci (GBS) strains were assessed. GBS isolated from invasive infections (virulent strains) presented an increased level of sialic acid content (1.4%) when compared with GBS isolated from asymptomatic patients (0.53%). Treatment of GBS strain 85634 with neuraminidase resulted in a decrease (about 25%) in the net negative surface charge as assessed by cell electrophoresis. This finding suggests that sialic acid residues are important anionogenic groups exposed on GBS cell surface. N-acetylneuraminic acid was the only sialic acid derivative characterized in the strain 85634 as evaluated by gas-liquid chromatography. GBS from different serotypes presented a hydrophobic index mean value of 0.9. Even though the sialic acid contributed effectively to the negative charge on GBS cell surface, no difference was observed in the hydrophobic index when virulent and avirulent strains were compared.

Effect of a phorbol ester on basic surface properties of trichomonads.

The effect of nanomolar concentrations of 12-O-tetradecanoilphorbol-13-acetate (TPA) on the cell surface of the urogenital parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus was evaluated by means of measurements of the parasites' surface tension, electrokinesis, lectin agglutination tests, and adhesion to inert substrates. TPA-treated parasites had their adhesion increased to both plastic and glass substrates. This was accompanied by increases in the parasites' net negative surface charge and also by changes in their surface tension. The lectin agglutination assays suggest that the increase in surface negativeness may be related in some extent to alterations in the oligosaccharide composition. Successive treatment of the microorganisms with TPA and sphingosine, a well-known competitive inhibitor of the phorbol ester active site, depressed the tendency of trichomonads to exhibit a phenotype of activated cells.

Surface charge and hydrophobicity of wild and mutant Crithidia fasciculata.

Surface charge of wild-type Crithidia fasciculata and three drug-resistant mutants (TR3, TFRR1, and FUR11) was studied by direct zeta-potential determination and ultrastructural cytochemistry. Surface tension was also investigated by measurements of the advancing contact angle formed by the protozoa monolayers with drops of liquids of different polarities. The individual zeta potential varies markedly among the C. fasciculata cells. The wild and FUR11 mutant strains displayed lower negative surface charge (-12.5 and -9.5 mV, respectively) as compared with the TR3 (-14.8 mV) and TFRR1 (-14.7 mV) mutant strains. Binding of cationized ferritin (CF) was observed at the cell surface of wild and mutant strains of C. fasciculata. Neuraminidase treatment reduced the negative surface charge in the TFRR1 and TR3 mutants in about 37 and 29%, respectively, whereas no significant change was observed with the wild and FUR11 mutant strains. These findings suggest that sialic acid residues are the major anionogenic groups on the surface of C. fasciculata. The density of sialic acid residues per cell in wild and mutant strains of C. fasciculata falls in a range of 1.4 x 10(4) to 3.6 x 10(4). Marked differences of hydrophobicity were also observed. For example, the TFRR1, FUR11, and TR3 drug-resistant mutant strains showed higher contact angle values (55.4, 54.2, and 49.3, respectively) than the wild-type (35.6), as assessed by alpha-bromonaphtalene.

Effect of estrogens on the adhesion of Trichomonas vaginalis to epithelial cells in vitro.

1. To further characterize the basic aspects of parasite-cell interaction, untreated and hormone-treated Madin-Darby canine kidney (MDCK) cells were allowed to interact with T. vaginalis. 2. Among the six hormones assayed, alpha-estradiol caused MDCK cells to become highly adhesive to T. vaginalis. Increases in parasite adhesion of 83% and 68% were obtained by previous incubation of the epithelial monolayers for 18 h with 1 ng/ml alpha-estradiol and 10 ng/ml 17-beta-estradiol, respectively. 3. The estrogenic response of MDCK cells was inhibited by tamoxifen, cycloheximide, puromycin, or 2-deoxy-D-glucose. 4. We conclude that MDCK cells possess receptors for estrogens and that T. vaginalis cytoadherence can be influenced by the hormonal cycle of its host.

Effect of estrogens on the adhesion of trichomonas vaginalis to epithelial cells in vitro

The feeding behavior of six triatomid species toward latex condoms filled with blood at 26 ñ 1 and 36 ñ 1 grade C observed for 4 h. The species studied were Triatoma infestans, Triatoma brasiliensis, Tritoma vitiiticeps, Triatoma pseudomaculata, Rhodnius and Panstrongylus, and 34 to 348 insects were studied in each group (average, 190). All the life stages of each species were used. Although most insects (80%, average for 6 species) preferred the warmer blood, 54% of the individuals of all species fed on blood kept at 26 grade C. For Triatoma pseudomaculata, a higher proportion of insects fed on blood at 26 grade C (92%) than on blood at 36 grade C (65%). These results suggest that bait containing insecticides which are effective through the digest tract may be useful in the control of these vectors even at room temperature

The interaction of Tritrichomonas foetus and Trichomonas vaginalis with macrophages.

The process of interaction between macrophages and Tritrichomonas foetus and Trichomonas vaginalis was analysed using light microscopy, scanning and transmission electron microscopy. The parasites attach to the macrophage surface and are ingested through a phagocytic process. Parasite-macrophage association index was higher for activated than for resident macrophages. Previous incubation of the parasites in the presence of Concanavalin A rendered their surface less negative and more hydrophobic, as evaluated by measurement of the zeta potential and contact angle, respectively. This treatment significantly increased parasite ingestion by resident, but not activated macrophages.

The surface free energy of Leishmania mexicana amazonensis.

Surface charge of Leishmania mexicana amazonensis was investigated by direct zeta-potential determination and ultrastructural cytochemistry, and its surface tension was studied by measurements of the advancing contact angle formed by the parasite monolayers with drops of liquids of different polarities. Both virulent and avirulent promastigotes exhibited negatively charged surfaces with a zeta-potential of about -15 mV. Treatment of these cells with trypsin, alkaline phosphatase, or phospholipase C rendered their surfaces less negatively charged, whereas neuraminidase did not alter the parasite negativeness. Cytochemically, we could observe a reduction in the cationized ferritin binding after the parasite treatment with each of the former enzymes, but not with neuraminidase. The surface free energy of parasites was calculated by taken to account the London dispersion, the Keeson dipole-dipole, and the Debye dipole-induced forces, as well as the surface polarity of the parasites and their zeta-potentials, by considering their adhesion to polystyrene surfaces. The delta G values of -6.4 and -18.1 mJ.m-2 were obtained for avirulent and virulent promstigotes, respectively.