Kelley R. Healey

Fks1 and Fks2 Are Functionally Redundant but Differentially Regulated in Candida glabrata: Implications for Echinocandin Resistance

ABSTRACT The echinocandins caspofungin, micafungin, and anidulafungin, inhibitors of cell wall β-1,3-glucan synthesis, were recently elevated to first-line agents for treating infections due to the azole-refractory yeast Candida glabrata. In Candida albicans, echinocandin resistance is strictly associated with mutations in Fks1, a large integral membrane protein and putative β-1,3-glucan synthase, while mutations in both Fks1 and its paralog Fks2 (but not Fks3) have been associated with resistance in C. glabrata. To further explore their function, regulation, and role in resistance, C. glabrata fks genes were disrupted and subjected to mutational analysis, and their differential regulation was explored. An fks1Δ fks2Δ double disruptant was not able to be generated; otherwise, all three single and remaining two double disruptants displayed normal growth and echinocandin susceptibility, indicating Fks1-Fks2 redundancy. Selection on echinocandin-containing medium for resistant mutants was dependent on strain background: only fks1Δ and fks1Δ fks3Δ strains consistently yielded mutants exhibiting high-level resistance, all with Fks2 hot spot 1 mutations. Thus, Fks1-Fks2 redundancy attenuates the rate of resistance; further analysis showed that it also attenuates the impact of resistance-conferring mutations. Growth of the fks1Δ and, especially, fks1Δ fks3Δ strains was specifically susceptible to the calcineurin inhibitor FK506. Relatedly, FK506 addition or calcineurin gene CMP2 disruption specifically reversed Fks2-mediated resistance of laboratory mutants and clinical isolates. RNA analysis suggests that transcriptional control is not the sole mechanism by which calcineurin modulates Fks2 activity.

CRS–MIS in Candida glabrata: sphingolipids modulate echinocandin–Fks interaction

Infections with the azole‐refractory yeast Candida glabrata are now commonly treated with the echinocandins caspofungin (CSF) or micafungin (MCF). True resistance (> 32‐fold decreased susceptibility) to these lipopeptide inhibitors of cell wall synthesis is rare and strictly associated with mutations in integral membrane proteins Fks1 or Fks2. In contrast, mutants exhibiting 4‐ to 32‐fold CSF reduced susceptibility (CRS) were readily selected in vitro, and surprisingly demonstrated 4‐ to 32‐fold MCF increased susceptibility (MIS). Sequencing and gene deletion demonstrated that CRS–MIS is Fks‐independent. To explore alternative mechanisms, we initially employed Saccharomyces cerevisiae, and observed that CRS was conferred by multiple mutations (fen1Δ, sur4Δ, cka2Δ and tsc10‐ts) disrupting sphingolipid biosynthesis. Following this lead, C. glabrata fen1Δ and cka2Δ deletants were constructed, and shown to exhibit CRS–MIS. Sphingolipid analysis of CRS–MIS laboratory mutants and clinical isolates demonstrated elevated dihydrosphingosine (DHS) and phytosphingosine (PHS) levels, and consistent with this sequencing revealed fen1, sur4, ifa38 and sur2 mutations. Moreover, exogenous DHS or PHS conferred a CRS–MIS phenotype on wild‐type C. glabrata. Exogenous PHS failed, however, to suppress CRS–MIS in a sur2 mutant blocked in conversion of DHS to PHS, implying that accumulation of these intermediates confers CRS–MIS. We conclude that membrane sphingolipids modulate echinocandin–Fks interaction.

Candida glabrata Mutants Demonstrating Paradoxical Reduced Caspofungin Susceptibility but Increased Micafungin Susceptibility

ABSTRACT Echinocandins, including caspofungin (CSP) and micafungin (MCF), are highly active versus Candida glabrata (MIC of ≤0.06 μg/ml). True resistance (MIC of ≥1 μg/ml) is a rare event and strictly associated with mutations in β-1,3-glucan synthase gene FKS1 or FKS2. In contrast, we show here that mutants exhibiting reduced susceptibility to CSP (CRS; MICs of 0.12 to 0.5 μg/ml) are readily selected in vitro and, paradoxically, demonstrate increased susceptibility to MCF (MIS) ranging from 4- to 32-fold. CRS-MIS mutants were generated from all 10 C. glabrata strains tested and were tentatively identified within a collection of clinical isolates. Intriguingly, sequencing and gene disruption demonstrated that CRS-MIS is Fks independent.

Sphingolipids Mediate Differential Echinocandin Susceptibility in Candida albicans and Aspergillus nidulans

ABSTRACT The cell wall synthesis-inhibiting echinocandins, including caspofungin and micafungin, play important roles in the treatment of candidiasis and aspergillosis. Previous studies revealed that, in the haploid yeast Candida glabrata, sphingolipid biosynthesis pathway mutations confer caspofungin reduced susceptibility (CRS) but micafungin increased susceptibility (MIS). Here, we describe one Candida albicans strain (of 10 tested) that similarly yields CRS-MIS mutants at relatively high frequency. Mutants demonstrated increased levels of long-chain bases (sphingolipid pathway intermediates) and, unique to this strain, loss of His104/Pro104 heterozygosity in the TSC13-encoded enoyl reductase. CRS-MIS was similarly observed in a C. albicans homozygous fen1Δ fen12Δ laboratory strain and in diverse wild-type strains following exogenous long-chain-base treatment. Analogous to these results, CRS-MIS was demonstrated in an Aspergillus nidulans basA mutant encoding defective sphingolipid C4-hydroxylase and in its wild-type parent exposed to long-chain bases. Sphingolipids likely modulate echinocandin interaction with their Fks membrane target in all susceptible fungi, with potential implications for optimizing therapy with existing antifungals and the development of novel agents.

Evaluation of Polymorphic Locus Sequence Typing for Candida glabrata Epidemiology

ABSTRACT The opportunistic yeast Candida glabrata is increasingly refractory to antifungal treatment or prophylaxis and relatedly is increasingly implicated in health care-associated infections. To elucidate the epidemiology of these infections, strain typing is required. Sequence-based typing provides multiple advantages over length-based methods, such as pulsed-field gel electrophoresis (PFGE); however, conventional multilocus sequence typing (targeting 6 conserved loci) and whole-genome sequencing are impractical for routine use. A commercial sequence-based typing service for C. glabrata that targets polymorphic tandem repeat-containing loci has recently been developed. These CgMT-J and CgMT-M services were evaluated with 56 epidemiologically unrelated isolates, 4 to 7 fluconazole-susceptible or fluconazole-resistant isolates from each of 5 center A patients, 5 matched pairs of fluconazole-susceptible/resistant isolates from center B patients, and 7 isolates from a center C patient who responded to then failed caspofungin therapy. CgMT-J and CgMT-M generated congruent results, resolving isolates into 24 and 20 alleles, respectively. Isolates from all but one of the center A patients shared the same otherwise rare alleles, suggesting nosocomial transmission. Unexpectedly, Pdr1 sequencing showed that resistance arose independently in each patient. Similarly, most isolates from center B also clustered together; however, this may reflect a dominant clone since their alleles were shared by multiple unrelated isolates. Although distinguishable by their echinocandin susceptibilities, all isolates from the center C patient shared alleles, in agreement with the previously reported relatedness of these isolates based on PFGE. Finally, we show how phylogenetic clusters can be used to provide surrogate parents to analyze the mutational basis for antifungal resistance.

Fungal Resistance to Echinocandins and the MDR Phenomenon in Candida glabrata

Candida glabrata has thoroughly adapted to successfully colonize human mucosal membranes and survive in vivo pressures. prior to and during antifungal treatment. Out of all the medically relevant Candida species, C. glabrata has emerged as a leading cause of azole, echinocandin, and multidrug (MDR: azole + echinocandin) adaptive resistance. Neither mechanism of resistance is intrinsic to C. glabrata, since stable genetic resistance depends on mutation of drug target genes, FKS1 and FKS2 (echinocandin resistance), and a transcription factor, PDR1, which controls expression of major drug transporters, such as CDR1 (azole resistance). However, another hallmark of C. glabrata is the ability to withstand drug pressure both in vitro and in vivo prior to stable “genetic escape”. Additionally, these resistance events can arise within individual patients, which underscores the importance of understanding how this fungus is adapting to its environment and to drug exposure in vivo. Here, we explore the evolution of echinocandin resistance as a multistep model that includes general cell stress, drug adaptation (tolerance), and genetic escape. The extensive genetic diversity reported in C. glabrata is highlighted.