An Azole-Tolerant Endosomal Trafficking Mutant of Candida albicans Is Susceptible to Azole Treatment in a Mouse Model of Vaginal Candidiasis

ABSTRACT We recently reported that a Candida albicans endosomal trafficking mutant continues to grow after treatment with the azole antifungals. Herein, we report that the vps21Δ/Δ mutant does not have a survival advantage over wild-type isolates after fluconazole treatment in a mouse model of vaginal candidiasis. Furthermore, loss of VPS21 does not synergize with established mechanisms of azole resistance, such as overexpression of efflux pumps or of Erg11p, the target enzyme of the azoles. In summary, although loss of VPS21 function enhances C. albicans survival after azole treatment in vitro, it does not seem to affect azole susceptibility in vivo.

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Toward Harmonization of Voriconazole CLSI and EUCAST Breakpoints for Candida albicans Using a Validated In Vitro Pharmacokinetic/Pharmacodynamic Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00170-20 ◽

2020 ◽

Vol 64 (6) ◽

Author(s):

Maria-Ioanna Beredaki ◽

Panagiota-Christina Georgiou ◽

Maria Siopi ◽

Lamprini Kanioura ◽

David Andes ◽

...

Keyword(s):

Candida Albicans ◽

Simulation Analysis ◽

Maximal Activity ◽

Wild Type ◽

Time Curve ◽

Unbound Fraction ◽

Content Type ◽

Interval Probability

ABSTRACT CLSI and EUCAST susceptibility breakpoints for voriconazole and Candida albicans differ by one dilution (≤0.125 and ≤0.06 mg/liter, respectively) whereas the epidemiological cutoff values for EUCAST (ECOFF) and CLSI (ECV) are the same (0.03 mg/liter). We therefore determined the pharmacokinetic/pharmacodynamic (PK/PD) breakpoints of voriconazole against C. albicans for both methodologies with an in vitro PK/PD model, which was validated using existing animal PK/PD data. Four clinical wild-type and non-wild-type C. albicans isolates (voriconazole MICs, 0.008 to 0.125 mg/liter) were tested in an in vitro PK/PD model. For validation purposes, mouse PK were simulated and in vitro PD were compared with in vivo outcomes. Human PK were simulated, and the exposure-effect relationship area under the concentration-time curve for the free, unbound fraction of a drug from 0 to 24 h (fAUC0–24)/MIC was described for EUCAST and CLSI 24/48-h methods. PK/PD breakpoints were determined using the fAUC0–24/MIC associated with half-maximal activity (EI50) and Monte Carlo simulation analysis. The in vitro 24-h PD EI50 values of voriconazole against C. albicans were 2.5 to 5 (1.5 to 17) fAUC/MIC. However, the 72-h PD were higher at 133 (51 to 347) fAUC/MIC for EUCAST and 94 (35 to 252) fAUC/MIC for CLSI. The mean (95% confidence interval) probability of target attainment (PTA) was 100% (95 to 100%), 97% (72 to 100%), 83% (35 to 99%), and 49% (8 to 91%) for EUCAST and 100% (97 to 100%), 99% (85 to 100%), 91% (52 to 100%), and 68% (17 to 96%) for CLSI for MICs of 0.03, 0.06, 0.125, and 0.25 mg/liter, respectively. Significantly, >95% PTA values were found for EUCAST/CLSI MICs of ≤0.03 mg/liter. For MICs of 0.06 to 0.125 mg/liter, trough levels 1 to 4 mg/liter would be required to attain the PK/PD target. A PK/PD breakpoint of C. albicans voriconazole at the ECOFF/ECV of 0.03 mg/liter was determined for both the EUCAST and CLSI methods, indicating the need for breakpoint harmonization for the reference methodologies.

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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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Loss of Sigma Factor RpoN Increases Intestinal Colonization of Vibrio parahaemolyticus in an Adult Mouse Model

Infection and Immunity ◽

10.1128/iai.01210-13 ◽

2013 ◽

Vol 82 (2) ◽

pp. 544-556 ◽

Cited By ~ 19

Author(s):

W. Brian Whitaker ◽

Gary P. Richards ◽

E. Fidelma Boyd

Keyword(s):

Mouse Model ◽

Sigma Factor ◽

Vibrio Parahaemolyticus ◽

Wild Type ◽

Carbon Utilization ◽

Content Type ◽

Wide Range ◽

Competition Assays

ABSTRACTVibrio parahaemolyticusis the leading cause of bacterial seafood-borne gastroenteritis worldwide, yet little is known about how this pathogen colonizes the human intestine. The alternative sigma factor RpoN/sigma-54 is a global regulator that controls flagellar synthesis, as well as a wide range of nonflagellar genes. We constructed an in-frame deletion mutation inrpoN(VP2670) inV. parahaemolyticusRIMD2210633, a clinical serogroup O3:K6 isolate, and examined the effectsin vivousing a streptomycin-treated mouse model of colonization. We confirmed that deletion ofrpoNrenderedV. parahaemolyticusnonmotile, and it caused reduced biofilm formation and an apparent defect in glutamine synthetase production. Inin vivocompetition assays between therpoNmutant and a wild-type RIMD2210633 strain marked with the β-galactosidase genelacZ(WBWlacZ), the mutant colonized significantly more proficiently. Intestinal persistence competition assays also demonstrated that therpoNmutant had enhanced fitness and outcompeted WBWlacZ. Mutants defective in the polar flagellum biosynthesis FliAP sigma factor also outcompeted WBWlacZ but not to the same level as therpoNmutant, which suggested that lack of motility is not the sole cause of the fitness effect. In anin vitrogrowth competition assay in mouse intestinal mucus, therpoNmutant also outcompeted the wild type and exhibited faster doubling times when grown in mucus and on individual components of mucus. Genes in the pathways for the catabolism of mucus sugars also had significantly higher expression levels in a ΔrpoNmutant than in the wild type. These data suggest that inV. parahaemolyticus, RpoN plays an important role in carbon utilization regulation, which may significantly affect host colonization.

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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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Vaginal Epithelial Cell-Derived S100 Alarmins Induced by Candida albicans via Pattern Recognition Receptor Interactions Are Sufficient but Not Necessary for the Acute Neutrophil Response during Experimental Vaginal Candidiasis

Infection and Immunity ◽

10.1128/iai.00861-13 ◽

2013 ◽

Vol 82 (2) ◽

pp. 783-792 ◽

Cited By ~ 38

Author(s):

Junko Yano ◽

Glen E. Palmer ◽

Karen E. Eberle ◽

Brian M. Peters ◽

Thomas Vogl ◽

...

Keyword(s):

Pattern Recognition ◽

Candida Albicans ◽

Epithelial Cells ◽

Biologically Active ◽

Polymorphonuclear Neutrophil ◽

Vaginal Candidiasis ◽

Content Type ◽

Vaginal Epithelial Cells

ABSTRACTVulvovaginal candidiasis (VVC), caused byCandida albicans, affects women worldwide. Animal and clinical studies suggest that the immunopathogenic inflammatory condition of VVC is initiated by S100 alarmins in response toC. albicans, which stimulate polymorphonuclear neutrophil (PMN) migration to the vagina. The purpose of this study was to extend previousin vitrodata and determine the requirement for the alarmin S100A8 in the PMN response and to evaluate pattern recognition receptors (PRRs) that initiate the response. For the former, PMN migration was evaluatedin vitroorin vivoin the presence or absence of S100 alarmins initiated by several approaches. For the latter, vaginal epithelial cells were evaluated for PRR expression andC. albicans-induced S100A8 and S100A9 mRNAs, followed by evaluation of the PMN response in inoculated PRR-deficient mice. Results revealed that, consistent with previously reportedin vitrodata, eukaryote-derived S100A8, but not prokaryote-derived recombinant S100A8, induced significant PMN chemotaxisin vivo. Conversely, a lack of biologically active S100A8 alarmin, achieved by antibody neutralization or by using S100A9−/−mice, had no effect on the PMN responsein vivo. In PRR analyses, whereas Toll-like receptor 4 (TLR4)- and SIGNR1-deficient vaginal epithelial cells showed a dramatic reduction inC. albicans-induced S100A8/S100A9 mRNAsin vitro, inoculated mice deficient in these PRRs showed PMN migration similar to that in wild-type controls. These results suggest that S100A8 alarmin is sufficient, but not necessary, to induce PMN migration during VVC and that the vaginal PMN response toC. albicansinvolves PRRs in addition to SIGNR1 and TLR4, or other induction pathways.

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The Novel Arylamidine T-2307 MaintainsIn VitroandIn VivoActivity against Echinocandin-Resistant Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04228-14 ◽

2014 ◽

Vol 59 (2) ◽

pp. 1341-1343 ◽

Cited By ~ 14

Author(s):

Nathan P. Wiederhold ◽

Laura K. Najvar ◽

Annette W. Fothergill ◽

Rosie Bocanegra ◽

Marcos Olivo ◽

...

Keyword(s):

Body Weight ◽

Candida Albicans ◽

Invasive Candidiasis ◽

Placebo Control ◽

The Novel ◽

Content Type ◽

Fungal Burden ◽

Potential Use

ABSTRACTWe evaluated thein vitroandin vivoactivities of the investigational arylamidine T-2307 against echinocandin-resistantCandida albicans. T-2307 demonstrated potentin vitroactivity, and daily subcutaneous doses between 0.75 and 6 mg/kg of body weight significantly improved survival and reduced fungal burden compared to placebo control and caspofungin (10 mg/kg/day) in mice with invasive candidiasis caused by an echinocandin-resistant strain. Thus, T-2307 may have potential use in the treatment of echinocandin-resistantC. albicansinfections.

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Antimicrobial Photodynamic Inactivation Inhibits Candida albicans Virulence Factors and ReducesIn VivoPathogenicity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01451-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 445-451 ◽

Cited By ~ 41

Author(s):

Ilka Tiemy Kato ◽

Renato Araujo Prates ◽

Caetano Padial Sabino ◽

Beth Burgwyn Fuchs ◽

George P. Tegos ◽

...

Keyword(s):

Candida Albicans ◽

Virulence Factors ◽

Sodium Dodecyl ◽

Systemic Infection ◽

Tube Formation ◽

Photodynamic Inactivation ◽

Content Type ◽

Daughter Cells

ABSTRACTThe objective of this study was to evaluate whetherCandida albicansexhibits altered pathogenicity characteristics following sublethal antimicrobial photodynamic inactivation (APDI) and if such alterations are maintained in the daughter cells.C. albicanswas exposed to sublethal APDI by using methylene blue (MB) as a photosensitizer (0.05 mM) combined with a GaAlAs diode laser (λ 660 nm, 75 mW/cm2, 9 to 27 J/cm2).In vitro, we evaluated APDI effects onC. albicansgrowth, germ tube formation, sensitivity to oxidative and osmotic stress, cell wall integrity, and fluconazole susceptibility.In vivo, we evaluatedC. albicanspathogenicity with a mouse model of systemic infection. Animal survival was evaluated daily. Sublethal MB-mediated APDI reduced the growth rate and the ability ofC. albicansto form germ tubes compared to untreated cells (P< 0.05). Survival of mice systemically infected withC. albicanspretreated with APDI was significantly increased compared to mice infected with untreated yeast (P< 0.05). APDI increasedC. albicanssensitivity to sodium dodecyl sulfate, caffeine, and hydrogen peroxide. The MIC for fluconazole forC. albicanswas also reduced following sublethal MB-mediated APDI. However, none of those pathogenic parameters was altered in daughter cells ofC. albicanssubmitted to APDI. These data suggest that APDI may inhibit virulence factors and reducein vivopathogenicity ofC. albicans. The absence of alterations in daughter cells indicates that APDI effects are transitory. The MIC reduction for fluconazole following APDI suggests that this antifungal could be combined with APDI to treatC. albicansinfections.

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Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

Infection and Immunity ◽

10.1128/iai.05773-11 ◽

2011 ◽

Vol 80 (1) ◽

pp. 3-13 ◽

Cited By ~ 27

Author(s):

Chen Li ◽

Kurniyati ◽

Bo Hu ◽

Jiang Bian ◽

Jianlan Sun ◽

...

Keyword(s):

Porphyromonas Gingivalis ◽

Biofilm Formation ◽

Adult Population ◽

Transcriptional Analysis ◽

Wild Type ◽

Content Type ◽

Multiple Organs ◽

Biochemical Analyses

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

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Reversal of Azole Resistance inCandida albicansby Sulfa Antibacterial Drugs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00701-17 ◽

2017 ◽

Vol 62 (3) ◽

Cited By ~ 15

Author(s):

Hassan E. Eldesouky ◽

Abdelrahman Mayhoub ◽

Tony R. Hazbun ◽

Mohamed N. Seleem

Keyword(s):

Treatment Options ◽

Antifungal Drugs ◽

Infection Model ◽

Azole Resistance ◽

Global Public Health ◽

Sulfa Drugs ◽

Antibacterial Drugs ◽

Content Type

ABSTRACTInvasive candidiasis presents an emerging global public health challenge due to the emergence of resistance to the frontline treatment options, such as fluconazole. Hence, the identification of other compounds capable of pairing with fluconazole and averting azole resistance would potentially prolong the clinical utility of this important group. In an effort to repurpose drugs in the field of antifungal drug discovery, we explored sulfa antibacterial drugs for the purpose of reversing azole resistance inCandida. In this study, we assembled and investigated a library of 21 sulfa antibacterial drugs for their ability to restore fluconazole sensitivity inCandida albicans. Surprisingly, the majority of assayed sulfa drugs (15 of 21) were found to exhibit synergistic relationships with fluconazole by checkerboard assay with fractional inhibitory concentration index (ΣFIC) values ranging from <0.0312 to 0.25. Remarkably, five sulfa drugs were able to reverse azole resistance in a clinically achievable range. The structure-activity relationships (SARs) of the amino benzene sulfonamide scaffold as antifungal agents were studied. We also identified the possible mechanism of the synergistic interaction of sulfa antibacterial drugs with azole antifungal drugs. Furthermore, the ability of sulfa antibacterial drugs to inhibitCandidabiofilm by 40%in vitrowas confirmed. In addition, the effects of sulfa-fluconazole combinations onCandidagrowth kinetics and efflux machinery were explored. Finally, using aCaenorhabditis elegansinfection model, we demonstrated that the sulfa-fluconazole combination does possess potent antifungal activityin vivo, reducingCandidain infected worms by ∼50% compared to the control.

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Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

10.1101/2021.04.05.438547 ◽

2021 ◽

Author(s):

Fatima Amanat ◽

Shirin Strohmeier ◽

Wen-Hsin Lee ◽

Sandhya Bangaru ◽

Andrew B Ward ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Dependent Manner ◽

Receptor Binding Domain ◽

Wild Type ◽

Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

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