Effect of temperature and water activity on the production of fumonisins by Aspergillus niger and different Fusariumspecies

Background

Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.

Results

The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.

Conclusion

The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.     

Evaluation of the V404I and V509M amino acid substitutions of <Emphasis Type="Italic">ERG11</Emphasis> gene in <Emphasis Type="Italic">Candida albicans</Emphasis> isolates by pyrosequencing

The molecular mechanisms underlying fluconazole resistance in C. albicans involve mutations and the overexpression of the ERG11 gene and membrane transport proteins. We examined the relationship between the reduced fluconazole susceptibility of C. albicans and mutations of V404I and V509M in the ERG11 gene in 182 C. albicans clinical isolates using the Pyrosequencing™ method. DNAs from these clinical isolates with different levels of in-vitro fluconazole susceptibility — one resistant, five susceptible dose-dependent (SDD), four trailer, and 172 susceptible — were analyzed. None of the fluconazole-susceptible, SDD, trailer or resistant isolates had mutations of V404I or V509M. Our results showed that no correlation can be found between the V404I or V509M mutation and fluconazole susceptibility in C. albicans.     

The new mutation L321F in Candida albicans ERG11 gene may be associated with fluconazole resistance

BackgroundFor many years fluconazole has been commonly used to treat Candida infections. However, the indiscriminate use of this antimycotic therapy has favored the emergence of resistant isolates. Mutations in the ERG11 gene have been described as one of the primary mechanisms of resistance in Candida species.AimsIn this study we investigated missense mutations in ERG11 genes of Candida albicans, Candida glabrata and Candida tropicalis isolates previously evaluated by susceptibility testing to fluconazole.MethodsScreening for these mutations was performed on 19 Candida clinical isolates (eight C. albicans, five C. glabrata and six C. tropicalis) resistant and susceptible to fluconazole. The ERG11 gene was amplified by PCR with specific primers for each Candida species and analyzed by automated sequencing.ResultsWe identified 14 different missense mutations, five of which had not been described previously. Among them, a new mutation L321F was identified in a fluconazole resistant C. albicans isolate and it was analyzed by a theoretical three-dimensional structure of the ERG11p.ConclusionThe L321F mutation in C. albicans ERG11 gene may be associated with fluconazole resistance.     

Proliferation of intracellular structure corresponding to reduced affinity of fluconazole for cytochrome P-450 in two low-susceptibility strains of Candida albicans isolated from a Japanese AIDS patient

Three Candida albicans isolates, TIMM 3164, 3165 and 3166 with reduced fluconazole susceptibility, were isolated from two Japanese AIDS patients. We earlier reported that a reduced intracellular accumulation of fluconazole in these isolates played an important role in the resistance mechanism of fluconazole, but we did not exclude the involvement of other factors. We here examined characteristics related to cytochrome P-450 (CYP), especially sterol 14alpha-demethylase encoded by the ERG11 gene which is the target molecule for fluconazole. In TIMM 3164 and 3165, the ergosterol synthesis by cell-free extracts was somewhat less susceptible to fluconazole, due to a decrease in fluconazole affinity for CYP. The nucleotide substitutions in the ERG11 gene were identified to result in three amino acid changes of K143R, E266D and V488I in TIMM 3164, and of E266D, V404L and V488I in TIMM 3165. These amino acid substitutions might contribute to the decreased affinity for CYP in both isolates. However, a single amino acid change, E266D, observed in TIMM 3166 was unrelated to the decreased affinity for CYP. The most prominent finding on the ultrastructure of TIMM 3164 and 3165 was the development of mesh membrane structures of the endoplasmic reticula, which is a location related to sterol synthesis. This phenomenon was not observed in the cells of TIMM 3166 or the susceptible control strains of ATCC 90028 and 10231. In addition to the reduced intracellular accumulation, the decreased affinity of fluconazole for CYP in TIMM 3164 and 3165 is assumed to be associated with the fluconazole-resistance phenotype.     

ABC transporters coupled with the elevated ergosterol contents contribute to the azole resistance and amphotericin B susceptibility

Most screening approaches produce compounds that target survival genes and are likely to generate resistance over time. Simply having more drugs does not address the potential emergence of resistance caused by target mutation, drug efflux pumps over-expression, and so on. There is a great need to explore new strategies to treat fungal infections caused by drug-resistant pathogens. In this study, we found that azole-resistant Candida albicans with CaCDR1 and CaCDR2 over-expression is hypersensitive against amphotericin B (AmB) by our high throughput synergy screening (HTSS). In contrast, Δcdr1 and Δcdr2 knockout strains were resistant to AmB. Moreover, clinical isolates with increased expression of CaCDR1 and CaCDR2 demonstrated susceptibility to AmB, which can also synergize with the efflux pumps inducer fluphenazine (FPZ). Finally, the increased drug susceptibility to AmB in azole-resistant C. albicans with drug efflux pumps over-expression was consistent with the elevated expression of CaERG11 and its associated ergosterols in clinical isolates. Our data implies that the level of ergosterol contents determines the susceptibility to azoles and AmB in C. albicans. Deep understanding of the above mechanisms would offer new hope to treat drug-resistant C. albicans.     

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.     

Synergistic Interactions of Eugenol-tosylate and Its Congeners with Fluconazole against Candida albicans

We previously reported the antifungal properties of a monoterpene phenol “Eugenol” against different Candida strains and have observed that the addition of methyl group to eugenol drastically increased its antimicrobial potency. Based on the results and the importance of medicinal synthetic chemistry, we synthesized eugenol-tosylate and its congeners (E1-E6) and tested their antifungal activity against different clinical fluconazole (FLC)- susceptible and FLC- resistant C. albicans isolates alone and in combination with FLC by determining fractional inhibitory concentration indices (FICIs) and isobolograms calculated from microdilution assays. Minimum inhibitory concentration (MIC) results confirmed that all the tested C. albicans strains were variably susceptible to the semi-synthetic derivatives E1-E6, with MIC values ranging from 1–62 μg/ml. The test compounds in combination with FLC exhibited either synergy (36%), additive (41%) or indifferent (23%) interactions, however, no antagonistic interactions were observed. The MICs of FLC decreased 2–9 fold when used in combination with the test compounds. Like their precursor eugenol, all the derivatives showed significant impairment of ergosterol biosynthesis in all C. albicans strains coupled with down regulation of the important ergosterol biosynthesis pathway gene-ERG11. The results were further validated by docking studies, which revealed that the inhibitors snugly fitting the active site of the target enzyme, mimicking fluconazole, may well explain their excellent inhibitory activity. Our results suggest that these compounds have a great potential as antifungals, which can be used as chemosensitizing agents with the known antifungal drugs.