Machaerium acutifolium lectin alters membrane structure and induces ROS production in Candida parapsilosis.

Lectins are a group of widely distributed and structurally heterogeneous proteins of nonimmune origin. These proteins have the ability to interact with glycans present on cell surfaces and elicit diverse biological activities. Machaerium acutifolium lectin (MaL) is an N-acetyl-D-glucosamine-binding lectin that exhibits antinociceptive activity via transient receptor potential cation channel subfamily V member 1 (TRPV1). Lectins that have the ability to recognize and interact with N-acetyl-D-glucosamine residues are potential candidates for studies of fungicidal activity. In this work, we show that MaL has antifungal activity against Candida species, and we describe its mode of action towards Candida parapsilosis. MaL inhibited the growth of C. albicans and C. parapsilosis. However, MaL was more potent against C. parapsilosis. The candidacidal mode of action of MaL on C. parapsilosis involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+-ATPase), induction of oxidative stress, and DNA damage. MaL also exhibited antibiofilm activity and noncytotoxicity to Vero cells. These results indicate that MaL is a promising candidate for the future development of a new, natural, and safe drug for the treatment of infections caused by C. parapsilosis.

Impact of manganese on biofilm formation and cell morphology of Candida parapsilosis clinical isolates with different biofilm forming abilities.

The commensal species Candida parapsilosis is an emerging human pathogen that has the ability to form biofilms. In this study, we explored the impact of the divalent cations cobalt (Co2+), copper (Cu2+), iron (Fe3+), manganese (Mn2+), nickel (Ni2+) and zinc (Zn2+) on biofilm formation of clinical isolates of C. parapsilosis with no, low and high biofilm forming abilities at 30 and 37°C. All strains besides one isolate showed a concentration-dependent enhancement of biofilm formation at 30°C in the presence of Mn2+ with a maximum at 2 mM. The biofilm forming ability of no and low biofilm forming isolates was >2-fold enhanced in the presence of 2 mM Mn2+, while the effect in high biofilm forming isolate was significantly less pronounced. Of note, cells in the biofilms of no and low biofilm forming strains differentiated into yeast and pseudohyphal cells similar in morphology to high biofilm formers. The biofilm transcriptional activator BCR1 has a dual developmental role in the absence and presence of 2 mM Mn2+ as it promoted biofilm formation of no biofilm forming strains, and, surprisingly, suppressed cells of no biofilm forming strains to develop into pseudohyphae and/or hyphae. Thus, environmental conditions can significantly affect the amount of biofilm formation and cell morphology of C. parapsilosis with Mn2+ to overcome developmental blocks to trigger biofilm formation and to partially relieve BCR1 suppressed cell differentiation.

The serine peptidase inhibitor TPCK induces several morphophysiological changes in the opportunistic fungal pathogen Candida parapsilosis sensu stricto.

Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.

Nitrogen source and pH interact and modulate lipase secretion in a non-clinical strain of Candida parapsilosis

Lipases (E.C. 3.1.1.3) are serine-hydrolases, and act on long chain fatty acid ester bonds. They exhibit specific and enantioselective activities, which are desirable for many industrial applications. This study aimed at screening and optimizing the production of lipases by wild yeast strains from a variety of substrates, as well as characterizing the enzyme. An initial selection was made in oxygenated oil-supplemented minimum medium, and the enzymatic activity of the supernatant was tested over p-nitrophenyl palmitate. One-hundred and twenty-four yeast strains from different substrates were tested, and twenty-three showed significantly higher lipolytic activity (p<0.01). One yeast in particular, QU110, showed best lipase production and therefore was selected for the optimization and characterization processes. This yeast exhibits enzyme secretion in initial pH 6.0, with olive oil and tryptone as carbon and nitrogen sources, respectively. There was a strong interaction between nitrogen source and initial pH, and pH 9.0seems to inhibit enzyme secretion. The crude enzyme (cell-free supernatant) shows stability in surfactants and n-hexane, but not in ethanol or methanol. A Response Surface Model was created and optimal enzyme activity conditions were observed at 36°C and pH 8.0. The lipase is appropriate for transesterification reactions, as the enzyme is more stable in strong apolar solvents than moderately apolar ones. Also, secretion by pH was not reported elsewhere, which should be further investigated and contribute for other yeast bioprocesses as well.

Direct observation of redox reactions in Candida parapsilosis ATCC 7330 by Confocal microscopic studies.

Confocal microscopic studies with the resting cells of yeast, Candida parapsilosis ATCC 7330, a reportedly versatile biocatalyst for redox enzyme mediated preparation of optically pure secondary alcohols in high optical purities [enantiomeric excess (ee) up to >99%] and yields, revealed that the yeast cells had large vacuoles under the experimental conditions studied where the redox reaction takes place. A novel fluorescence method was developed using 1-(6-methoxynaphthalen-2-yl)ethanol to track the site of biotransformation within the cells. This alcohol, itself non-fluorescent, gets oxidized to produce a fluorescent ketone, 1-(6-methoxynaphthalen-2-yl)ethanone. Kinetic studies showed that the reaction occurs spontaneously and the products get released out of the cells in less time [5 mins]. The biotransformation was validated using HPLC.