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.

Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases.

The enzymes dolichol phosphate glucose synthase and dolichol phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans. Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to dolichol phosphate glucose and minor amounts of glucoproteins in the absence of exogenous dolichol phosphate. Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme. Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin. On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40. This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound. DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P. Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases.

Processing of the Man(10)GlcNAc (M(10)) oligosaccharide by alpha-mannosidases from Candida albicans.

The hydrolysis of Man(10)GlcNAc (M(10)) by purified alpha-mannosidases and its further processing by a mixed membrane preparation from Candida albicans were studied. Incubation of the oligosaccharide with purified alpha-mannosidases I (E-I) or II (E-II) from C. albicans released 1 and 2 mol of mannose per mol of M(10), respectively. This treatment converted M(10) into an acceptor substrate of further mannose residues from GDP-Man as catalyzed by membrane-bound mannosyltransferases. Elongation of E-I- or E-II-trimmed M(10) yielded a low molecular mass product (14-17 mannose residues added), and in the case of E-II, a minor amount of an additional product of a higher molecular mass. Our results indicate that purified alpha-mannosidases participate in N-glycan processing in C. albicans.

Partial purification and characterization of dolichol phosphate mannose synthase from Entamoeba histolytica.

Dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis, was partially purified from E.histolytica by hydrophobic interaction and affinity chromatography with octyl Sepharose CL-4B and Affi-Gel 501, respectively. Reducing agents, particularly dithiothreitol, positively influenced enzyme activity and stability, indicating a role of sulfhydryl groups on the transferase function. Activity did not depend on phospholipids; however, it was significantly stimulated by phosphatidylethanolamine and to a lower extent by other common phospholipids. Mixtures consisting of activating phospholipids did not exert an additive effect. In vitro phosphorylation with a cAMP-dependent protein kinase resulted in enzyme activation. This alteration was not associated with a change in the K(m) for the substrate but rather with a 2.6-fold increase in V(max). Phosphorylation in the presence of [gamma-(32)P]ATP resulted in strong labeling of two polypeptides, one of which exhibited the molecular mass reported for the enzyme from other organisms. Whether phosphorylation functions in vivo as a mechanism of regulation of dolichol phosphate mannose synthesis in E.histolytica remains to be determined.

Purification and biochemical characterization of two soluble alpha-mannosidases from Candida albicans.

Two soluble alpha-mannosidases, E-I and E-II, were purified from C. albicans yeast cells by a three-step procedure consisting of size exclusion and ion exchange chromatographies in Sepharose CL6B and Mono Q columns, respectively, and preparative nondenaturing electrophoresis. E-I and E-II migrated as monomeric polypeptides of 54.3 and 93.3 kDa in SDS-PAGE, respectively. Some biochemical properties of purified enzymes were investigated by using 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside as substrates. Hydrolysis of both substrates by either enzyme was optimum at pH 6.0 with 50 mM Mes-Tris buffer and at 42 degrees C. Apparent Kmvalues for hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside by E-I were 0.83 microM and 2. 4 mM, respectively. Corresponding values for E-II were 0.25 microM and 1.86 mM. Swansonine and deoxymannojirimicin strongly inhibited the hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside by both enzymes. On the contrary, hydrolysis of p-nitrophenyl-alpha-D-mannopyranoside by E-I and E-II was slightly stimulated or not affected, respectively, by both inhibitors. E-I and E-II did not depend on metal ions although activity of the latter was slightly stimulated by Mn2+and Ca2+in the range of 0.5-2 mM. At the same concentrations, Mg2+was slightly inhibitory of both enzymes. Substrate specificity experiments revealed that both E-I and E-II preferentially cleaved alpha-1,6 and alpha-1,3 linkages, respectively.

Biosynthesis of glycoproteins in Candida albicans: solubilization and partial characterization of dolichol phosphate mannose synthase and protein mannosyl transferases.

Incubation of a mixed membrane fraction isolated from C. albicans yeast cells with Nonidet P-40 at a detergent/protein ratio as low of 0.025 (0.016-0.019%, w/v) yielded a soluble fraction that catalyzed the transfer of mannose from GDP-[14C] Man into dolichol phosphate mannose and from this intermediate into mannoproteins. Over 95% of the sugar in mannoproteins was O-linked as judged from its release after beta-elimination. Mannose was identified as the sole product after this treatment. Transfer activity did not depend on exogenous lipid acceptor indicating that the latter was solubilized along with the mannosyl transferases. Synthesis of mannolipid and mannoproteins occurred at optima temperatures of 20 degrees C, and 37 degrees C, respectively, and at a pH in the range of 7.5-9.5. Mannosyl transfer into the mannolipid was stimulated by Mg2+ and inhibited by Ca2+ and Mn2+ whereas mannoprotein labeling was stimulated by Mn2+ and to a lower extent by Mg2+. When measured as a function of substrate concentration, the synthesis of the mannolipid was a nearly linear function of GDP-Man concentration in the range of 5 to 32 microM whereas protein mannosylation exhibited hyperbolic kinetics with saturation reached at about 10 microM. The solubilized preparation was able to utilize an exogenous source of mannolipid as sugar donor for protein mannosylation. Dinucleotides and, to a higher extent trinucleotides, inhibited mannosyl transfer into the mannolipid and hence into mannoproteins.

Biosynthesis of glycoproteins in Candida albicans: biochemical characterization of dolichol phosphate glucose synthase.

A mixed membrane fraction isolated from C. albicans yeast cells catalyzed the transfer of glucose from UDP-Glc into three classes of endogenous acceptors: glucolipid, glycoprotein and lipid-linked oligosaccharides. About 80% of the total radioactivity transferred into these products corresponded to the glucolipid which was identified as dolichol phosphate glucose by several criteria. The remainder was detected in about equal proportions in the other two fractions. Conditions that stimulated or inhibited glucolipid synthesis did not affect the extent of glycoprotein labeling. The synthesis of dolichol phosphate glucose exhibited a K(m) of 104 microM UDP-Glc and was stimulated by Mg2+ but not by Mn2+ or Ca2+. The latter cations were, however, better stimulators of glycoprotein labeling than Mg2+. Most nucleotides strongly inhibited the synthesis of dolichol phosphate glucose, UMP being a competitive inhibitor with a Ki of 100 microM. The dolichol phosphate glucose synthase reaction was reversed about 57% by 0.62 mM UDP but not by UMP.

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.

Entamoeba histolytica: solubilization and biochemical characterization of dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis.

Sequential treatment of trophozoite membranes with the nonionic detergents Brij 35 and Igepal CA-630 released a soluble fraction that efficiently catalyzed the transfer of mannose from GDP-Man into a mannolipid that was identified as dolichol phosphate mannose (Dol-P-Man) by several criteria. The transfer reaction occurred only in the presence of exogenously added dolichol monophosphate (Dol-P). Plots of enzyme velocity versus Dol-P and GDP-Man concentrations revealed sigmoidal and hyperbolic kinetics, respectively. Values of S0.5 for Dol-P and K(m) for GDP-Man were 15 micrograms/ml and 4.1 microM, respectively. The solubilized fraction failed to transfer the label into other products such as lipid-linked oligosaccharides and glycoproteins. The optimum pH was 7.5-8.0 in potassium phosphate or Tris/HCl buffers and the enzyme required either Mg2+ or Mn2+. The latter was more effective but in a narrower range of concentrations. The transferase was inhibited by a number of nucleotides the strongest being GMP, GDP, and GTP. When assayed in the reverse direction, however, the enzyme catalyzed the transfer of mannose from Dol-P-Man back into GDP-Man as a function of increasing concentrations of GDP. Mg2+ was a better activator of the reverse reaction than Mn2+, which reached up to 60% at 2 mM GDP. These results suggest that some of the enzyme catalytic properties may change depending on the direction of the transfer reaction.

Identification and partial characterization of three chitinase forms in Entamoeba invadens with emphasis on their inhibition by allosamidin.

Three chitinase forms were identified in Entamoeba invadens cysts following fractionation of a soluble fraction by anionic exchange, size exclusion and hydroxyapatite adsorption chromatographies. The enzymes, named here as A, B and B', showed molecular weights of 64, 33.4 and 33.4 kDa, respectively, as measured by gel filtration. Comparison of their levels of specific activity in partially purified samples revealed chitinase A as the major species. Chitinase B' was a minor component of the chitinolytic complex. Whereas some properties were common to the three forms, analysis of other parameters revealed significant catalytic site-related differences. Accordingly, the three chitinases hydrolyzed the fluorogenic substrate 4-methylumbelliferyl chitotriose with typical Michaelian kinetics and Km values of 4.5, 11.8 and 3.8 microM for A, B and B', respectively. Allosamidin strongly inhibited the three enzyme forms with different kinetics. Dixon plots revealed competitive-type inhibition and Ki values of 10.0, 2.3 and 10.8 nM for A, B and B', respectively. Km/Ki ratios indicated 450-, 350- and 5130-fold higher affinity for the inhibitor over the substrate for the A, B and B' forms, respectively. Results are discussed in terms of the possibility that the three chitinase species correspond to different enzyme proteins.

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.

Biosynthesis of glycoproteins in Candida albicans: activity of dolichol phosphate mannose synthase and protein mannosylation in a mixed membrane fraction.

A mixed membrane fraction (MMF) was isolated from yeast cells of Candida albicans with the ability to synthesize dolichol phosphate mannose (Dol-P-Man) from GDP-Man and dolichol phosphate (Dol-P) and transfer the sugar to proteins. Temperature of incubation (20-37 degrees C) did not affect the synthesis of Dol-P-Man but protein mannosylation occurred better at physiological temperatures (28 degrees C and 37 degrees C). Most of the sugar (87-93%) in the mannoproteins was O-linked as judged by its release by beta-elimination. Mannose was identified as the sole product after this treatment. Following incubation of MMF with the sugar donor, parallel levels of Dol-P-Man and mannosylated proteins were detected up to 30 min. Thereafter, Dol-P-Man levels reached a steady value whereas mannoproteins rapidly accumulated. Lipid-linked oligosaccharides were also detected in incubation mixtures, though in much lower amounts than those of Dol-P-Man or mannoproteins. Dol-P-Man synthase activity increased proportionally in response to increasing concentrations of either of the two enzyme substrates. A Km value of 0.36 microM for GDP-Man was calculated. MMF failed to use exogenous Dol-P-Man for protein glycosylation. Specific inhibition of Dol-P-Man synthesis with amphomycin was concomitant with a parallel decrease in protein mannosylation, indicating that most of the sugar is transferred to protein via the carrier lipid. Results are discussed in terms of the role of Dol-P-Man in protein glycosylation in C. albicans.

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.

Encystation in Entamoeba invadens.

Knowledge of the biology and pathogenesis of the human parasite Entamoeba histolytica has been limited by the lack of efficient procedures to induce axenic encystation in the laboratory. However, such methods have been developed for E. invadens, a reptilian parasite, for which encystation can be induced by mechanisms that are as yet poorly defined. This has allowed the analysis of some morphological, physiological and biochemical events that accompany differentiation into cysts. Elucidation of these changes will lead to a better understanding of the process and therefore to the possibility of controlling it. Here, Everardo López-Romero and Julio Cézar Villagómez-Castro emphasize the metabolism of cyst-wall polymers as a potential target to inhibit cyst formation with specific drugs that would be in principle, harmless to the host.

Nikkomycin-resistant mutants of Mucor rouxii: physiological and biochemical properties.

We isolated three nikkomycin-resistant mutants of the dimorphic fungus M. rouxii which were physiologically characterized regarding their response to yeast-phase inducing conditions and their sensitivity to bacilysin. Mutant strains G21 and G23, showed a qualitatively normal, though delayed, dimorphic transition and partial cross-resistance to bacilysin. Mutant strain G27 showed an altered dimorphism, producing a high proportion (50%) of hyphal cells, and a wild-type sensitivity to bacilysin. Cell-free extracts from this mutant exhibited an activity of both basal and protease-activated chitin synthetase which was overexpressed as compared with the parental strain and mutants G21 and G23. Results are discussed in terms of the different genetic background of the mutants.

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.

Chitinase activity in germinating cells of Mucor rouxii.

Chitinase activity in germinating cells (4 h cultures) of Mucor rouxii was studied. The enzyme activity was recovered in a high speed supernatant of cell homogenates. No activity was detected in the mixed membrane fraction or in the cell walls. Maximum activity was observed at pH 7.6 and at 30-35 degrees C using the chromogenic assay with chitin azure. The latter was digested by GS-chitinase in a manner dependent on substrate concentration and time of incubation. As with other chitinases, GS-chitinase was much more effective against nascent than against preformed chitin. The main product of nascent chitin digestion was diacetylchitobiose, although significant amounts of the trimer were also detected in the hydrolyzates. Allosamidin, an insect and fungal chitinase inhibitor, strongly inhibited hydrolysis of nascent chitin but not of chitin azure by GS-chitinase. The drug failed to inhibit the germination and the ensuing growth of the fungus. Results are discussed in terms of the possible role of GS-chitinase in germination.

Chitin synthetase activity in a developmental mutant of Phycomyces blakesleeanus.

Chitin synthetase activity was analyzed in vitro and in vivo in two morphogenetic stages, namely, dormant spore cells and germlings of the wild type strain and the developmental mutant S356 of Phycomyces blakesleeanus. In vitro experiments showed a much higher specific activity in dormant spores of the mutant strain than in those of the wild-type. This difference was restricted to the dormant spore phase since germlings exhibited comparable levels of activity to those detected in the wild-type strain. Although no correlation was observed between chitin synthesis in vitro and in vivo in mutant spores, germination of these cells was accompanied by an earlier expression of chitin synthetase in vivo. Germination of mutant spores in liquid medium produced morphologically aberrant germlings. Contrary to the extended mycelial growth of the wild-type strain in solid medium, the mutant grew with a typical colonial morphology. Results are discussed in relation to the possible basis of the mutant phenotype.

[Antigens specific to pre-cysts and in vivo chitin synthetase activity in Entamoeba invadens]. / Anígenos específicos de prequistes y activadad in vivo de quitina sintetasa en Entamoeba invadens.

In this paper we studied the transformation of a trophozoite into a cyst in Entamoeba, using E. invadens as a model. We had the following objectives: a) identification of precyst-specific proteins (P), by a monoclonal antibody against E. invadens and heterologous polyclonal antibodies against cellular (165) fractions of Mucar rouxii, which are chitin synthetase activity rich; and b) in vivo determination of the time required for the expression of activity of the chitin synthetase during encystment. We found P markers which are not found in either trophozoites nor cysts. Monoclonal F507 antibody recognized a 33 kDa protein in P and the polyclonal anti-16S antibodies reacted with a 90 kDa protein to P. Even though the 33 and 90 kDa proteins have a different molecular weight from the chitin synthetase described in fungi (57 kDa and 65 kDa) and yeasts (63 kDa), we conclude that these proteins are specific to P and that the 90 kDa one shares epitopes with chitosomal fractions of M. rouxii. Also, the mayor accumulation of alkali-resistant material, sensitive to chitinase, occurred during the formation of P, between 40 and 50 hours post incubation, during encystment. One may conclude that chitin polymers are synthesized during the P phase.