Persistent Candida albicans colonization and molecular mechanisms of azole resistance in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients.

Objectives: Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS-I) suffer from chronic candidosis caused mainly by Candida albicans, and repeated courses of azole antifungals have led to the development of resistance in the APECED patient population in Finland. The aim of our study was to address whether the patients are persistently colonized with the same or genetically closely related strains, whether epidemic strains are present and which molecular mechanisms account for azole resistance. Methods: Sets of C. albicans (n?=?19) isolates from nine APECED patients reported with decreased susceptibility to fluconazole isolated up to 9 years apart were included. The strains were typed by multilocus sequence typing. CDR1/2, MDR1 and ERG11 mRNA expression was analysed by northern blotting and Cdr1, Cdr2 and Mdr1 protein expression by western blotting, and TAC1 and ERG11 genes were sequenced. Results: All seven patients with multiple C. albicans isolates analysed were persistently colonized with the same or a genetically closely related strain for a mean of 5 years. All patients were colonized with different strains and no epidemic strains were found. The major molecular mechanisms behind the azole resistance were mutations in TAC1 contributing to overexpression of CDR1 and CDR2. Six new TAC1 mutations were found, one of which (N740S) is likely to be a gain-of-function mutation. Most isolates were found to have gained multiple TAC1 and ERG11 point mutations. Conclusions: Despite clinically successful treatment leading to relief of symptoms, colonization by C. albicans strains is persistent within APECED patients. Microevolution and point mutations occur within strains, leading to the development of azole-resistant isolates.

Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models.

The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt.

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis.

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa's ability to colonize the GI tract but does decrease P. aeruginosa's cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.

Stepwise emergence of azole, echinocandin and amphotericin B multidrug resistance in vivo in Candida albicans orchestrated by multiple genetic alterations.

OBJECTIVES: The objective of this study was to characterize the underlying molecular mechanisms in consecutive clinical Candida albicans isolates from a single patient displaying stepwise-acquired multidrug resistance. METHODS: Nine clinical isolates (P-1 to P-9) were susceptibility tested by EUCAST EDef 7.2 and Etest. P-4, P-5, P-7, P-8 and P-9 were available for further studies. Relatedness was evaluated by MLST. Additional genes were analysed by sequencing (including FKS1, ERG11, ERG2 and TAC1) and gene expression by quantitative PCR (CDR1, CDR2 and ERG11). UV-spectrophotometry and GC-MS were used for sterol analyses. In vivo virulence was determined in the insect model Galleria mellonella and evaluated by log-rank Mantel-Cox tests. RESULTS: P-1 + P-2 were susceptible, P-3 + P-4 fluconazole resistant, P-5 pan-azole resistant, P-6 + P-7 pan-azole and echinocandin resistant and P-8 + P-9 MDR. MLST supported genetic relatedness among clinical isolates. P-4 harboured four changes in Erg11 (E266D, G307S, G450E and V488I), increased expression of ERG11 and CDR2 and a change in Tac1 (R688Q). P-5, P-7, P-8 and P-9 had an additional change in Erg11 (A61E), increased expression of CDR1, CDR2 and ERG11 (except for P-7) and a different amino acid change in Tac1 (R673L). Echinocandin-resistant isolates harboured the Fks1 S645P alteration. Polyene-resistant P-8 + P-9 lacked ergosterol and harboured a frameshift mutation in ERG2 (F105SfsX23). Virulence was attenuated (but equivalent) in the clinical isolates, but higher than in the azole- and echinocandin-resistant unrelated control strain. CONCLUSIONS: C. albicans demonstrates a diverse capacity to adapt to antifungal exposure. Potentially novel resistance-inducing mutations in TAC1, ERG11 and ERG2 require independent validation.

RNA enrichment method for quantitative transcriptional analysis of pathogens in vivo applied to the fungus Candida albicans.

UNLABELLED: In vivo transcriptional analyses of microbial pathogens are often hampered by low proportions of pathogen biomass in host organs, hindering the coverage of full pathogen transcriptome. We aimed to address the transcriptome profiles of Candida albicans, the most prevalent fungal pathogen in systemically infected immunocompromised patients, during systemic infection in different hosts. We developed a strategy for high-resolution quantitative analysis of the C. albicans transcriptome directly from early and late stages of systemic infection in two different host models, mouse and the insect Galleria mellonella. Our results show that transcriptome sequencing (RNA-seq) libraries were enriched for fungal transcripts up to 1,600-fold using biotinylated bait probes to capture C. albicans sequences. This enrichment biased the read counts of only ~3% of the genes, which can be identified and removed based on a priori criteria. This allowed an unprecedented resolution of C. albicans transcriptome in vivo, with detection of over 86% of its genes. The transcriptional response of the fungus was surprisingly similar during infection of the two hosts and at the two time points, although some host- and time point-specific genes could be identified. Genes that were highly induced during infection were involved, for instance, in stress response, adhesion, iron acquisition, and biofilm formation. Of the in vivo-regulated genes, 10% are still of unknown function, and their future study will be of great interest. The fungal RNA enrichment procedure used here will help a better characterization of the C. albicans response in infected hosts and may be applied to other microbial pathogens. IMPORTANCE: Understanding the mechanisms utilized by pathogens to infect and cause disease in their hosts is crucial for rational drug development. Transcriptomic studies may help investigations of these mechanisms by determining which genes are expressed specifically during infection. This task has been difficult so far, since the proportion of microbial biomass in infected tissues is often extremely low, thus limiting the depth of sequencing and comprehensive transcriptome analysis. Here, we adapted a technology to capture and enrich C. albicans RNA, which was next used for deep RNA sequencing directly from infected tissues from two different host organisms. The high-resolution transcriptome revealed a large number of genes that were so far unknown to participate in infection, which will likely constitute a focus of study in the future. More importantly, this method may be adapted to perform transcript profiling of any other microbes during host infection or colonization.

Adaptation of a Gaussia princeps Luciferase reporter system in Candida albicans for in vivo detection in the Galleria mellonella infection model.

For the past 10 years, mini-host models and in particular the greater wax moth Galleria mellonella have tended to become a surrogate for murine models of fungal infection mainly due to cost, ethical constraints and ease of use. Thus, methods to better assess the fungal pathogenesis in G. mellonella need to be developed. In this study, we implemented the detection of Candida albicans cells expressing the Gaussia princeps luciferase in its cell wall in infected larvae of G. mellonella. We demonstrated that detection and quantification of luminescence in the pulp of infected larvae is a reliable method to perform drug efficacy and C. albicans virulence assays as compared to fungal burden assay. Since the linearity of the bioluminescent signal, as compared to the CFU counts, has a correlation of R(2) = 0.62 and that this method is twice faster and less labor intensive than classical fungal burden assays, it could be applied to large scale studies. We next visualized and followed C. albicans infection in living G. mellonella larvae using a non-toxic and water-soluble coelenterazine formulation and a CCD camera that is commonly used for chemoluminescence signal detection. This work allowed us to follow for the first time C. albicans course of infection in G. mellonella during 4 days.

Antifungal activity against planktonic and biofilm Candida albicans in an experimental model of foreign-body infection.

OBJECTIVES: The treatment of Candida implant-associated infections remains challenging. We investigated the antifungal activity against planktonic and biofilm Candida albicans in a foreign-body infection model. METHODS: Teflon cages were subcutaneously implanted in guinea pigs, infected with C. albicans (ATCC 90028). Animals were treated intraperitoneally 12 h after infection for 4 days once daily with saline, fluconazole (16 mg/kg), amphotericin B (2.5 mg/kg), caspofungin (2.5 mg/kg) or anidulafungin (20 mg/kg). Planktonic Candida was quantified, the clearance rate and cure rate determined. RESULTS: In untreated animals, planktonic Candida was cleared from cage fluid in 25% (infected with 4.5 × 10(3) CFU/cage), 8% (infected with 4.8 × 10(4) CFU/cage) and 0% (infected with 6.2 × 10(5) CFU/cage). Candida biofilm persisted on all explanted cages. Compared to untreated controls, caspofungin reduced the number of planktonic C. albicans to 0.22 and 0.0 CFU/ml, respectively, and anidulafungin to 0.11 and 0.13 CFU/ml, respectively. Fluconazole cured 2/12 cages (17%), amphotericin B and anidulafungin 1/12 cages (8%) and caspofungin 3/12 cages (25%). CONCLUSION: Echinocandins showed superior activity against planktonic C. albicans. Caspofungin showed the highest cure rate of C. albicans biofilm. However, no antifungal exceeded 25% cure rate, demonstrating the difficulty of eradicating Candida biofilms from implants.

Estudos de QSAR 2D baseados em descritores topológicos e fragmentos moleculares para uma série de derivados azólicos ativos contra Candida albicans

Azole derivatives are the main therapeutical resource against Candida albicans infection in immunocompromised patients. Nevertheless, the widespread use of azoles has led to reduced effectiveness and selection of resistant strains. In order to guide the development of novel antifungal drugs, 2D-QSAR models based on topological descriptors or molecular fragments were developed for a dataset of 74 molecules. The optimal fragment-based model (r² = 0.88, q² = 0.73 and r²pred = 0.62 with 6PCs) and descriptor-based model (r² = 0.82, q² = 0.79 and r²pred = 0.70 with 2 PCs), when analysed synergically, suggested that the triazolone ring and lipophilic properties are both important to antifungal activity.

Lactobacillus rhamnosus pode alterar a virulência de Candida albicans

OBJETIVO: Avaliar a influência de Lactobacillus rhamnosusna expressão dos fatores de virulência de Candida albicans in vitro.MÉTODOS: Uma suspensão de L. rhamnosusfoi inicialmente cultivada em ágar MRS. No dia seguinte, foi adicionado ágar Sabouraud dextrose sobre o crescimento dos lactobacilos e C. albicansfoi cultivada, por 24, 48 e 72 horas. As cepas de Candidaforam então isoladas para investigação da capacidade de formação de biofilme, por meio do cultivo em placas de 96 poços e leitufra das densidades ópticas e contagem de unidades formadoras de colônia por mL (UFC/mL). Também se investigou a capacidade de formação de tubo germinativo, após incubação em soro de cavalo e contagem em 200 células. Os resultados foram comparados aos observados nas cepas de Candidacultivadas na ausência de L. rhamnosus, utilizando o teste tde Student para análise estatística.RESULTADOS: Observou-se uma redução significativa no crescimento de C. albicans na presença de lactobacilos após 24, 48 ou 72 horas. Também foi observada redução significativa na formação de tubo germinativo após a interação por 48 ou 72 horas. Quanto à formação de biofilme, não foi observada diferença significante entre as cepas de Candidacultivadas na presença ou na ausência de lactobacilos.CONCLUSÃO: Os resultados sugerem que L. rhamnosusé capaz de influenciar significativamente o crescimento e a expressão de fatores de virulência de C. albicans in vitro, podendo interferir na patogenicidade desses micro-organismos.

Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans

The respiration, membrane potential (Dy), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 µM) in the presence of 0.05% BSA. Mitochondria in situ generated and sustained stable mitochondrial Dy respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 µM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 µM) inhibited respiration by 30% and 2 µM antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85%. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of Dy induced by 5 mM ATP and 0.5% BSA, and Dy decrease induced by 10 µM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death.

Trailing end-point phenotype antibiotic-sensitive strains of Candida albicans produce different amounts of aspartyl peptidases

Candida albicans is an opportunistic fungal pathogen that causes severe systemic infections in immunosuppressed individuals. C. albicans resistance to antifungal drugs is a severe problem in patients receiving prolonged therapy. Moreover, trailing yeast growth, which is defined as a resistant MIC after 48 h of incubation with triazole antifungal agents but a susceptible MIC after 24 h, has been noted in tests of antifungal susceptibility against some C. albicans isolates. In this context, we recently noticed this phenomenon in our routine susceptibility tests with fluconazole/itraconazole and C. albicans clinical isolates. In the present study, we investigated the production of cell-associated and secreted aspartyl peptidases (Saps) in six trailing clinical isolates of C. albicans, since this class of hydrolytic enzymes is a well-known virulence factor expressed by this fungal pathogen. Sap2, which is the best-studied member of the Sap family, was detected by flow cytometry on the cell surface of yeasts and as a 43-kDa polypeptide in the culture supernatant, as demonstrated by Western blotting assay using an anti-Sap1-3 polyclonal antibody. Released aspartyl peptidase activity was measured with BSA hydrolysis and inhibited by pepstatin A, showing distinct amounts of proteolytic activity ranging from 5.7 (strain 44B) to 133.2 (strain 11) arbitrary units. Taken together, our results showed that trailing clinical isolates of C. albicans produced different amounts of both cellular and secreted aspartyl-type peptidases, suggesting that this phenotypic feature did not generate a regular pattern regarding the expression of Sap.

Método inmunoenzimático para investigar antígenos de Candida albicans

Tesis (Maestría en Ciencias con Especialidad en Microbiología Médica) UANL

Efecto de hormonas esteroides sobre la micelización de Candida albicans.

Tesis (Maestría en Ciencias con Especialidad en Microbiología Médica) UANL