The Function Of Calcineurin Pathway And Its Target Gene RTA2 On Fluconazole Resistance In Candida Albicans

Candida albicans is primarily a common invasive pathogen causing infections in humans with compromised immune defenses. Long-term use of azoles, in particular fluconazole (FLC) aggravates tolerance with multiple mechanisms, which complicates clinical treatment. Calcineurin signaling pathway, as one of the most important virulence factors, has been reported to be related to the azole resistance in C. albicans. Our previous study has demonstrated in vitro that RTA2 is an essential gene in calcineurin pathway participating in azole resistance with gene manipulation techniques, whereas there are still no further proofs of RTA2 contributing to azole tolerance in vivo. Therefore, this study explored the role of calcineurin pathway and RTA2 on tolerance to FLC in C. albicans with in vitro and in vivo methods.To demonstrate the impact of calcinerin pathway on the FLC resistance in C. albicans, this study collected 45 clinical isolates from different areas and different patients. The results of the broth microdilution assay showed the susceptibility to FLC in 37.8% (17/45) of the isolates turned from susceptibility to dose-dependent susceptible, while 13.3% (6/45) turned from susceptible to drug resistant after the addition of ionized calcium with the similar concentration in human serum. On the other hand, the decrease of susceptibility to FLC may means the discrepancy of real susceptibility in vivo and susceptibility tested in vitro. Then, real-time RT-PCR assay were conducted to test the gene RTA2 expression profiles in these clinical isolates before and after adding ionized calcium. Results showed that the RTA2 expression of 66.7% isolates increased, indicating that addition of ionized calcium up-regulated RTA2 in a calcineurin-dependent manner.To investigate whether or not susceptibility is identitical in C. albicans in vitro and in vivo, we classified the MIC values and RTA2 expression to explore the impact on virulence and FLC resistance formation. We tested the virulence and therapeutic effect of FLC of 8 clinical isolates, strains with RTA2 deletion, recovery, CRZ1 deletion, recovery and wild-type strains in a mice systemic infection model by intravenous tail injection. FLC significantly prolonged the survival time of mice infected with the isolates in low increase of the MIC values and invariable RTA2 expression compared with those in high increase of MICs and up-regulated RTA2 expression. The survival of mice infected with deletion of RTA2 or CRZ1 strains and then treated with FLC was significantly more than strains with genes reintroduced and the wild-type. Pathological examination and the colony forming unit assay further confirmed the findings of survival analysis. In addition, the survival time of mice treated without FLC showed no difference. All the results demonstrated that the gene RTA2 is not relevant with virulence in vivo, however, it is associated with the susceptibility to FLC and contributed to the FLC tolerance in calcineurin-dependent manner.To clarify the reason of invariable RTA2 expression in a small part of clinical isolates after the addition of calcium ions, we sequenced the upstream 1500bp of RTA2 promoter domain, and then found six mutant sites. Different size of RTA2 promoter domain containing the diverse mutant sites were fused to the RLUC reporter plasmid pBES116-rluc-act1 and transformed in RTA2 knocked-out strain, JXM101. Proteins were extracted and then mixed with the substrate for luciferase. Then we found that the mutants with -531bp and -652bp showed no enzyme activities, implying that these two sites may cause the failure of transcription factor Crz1p of calcineurin pathway indentifying the promotor of RTA2.In conclusion, this study deepens the understanding of the crucial role of calcineurin pathway and target gene RTA2 on the resistance in C. albicans.