Dataset Information

Spectrum of activity and mechanisms of azole-bisphosphonate synergy in pathogenic Candida.

ABSTRACT: Candidiasis places a significant burden on human health and can range from common superficial vulvovaginal and oral infections to invasive diseases with high mortality. The most common Candida species implicated in human disease is Candida albicans, but other species like Candida glabrata are emerging. The use of azole antifungals for treatment is limited by increasing rates of resistance. This study explores repositioning bisphosphonates, which are traditionally used for osteoporosis, as antifungal synergists that can improve and revitalize the use of azoles. Risedronate, alendronate, and zoledronate (ZOL) were tested against isolates from six different species of Candida, and ZOL produced moderate antifungal activity and strong synergy with azoles like fluconazole (FLC), particularly in C. glabrata. FLC:ZOL combinations had increased fungicidal and antibiofilm activity compared to either drug alone, and the combination prevented the development of antifungal resistance. Mechanistic investigations demonstrated that the synergy was mediated by the depletion of squalene, resulting in the inhibition of ergosterol biosynthesis and a compromised membrane structure. In C. glabrata, synergy compromised the function of membrane-bound multidrug transporters and caused an accumulation of reactive oxygen species, which may account for its acute sensitivity to FLC:ZOL. The efficacy of FLC:ZOL in vivo was confirmed in a Galleria mellonella infection model, where combinations improved the survival of larvae infected with C. albicans and C. glabrata to a greater extent than monotherapy with FLC or ZOL, and at reduced dosages. These findings demonstrate that bisphosphonates and azoles are a promising new combination therapy for the treatment of topical candidiasis.

Importance

Candida is a common and often very serious opportunistic fungal pathogen. Invasive candidiasis is a prevalent cause of nosocomial infections with a high mortality rate, and mucocutaneous infections significantly impact the quality of life of millions of patients a year. These infections pose substantial clinical challenges, particularly as the currently available antifungal treatment options are limited in efficacy and often toxic. Azoles are a mainstay of antifungal therapy and work by targeting the biosynthesis of ergosterol. However, there are rising rates of acquired azole resistance in various Candida species, and some species are considered intrinsically resistant to most azoles. Our research demonstrates the promising therapeutic potential of synergistically enhancing azoles with non-toxic, FDA-approved bisphosphonates. Repurposing bisphosphonates as antifungal synergists can bypass much of the drug development pipeline and accelerate the translation of azole-bisphosphonate combination therapy.

SUBMITTER: Kane A

PROVIDER: S-EPMC11237636 | biostudies-literature | 2024 Jun

REPOSITORIES: biostudies-literature

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Spectrum of activity and mechanisms of azole-bisphosphonate synergy in pathogenic Candida.

Kane Aidan A Dinh Hue H Campbell Leona L Cain Amy K AK Hibbs David D Carter Dee D

Microbiology spectrum 20240502 6

Candidiasis places a significant burden on human health and can range from common superficial vulvovaginal and oral infections to invasive diseases with high mortality. The most common Candida species implicated in human disease is Candida albicans, but other species like Candida glabrata are emerging. The use of azole antifungals for treatment is limited by increasing rates of resistance. This study explores repositioning bisphosphonates, which are traditionally used for os ...[more]

PMID: 38695556

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Project description:Opportunistic pathogens of the genus Candida reign as the leading cause of mycotic disease and are associated with mortality rates greater than 40%, even with antifungal intervention. This is in part due to the limited arsenal of antifungals available to treat systemic fungal infections. Azoles have been the most widely deployed class of antifungal drug for decades and function by targeting the biosynthesis of ergosterol, a key component of the fungal cell membrane. However, their utility is compromised by their fungistatic nature, which favors the development of resistance. Combination therapy has the potential to confer enhanced efficacy as well as mitigate the evolution of resistance. Previously, we described the generation of structurally diverse macrocyclic peptides with a 1,3,4-oxadiazole and an endocyclic amine grafted within the peptide backbone. Importantly, this noncanonical backbone displayed high membrane permeability, an important attribute for compounds that need to permeate across the fungal cell wall and membrane in order to reach their intracellular target. Here, we explored the bioactivity of this novel chemical scaffold on its own and in combination with the azole fluconazole. Although few of the oxadiazole-containing macrocyclic peptides displayed activity against Candida albicans on their own, many increased the efficacy of fluconazole, resulting in a synergistic combination that was independent of efflux inhibition. Interestingly, these molecules also enhanced azole activity against several non-albicans Candida species, including the azole-resistant pathogens Candida glabrata and Candida auris This work characterizes a novel chemical scaffold that possesses azole-potentiating activity against clinically important Candida species.IMPORTANCE Fungal infections, such as those caused by pathogenic Candida species, pose a serious threat to human health. Treating these infections relies heavily on the use of azole antifungals; however, resistance to these drugs develops readily, demanding novel therapeutic strategies. This study characterized the antifungal activity of a series of molecules that possess unique chemical attributes and the ability to traverse cellular membranes. We observed that many of the compounds increased the activity of the azole fluconazole against Candida albicans, without blocking the action of drug efflux pumps. These molecules also increased the efficacy of azoles against other Candida species, including the emerging azole-resistant pathogen Candida auris Thus, we describe a novel chemical scaffold with broad-spectrum bioactivity against clinically important fungal pathogens.

Project description:Candida auris is an emerging yeast pathogen of major concern because of its ability to cause hospital outbreaks of invasive candidiasis and to develop resistance to antifungal drugs. A majority of C. auris isolates are resistant to fluconazole, an azole drug used for the treatment of invasive candidiasis. Mechanisms of azole resistance are multiple, including mutations in the target gene ERG11 and activation of the transcription factors Tac1b and Mrr1, which control the drug transporters Cdr1 and Mdr1, respectively. We investigated the role of the transcription factor Upc2, which is known to regulate the ergosterol biosynthesis pathway and azole resistance in other Candida spp. Genetic deletion and hyperactivation of Upc2 by epitope tagging in C. auris resulted in drastic increases and decreases in susceptibility to azoles, respectively. This effect was conserved in strains with genetic hyperactivation of Tac1b or Mrr1. Reverse transcription PCR analyses showed that Upc2 regulates ERG11 expression and also activates the Mrr1/Mdr1 pathway. We showed that upregulation of MDR1 by Upc2 could occur independently from Mrr1. The impact of UPC2 deletion on MDR1 expression and azole susceptibility in a hyperactive Mrr1 background was stronger than that of MRR1 deletion in a hyperactive Upc2 background. While Upc2 hyperactivation resulted in a significant increase in the expression of TAC1b, CDR1 expression remained unchanged. Taken together, our results showed that Upc2 is crucial for azole resistance in C. auris, via regulation of the ergosterol biosynthesis pathway and activation of the Mrr1/Mdr1 pathway. Notably, Upc2 is a very potent and direct activator of Mdr1.IMPORTANCECandida auris is a yeast of major medical importance causing nosocomial outbreaks of invasive candidiasis. Its ability to develop resistance to antifungal drugs, in particular to azoles (e.g., fluconazole), is concerning. Understanding the mechanisms of azole resistance in C. auris is important and may help in identifying novel antifungal targets. This study shows the key role of the transcription factor Upc2 in azole resistance of C. auris and shows that this effect is mediated via different pathways, including the regulation of ergosterol biosynthesis and also the direct upregulation of the drug transporter Mdr1.

Project description:TAC1 (for transcriptional activator of CDR genes) is critical for the upregulation of the ABC transporters CDR1 and CDR2, which mediate azole resistance in Candida albicans. While a wild-type TAC1 allele drives high expression of CDR1/2 in response to inducers, we showed previously that TAC1 can be hyperactive by a gain-of-function (GOF) point mutation responsible for constitutive high expression of CDR1/2. High azole resistance levels are achieved when C. albicans carries hyperactive alleles only as a consequence of loss of heterozygosity (LOH) at the TAC1 locus on chromosome 5 (Chr 5), which is linked to the mating-type-like (MTL) locus. Both are located on the Chr 5 left arm along with ERG11 (target of azoles). In this work, five groups of related isolates containing azole-susceptible and -resistant strains were analyzed for the TAC1 and ERG11 alleles and for Chr 5 alterations. While recovered ERG11 alleles contained known mutations, 17 new TAC1 alleles were isolated, including 7 hyperactive alleles with five separate new GOF mutations. Single-nucleotide-polymorphism analysis of Chr 5 revealed that azole-resistant strains acquired TAC1 hyperactive alleles and, in most cases, ERG11 mutant alleles by LOH events not systematically including the MTL locus. TAC1 LOH resulted from mitotic recombination of the left arm of Chr 5, gene conversion within the TAC1 locus, or the loss and reduplication of the entire Chr 5. In one case, two independent TAC1 hyperactive alleles were acquired. Comparative genome hybridization and karyotype analysis revealed the presence of isochromosome 5L [i(5L)] in two azole-resistant strains. i(5L) leads to increased copy numbers of azole resistance genes present on the left arm of Chr 5, among them TAC1 and ERG11. Our work shows that azole resistance was due not only to the presence of specific mutations in azole resistance genes (at least ERG11 and TAC1) but also to their increase in copy number by LOH and to the addition of extra Chr 5 copies. With the combination of these different modifications, sophisticated genotypes were obtained. The development of azole resistance in C. albicans is therefore a powerful instrument for generating genetic diversity.

Dataset Information

Spectrum of activity and mechanisms of azole-bisphosphonate synergy in pathogenic Candida.

Importance

Publications

Spectrum of activity and mechanisms of azole-bisphosphonate synergy in pathogenic <i>Candida</i>.

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