Arnaldo Feitosa Braga Andrade

Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route

Sialo- and asialoglycoconjugates were isolated from Trypanosoma cruzi epimastigotes and their composition determined. Sialoglycoconjugates bound to wheat germ agglutinin (WGA)-Sepharose and were precipitated by concanavalin A, Wistaria floribunda hemagglutinin and WGA. Asialoglycoconjugate bound to concanavalin A-Sepharose and precipitated with concanavalin-A and W. floribunda hemagglutinin but not with WGA. Cells grown in the presence of fetal calf serum were agglutinated by WGA but not by peanut agglutinin. The reverse was true for cells grown without fetal calf serum. Neuraminidase-treated cells incorporated sialic acid or its 7-carbon analog, 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid (AcNeu7) from sialylated compounds such as fetuin or sialyl-lactose but did not incorporate free sialic acid. Restoration of the WGA sialylreceptors in neuraminidase-treated cells, as determined by cell agglutination with WGA, was also obtained by incubation with fetuin or sialyl-lactose but not with free sialic acid. Moreover, restoration of agglutinability by WGA in neuraminidase-treated cells or cells grown in medium without fetal calf serum occurred equally well in energy-rich or energy-depleted cells. A transglycosilase reaction for sialic acid incorporation in T. cruzi epimastigotes is suggested.

Effects of iron limitation on adherence and cell surface carbohydrates of Corynebacterium diphtheriae strains.

ABSTRACT Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment. The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of Corynebacterium diphtheriae subsp. mitis and evaluated their adherence to human group B erythrocytes and HEp-2 cells. A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl3. The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively. Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells. Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions. Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface. Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in d-mannose and d-galactose residues on the surfaces of both strains. Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of C. diphtheriae surface carbohydrate moieties. The significant changes in the degree of lectin binding specific for d-mannose, d-galactose and sialic acid residues may have an effect on binding of host cells. The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems. For C. diphtheriae, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes.

Cell Surface Hydrophobicity of Sucrose Fermenting and Nonfermenting Corynebacterium diphtheriae Strains Evaluated by Different Methods

Abstract.Corynebacterium diphtheriae strains expressed variation in hydrophobic characteristics dependent on the method used. Results of single assays are not a reliable representation of C. diphtheriae hydrophobicity. All 12 strains adhered to polystyrene surfaces; three showed spontaneous aggregation (SA) in Trypticase Soy Broth (TSB) medium, and eight exhibited autoagglutination in phosphate-buffered saline (PBS; AA-positive). The salt aggregation test (SAT) values ≤0.002 or ≥1.6 represented breakpoints for groups of strains with differing hydrophobicity. C. diphtheriae strains showed affinity towards n-hexadecane. Percentages of adhesion varied from 31% to 63% and were not directly related to morphological n-hexadecane adhesion patterns. Diffuse and localized adhesion patterns were noted predominantly among sucrose-positive and sucrose-negative strains, respectively. Strains of the sucrose-negative biotype expressed a higher degree of hydrophobicity. The choice of the growth medium influenced the hydrophobicity, not the hemagglutinating activity (HA) of C. diphtheriae. Heating bacterial suspensions at 121°C decreased both HA and hydrophobicity of three strains. However, hydrophobins and hemagglutinins were trypsin and detergent resistant. The treatment of microorganisms with Clostridium perfringens neuraminidase increased the hydrophobicity but not the HA titers of strains tested. Hemagglutinins were partially responsible for hydrophobicity. Hydrophilic AA-negative strains adhered strongly to glass but expressed weak HA. Sialylglycoconjugates functioned as hydrophilins on C. diphtheriae surfaces.

Peptostreptococcus micros in Primary Endodontic Infections as Detected by 16S rDNA-based Polymerase Chain Reaction

A 16S rDNA-based polymerase chain reaction (PCR) method was used to detect Peptostreptococcus micros in primary root canal infections. Samples were collected from 50 teeth having carious lesions, necrotic pulps, and different forms of periradicular diseases. DNA extracted from the samples was amplified using the PCR assay, which yielded a specific fragment of P. micros 16S rDNA. P. micros was detected in 6 of 22 root canals associated with asymptomatic chronic periradicular lesions (27.3%), 2 of 8 teeth with acute apical periodontitis (25%), and 6 of 20 cases of acute periradicular abscess (30%). In general, P. micros was found in 14 of 50 cases (28%). There was no correlation between the presence of P. micros and the occurrence of symptoms. Findings suggested that P. micros can be involved in the pathogenesis of different forms of periradicular lesions.

Pyogenic Liver Abscess Due to Rhodococcus equi in an Immunocompetent Host

ABSTRACT A case of pyogenic liver abscess (PLA) due to Rhodococcus equi in an immunocompetent individual was successfully treated by combining surgery and antibiotics. The R. equi-targeted antimicrobial agents erythromycin and rifampin were used only after surgical resection of the lesion and identification of the infective organism.

Lectin-binding properties of different Leishmania species.

Abstract Carbohydrate cell-surface residues on stationary promastigotes of 19 isolates of Leishmania were studied with a panel of 27 highly purified lectins, which were specific for N-acetyl-D-glucosamine, D-mannose, L-fucose, D-galactose, N-acetyl-D-galactosamine, and sialic acid. The specificity of the cell-surface carbohydrates was analyzed by agglutination and radioiodinated lectin-binding assays. L. (L.) amazonensis and L. (L.) donovani were agglutinated by 12 and 10 of the 27 lectins used, respectively. Artocarpus integrifolia lectin (Jacalin) was incapable of agglutinating the tested species of the donovani complex, and this result was confirmed by radioiodinated Jacalin-binding assays. Jacalin had an average of 3.8 × 106 receptors/L. (L) amazonensis promastigote and bound with an association constant of 5 × 106 M−1.

Cell surface carbohydrates and in vivo infectivity of glucantime-sensitive and resistant Leishmania (Viannia) guyanensis cell lines

Abstract. The cell surface plays an important role in the interaction of parasites with their hosts. Drug resistance in the protozoan Leishmania may involve changes in cell-surface composition, although it is not known whether infectivity is also affected. One sensitive and two glucantime-resistant lines of Leishmania (Viannia) guyanensis previously isolated were inoculated into hamsters. The sensitive line caused the disease to manifest earlier than the resistant lines. Imprinting analyses of infected macrophages showed that the sensitive line was more infective than the resistant cell lines. In vitro drug resistance was evaluated and the comparative analyses of dose–response curves showed that the susceptibility pattern of the sensitive line did not change after passage in animals, but a decrease in drug resistance was observed in resistant cell lines recovered from the mammalian host. Cell surface carbohydrates of sensitive and resistant cell lines were analysed before and after passage in animals by agglutination tests with several plant lectins. Passage in animals changed the agglutination pattern for many lectins from all three cell lines. Loss of reactivity to lectins seemed to be correlated with a decrease in infectivity of the parasite-resistant cell lines. This study opens possibilities for exploring the relationship between drug susceptibility, infectivity and surface carbohydrate composition of protozoan parasites.

Involvement of the macrophage mannose-6-phosphate receptor in the recognition of Leishmania mexicana amazonensis.

Significant differences were found in the ability of resident mouse peritoneal macrophages to ingest amastigote and promastigote forms of Leishmania mexicana amazonensis. Differences in the association index of the parasites to the macrophages were also found between infective and non-infective promastigotes. Evidence was obtained suggesting that the macrophage receptor, which recognizes mannose-6-phosphate-containing units found in lysosomal enzymes, is involved in the association with the macrophage of promastigotes, but not of amastigotes. Addition of mannose-6-phosphate, its structural analogue fructose-1-phosphate, Hansenula holstii phosphomannan or the mannose-6-phosphate-containing lysosomal enzyme α-D-mannosidase to the interaction medium, markedly inhibits the association of the parasites with macrophages.

Sialic acid as receptor of Bacteroides fragilis lectin-like adhesin.

It was observed that sialic acid and macromolecules rich in this sugar were able to inhibit the hemagglutination activity (HA) of Bacteroides fragilis strains in low concentrations. Reversion of the HA and also of the adsorption to beads of Sepharose coupled to bovine submaxillary mucin, by this sugar residue corroborated the recognition capacity of the bacterial lectin-like adhesin. However, when erythrocytes were treated with clostridial neuraminidase, an increase in the HA of some strains was observed. Protease treatment of erythrocytes abolished the HA, indicating that cell receptors of B. fragilis are probably a glycoprotein moiety.

Occurrence ofN-acetyl-andN-O-diacetyl-neuraminic acid derivatives in wild and mutantCrithidia fasciculata

The cell-surface expression of sialic acids in wild-typeCrithidia fasciculata and three drug-resistant mutants (FUR11, TR3, and TFRR1) was analyzed using fluorescein-labeledLimulus polyphemus agglutinin (LPA) binding, glycosidase of known sugar specificity, and thin-layer chromatography (TLC). Gas-liquid chromatography-mass spectrometry (GC-MS) analysis using both electron-impact (EI-MS) and chemical ionization (CI-MS) by isobutane with selected ion monitoring (SIM) was also used. The surface location of sialic acid was inferred from LPA binding to whole cells abrogated by previous treatment with neuraminidase. An exception occurred with the TFRR1 strain, which after incubation with neuraminidase showed increased reactivity with the fluorescent lectin. BothN-acetyl- andN-O-diacetyl-neuraminic acids were identified in the flagellates by TLC, with a clear predominance being noted for the former derivative. However, the content ofN-O-diacetyl-neuraminic acid was preferentially found in the TFRR1 strain. The GC-MS analysis of the acidic component of the TERR1 mutant strain confirmed the occurrence ofN-acetyl-neuraminic acid (Neu5Ac) by the presence of the diagnostic ions (m/z values: 684 and 594 for CI-MS and 478, 298, and 317 for EI-MS) and also by comparison with the standard Neu5Ac retention time. GC-MS analysis also showed fragments (m/z values: 654 and 564 for CI-MS and 594, 478, 298 and 317 for EI-MS) expected for the 7-O- and 9-O-acetyl-N-acetyl-neuraminic acids (Neu5,7Ac2 and Neu 5,9Ac2, respectively).