Fabrice Pagniez

Amino acid substitutions in the Candida albicans sterol Δ5,6-desaturase (Erg3p) confer azole resistance: characterization of two novel mutants with impaired virulence

OBJECTIVES To determine the mechanisms responsible for fluconazole resistance in two Candida albicans isolates (CAAL2 and CAAL76) recovered from two hospitalized patients after fluconazole prophylaxis. METHODS MICs of fluconazole and voriconazole were determined by the broth microdilution method (CLSI M27-A3), and by Etest(®) for amphotericin B. RNA expression levels of CDR1, MDR1 and ERG11 were determined by RT-PCR. Mutations in ERG11 and ERG3 were investigated by amplification and sequencing. Sterol membrane profiles were determined by gas chromatography-mass spectrometry (GC-MS). In vivo virulence was determined in a murine model of invasive candidiasis. RESULTS Both isolates displayed azole cross-resistance and reduced susceptibility to amphotericin B, and are novel Δ(5,6)-desaturase (Erg3p) mutants. CAAL2 harbours a new amino acid substitution (L193R), whereas a 13 bp deletion leading to a truncated Erg3p (Δ366-378) was found in CAAL76. Both genetic alterations impaired Erg3p function as shown by GC-MS in these isolates (ergosterol content below 10%, and accumulation of ergosta-7,22-dienol above 40%). In vivo, in a murine model of invasive candidiasis, both CAAL2 and CAAL76 exhibited a significant trend toward reduced virulence, which seems to be linked to a reduced capacity for hyphal growth. CONCLUSIONS These findings demonstrate the critical role of residue 193 in Erg3p function and azole resistance. We suggest that this attenuated in vivo virulence phenotype could be linked to lower potential for hyphal growth. Taken together, our findings highlight the fact that erg3 mutants must be considered in future studies aiming at investigating azole antifungal drug resistance.

Synthesis and antifungal activity of new 1-halogenobenzyl-3-imidazolylmethylindole derivatives

A series of 1-benzyl-3-(imidazol-1-ylmethyl)indole derivatives 35-46 were prepared under mild reaction conditions and tested for their antifungal activity. Pharmacomodulation at N(1), C(2) and C(5) of the indole ring and at the level of the alkyl chain (R(1)) was carried out starting from the corresponding 3-acylindoles 6, 7 or 3-formylindoles 11-22. Target imidazolyl compounds 35-46 were obtained in satisfactory yields by CO(2) elimination from the intermediate carbamates. All of the compounds were evaluated in vitro against two human fungal pathogens, Candida albicans (CA980001) and Aspergillus fumigatus (AF980003); amphotericin B, fluconazole and itraconazole were used as references. Seven out of 27 compounds (35b, 35e, 35g, 35h, 36a, 38a and especially 40a) exerted significant antifungal activity against C. albicans, with MIC in the range of 1-6 microg mL(-1). As regards inhibitory activity against A. fumigatus, the MIC figures of most of our compounds were in excess of 20 microg mL(-1) in contrast to the reference drugs, amphotericin B and itraconazole, whose MIC(90) and MIC(80) values were 0.14 and 0.50 microg mL(-1), respectively. The most potent compound, 45a, exhibited MIC value (8 +/- 1 microg mL(-1)) 16-fold higher than that of itraconazole.

Synthesis and structure-activity relationships of 2-phenyl-1-[(pyridinyl- and piperidinylmethyl)amino]-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents.

Continuous efforts on the synthesis and structure-activity relationships (SARs) studies of modified 1-benzylamino-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents, allowed identification of new 1-[(pyridinyl- and piperidinylmethyl)amino] derivatives with MIC(80) values ranging from 1410.0 to 23.0ngmL(-1) on Candidaalbicans. These results confirmed both the importance of pi-pi stacking and hydrogen bonding interactions in the active site of CYP51-C. albicans.

Synthesis and biological evaluation of 2,3-diarylimidazo[1,2-a]pyridines as antileishmanial agents

A novel series of 2,3-diarylimidazo[1,2-a]pyridines was synthesized and evaluated for their antileishmanial activities. Four derivatives exhibited good activity against the promastigote and intracellular amastigote stages of Leishmania major, coupled with a low cytotoxicity against the HeLa human cell line. The impact of compound lipophilicity on antiparasitic activities was investigated by Log D comparison. Although LmCK1 could be the parasitic target for three compounds (13, 18, 21), the inhibition of another target is under study to explain the antileishmanial effect of the most promising compounds.

Comparison of three methods to study biofilm formation by clinical strains of Escherichia coli.

Biofilm formation seems to be a key factor in many bacterial infections, particularly those involving prosthetic implants or urinary catheters, where Escherichia coli is frequently involved. We have determined the ability to form biofilm in vitro of 34 E. coli isolates by 3 different methods (crystal violet staining, BioFilm Ring Test®, and resazurin assay) and tried to correlate biofilm production with phylogenetic background and with the presence of different genes involved in biofilm synthesis. Only 3 isolates (including positive control E. coli ATCC 25922) were classified as strong biofilm producers (1B1, 1D, and 1B2 = control) by the 3 methods, 2 isolates by 2 different methods, and 5 additional isolates by only 1 method. All isolates possessed the csgA gene belonging to the csgABC operon encoding curli, and its regulator csgD. By contrast, only 76% possessed pgaA gene which is part of the pgaABCD operon encoding a polysaccharide adhesin. Interestingly, one of the strong biofilm producers did not harbor pgaA. In the second part, we have selected 5 specific isolates to study the impact of various experimental conditions on biofilm formation. For all these isolates, biofilm production was decreased in anaerobiosis and increased in LB medium compared with brain heart infusion medium, but at various degrees for the different isolates. These results underline the problems encountered in comparing the different published studies using various methods to study biofilm formation in vitro and the great need of standardization.

Design, synthesis, and evaluation of 1-(N-benzylamino)-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents.

A series of 1-(N-benzylamino)-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols 6a-c, 7a-c, 8a, and 9a were prepared in five steps and evaluated for their antifungal activity. The most active compound 7b was docked into a home-made 3D model of the targeted enzyme confirming the importance of Tyr118, His377, and Ser378 residues in its binding mode.

Amino Acid Substitutions at the Major Insertion Loop of Candida albicans Sterol 14alpha-Demethylase Are Involved in Fluconazole Resistance

Background In the fungal pathogen Candida albicans, amino acid substitutions of 14alpha-demethylase (CaErg11p, CaCYP51) are associated with azole antifungals resistance. This is an area of research which is very dynamic, since the stakes concern the screening of new antifungals which circumvent resistance. The impact of amino acid substitutions on azole interaction has been postulated by homology modeling in comparison to the crystal structure of Mycobacterium tuberculosis (MT-CYP51). Modeling of amino acid residues situated between positions 428 to 459 remains difficult to explain to date, because they are in a major insertion loop specifically present in fungal species. Methodology/Principal Finding Fluconazole resistance of clinical isolates displaying Y447H and V456I novel CaErg11p substitutions confirmed in vivo in a murine model of disseminated candidiasis. Y447H and V456I implication into fluconazole resistance was then studied by site-directed mutagenesis of wild-type CaErg11p and by heterogeneously expression into the Pichia pastoris model. CLSI modified tests showed that V447H and V456I are responsible for an 8-fold increase in fluconazole MICs of P. pastoris mutants compared to the wild-type controls. Moreover, mutants showed a sustained capacity for producing ergosterol, even in the presence of fluconazole. Based on these biological results, we are the first to propose a hybrid homology structure-function model of Ca-CYP51 using 3 different homology modeling programs. The variable position of the protein insertion loop, using different liganded or non-liganded templates of recently solved CYP51 structures, suggests its inherent flexibility. Mapping of recognized azole-resistant substitutions indicated that the flexibility of this region is probably enhanced by the relatively high glycine content of the consensus. Conclusions/Significance The results highlight the potential role of the insertion loop in azole resistance in the human pathogen C. albicans. This new data should be taken into consideration for future studies aimed at designing new antifungal agents, which circumvent azole resistance.

Discovery of a Novel Broad-Spectrum Antifungal Agent Derived from Albaconazole

Synthesis of a strict structural analogue of albaconazole in which the quinazolinone ring is fused by a thiazole moiety led to the discovery of a new triazole with broad-spectrum antifungal activity. Compound I exhibited high in vitro activity against pathogenic Candida species and filamentous fungi and showed preliminary in vivo antifungal efficacy in a mice model of systemic candidiasis.

Design of new antifungal agents: synthesis and evaluation of 1-[(1H-indol-5-ylmethyl)amino]-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols.

We previously reported on the design and synthesis of 1-[((hetero)aryl- or piperidinylmethyl)amino]-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols showing various degrees of antifungal activity against Candida albicans and Aspergillus fumigatus strains. Now we have identified a series of 1-[(1H-indol-5-ylmethyl)amino] derivatives which exhibited potent MICs (<65 ng mL(-1)) against C. albicans strain. The synthesis and SAR behind the indole scaffold will be discussed.

Deciphering azole resistance mechanisms with a focus on transcription factor-encoding genes TAC1, MRR1 and UPC2 in a set of fluconazole-resistant clinical isolates of Candida albicans

Several and often combined mechanisms can lead to acquired azole resistance in Candida albicans and subsequent therapeutic failure. The aim of this study was to provide a complete overview of the molecular basis of azole resistance in a set of six C. albicans clinical isolates recovered from patients who failed azole therapy. For this purpose, expression levels of CDR1, MDR1 and ERG11 were investigated by reverse transcription PCR (RT-PCR) together with amplification and sequencing of the genes encoding their transcription factors TAC1, MRR1 and UPC2. In all, the data underline that azole resistance in this set of clinical isolates results from distinct, often combined, mechanisms, being mostly driven by CDR1 and/or MDR1 active efflux. We show that gain-of-function (GOF) mutations in the transcription-factor-encoding genes TAC1, MRR1 and UPC2 are a common event in azole-resistant C. albicans clinical isolates. In addition, together with the finding that these genes are highly permissive to nucleotide changes, we describe several novel mutations that could act as putative GOF mutations involved in fluconazole resistance.