Identification and functional characterization of <Emphasis Type="Italic">Candida albicans CDC4</Emphasis>

Summary The CDC4 gene of Saccharomyces cerevisiae encodes an essential function that is required for G1-S and G2-M transitions during mitosis and at various stages during meiosis. We have isolated a functional homologue of CDC4 (CaCDC4) from the pathogenic yeast Candida albicans by complementing the S. cerevisiae cdc4-3 mutation with CaCDC4 expressed from its own promoter on a single-copy vector. The predicted product of CaCDC4 has 37% overall identity to the S. cerevisiae Cdc4 protein, although this identity is biased towards the C-terminal region of the two proteins which contains eight copies of the degenerate WD-40 motif, an element found in proteins that regulate diverse biological processes and an F-box domain proximal to the first iteration of the WD-40 motif. Both the F-box domain and WD-40 motifs appear necessary for the mitotic functions of Cdc4 in both yeasts. In contrast to its conserved role in mitosis, C. albicans CDC4 is unable to rescue the meiotic deficiency in a S. cerevisiae cdc4 homozygous diploid under restrictive conditions, even when expressed from an efficient S. cerevisiae promoter. In opposition to S. cerevisiae CDC4 being essential, C. albicans CDC4 appears to be nonessential and in its absence is critical for filamentous growth in C. albicans.     

Dissection of the Candida albicans Cdc4 protein reveals the involvement of domains in morphogenesis and cell flocculation

Background

CDC4, which encodes an F-box protein that is a member of the Skp1-Cdc53/Cul1-F-box (SCF) ubiquitin E3 ligase, was initially identified in the budding yeast Saccharomyces cerevisiae as an essential gene for progression through G1-S transition of the cell cycle. Although Candida albicans CDC4 (CaCDC4) can release the mitotic defect caused by the loss of CDC4 in S. cerevisiae, CaCDC4 is nonessential and suppresses filamentation.

Results

To further elucidate the function of CaCDC4, a C. albicans strain, with one CaCDC4 allele deleted and the other under the repressible C. albicans MET3 promoter (CaMET3p) control, was made before introducing cassettes capable of doxycycline (Dox)-induced expression of various C. albicans Cdc4 (CaCdc4) domains. Cells from each strain could express a specific CaCdc4 domain under Dox-induced, but CaMET3-CaCDC4 repressed conditions. Cells expressing domains without either the F-box or WD40-repeat exhibited filamentation and flocculation similarly to those lacking CaCDC4 expression, indicating the functional essentiality of the F-box and WD40-repeat. Notably, cells expressing the N-terminal 85-amino acid truncated CaCdc4 partially reverse the filament-to-yeast and weaken the ability to flocculate compared to those expressing the full-length CaCdc4, suggesting that N-terminal 85-amino acid of CaCdc4 regulates both morphogenesis and flocculation.

Conclusions

The F-box and the WD40-repeat of CaCdc4 are essential in inhibiting yeast-to-filament transition and flocculation. The N-terminal region (1–85) of CaCdc4 also has a positive role for its function, lost of which impairs both the ability to flocculate and to reverse filamentous growth in C. albicans.     

Isolation of anti-Candida albicans compounds from Markhamia obtusifolia (Baker) Sprague (Bignoniaceae)

An increase in clinical cases of Candidiosis globally as well as fungal resistance to drugs prompted the search for novel anti-Candida albicans agents from plant sources. Leaf extracts of Markhamia obtusifolia were screened for activity against C. albicans in vitro. An acetone extract obtained following serial exhaustive extraction contained mainly the active components with at least four active zones on the bioautogram. Bioassay guided fractionation of this extract led to the isolation of three compounds which inhibited the growth of three C. albicans strains. Based on spectroscopy studies (NMR and MS), the compounds were identified as 3β-hydroxyurs-12-en-28-oic acid, ursolic acid (1) 3β, 19α-dihydroxyurs-12-en-28-oic acid, pomolic acid (2) and 2β, 3β, 19α -trihydroxy-urs-12-en-28-oic acid, 2-epi-tormentic acid (3). The most active compound was 3β, 19α-dihydroxy-12-ursen-28-oic acid (2) with a minimum inhibitory concentration (MIC) value of 12.5 µg/mL for C. albicans isolated from dog and 25.0 µg/mL for C. albicans from cat and ATCC 90028 at 24 h following incubation. However, at 48 h of incubation MICs were > 400 µg/mL for all the three compounds isolated. This study indicated that M. obtusifolia could be a potential source of active principles against C. albicans.     

Candida albicans NADPH-P450 reductase: Expression, purification, and characterization of recombinant protein

Candida albicans is responsible for serious fungal infections in humans. Analysis of its genome identified NCP1 gene coding for a putative NADPH-P450 reductase (NPR) enzyme. This enzyme appears to supply reducing equivalents to cytochrome P450 or heme oxygenase enzymes for fungal survival and virulence. In this study, we report the characterization of the functional features of NADPH-P450 reductase from C. albicans. The recombinant C. albicans NPR protein harboring a 6×(His)-tag was expressed heterologously in Escherichia coli, and was purified. Purified C. albicans NPR has an absorption maximum at 453 nm, indicating the feature of an oxidized flavin cofactor, which was decreased by the addition of NADPH. It also evidenced NADPH-dependent cytochrome c or nitroblue tetrazolium reducing activity. This purified reductase protein was successfully able to substitute for purified mammalian NPR in the reconstitution of the human P450 1A2-catalyzed O-deethylation of 7-ethoxyresorufin. These results indicate that purified C. albicans NPR is an orthologous reductase protein that supports cytochrome P450 or heme oxygenase enzymes in C. albicans.     

Non-lethal Candida albicans cph1/cph1 efg1/efg1 mutant partially protects mice from systemic infections by lethal wild-type cells

Although Candida albicans cph1/cph1 efg1/efg1 mutant cells are not lethal to mice, they proliferated in infected mice instead of simply being cleared by the host immune system. Here, we have shown that the cph1/cph1 efg1/efg1 mutant partially protects mice from systemic infections by the lethal wild-type Candida albicans cells. Our results further indicate that a second dose of the cph1/cph1 efg1/efg1 mutant did not boost the degree of protection.     

Candida albicans CDK1 and CYB1: cDNA homologues of the cdc2/CDC28 and cdcl3/CLB1/CLB2 cell cycle control genes

Major transitions in the eukaryotic cell cycle are regulated by the cyclin-dependent protein kinases (CDK). In particular, the G2/M transition is initiated by the activity of a complex formed by a CDK of the Cdc2/Cdc28 family and B-type cyclins of the Cdc13/Clb family in the yeasts, Schizosaccharomyces pombe (Sp) and Saccharomyces cerevisiae (Sc). To study the molecular mechanisms that control the G2/M transition in the dimorphic pathogenic yeast, Candida albicans, we have cloned and characterized cDNAs corresponding to CDK1 and CYB1. The CDK1 cDNA encodes a 317-amino-acid (aa) protein that shares 76.8 and 62.3% identity with the Sc CDC28 and Sp cdc2 gene products, respectively. The CYB1 cDNA encodes a 493-aa protein that is 34.8, 34.4 and 35.5% identical to Sc Clbl and Clb2, and to Sp Cdc13, respectively. Cyb1 contains characteristic mitotic destruction and cyclin boxes. The CDK1 and CYB1 cDNAs are functional homologues, as they are able to complement Sp cdc2 and cdc13 temperature-sensitive (ts) mutations, respectively, and their gene products interact in vivo in Sc to form an active histone H1 kinase.     

Plagiochin E,an antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway

Background

Plagiochin E (PLE) is an antifungal active macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. To elucidate the mechanism of action, previous studies revealed that the antifungal effect of PLE was associated with the accumulation of ROS, an important regulator of apoptosis in Candida albicans. The present study was designed to find whether PLE caused apoptosis in C. albicans.

Methods

We assayed the cell cycle by flow cytometry using PI staining, observed the ultrastructure by transmission electron microscopy, studied the nuclear fragmentation by DAPI staining, and investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V staining. The effect of PLE on expression of CDC28, CLB2, and CLB4 was determined by RT-PCR. Besides, the activity of metacaspase was detected by FITC-VAD-FMK staining, and the release of cytochrome c from mitochondria was also determined. Furthermore, the effect of antioxidant L-cysteine on PLE-induced apoptosis in C. albicans was also investigated.

Results

Cells treated with PLE showed typical markers of apoptosis: G₂/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. The expression of CDC28, CLB2, and CLB4 was down-regulated by PLE, which may contribute to PLE-induced G₂/M cell cycle arrest. Besides, PLE promoted the cytochrome c release and activated the metacaspase, which resulted in the yeast apoptosis. The addition of L-cysteine prevented PLE-induced nuclear fragmentation, phosphatidylserine exposure, and metacaspase activation, indicating the ROS was an important mediator of PLE-induced apoptosis.

Conclusions

PLE induced apoptosis in C. albicans through a metacaspase-dependent apoptotic pathway.

General significance

In this study, we reported for the first time that PLE induced apoptosis in C. albicans through activating the metacaspase. These results would conduce to elucidate its underlying antifungal mechanism.     

Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography

Immobilised metal affinity chromatography (IMAC) is the most widely used technique for single-step purification of recombinant proteins. However, despite its use in the purification of heterologue proteins in the eubacteria Escherichia coli for decades, the presence of native E. coli proteins that exhibit a high affinity for divalent cations such as nickel, cobalt or copper has remained problematic. This is of particular relevance when recombinant molecules are not expressed at high levels or when their overexpression induces that of native bacterial proteins due to pleiotropism and/or in response to stress conditions. Identification of such contaminating proteins is clearly relevant to those involved in the purification of histidine-tagged proteins either at small/medium scale or in high-throughput processes. The work presented here reviews the native proteins from E. coli most commonly co-purified by IMAC, including Fur, Crp, ArgE, SlyD, GlmS, GlgA, ODO1, ODO2, YadF and YfbG. The binding of these proteins to metal-chelating resins can mostly be explained by their native metal-binding functions or their possession of surface clusters of histidine residues. However, some proteins fall outside these categories, implying that a further class of interactions may account for their ability to co-purify with histidine-tagged proteins. We propose a classification of these E. coli native proteins based on their physicochemical, structural and functional properties.     

Morphogenesis beyond Cytokinetic Arrest in Saccharomyces cerevisiae

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.     

Ung1p-mediated uracil-base excision repair in mitochondria is responsible for the petite formation in thymidylate deficient yeast

The budding yeast CDC21 gene, which encodes thymidylate synthase, is crucial in the thymidylate biosynthetic pathway. Early studies revealed that high frequency of petites were formed in heat-sensitive cdc21 mutants grown at the permissive temperature. However, the molecular mechanism involved in such petite formation is largely unknown. Here we used a yeast cdc21-1 mutant to demonstrate that the mutant cells accumulated dUMP in the mitochondrial genome. When UNG1 (encoding uracil-DNA glycosylase) was deleted from cdc21-1, we found that the ung1Δ cdc21-1 double mutant reduced frequency of petite formation to the level found in wild-type cells. We propose that the initiation of Ung1p-mediated base excision repair in the uracil-laden mitochondrial genome in a cdc21-1 mutant is responsible for the mitochondrial petite mutations.     

The Dad1 subunit of the yeast kinetochore Dam1 complex is an intrinsically disordered protein

The Dam1 complex is an important part of the yeast kinetochore. It mediates attachment of the chromosome to the mitotic spindle and is involved in chromatid separation initiated at anaphase. It is comprised of 10 individual subunits and has been observed to oligomerize in various ways as it interacts with microtubules, including forming a ring. This work explores the biochemical and biophysical properties of Dad1, one of the Dam1 complex subunits from the human fungal pathogen Candida albicans. Unlike its Saccharomyces cerevisiae counterpart, C. albicans Dad1 can be expressed as a soluble protein in Escherichia coli. Analysis of this protein’s hydrodynamic properties, thermostability and primary sequence have been conducted. As a result, we conclude that isolated Dad1 is an intrinsically disordered protein.     

A Highlights from MBoC Selection: Cdc1 removes the ethanolamine phosphate of the first mannose of GPI anchors and thereby facilitates the integration of GPI proteins into the yeast cell wall

Temperature-sensitive cdc1^ts mutants are reported to stop the cell cycle upon a shift to 30°C in early G2, that is, as small budded cells having completed DNA replication but unable to duplicate the spindle pole body. A recent report showed that PGAP5, a human homologue of CDC1, acts as a phosphodiesterase removing an ethanolamine phosphate (EtN-P) from mannose 2 of the glycosylphosphatidylinositol (GPI) anchor, thus permitting efficient endoplasmic reticulum (ER)-to-Golgi transport of GPI proteins. We find that the essential CDC1 gene can be deleted in mcd4∆ cells, which do not attach EtN-P to mannose 1 of the GPI anchor, suggesting that Cdc1 removes the EtN-P added by Mcd4. Cdc1-314^ts mutants do not accumulate GPI proteins in the ER but have a partial secretion block later in the secretory pathway. Growth tests and the genetic interaction profile of cdc1-314^ts pinpoint a distinct cell wall defect. Osmotic support restores GPI protein secretion and actin polarization but not growth. Cell walls of cdc1-314^ts mutants contain large amounts of GPI proteins that are easily released by β-glucanases and not attached to cell wall β1,6-glucans and that retain their original GPI anchor lipid. This suggests that the presumed transglycosidases Dfg5 and Dcw1 of cdc1-314^ts transfer GPI proteins to cell wall β1,6-glucans inefficiently.     

Isolation and Characterization of New Alleles of the Cyclin-Dependent Kinase Gene CDC28 with Cyclin-Specific Functional and Biochemical Defects

The G₁ cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits.     

Geranylgeranyltransferase I of Candida albicans: null mutants or enzyme inhibitors produce unexpected phenotypes

Geranylgeranyltransferase I (GGTase I) catalyzes the transfer of a prenyl group from geranylgeranyl diphosphate to the carboxy-terminal cysteine of proteins with a motif referred to as a CaaX box (C, cysteine; a, usually aliphatic amino acid; X, usually L). The alpha and beta subunits of GGTase I from Saccharomyces cerevisiae are encoded by RAM2 and CDC43, respectively, and each is essential for viability. We are evaluating GGTase I as a potential target for antimycotic therapy of the related yeast, Candida albicans, which is the major human pathogen for disseminated fungal infections. Recently we cloned CaCDC43, the C. albicans homolog of S. cerevisiae CDC43. To study its role in C. albicans, both alleles were sequentially disrupted in strain CAI4. Null Cacdc43 mutants were viable despite the lack of detectable GGTase I activity but were morphologically abnormal. The subcellular distribution of two GGTase I substrates, Rho1p and Cdc42p, was shifted from the membranous fraction to the cytosolic fraction in the cdc43 mutants, and levels of these two proteins were elevated compared to those in the parent strain. Two compounds that are potent GGTase I inhibitors in vitro but that have poor antifungal activity, J-109,390 and L-269,289, caused similar changes in the distribution and quantity of the substrate. The lethality of an S. cerevisiae cdc43 mutant can be suppressed by simultaneous overexpression of RHO1 and CDC42 on high-copy-number plasmids (Y. Ohya et al., Mol. Biol. Cell 4:1017, 1991; C. A. Trueblood, Y. Ohya, and J. Rine, Mol. Cell. Biol. 13:4260, 1993). Prenylation presumably occurs by farnesyltransferase (FTase). We hypothesize that Cdc42p and Rho1p of C. albicans can be prenylated by FTase when GGTase I is absent or limiting and that elevation of these two substrates enables them to compete with FTase substrates for prenylation and thus allows sustained growth.     

Inhibition of Candida albicans biofilm formation and yeast-hyphal transition by 4-hydroxycordoin

Candida albicans distinguishing features such as dimorphism and biofilm formation are thought to play a key role in oral tissue invasion and resistance to host defences and antifungal agents. In this study, we investigated the effect of 4-hydroxycordoin, a natural isopentenyloxychalcone, on growth, biofilm formation and yeast-hyphal transition of C. albicans. Serial dilutions of 4-hydroxycordoin in YNB medium were prepared in microplates to determine minimal inhibitory concentrations (MIC) and effects on biofilm formation for two strains of C. albicans. 4-Hydroxycordoin at up to 200 μg/ml had no effect on growth of C. albicans. Biofilm formation was strongly inhibited (>85%) by 4-hydroxycordoin at 20 μg/ml, while concentrations ranging from 50 to 200 μg/ml caused a significant inhibition of yeast-hyphal transition, as determined by microscopic observation. In conclusion, 4-hydroxycordoin exerts inhibitory effects on two important virulence factors of C. albicans: biofilm formation or yeast-hyphal transition. This suggests that 4-hydroxycordoin may have a therapeutic potential for C. albicans infections.     

The Telomere Capping Complex CST Has an Unusual Stoichiometry,Makes Multipartite Interaction with G-Tails,and Unfolds Higher-Order G-Tail Structures

The telomere-ending binding protein complex CST (Cdc13-Stn1-Ten1) mediates critical functions in both telomere protection and replication. We devised a co-expression and affinity purification strategy for isolating large quantities of the complete Candida glabrata CST complex. The complex was found to exhibit a 2∶4∶2 or 2∶6∶2 stoichiometry as judged by the ratio of the subunits and the native size of the complex. Stn1, but not Ten1 alone, can directly and stably interact with Cdc13. In gel mobility shift assays, both Cdc13 and CST manifested high-affinity and sequence-specific binding to the cognate telomeric repeats. Single molecule FRET-based analysis indicates that Cdc13 and CST can bind and unfold higher order G-tail structures. The protein and the complex can also interact with non-telomeric DNA in the absence of high-affinity target sites. Comparison of the DNA–protein complexes formed by Cdc13 and CST suggests that the latter can occupy a longer DNA target site and that Stn1 and Ten1 may contact DNA directly in the full CST–DNA assembly. Both Stn1 and Ten1 can be cross-linked to photo-reactive telomeric DNA. Mutating residues on the putative DNA–binding surface of Candida albicans Stn1 OB fold domain caused a reduction in its crosslinking efficiency in vitro and engendered long and heterogeneous telomeres in vivo, indicating that the DNA–binding activity of Stn1 is required for telomere protection. Our data provide insights on the assembly and mechanisms of CST, and our robust reconstitution system will facilitate future biochemical analysis of this important complex.     

Cdc24, the GDP-GTP exchange factor for Cdc42, is required for invasive hyphal growth of Candida albicans

Candida albicans, the most common human fungal pathogen, is particularly problematic for immunocompromised individuals. The reversible transition of this fungal pathogen to a filamentous form that invades host tissue is important for its virulence. Although different signaling pathways such as a mitogen-activated protein kinase and a protein kinase A cascade are critical for this morphological transition, the function of polarity establishment proteins in this process has not been determined. We examined the role of four different polarity establishment proteins in C. albicans invasive growth and virulence by using strains in which one copy of each gene was deleted and the other copy expressed behind the regulatable promoter MET3. Strikingly, mutants with ectopic expression of either the Rho G-protein Cdc42 or its exchange factor Cdc24 are unable to form invasive hyphal filaments and germ tubes in response to serum or elevated temperature and yet grow normally as a budding yeast. Furthermore, these mutants are avirulent in a mouse model for systemic infection. This function of the Cdc42 GTPase module is not simply a general feature of polarity establishment proteins. Mutants with ectopic expression of the SH3 domain containing protein Bem1 or the Ras-like G-protein Bud1 can grow in an invasive fashion and are virulent in mice, albeit with reduced efficiency. These results indicate that a specific regulation of Cdc24/Cdc42 activity is required for invasive hyphal growth and suggest that these proteins are required for pathogenicity of C. albicans.     

Baicalin prevents Candida albicans infections via increasing its apoptosis rate

Background

These experiments were employed to explore the mechanisms underlying baicalin action on Candida albicans.

Methodology and principal findings

We detected the baicalin inhibition effects on three isotope-labeled precursors of ³H-UdR, ³H-TdR and ³H-leucine incorporation into C. albicans using the isotope incorporation technology. The activities of Succinate Dehydrogenase (SDH), cytochrome oxidase (CCO) and Ca²⁺–Mg²⁺ ATPase, cytosolic Ca²⁺ concentration, the cell cycle and apoptosis, as well as the ultrastructure of C.albicans were also tested. We found that baicalin inhibited ³H-UdR, ³H-TdR and ³H-leucine incorporation into C.albicans (P < 0.005). The activities of the SDH and Ca²⁺–Mg²⁺ ATPase of C.albicans in baicalin groups were lower than those in control group (P < 0.05). Ca²⁺ concentrations of C. albicans in baicalin groups were much higher than those in control group (P < 0.05). The ratio of C.albicans at the G0/G1 stage increased in baicalin groups in dose dependent manner (P < 0.01). There were a significant differences in the apoptosis rate of C.albicans between baicalin and control groups (P < 0.01). After 12–48 h incubation with baicalin (1 mg/ml), C. albicans shown to be markedly damaged under transmission electron micrographs.

Innovation and significance

Baicalin can increase the apoptosis rate of C. albicans. These effects of Baicalin may involved in its inhibiting the activities of the SDH and Ca²⁺–Mg²⁺ ATPase, increasing cytosolic Ca²⁺ content and damaging the ultrastructure of C. albicans.     

Development of an aggregation assay to screen FimH antagonists

α-d-Mannopyranosides are potent FimH antagonists, which inhibit the adhesion of Escherichia coli to highly mannosylated uroplakin Ia on the urothelium and therefore offer an efficient therapeutic opportunity for the treatment and prevention of urinary tract infection. For the evaluation of the therapeutic potential of FimH antagonists, their effect on the disaggregation of E. coli from Candida albicans and guinea pig erythrocytes (GPE) was studied.The mannose-specific binding of E. coli to yeast cells and erythrocytes is mediated by type 1 pili and can be monitored by aggregometry. Maximal aggregation of C. albicans or GPE to E. coli is reached after 600 s. Then the FimH antagonist was added and disaggregation determined by light transmission over a period of 1400 s. A FimH-deleted mutant of E. coli, which does not induce any aggregation, was used in a control experiment. The activities of FimH antagonists are expressed as IC₅₀s, the half maximal inhibitory concentration of the disaggregation potential. n-Heptyl α-d-mannopyranoside (1) was used as a reference compound and exhibits an IC₅₀ of 77.14 μM , whereas methyl α-d-mannopyranoside (2) does not lead to any disaggregation at concentrations up to 800 μM. o-Chloro-p-[N-(2-ethoxy-3,4-dioxocyclobut-1-enyl)amino]phenyl α-d-mannopyranoside (3) shows a 90-fold and 2-chloro-4-nitrophenyl α-d-mannopyranoside (4) a 6-fold increased affinity compared to 1. Finally, 4-nitrophenyl α-d-mannopyranoside (5) exhibits an activity similar to 1. As negative control, d-galactose (6) was used.The standardized aggregation assay generates concentration-dependent, reproducible data allowing the evaluation of FimH antagonists according to their potency to inhibit E. coli adherence and can therefore be employed to select candidates for experimental and clinical studies for treatment and prevention of urinary tract infections.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.