Amyloid of the Candida albicans Ure2p prion domain is infectious and has an in-register parallel β-sheet structure

Ure2p of Candida albicans (Ure2(albicans) or CaUre2p) can be a prion in Saccharomyces cerevisiae, but Ure2p of Candida glabrata (Ure2(glabrata)) cannot, even though the Ure2(glabrata) N-terminal domain is more similar to that of the S. cerevisiae Ure2p (Ure2(cerevisiae)) than Ure2(albicans) is. We show that the N-terminal N/Q-rich prion domain of Ure2(albicans) forms amyloid that is infectious, transmitting [URE3alb] to S. cerevisiae cells expressing only C. albicans Ure2p. Using solid-state nuclear magnetic resonance of selectively labeled C. albicans Ure2p(1-90), we show that this infectious amyloid has an in-register parallel β-sheet structure, like that of the S. cerevisiae Ure2p prion domain and other S. cerevisiae prion amyloids. In contrast, the N/Q-rich N-terminal domain of Ure2(glabrata) does not readily form amyloid, and that formed upon prolonged incubation is not infectious.     

NMR characterization of the interactions between lyso-GM1 aqueous micelles and amyloid β

Gangliosides are targets for a variety of pathologically relevant proteins, including amyloid β (Aβ), an important component implicated in Alzheimer’s disease (AD). To provide a structural basis for this pathogenic interaction associated with AD, we conducted NMR analyses of the Aβ interactions with gangliosides using lyso-GM1 micelles as a model system. Our NMR data revealed that the sugar-lipid interface is primarily perturbed upon binding of Aβ to the micelles, underscoring the importance of the inner part of the ganglioside cluster for accommodating Aβ in comparison with the outer carbohydrate branches that provide microbial toxin- and virus-binding sites.     

Characterizing the role of cell-wall β-1,3-exoglucanase Xog1p in Candida albicans adhesion by the human antimicrobial peptide LL-37

Candida albicans is the major fungal pathogen of humans. Its adhesion to host-cell surfaces is the first critical step during mucosal infection. Antimicrobial peptides play important roles in the first line of mucosal immunity against C. albicans infection. LL-37 is the only member of the human cathelicidin antimicrobial peptide family and is commonly expressed in various tissues, including epithelium. We previously showed that LL-37 significantly reduced C. albicans adhesion to plastic, oral epidermoid OECM-1 cells, and urinary bladders of female BALB/c mice. The inhibitory effect of LL-37 on cell adhesion occurred via the binding of LL-37 to cell-wall carbohydrates. Here we showed that formation of LL-37-cell-wall protein complexes potentially inhibits C. albicans adhesion to polystyrene. Using phage display and ELISA, we identified 10 peptide sequences that could bind LL-37. A BLAST search revealed that four sequences in the major C. albicans cell-wall β-1,3-exoglucanase, Xog1p, were highly similar to the consensus sequence derived from the 10 biopanned peptides. One Xog1p-derived peptide, Xog1p(90-115), and recombinant Xog1p associated with LL-37, thereby reversing the inhibitory effect of LL-37 on C. albicans adhesion. LL-37 reduced Xog1p activity and thus interrupted cell-wall remodeling. Moreover, deletion of XOG1 or another β-1,3-exoglucanase-encoding gene EXG2 showed that only when XOG1 was deleted did cellular exoglucanase activity, cell adhesion and LL-37 binding decrease. Antibodies against Xog1p also decreased cell adhesion. These data reveal that Xog1p, originally identified from LL-37 binding, has a role in C. albicans adhesion to polystyrene and, by inference, attach to host cells via direct or indirect manners. Compounds that target Xog1p might find use as drugs that prevent C. albicans infection. Additionally, LL-37 could potentially be used to screen for other cell-wall components involved in fungal cell adhesion.     

LL37 and hBD-3 elevate the β-1,3-exoglucanase activity of Candida albicans Xog1p, resulting in reduced fungal adhesion to plastic

The opportunistic fungus Candida albicans causes oral thrush and vaginal candidiasis, as well as candidaemia in immunocompromised patients including those undergoing cancer chemotherapy, organ transplant and those with AIDS. We previously found that the AMPs (antimicrobial peptides) LL37 and hBD-3 (human β-defensin-3) inhibited C. albicans viability and its adhesion to plastic. For the present study, the mechanism by which LL37 and hBD-3 reduced C. albicans adhesion was investigated. After AMP treatment, C. albicans adhesion to plastic was reduced by up to ~60% and was dose-dependent. Our previous study indicated that LL37 might interact with the cell-wall β-1,3-exoglucanase Xog1p, which is involved in cell-wall β-glucan metabolism, and consequently the binding of LL37 or hBD-3 to Xog1p might cause the decrease in adhesion. For the present study, Xog1p(41-438)-6H, an N-terminally truncated, active, recombinant construct of Xog1p and Xog1p fragments were produced and used in pull-down assays and ELISA in vitro, which demonstrated that all constructs interacted with both AMPs. Enzymatic analyses showed that LL37 and hBD-3 enhanced the β-1,3-exoglucanase activity of Xog1p(41-438)-6H approximately 2-fold. Therefore elevated Xog1p activity might compromise cell-wall integrity and decrease C. albicans adhesion. To test this hypothesis, C. albicans was treated with 1.3?μM Xog1p(41-438)-6H and C. albicans adhesion to plastic decreased 47.7%. Taken together, the evidence suggests that Xog1p is one of the LL37/hBD-3 targets, and elevated β-1,3-exoglucanase activity reduces C. albicans adhesion to plastic.     

Allosteric interactions between the oxytocin receptor and the β2-adrenergic receptor in the modulation of ERK1/2 activation are mediated by heterodimerization

The oxytocin receptor (OTR) and the β₂-adrenergic receptor (β₂AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and β₂AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/β₂AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and β₂AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the β₂AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third β₂AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, β₂AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and β₂AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and β₂AR in the context of a new heterodimer pair lie at the heart of the allosteric effects.     

Candida albicans Utilizes a Modified β-Oxidation Pathway for the Degradation of Toxic Propionyl-CoA

Propionyl-CoA arises as a metabolic intermediate from the degradation of propionate, odd-chain fatty acids, and some amino acids. Thus, pathways for catabolism of this intermediate have evolved in all kingdoms of life, preventing the accumulation of toxic propionyl-CoA concentrations. Previous studies have shown that fungi generally use the methyl citrate cycle for propionyl-CoA degradation. Here, we show that this is not the case for the pathogenic fungus Candida albicans despite its ability to use propionate and valerate as carbon sources. Comparative proteome analyses suggested the presence of a modified β-oxidation pathway with the key intermediate 3-hydroxypropionate. Gene deletion analyses confirmed that the enoyl-CoA hydratase/dehydrogenase Fox2p, the putative 3-hydroxypropionyl-CoA hydrolase Ehd3p, the 3-hydroxypropionate dehydrogenase Hpd1p, and the putative malonate semialdehyde dehydrogenase Ald6p essentially contribute to propionyl-CoA degradation and its conversion to acetyl-CoA. The function of Hpd1p was further supported by the detection of accumulating 3-hydroxypropionate in the hpd1 mutant on propionyl-CoA-generating nutrients. Substrate specificity of Hpd1p was determined from recombinant purified enzyme, which revealed a preference for 3-hydroxypropionate, although serine and 3-hydroxyisobutyrate could also serve as substrates. Finally, virulence studies in a murine sepsis model revealed attenuated virulence of the hpd1 mutant, which indicates generation of propionyl-CoA from host-provided nutrients during infection.     

Synthesis and anti-inflammatory evaluations of β-lapachone derivatives

β-Lapachone (β-LAPA), a natural product from the lapacho tree in South America, is a potential chemotherapeutic agent that exhibit a wide variety of pharmacological effects such as anti-virus, anti-parasitic, anti-cancer, and anti-inflammatory activities. In order to discover novel anti-inflammatory agents, we have synthesized a series of β-LAPA derivatives for evaluation. Among them, 4-(4-methoxyphenoxy)naphthalene-1,2-dione (6b) was found to be able to inhibit NO and TNF-α released in LPS-induced Raw 264.7 cells. Inhibition of iNOS and COX-2 was also observed in compound 6b treated cells. Mechanism studies indicated that 6b exhibited anti-inflammatory properties by suppressing the release of pro-inflammatory factors through down-regulating NF-κB activation. In addition, it suppressed NF-κB translocation by inhibiting the phosphorylation of p38 kinase. Our results also indicate that the inhibitory effect of 6b on LPS-stimulated inflammatory mediator production in Raw 264.7 cell is associated with the suppression of the NF-κB and MAPK signaling pathways. A low cytotoxicity (IC₅₀ = 31.70 μM) and the potent anti-inflammatory activity exhibited by compound 6b make this compound a potential lead for developing new anti-inflammatory agents. Further structural optimization of compound 6b is on-going.     

Functional interactions between the oxytocin receptor and the β2-adrenergic receptor: implications for ERK1/2 activation in human myometrial cells

The Gq-coupled oxytocin receptor (OTR) and the Gs-coupled β₂-adrenergic receptor (β₂AR) are both expressed in myometrial cells and mediate uterine contraction and relaxation, respectively. The two receptors represent important pharmacological targets as OTR antagonists and β₂AR agonists are used to control pre-term uterine contractions. Despite their physiologically antagonistic effects, both receptors activate the MAP kinases ERK1/2, which has been implicated in uterine contraction and the onset of labor. To determine the signalling pathways involved in mediating the ERK1/2 response, we assessed the effect of blockers of specific G protein-associated pathways. In human myometrial hTERT-C3 cells, inhibition of Gαi as well as inhibition of the Gαq/PKC pathway led to a reduction of both OTR- and β₂AR-mediated ERK1/2 activation. The involvement of Gαq/PKC in β₂AR-mediated ERK1/2 induction was unexpected. To test whether the emergence of this novel signalling mechanism was dependent on OTR expression in the same cell, we conducted experiments in HEK 293 cells that were transfected with the β₂AR alone or co-transfected with the OTR. Using this approach, we found that β₂AR-mediated ERK1/2 responses became sensitive to PKC inhibition only in cells co-transfected with the OTR. Inhibitor studies indicated the involvement of an atypical PKC isoform in this process. We confirmed the specific involvement of PKCζ in this pathway by assessing PKCζ translocation to the cell membrane. Consistent with our inhibitor studies, we found that β₂AR-mediated PKCζ translocation was dependent on co-expression of OTR. The present demonstration of a novel β₂AR-coupled signalling pathway that is dependent on OTR co-expression is suggestive of a molecular interaction between the two receptors.     

Members 5 and 6 of the Candida albicans BMT family encode enzymes acting specifically on β-mannosylation of the phospholipomannan cell-wall glycosphingolipid

A family of nine genes encoding proteins involved in the synthesis of β-1,2 mannose adhesins of Candida albicans has been identified. Four of these genes, BMT1-4, encode enzymes acting stepwise to add β-mannoses on to cell-wall phosphopeptidomannan (PPM). None of these acts on phospholipomannan (PLM), a glycosphingolipid member of the mannose-inositol-phosphoceramide family, which contributes with PPM to β-mannose surface expression. We show that deletion of BMT5 and BMT6 led to a dramatic reduction of PLM glycosylation and accumulation of PLM with a truncated β-oligomannoside chain, respectively. Disruptions had no effect on sphingolipid biosynthesis and on PPM β-mannosylation. β-Mannose surface expression was not affected, confirming that β-mannosylation is a process based on specificity of acceptor molecules, but liable to global regulation.     

Distinct interactions between the human adrenergic β2 receptor and Gαs—an in silico study

The aim of this study was to perform an in silico analysis of the interaction of the human β₂ adrenergic receptor with Gα_s. In a first step, a systematic surface-interaction-scan between the inactive or active human β₂ adrenergic receptor and Gα_s was performed in order to gain knowledge about energetically preferred areas on the potential energy surface. Subsequently, two energetically favored regions for the active human β₂ adrenergic receptor–Gα_s complex were identified. Two representative complex structures were put into a POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine) bilayer and solvated in order to perform molecular dynamic simulations. The simulations revealed that both conformations, which have comparable potential energy, are stable. A mean number of about 14 hydrogen bonds was observed between the active receptor and Gα_s for both conformations. Based on these results, two energetically favored β₂–Gα_scomplexes can be proposed.     

Antibiotic tetaine — a selective inhibitor of chitin and mannoprotein biosynthesis in Candida albicans

The antibiotic tetaine inhibits in Candida albicans the biosynthesis of two important cell wall constituents, chitin and mannoprotein. This effect is a consequence of inactivation of the enzyme glucosamine-6-phosphate synthetase. Due to the lack of glucosamine-6-phosphate the effective secretion of mannoprotein enzymes, acid phosphatase and invertase, by Candida albicans spheroplasts is inhibited. In the presence of tetaine, probably a modified mannoprotein, lacking a branched polymannan, is synthesized. The antibiotic action decreases the viability of Candida albicans cells, especially that of mycelial forms of this fungus.Abbreviations GlcNAc N-acetyl-d-glucosamine - GlcN-6-P d-glucosamine-6-phosphate - ManNAc N-acetyl-d-mannosamine - -MM -methylmannoside - EGTA 1,2 di/2-aminoethoxy/ethane - N,N,N,N tetra-acetic acid     

Heterologous expression and characterization of the sterol 14α-demethylase CYP51F1 from Candida albicans

Lanosterol 14α-demethylase (CYP51F1) from Candida albicans is known to be an essential enzyme in fungal sterol biosynthesis. Wild-type CYP51F1 and several of its mutants were heterologously expressed in Escherichia coli, purified, and characterized. It exhibited a typical reduced CO-difference spectrum with a maximum at 446 nm. Reconstitution of CYP51F1 with NADPH-P450 reductase gave a system that successfully converted lanosterol to its demethylated product. Titration of the purified enzyme with lanosterol produced a typical type I spectral change with K_d = 6.7 μM. The azole antifungal agents econazole, fluconazole, ketoconazole, and itraconazole bound tightly to CYP51F1 with K_d values between 0.06 and 0.42 μM. The CYP51F1 mutations F105L, D116E, Y132H, and R467K frequently identified in clinical isolates were examined to determine their effect on azole drug binding affinity. The azole K_d values of the purified F105L, D116E, and R467K mutants were little altered. A homology model of C. albicans CYP51F1 suggested that Tyr132 in the BC loop is located close to the heme in the active site, providing a rationale for the modified heme environment caused by the Y132H substitution. Taken together, functional expression and characterization of CYP51F1 provide a starting basis for the design of agents effective against C. albicans infections.     

Phr1p, a glycosylphosphatidylinsitol-anchored β(1,3)-glucanosyltransferase critical for hyphal wall formation, localizes to the apical growth sites and septa in Candida albicans

Cell wall biogenesis is a dynamic process relying on the coordinated activity of several extracellular enzymes. PHR1 is a pH-regulated gene of Candida albicans encoding a glycosylphosphatidylinositol-anchored β(1,3)-glucanosyltransferase of family GH72 which acts as a cell wall remodelling enzyme and is crucial for morphogenesis and virulence. In order to explore the function of Phr1p, we obtained a green fluorescent protein (GFP) fusion to determine its localization. During induction of vegetative growth, Phr1p-GFP was concentrated in the plasma membrane of the growing bud, in the mother-bud neck, and in the septum. Phr1p-GFP was recovered in the detergent-resistant membranes indicating its association with the lipid rafts as the wild type Phr1p. Upon induction of hyphal growth, Phr1p-GFP highly concentrated at the apex of the germ tubes and progressively distributed along the lateral sides of the hyphae. Phr1p-GFP also labelled the hyphal septa, where it colocalized with chitin. Localization to the hyphal septa was perturbed in nocodazole-treated cells, whereas inhibition of actin polymerization hindered the apical localization. Electron Microscopy analysis of the hyphal wall ultrastructure of a PHR1 null mutant showed loss of compactness and irregular organization of the surface layer. These observations indicate that Phr1p plays a crucial role in hyphal wall formation, a highly regulated process on which morphogenesis and virulence rely.     

Carbonic anhydrase inhibitors. Inhibition and homology modeling studies of the fungal β-carbonic anhydrase from Candida albicans with sulfonamides

The β-carbonic anhydrase (CA, EC 4.2.1.1) from the fungal pathogen Candida albicans (Nce103) is involved in a CO₂ sensing pathway critical for the pathogen life cycle and amenable to drug design studies. Herein we report an inhibition study of Nce103 with a library of sulfonamides and one sulfamate, showing that Nce103, similarly to the related enzyme from Cryptococcus neoformans Can2, is inhibited by these compounds with K_Is in the range of 132 nM–7.6 μM. The best Nce103 inhibitors were acetazolamide, methazolamide, bromosulfanilamide, and 4-hydroxymethylbenzenesulfonamide (K_Is < 500 nM). A homology model was generated for Nce103 based on the crystal structure of Can2. The model shows that compounds with zinc-binding groups incorporating less polar moieties and compact scaffolds generate stronger Nce103 inhibitors, whereas highly polar zinc-binding groups and bulkier compounds appear more promising for the specific inhibition of Can2. Such compounds may be useful for the design of antifungal agents possessing a new mechanism of action.     

Study of the mechanism of ε-poly-l-lysine as an antifungal on Candida albicans and Saccharomyces cerevisiae

The antimicrobial activity of ε-poly-l-lysine (EPL) has been documented, but its antifungal activity on yeast is not well defined and its mechanism of action has been vaguely explained. Our studies revealed that on both, Candida albicans and Saccharomyces cerevisiae, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were 250 μg·mL^?1; EPL produced a K⁺ and Ca²⁺ efflux, and at higher concentrations also an efflux of material absorbing at 260 nm, small peptides, and phosphate is produced, along with the inhibition of fermentation and extracellular acidification and respiration. Moreover, growth was inhibited, reactive oxygen species (ROS) production increased, and cell viability decreased. The polycation also produced plasma membrane potential hyperpolarization. The effects were dependent both on the cell quantity and polycation concentration, as well as the media used. The plasma membrane disruption was confirmed by TEM and PI staining.