Comparative pathogenicity of auxotrophic mutants of Candida albicans

An induced mutant of Candida albicans with greatly decreased virulence for mice is described. The mutant was one of five auxotrophic mutants obtained by ultraviolet irradiation of a clinical isolate (strain MY 1044). The five mutants included two methionine auxotrophs, one methionine–cysteine auxotroph, one temperature-sensitive serine auxotroph, and one auxotroph with unknown growth requirements. Each of the mutants produced normal mycelium and had a normal profile of susceptibility to four antifungal drugs. The virulence of each mutant was compared with the parent strain by LD50 determination in mice. Four of the five auxotrophs exhibited LD50's that were not significantly different from the parent strain (mean LD50 = 7.5 × 105 cells). However, the temperature-sensitive serine auxotroph was significantly less virulent than the parent strain (LD50 > 107 cells), even though it grew well in vivo and in mouse serum at 37 °C in vitro. Use of this mutant in conjunction with its "isogenic" parent should help to elucidate true virulence factors in C. albicans.

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Azole Resistance by Loss of Function of the Sterol Δ5,6-Desaturase Gene (ERG3) in Candida albicans Does Not Necessarily Decrease Virulence

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05720-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 1960-1968 ◽

Cited By ~ 53

Author(s):

L. A. Vale-Silva ◽

A. T. Coste ◽

F. Ischer ◽

J. E. Parker ◽

S. L. Kelly ◽

...

Keyword(s):

Candida Albicans ◽

Premature Stop Codon ◽

Antifungal Drugs ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Wild Type ◽

Disseminated Candidiasis ◽

Double Base

ABSTRACTThe inactivation ofERG3, a gene encoding sterol Δ5,6-desaturase (essential for ergosterol biosynthesis), is a known mechanism ofin vitroresistance to azole antifungal drugs in the human pathogenCandida albicans. ERG3inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified aC. albicansclinical isolate (VSY2) with high-level resistance to azole drugsin vitroand an absence of ergosterol but normal filamentation. Sequencing ofERG3in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (erg3-1) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming thatERG3inactivation was the mechanism of azole resistance. Additionally, the replacement of bothERG3alleles byerg3-1in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinicalERG3mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole bothin vitroandin vivo, theERG3-derived mutant of SC5314 was resistant onlyin vitroand was less virulent than the wild type. This suggests that VSY2 compensated for thein vivofitness defect ofERG3inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation ofERG3does not necessarily affect filamentation and virulence.

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Effect of Prolonged Fluconazole Treatment on Candida albicans in Diffusion Chambers Implanted into Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.10.3175-3179.2002 ◽

2002 ◽

Vol 46 (10) ◽

pp. 3175-3179

Author(s):

Peter G. Sohnle ◽

Beth L. Hahn

Keyword(s):

Candida Albicans ◽

Inflammatory Response ◽

Inflammatory Cells ◽

Model System ◽

Antifungal Drugs ◽

Yeast Cells ◽

In Vitro Susceptibility ◽

Bonferroni Test

ABSTRACT Fluconazole is an azole agent with primarily fungistatic activity in standard in vitro susceptibility tests. The present study was undertaken to develop a diffusion chamber model system in mice in order to study the in vivo effects of prolonged fluconazole treatment on Candida albicans. Chambers containing 100 C. albicans yeast cells were implanted subcutaneously on the flanks of C57BL/6 mice and were then retrieved 6 or 14 weeks later (after fluconazole treatment for 4 or 12 weeks, respectively). Leukocyte counts demonstrated that implantation of the chambers did elicit an inflammatory response but that only small numbers of inflammatory cells were able to enter the chamber interior. Treatment with fluconazole at 10 mg/kg of body weight/day for 12 weeks not only reduced the numbers of viable organisms within the chambers compared to those in untreated mice (mean ± standard deviation of log10 CFU of 0.7 ± 1.2 versus 2.3 ± 2.0; P < 0.001 by the Bonferroni test) but also increased the numbers of chambers that became sterile over the treatment period (14 of 16 versus 6 of 19; P = 0.0009 by the chi-square test). However, treatment for only 4 weeks had minimal effects on the numbers of chamber CFU, and none of the chambers became sterile during this period. Distribution of retrieved organisms between interior fluid and the chamber filters was approximately equal in all the treatment groups. This model system appears to be useful for evaluating the effects of antifungal drugs over prolonged periods in vivo. Its use in the present study demonstrates that fluconazole can increase the rate of sterilization of C. albicans foci that are protected from the host's inflammatory response.

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Candida albicans PEP12 Is Required for Biofilm Integrity and In Vivo Virulence

Eukaryotic Cell ◽

10.1128/ec.00295-09 ◽

2009 ◽

Vol 9 (2) ◽

pp. 266-277 ◽

Cited By ~ 25

Author(s):

Suresh K. A. Palanisamy ◽

Melissa A. Ramirez ◽

Michael Lorenz ◽

Samuel A. Lee

Keyword(s):

Candida Albicans ◽

Lipolytic Activity ◽

Infection Model ◽

Temperature Sensitive ◽

Null Mutant ◽

Macrophage Infection ◽

Content Type ◽

Vacuolar Acidification

ABSTRACT To investigate the role of the prevacuolar secretion pathway in biofilm formation and virulence in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae prevacuolar trafficking gene PEP12. C. albicans PEP12 encodes a deduced t-SNARE that is 28% identical to S. cerevisiae Pep12p, and plasmids bearing C. albicans PEP12 complemented the abnormal vacuolar morphology and temperature-sensitive growth of an S. cerevisiae pep12 null mutant. The C. albicans pep12 Δ null mutant was defective in endocytosis and vacuolar acidification and accumulated 40- to 60-nm cytoplasmic vesicles near the plasma membrane. Secretory defects included increased extracellular proteolytic activity and absent lipolytic activity. The pep12Δ null mutant was more sensitive to cell wall stresses and antifungal agents than the isogenic complemented strain or the control strain DAY185. Notably, the biofilm formed by the pep12Δ mutant was reduced in overall mass and fragmented completely upon the slightest disturbance. The pep12Δ mutant was markedly reduced in virulence in an in vitro macrophage infection model and an in vivo mouse model of disseminated candidiasis. These results suggest that C. albicans PEP12 plays a key role in biofilm integrity and in vivo virulence.

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Distinct Roles of Candida albicans Drug Resistance Transcription FactorsTAC1,MRR1, andUPC2in Virulence

Eukaryotic Cell ◽

10.1128/ec.00245-13 ◽

2013 ◽

Vol 13 (1) ◽

pp. 127-142 ◽

Cited By ~ 48

Author(s):

Andrea Lohberger ◽

Alix T. Coste ◽

Dominique Sanglard

Keyword(s):

Candida Albicans ◽

Target Genes ◽

Negative Impact ◽

Antifungal Drugs ◽

Wild Type ◽

Drug Pressure ◽

Content Type ◽

Negative Effect

ABSTRACTAzoles are widely used in antifungal therapy in medicine. Resistance to azoles can occur inCandida albicansprincipally by overexpression of multidrug transporter geneCDR1,CDR2, orMDR1or by overexpression ofERG11, which encodes the azole target. The expression of these genes is controlled by the transcription factors (TFs)TAC1(involved in the control ofCDR1andCDR2),MRR1(involved in the control ofMDR1), andUPC2(involved in the control ofERG11). Several gain-of-function (GOF) mutations are present in hyperactive alleles of these TFs, resulting in the overexpression of target genes. While these mutations are beneficial toC. albicanssurvival in the presence of the antifungal drugs, their effects could potentially alter the fitness and virulence ofC. albicansin the absence of the selective drug pressure. In this work, the effect of GOF mutations onC. albicansvirulence was addressed in a systemic model of intravenous infection by mouse survival and kidney fungal burden assays. We engineered a set of strains with identical genetic backgrounds in which hyperactive alleles were reintroduced in one or two copies at their genomic loci. The results obtained showed that neitherTAC1norMRR1GOF mutations had a significant effect onC. albicansvirulence. In contrast, the presence of two hyperactiveUPC2alleles inC. albicansresulted in a significant decrease in virulence, correlating with diminished kidney colonization compared to that by the wild type. In agreement with the effect on virulence, the decreased fitness of an isolate withUPC2hyperactive alleles was observed in competition experiments with the wild typein vivobut notin vitro. Interestingly,UPC2hyperactivity delayed filamentation ofC. albicansafter phagocytosis by murine macrophages, which may at least partially explain the virulence defects. Combining theUPC2GOF mutation with another hyperactive TF did not compensate for the negative effect ofUPC2on virulence. In conclusion, among the major TFs involved in azole resistance, onlyUPC2had a negative impact on virulence and fitness, which may therefore have consequences for the epidemiology of antifungal resistance.

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Repositioning Lopinavir, an HIV Protease Inhibitor, as a Promising Antifungal Drug: Lessons Learned from Candida albicans—In Silico, In Vitro and In Vivo Approaches

Journal of Fungi ◽

10.3390/jof7060424 ◽

2021 ◽

Vol 7 (6) ◽

pp. 424

Author(s):

André L. S. Santos ◽

Lys A. Braga-Silva ◽

Diego S. Gonçalves ◽

Lívia S. Ramos ◽

Simone S. C. Oliveira ◽

...

Keyword(s):

Candida Albicans ◽

Protease Inhibitor ◽

In Silico ◽

Neutral Lipids ◽

Hydrolytic Enzymes ◽

Lessons Learned ◽

Antifungal Drugs ◽

Dependent Manner

The repurposing strategy was applied herein to evaluate the effects of lopinavir, an aspartic protease inhibitor currently used in the treatment of HIV-infected individuals, on the globally widespread opportunistic human fungal pathogen Candida albicans by using in silico, in vitro and in vivo approaches in order to decipher its targets on fungal cells and its antifungal mechanisms of action. Secreted aspartic proteases (Saps) are the obviously main target of lopinavir. To confirm this hypothesis, molecular docking assays revealed that lopinavir bound to the Sap2 catalytic site of C. albicans as well as inhibited the Sap hydrolytic activity in a typically dose-dependent manner. The inhibition of Saps culminated in the inability of C. albicans yeasts to assimilate the unique nitrogen source (albumin) available in the culture medium, culminating with fungal growth inhibition (IC50 = 39.8 µM). The antifungal action of lopinavir was corroborated by distinct microscopy analyses, which evidenced drastic and irreversible changes in the morphology that justified the fungal death. Furthermore, our results revealed that lopinavir was able to (i) arrest the yeasts-into-hyphae transformation, (ii) disturb the synthesis of neutral lipids, including ergosterol, (iii) modulate the surface-located molecules, such as Saps and mannose-, sialic acid- and N-acetylglucosamine-containing glycoconjugates, (iv) diminish the secretion of hydrolytic enzymes, such as Saps and esterase, (v) negatively influence the biofilm formation on polystyrene surface, (vi) block the in vitro adhesion to epithelial cells, (vii) contain the in vivo infection in both immunocompetent and immunosuppressed mice and (viii) reduce the Sap production by yeasts recovered from kidneys of infected animals. Conclusively, the exposed results highlight that lopinavir may be used as a promising repurposing drug against C. albicans infection as well as may be used as a lead compound for the development of novel antifungal drugs.

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Multi-epitope Peptide Vaccine Prediction for Candida albicans targeting Pyruvate Kinase Protein; an Immunoinformatics Approach

10.1101/758920 ◽

2019 ◽

Author(s):

Anfal Osama Mohamed Sati ◽

Abdelrahman Hamza Abdelmoneim Hamza ◽

Enas Dawoud Khairi Dawoud ◽

Tebyan Ameer Abdelhameed Abbas ◽

Fatima Abdelrahman Bshier Abdelrahman ◽

...

Keyword(s):

Candida Albicans ◽

Pyruvate Kinase ◽

Homology Modelling ◽

Peptide Vaccine ◽

Antifungal Drugs ◽

Multiple Sequence ◽

Mhc Molecules ◽

Wide Range

AbstractThe fungus Candida albicans is an opportunistic pathogen that causes a wide range of infections. It’s the primary cause of candidiasis and the fourth most common cause of nosocomial infection. In addition, disseminated invasive candidiasis which is a major complication of the disease has an estimated mortality rate of 40%-60% even with the use of antifungal drugs. Over the last decades, several different anti-Candida vaccines have been suggested with different strategies for immunization against candidiasis such as, live-attenuated fungi, recombinant proteins, and glycoconjugates but none has been approved by the FDA, yet. This study aims to introduce a new possible vaccine for C. albicans through analyzing peptides of its pyruvate kinase (PK) protein as an immunogenic stimulant computationally.A total number of 28 C. albicans, pyruvate kinase proteins were obtained from NCBI on the 9th of February 2019 and were subjected to multiple sequence alignment using Bioedit for conservancy. The main analytical tool was IEDB, Chimera for homology modelling, and MOE for docking.Among the tested peptides, fifteen promising T-cell peptides were predicted. Five peptides were more important than the others (HMIFASFIR, YRGVYPFIY, AVAAVSAAY, LRWAVSEAV, and IFASFIRTA) They show high Binding Affinity to MHC molecules, low binding energy required indicating more stable bonds, and their ideal length of nine peptides. (PTRAEVSDV) peptide is the most promising linear B-cell peptide due to its physiochemical parameters and optimal length (nine amino acids). It’s highly recommended to have these five strong candidates in future in vivo and in vitro analysis studies.

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Lactobacillus rhamnosusLcr35®as an effective treatment for preventingCandida albicansinfection in preclinical models: first mechanistical insights

10.1101/612481 ◽

2019 ◽

Author(s):

Cyril Poupet ◽

Taous Saraoui ◽

Philippe Veisseire ◽

Muriel Bonnet ◽

Caroline Dausset ◽

...

Keyword(s):

Candida Albicans ◽

Lactobacillus Rhamnosus ◽

Probiotic Strain ◽

Mapk Signaling ◽

Antifungal Drugs ◽

Laboratory Animals ◽

Preclinical Models ◽

C Elegans

AbstractThe increased recurrence ofCandida albicansinfections is associated with greater resistance to antifungal drugs. This involves the establishment of alternative therapeutic protocols such as the probiotic microorganisms whose antifungal potential has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding the mechanisms of action of probiotic microorganisms has become a strategic need for the development of new therapeutics for humans. In this study, we investigated the prophylactic anti-Candida albicansproperties ofLactobacillus rhamnosusLcr35®using thein vitroCaco-2 cells model and thein vivo Caenorhabditis elegansmodel. On Caco-2 cells, we showed that the strain Lcr35®significantly inhibited the growth of the pathogen (~2 log CFU.mL−1) and its adhesion (150 to 6,300 times less). Moreover, on the top of having a prolongevity activity in the nematode, Lcr35®protects the animal from the fungal infection even if the yeast is still detectable in its intestine. At the mechanistic level, we noticed the repression of genes of the p38 MAPK signaling pathway and genes involved in the antifungal response induced by Lcr35®suggesting that the pathogen no longer appears to be detected by the worm immune system. However, the DAF-16 / FOXO transcription factor, implicated in the longevity and antipathogenic response ofC. elegans, is activated by Lcr35®. These results suggest that the probiotic strain acts by stimulating its host via DAF-16, but also by suppressing the virulence of the pathogen.

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In VitroActivity of Miltefosine against Candida albicans under Planktonic and Biofilm Growth Conditions andIn VivoEfficacy in a Murine Model of Oral Candidiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01890-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7611-7620 ◽

Cited By ~ 30

Author(s):

Taissa Vieira Machado Vila ◽

Ashok K. Chaturvedi ◽

Sonia Rozental ◽

Jose L. Lopez-Ribot

Keyword(s):

Candida Albicans ◽

Murine Model ◽

Biofilm Formation ◽

Pathogenic Fungus ◽

Oral Candidiasis ◽

Antifungal Drugs ◽

Drug Candidate ◽

Content Type

ABSTRACTThe generation of a new antifungal againstCandida albicansbiofilms has become a major priority, since biofilm formation by this opportunistic pathogenic fungus is usually associated with an increased resistance to azole antifungal drugs and treatment failures. Miltefosine is an alkyl phospholipid with promising antifungal activity. Here, we report that, when tested under planktonic conditions, miltefosine displays potentin vitroactivity against multiple fluconazole-susceptible and -resistantC. albicansclinical isolates, including isolates overexpressing efflux pumps and/or with well-characterized Erg11 mutations. Moreover, miltefosine inhibitsC. albicans biofilm formation and displays activity against preformed biofilms. Serial passage experiments confirmed that miltefosine has a reduced potential to elicit resistance, and screening of a library ofC. albicanstranscription factor mutants provided additional insight into the activity of miltefosine againstC. albicansgrowing under planktonic and biofilm conditions. Finally, we demonstrate thein vivoefficacy of topical treatment with miltefosine in the murine model of oropharyngeal candidiasis. Overall, our results confirm the potential of miltefosine as a promising antifungal drug candidate, in particular for the treatment of azole-resistant and biofilm-associated superficial candidiasis.

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