Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells

Pseudallescheria boydii is an emerging fungal pathogen that has a worldwide distribution. Virulence mechanisms of P. boydii are largely unknown. We studied the interaction between P. boydii and HEp2 cells and demonstrated that conidia of P. boydii attached to, and were ingested by, HEp2 cells in a time-dependent process. After 2 h of interaction, the conidia produced a germ-tube like projection, which was able to penetrate the epithelial cell membrane. Recently, our group characterized a peptidorhamnomannan (PRM) antigen on the cell surface of P. boydii. In order to better understand the role played by this surface glycoconjugate during cell adhesion and endocytosis, inhibition assays were performed using intact PRM and anti-PRM polyclonal antibody. When HEp2 cells were pre-treated with whole PRM molecule, the adhesion and endocytic indices were, respectively, 50% and 60% lower than in non-treated epithelial cells. Moreover, when the conidial cells were pre-incubated with anti-PRM antibodies, the adherence and endocytosis processes were inhibited in a dose-dependent manner. As PRM influenced the conidia P. boydii-HEp2 cell interaction, we also performed inhibition assays in order to observe which PRM moieties could be involved in this process. Treatment of PRM with proteinase K promoted a slight inhibition of adhesion. However, the de-O-glycosylated PRM molecule as well as the monosaccharide mannose was able to efficiently inhibit the adhesion and endocytic processes. In addition, our results indicate for the first time that P. boydii PRM binds to a polypeptide of 25 kDa on the HEp2 cell surface.     

Hypoxic rise in cytosolic calcium and renal proximal tubule injury mediated by a nickel-sensitive pathway

In the kidney, cell injury resulting from ischemia and hypoxia is thought to be due, in part, to increased cytosolic Ca(2+) levels, [Ca(2+)]i, leading to activation of lytic enzymes, cell dysfunction, and necrosis. We report evidence of a progressive and exponential increase in [Ca(2+)]i (from 245 +/- 10 to 975 +/- 100 nM at 45 mins), cell permeabilization and propidium iodide (PI) staining of the nucleus, and partial loss of cell transport functions such as Na(+)-gradient-dependent uptakes of (14)C-alpha-methylglucopyranoside and inorganic phosphate ((32)Pi) in proximal convoluted tubules of adult rabbits subjected to hypoxia. The rise in [Ca(2+)]i depended on the presence of extracellular [Ca(2+)] and could be blocked by 50 microM Ni(2+)but not by verapamil (100 microM). Presence of 50 microM Ni(2+) also reduced the hypoxia-induced morphological and functional injuries. We also used HEK 293 cells, a kidney cell line, incubated in media without glucose and exposed for 3.5 hrs to 1% O(2)-5% CO(2) and then returned to glucose-containing media for another 3.5 hrs in an air-5% CO(2) atmosphere and finally exposed for 1 min to media containing 1 microM PI. NiCl(2) (50 microM) or pentobarbital (300 microM) more than phenobarbital (1.5 mM), when present in the incubation medium during both the hypoxic and the reoxygenation periods, induced significant (P < 0.001) reductions in the number of cell nuclei stained with PI, similar to their relative potency as inhibitors of T channels. Our findings indicate that hypoxia-induced alterations in calcium level and subsequent cell injury in the proximal convoluted tubule and in HEK cells involve a nickel-sensitive and dihydropyridine insensitive pathway or channel.     

Synthetic arylquinuclidine derivatives exhibit antifungal activity against Candida albicans, Candida tropicalis and Candida parapsilopsis

Background

Severely burned patients may develop life-threatening nosocomial infections due to Pseudomonas aeruginosa, which can exhibit a high-level of resistance to antimicrobial drugs and has a propensity to cause nosocomial outbreaks. Antiseptic and topical antimicrobial compounds constitute major resources for burns care but in vitro testing of their activity is not performed in practice.

Results

In our burn unit, a P. aeruginosa clone multiresistant to antibiotics colonized or infected 26 patients over a 2-year period. This resident clone was characterized by PCR based on ERIC sequences. We investigated the susceptibility of the resident clone to silver sulphadiazine and to the main topical antimicrobial agents currently used in the burn unit. We proposed an optimized diffusion assay used for comparative analysis of P. aeruginosa strains. The resident clone displayed lower susceptibility to silver sulphadiazine and cerium silver sulphadiazine than strains unrelated to the resident clone in the unit or unrelated to the burn unit.

Conclusions

The diffusion assay developed herein detects differences in behaviour against antimicrobials between tested strains and a reference population. The method could be proposed for use in semi-routine practice of medical microbiology.     

Cellular characterisation of Candida tropicalis presenting fluconazole-related trailing growth

We assessed fluconazole susceptibility in 52 Candida tropicalis clinical strains using seven antifungal susceptibility methods, including broth microdilution (BMD) [standard M27 A3 (with neutral and acid pH), ATB Fungus 3, Vitek 2 system and flow cytometric analysis] and agar-based methods (disk diffusion and E-test). Trailing growth, detection of cell-associated secreted aspartic proteases (Saps) and morphological and ultrastructural traits of these clinical strains were also examined. The ranges of fluconazole 24 h-minimum inhibitory concentration (MIC) values were similar among all methods. The essential agreement among the methods used for MIC determinations was excellent and all methods categorised all strains as susceptible, except for one strain that showed a minor error. The presence of the trailing effect was assessed by six methods. Trailing positivity was observed for 86.5-100% of the strains. The exception was the BMD-Ac method where trailing growth was not observed. Morphological and ultrastructural alterations were detected in C. tropicalis trailing cells, including mitochondrial swelling and cell walls with irregular shapes. We tested the production of Saps in 13 C. tropicalis strains expressing trailing growth through flow cytometry. Our results showed that all of the C. tropicalis strains up-regulated surface Sap expression after 24 h or 48 h of exposure to fluconazole, which was not observed in untreated yeast strains. We concluded that C. tropicalis strains expressing trailing growth presented some particular features on both biological and ultrastructural levels.     

Identification, antifungal susceptibility and scanning electron microscopy of a keratinolytic strain of Rhodotorula mucilaginosa: a primary causative agent of onychomycosis

Onychomycosis is a dermatological problem of high prevalence that mainly affects the hallux toenail. Onychomycosis caused by the yeast Rhodotorula mucilaginosa was identified using colony morphology, light microscopy, urease and carbohydrate metabolism in a 57-year-old immunocompetent patient from Rio de Janeiro, Brazil. High-resolution scanning electron microscopy of nail fragments, processed by a noncoating method, led to the observation with fine detail of the structures of both nail and fungus involved in the infection. Yeasts were mainly found inside grooves in the nail. Budding yeasts presented a spiral pattern of growth and blastoconidia were found in the nail groove region. Keratinase assays and keratin enzymography revealed that this isolate was highly capable of degrading keratin. Antifungal susceptibility tests showed that the fungus was susceptible to low concentrations of amphotericin B and 5-flucytosine and resistant to high concentrations of fluconazole, itraconazole, voriconazole and terbinafine. These findings showed data for the first time concerning the interaction of R. mucilaginosa in toenail infection and suggest that this emerging yeast should also be considered an opportunistic primary causative agent of onychomycosis.     

Morphometric and densitometric study of the biogenesis of electron-dense granules in Fonsecaea pedrosoi

Fonsecaea pedrosoi is a polymorphic pathogenic fungus, etiological agent of chromoblastomycosis, that synthesizes a melanin-like pigment. Although this pigment has been described as a component of the outer layers of the cell wall, electron-dense cytoplasmic bodies have also been visualized. In this work, we have correlated the appearance of intracellular electron-dense granules with the melanization process in F. pedrosoi. For this, conidial forms were grown under conditions where melanin was not synthesized. Afterwards, cells were incubated in Hank's medium supplemented with bovine fetal serum, at 37 degrees C, to stimulate the pigment production. The genesis of cytoplasmic bodies, with different stages of electron density, was demonstrated by transmission electron microscopy. The appearance of fungal acidic compartments, visualized by confocal laser scanning microscopy in cells stained with acridine orange, was time coincident with the formation of electron-dense granules observed by transmission electron microscopy. The quantification of granule numbers as well as morphometric and densitometric studies were performed.     

Sodium Butyrate Induces Apoptosis in MSN Neuroblastoma Cells in a Calcium Independent Pathway

Sodium butyrate (NaBt), a histone deacetylase inhibitor, can cause apoptosis in a number of cancer cells. However, the mechanism of this action is poorly understood. Increased intracellular [Ca²⁺] level has been suggested as a likely mechanism, but there is little corroborating data. In this report we provide evidence that NaBt-treated MSN neuroblastoma cells undergo massive apoptosis in the presence of serum and regardless of external or internal [Ca²⁺] levels. Presented data suggest that apoptotic effect of NaBt is both time- and dose-dependent (LD₅₀ 1 mM); and that, presence of serum or cAMP, a second messenger molecule that modulates the apoptotic program in a wide variety of cells could not circumvent the apoptotic effect of NaBt. Our findings suggest that NaBt-induced apoptosis in MSN neuroblastoma cells occurs via a pathway that is independent of Ca²⁺flux, intracellular [Ca²⁺] or cAMP levels. Further, we also present data that exclude a role for PKC or histones acetylation.Special issue dedicated to Lawrence F. Eng     

Chromoblastomycosis: a retrospective study of 325 cases on Amazonic Region (Brazil)

A retrospective study of 325 cases of chromoblastomycosis diagnosed in the last 55 years in the Amazon region was carried out by the main Mycology services of the state of Pará, Brazil (Department of Tropical Pathology – UFPA and Mycology Department of the Evandro Chagas Institute/FNS). The data obtained showed that: (a) the main age group affected by the diseases range from 41 to 70 years-old, (b) 86.1% of the patients were agricultural-workers, (c) 93.2% of them were males and (d) 80.7% showed lesions on the lower limbs (feet and legs). The diagnosis of 62% of the cases was confirmed by laboratory studies considering the tissue form in histopathological analysis. In 24% of patients (78 cases), the etiological agent was isolated and identified through culture. Fonsecaea pedrosoi was present in 77 cases and Phialophora verucosa in only one case. This revised version was published online in June 2006 with corrections to the Cover Date.