Julio C. Villagómez-Castro

Sporothrix schenckii complex and sporotrichosis, an emerging health problem.

Sporothrix schenckii, now named the S. schenckii species complex, has largely been known as the etiological agent of sporotrichosis, which is an acute or chronic subcutaneous mycosis of humans and other mammals. Gene sequencing has revealed the following species in the S. schenckii complex: Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. The increasing number of reports of Sporothrix infection in immunocompromised patients, mainly the HIV-infected population, suggests sporotrichosis as an emerging global health problem concomitant with the AIDS pandemic. Molecular studies have demonstrated a high level of intraspecific variability. Components of the S. schenckii cell wall that act as adhesins and immunogenic inducers, such as a 70-kDa glycoprotein, are apparently specific to this fungus. The main glycan peptidorhamnomannan cell wall component is the only O-linked glycan structure known in S. schenckii. It contains an α-mannobiose core followed by one α-glucuronic acid unit, which may be mono- or di-rhamnosylated. The oligomeric structure of glucosamine-6-P synthase has led to a significant advance in the development of antifungals targeted to the enzymes catalytic domain in S. schenckii.

Protein glycosylation in Candida

Candidiasis is a significant cause of invasive human mycosis with associated mortality rates that are equivalent to, or worse than, those cited for most cases of bacterial septicemia. As a result, considerable efforts are being made to understand how the fungus invades host cells and to identify new targets for fungal chemotherapy. This has led to an increasing interest in Candida glycobiology, with an emphasis on the identification of enzymes essential for glycoprotein and adhesion metabolism, and the role of N- and O-linked glycans in host recognition and virulence. Here, we refer to studies dealing with the identification and characterization of enzymes such as dolichol phosphate mannose synthase, dolichol phosphate glucose synthase and processing glycosidases and synthesis, structure and recognition of mannans and discuss recent findings in the context of Candida albicans pathogenesis.

Candida species: new insights into biofilm formation

Biofilms of Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis are associated with high indices of hospital morbidity and mortality. Major factors involved in the formation and growth of Candida biofilms are the chemical composition of the medical implant and the cell wall adhesins responsible for mediating Candida-Candida, Candida-human host cell and Candida-medical device adhesion. Strategies for elucidating the mechanisms that regulate the formation of Candida biofilms combine tools from biology, chemistry, nanoscience, material science and physics. This review proposes the use of new technologies, such as synchrotron radiation, to study the mechanisms of biofilm formation. In the future, this information is expected to facilitate the design of new materials and antifungal compounds that can eradicate nosocomial Candida infections due to biofilm formation on medical implants. This will reduce dissemination of candidiasis and hopefully improve the quality of life of patients.

The effect of biomaterials and antifungals on biofilm formation by Candida species: a review

Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis are able to form biofilms on virtually any biomaterial implanted in a human host. Biofilms are a primary cause of mortality in immunocompromised and hospitalized patients, as they cause recurrent and invasive candidiasis, which is difficult to eradicate. This is due to the fact that the biofilm cells show high resistance to antifungal treatments and the host defense mechanisms, and exhibit an excellent ability to adhere to biomaterials. Elucidation of the mechanisms of antifungal resistance in Candida biofilms is of unquestionable importance; therefore, this review analyzes both the chemical composition of biomaterials used to fabricate the medical devices, as well as the Candida genes and proteins that confer drug resistance.

Chitinase activity in encysting Entamoeba invadens and its inhibition by allosamidin.

Chitinase activity was measured in extracts of Entamoeba invadens cells as a function of time of encystation in axenic conditions using 4-MU(Ch)3 as substrate. Encystment was paralleled by chitinase activity which showed a peak after about 72 h of cultivation where cysts accounted for 63% of cell population. Thereafter, activity fell off rapidly, whereas encystment continued, reaching 80% at the end of the experiment (96 h). Comparison of activity between cysts and the total cell population in 48- and 72-h-old encysting cultures suggested that chitinase may start to accumulate in the pre-cyst forms. About 70% of the enzyme was recovered in the supernatant following low-speed centrifugation of whole extracts. Most of this activity represented soluble chitinase since it was not sedimented by further centrifugation at 105,000 x g. A minor proportion of enzyme activity remained associated to the buffer-washed, high-speed sediment. In addition to 4-MU(Ch)3, chitinase activity was also measured following the hydrolysis of other substrates such as nascent, preformed or colloidal chitin. Like other chitinases, the cyst enzyme preferred nascent over preformed chitin as substrate. Digestion of the former yielded GlcNAc and minor amounts of (GlcNAc)2 as products. Allosamidin strongly inhibited hydrolysis of the fluorogenic substrate by the amebic chitinase in vitro with a Ki of 0.065 microM. IC50 values were 0.085 microM and 0.16 microM at 5 microM and 10 microM 4-MU(Ch)3, respectively. When added to the axenic medium, the drug markedly retarded encystment though it was partially recovered after longer periods of incubation.

Isolation and some properties of a glycoprotein of 70 kDa (Gp70) from the cell wall of Sporothrix schenckii involved in fungal adherence to dermal extracellular matrix

Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis and an emerging disease in immunocompromised patients. Adherence to target cells is a prerequisite for fungal dissemination and systemic complications. However, information on the cell surface components involved in this interaction is rather scarce. In this investigation, the extraction of isolated cell walls from the yeast phase of S. schenckii with SDS and separation of proteins by SDS-PAGE led to the identification of a periodic acid-Schiff (PAS)-reacting 70 kDa glycoprotein (Gp70) that was purified by elution from electrophoresis gels. The purified glycopeptide exhibited a pI of 4.1 and about 5.7% of its molecular mass was contributed by N-linked glycans with no evidence for O-linked oligosaccharides. Confocal analysis of immunofluorescence assays with polyclonal antibodies directed towards Gp70 revealed a rather uniform distribution of the antigen at the cell surface with no distinguishable differences among three different isolates. Localization of Gp70 at the cell surface was confirmed by immunogold staining. Gp70 seems specific for S. schenckii as no immunoreaction was observed in SDS-extracts from other pathogenic and non-pathogenic fungi. Yeast cells of the fungus abundantly adhered to the dermis of mouse tails and the anti-Gp70 serum reduced this process in a concentration-dependent manner. Results are discussed in terms of the potential role of Gp70 in the host-pathogen interaction.

Identification and characterisation of early reactions of asparagine-linked oligosaccharide assembly in Entamoeba histolytica.

Sequential incubation of a mixed membrane fraction isolated from Entamoeba histolytica trophozoites with the nonionic detergents Brij 35 and Igepal CA-630 rendered a soluble fraction with the ability to transfer N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to dolichol phosphate to form a lipid saccharide that was identified as a mixture of dolichol-P-P-GlcNAc and dolichol-P-P-(GlcNAc)2 as follows. (a) The reaction occurred only in the presence of exogenously added dolichol phosphate and was strongly inhibited by tunicamycin and amphomycin; (b) Over 90% of the aminosugar moiety of the lipid saccharide was released by mild acid hydrolysis and was identified as a mixture of GlcNAc and diacetylchitobiose [(GlcNAc)2]; (c) Time course experiments revealed that dolichol-P-P-(GlcNAc)2 accumulated at the expense of a parallel decrease in dolichol-P-P-GlcNAc revealing the tandem operation of UDPGlcNAc:dolichol-P GlcNAc-1-P transferase and UDPGlcNAc:dolichol-P GlcNAc transferase. Mg2+ and to a lower extent Mn2+ were required for catalytic activity and were optimal at 2.5 mM and 1.25 mM, respectively. Common phospholipids with different head groups failed to increase catalytic activity and phosphatidylglycerol was inhibitory. At low concentration, nucleotides such as ATP, GMP and GTP brought about stimulations of 24-54% but higher concentrations were inhibitory. Others were inhibitory at all concentrations the strongest being those containing a uridine base.

Partial Purification and Characterization of Ornithine Decarboxylase from Entamoeba histolytica

Multiplication of E. histolytica was accompanied by a parallel increase in ornithine decarboxylase (ODC) specific activity up to 72 h of cultivation in TYI-S-33 medium. Thereafter, activity rapidly decayed whereas growth continued for another 24 h before entering into the stationary growth phase. ODC was very unstable. Partial purification (14-fold) of the enzyme was achieved by a three-step procedure involving high-speed centrifugation, gel filtration and adsorption to hydroxylapatite. The partially purified enzyme (Mr 211 kDa) revealed maximum activity at pH 8.5-9.0 and a sigmoidal response to substrate concentration. An S0.5 value of 1.0 mM ornithine was estimated. Although ODC did not exhibit an absolute dependence on pyridoxal phosphate (PLP), addition of PLP increased catalytic activity about 4-fold, with an S0.5 value of 45 microM. Evolution of 14CO2 from ornithine was markedly inhibited by polyamines in the following increasing order of effectiveness: putrescine > spermidine > spermine. The substrate analogs alpha-methylornithine and alpha-difluoromethylornithine had no effect on enzyme activity and cell growth. In contrast, 1,3-diaminopropane and 2,4-diamino-2-butanone, 2 putrescine analogs, severely inhibited both enzyme activity and amoeba multiplication. Results are discussed in terms of the role of ODC in the amoeba proliferation.

Ornithine decarboxylase activity in Entamoeba invadens.

Growth of E. invadens was paralleled by a concomitant increase in ornithine decarboxylase activity which peaked after 5 days of cultivation in TYI-S-33 medium. Over this period, enzyme activity increased about nine-fold with respect to that present at the start of incubation. Thereafter and coinciding with the onset of the stationary growth phase, enzyme activity started to decline reaching trace levels after 8 days of cultivation. Most of the enzyme remained soluble following centrifugation of amoeba homogenates at 105,000 g. alpha-Difluoromethylornithine failed to affect ornithine decarboxylase activity in vitro and amoeba growth. The enzyme was markedly inhibited by polyamines (putrescine, spermidine and spermine) and 1,4-diamino-2-butanone, a putrescine-analog. The latter arrested proliferation of cells, an effect that could not be reversed by polyamines which by themselves also inhibited growth to a low but significant extent. Our results indicate that polyamine biosynthesis from ornithine is required for growth of E. invadens and that this function is rapidly abolished following entry into the stationary growth phase.

Identification of Candida albicans heat shock proteins and Candida glabrata and Candida krusei enolases involved in the response to oxidative stress

In the past two decades, Candida species have become the second leading cause of invasive mycosis in immunocompromised patients. In order to colonize their hosts, these microorganisms express adhesins and cell wall proteins that allow them to adhere and neutralize the reactive oxygen species produced by phagocytic cells during the respiratory burst. However, the precise mechanism by which Candida cell wall proteins change their expression in response to oxidative stress has not been described. In an attempt to understand this change in response to oxidative stress, in this study, three Candida species, namely, C. albicans, C. glabrata and C. krusei, were exposed to increasing concentrations of H2O2 and induced cell wall proteins were identified by two-dimensional gel electrophoresis and peptide mass fingerprinting. Sequence analysis of differential proteins led to the identification of two heat-shock proteins in C. albicans, two enolases in C. glabrata and one enolase in C. krusei. Enolases may be involved in the protection of pathogenic cells against oxidative stress as suggested by the decrease in their expression when they were exposed to high concentrations of H2O2. To our knowledge, this is the first demonstration that expression of these proteins changes in response to oxidative stress in different Candida species. This knowledge can eventually facilitate both an early diagnosis and a more efficient treatment of this mycosis.