Calcium-Activated-Calcineurin Reduces the In Vitro and In Vivo Sensitivity of Fluconazole to Candida albicans via Rta2p

et al. (2012) Calcium-Activated-Calcineurin Reduces the In Vitro and In Vivo Sensitivity of Fluconazole to Candida albicans via Rta2p. PLoS ONE 7(10): e48369. doi:10.1371/journal.pone.0048369 Calcium-Activated-Calcineurin Reduces the In Vitro and In Vivo Sensitivity of Fluconazole to Candida albicans via Rta2p Yu Jia 0 Ren-Jie Tang 0 Lin Wang 0 Xiang Zhang 0 Ying Wang 0 Xin-Ming Jia 0 Yuan-Ying Jiang 0 Gustavo Henrique Goldman, Universidade de Sao Paulo, Brazil 0 1 School of Pharmacy, Second Military Medical University , Shanghai , China , 2 Department of Immunology, Tongji University School of Medicine , Shanghai , China , 3 Department of Neurosurgery, Tenth People's Hospital, Tongji University School of Medicine , Shanghai , China , 4 Department of Dermatology, Changhai Hospital, Second Military Medical University , Shanghai , China Due to the emergence of drug-resistance, first-line therapy with fluconazole (FLC) increasingly resulted in clinical failure for the treatment of candidemia. Our previous studies found that in vitro RTA2 was involved in the calcineurin-mediated resistance to FLC in C. albicans. In this study, we found that calcium-activated-calcineurin significantly reduced the in vitro sensitivity of C. albicans to FLC by blocking the impairment of FLC to the plasma membrane via Rta2p. Furthermore, we found that RTA2 itself was not involved in C. albicans virulence, but the disruption of RTA2 dramatically increased the therapeutic efficacy of FLC in a murine model of systemic candidiasis. Conversely, both re-introduction of one RTA2 allele and ectopic expression of RTA2 significantly reduced FLC efficacy in a mammalian host. Finally, we found that calciumactivated-calcineurin, through its target Rta2p, dramatically reduced the efficacy of FLC against candidemia. Given the critical roles of Rta2p in controlling the efficacy of FLC, Rta2p can be a potential drug target for antifungal therapies. - Funding: This study was supported by the National Science Foundation of China (30900055, 30825041 and 90913008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Candida species are serious human fungal pathogens among immuno-compromised individuals such as patients with AIDS and patients undergoing organ transplantation, parenteral nutrition and radiation treatment for cancer [1]. Candida blood stream infections (candidemia) are life threatening among hospitalized immune-compromised patients, including neonates [2,3]. Despite available therapy options, the mortality rate directly attributable to candidemia is high, ranging from 32% to 54%, depending on the population and the species studied [4,5,6]. Today, candidemia leads to additional ICU length of stay and costs, the annual treatment cost is at least 16.2 million [7,8]. Although infections with non-albicans Candida species have emerged in recent years, Candida albicans is still responsible for the majority of cases [9,10]. To date, only four classes of antifungal drugs are clinically available for treatment of systemic fungal infections: polyenes (amphotericin B), pyrimidines (particularly 5-fluorocytosine), azoles (e.g. fluconazole, FLC) and echinocandins (e.g. caspofungin) [11,12]. Among these, azole antifungal drugs, such as FLC, are commonly used in the treatment of invasive candidiasis, and the mechanism of action of these drugs involves the blockade of ergosterol synthesis through the competitive inhibition of 14ademethylase, which is encoded by ERG11 gene. Consequently, the inhibition of the enzyme leads to accumulation of 14-methylated sterols that can cause disruption of fungal membranes [13]. Although these drugs have advanced the management of fungal infections, the rate of therapeutic failure remains high. Most importantly, the small number of treatment options available has resulted in wide-spread drug resistance in pathogenic species. For each of the four classes of antifungals drugs (polyenes, pyrimidines, azoles, echinocandins), drug-resistant clinical strains has been reported [14]; Azole-resistant Candida spp. is now particularly common among isolates from HIV-positive patients [15]. Therefore, the need of developing new antifungal strategies is pressing. Inhibition of calcineurin-mediated azole resistance has been proposed as a novel therapeutic approach [16]. Although the precise mechanisms involved remain elusive, extensive experiments have further confirmed the benefits of such combinatorial approaches. For instance, inhibitors of calcineurin (CsA, FK506) or inhibitors of calcineurin adaptor protein HSP90 (geldanamycin, radicicol) can chemosensitize C. albicans cells to azoles [17,18,19]. Although these examples sufficiently demonstrate the potential for combination antifungal therapy, suppression of the human immune system by CsA/FK506 currently precludes the use of such inhibitors in clinics [20,21]. While a non-immunosuppressive FK506 analogue (L-685, 818) has been identified, proprietary restrictions have currently prevented further testing [20]. Given the importance of serious mycoses in clinical medicine and the need of alternative therapies for use in patients with fungal infections, research designed to identify new antifungal targets remains a priority. We previously established that calcium-upregulated expression of RTA2 dramatically reduced the sensitivity of C. albicans to FLC by blocking its impairment to the fungal plasma membrane [22]. The expression of RTA2 was under the control of the calciumactivated-calcineurin via its transcriptional factor Crz1p in C. albicans [22]. However, it is unclear whether calcium-activatedcalcineurin mediates in vitro and in vivo responses to FLC via Rta2p in C. albicans. Moreover, the therapeutic potential of targeting Rta2p remains elusive. Here, we investigated the possible influences of RTA2 expression on the in vitro and in vivo responses to FLC mediated by the calcium-activated-calcineurin in C. albicans. Calcium-activated-calcineurin Dramatically Reduces in vitro Sensitivity to FLC via Rta2p in C. albicans It has been well documented that the phosphatase activity of calcineurin, consisted of a catalytic subunit A (encoded by CNA) and a regulatory subunit B (encoded by CNB1), can be activated by calcium ions and then regulate its target gene expression via transcriptional factor Crz1p [23]. In order to identify whether calcium-activated-calcineurin mediates the in vitro response to FLC via Rta2p in C. albicans, we sequentially disrupted two alleles of RTA2 gene in either cnaD/D or crz1D/D null mutant strains (Table 1), which were verified by Southern blot analysis with a probe specific to the RTA2 promoter (Figure S1). And then RTA2, CNA or CRZ1 was selectively re-introduced into either cnaD/D rta2D/D or crz1D/D rta2 (...truncated)