Molecular mechanism of azoles resistant Candida albicans in a patient with chronic mucocutaneous candidiasis

Chronic mucocutaneous candidiasis (CMC) is a heterogeneous disease in children featured by persistent and recurrent infections of skin, nails and mucous membranes caused mainly by Candida albicans [1, 2]. The factors that predispose host to CMC infection could be autosomal or acquisitive. Increasing evidence indicates that some immunologic and hormonal abnormalities are associated with CMC due to changes of cellular immunity, which then lead to subsequent autoimmune endocrine disorders [3, 4]. To date, heterozygous gain-of-function (GOF) mutations of signal transducer and activator of transcription 1 (STAT1) have been identified as a cause of CMC [5‐7]. Afterwards, many studies have proved that mutations at different sites of STAT1 are associated with the pathogenesis of CMC [8‐10].

Currently, azole anti-fungal drugs have been widely used for treating CMC as they show satisfactory bio-availability and safety. Unfortunately, there are an increasing number of resistant strains of Candida spp. in the presence of long-term azoles exposure. The potential mechanisms are associated with up-regulation of pharmaceutical transporters, over-expression or alteration of the drug target, as well as cellular changes caused, in some cases, by non-target effects induced by stress response [11].

Candida are normally non-pathogenic microorganisms in human beings. However, they are the major opportunistic fungal pathogens causing surface and invasive infections in individuals, which is associated with significant morbidity and mortality [9]. Immunosuppressive therapy, mucous damage, indwelling catheters, and prolonged hospital stay are the risk factors for Candida infection [14]. Besides, genetic factors must play a role in the pathogenesis of Candida infections, especially invasive candidiasis and CMC.

Several genes affecting the anti-fungal immunity have been reported to be associated with the pathogenesis of CMC, including AIRE, CLEC7A, CARD9, IL17RA, IL17F, IL2Rα, Dectin-1, STAT1 and STAT3 [5‐7, 15‐20]. STAT1, one of the seven transcription factors of the STAT family, is the major signaling components of interferon responses. In the presence of interferon binding to its receptor, STAT1 is activated by tyrosine phosphorylation [21]. Upon forming a dimer, STAT1 enters the nucleus and triggers the transcription of its targets. Such process plays a pivotal role in the defense against pathogens. The mutations of STAT1 were considered as a GOF type because of a gain of phosphorylation and a loss of nuclear dephosphorylation. In a previous study, Van de Veerdonk et al. [6] demonstrated that patients with heterozygous missense mutations in the coiled-coil domain of STAT1 had deficiencies in mounting TH1 and TH17 responses due to defective IL-12 receptor and IL-23 receptor signaling pathways [7]. S. Takezaki et al. [8] firstly reported that the GOF mutations of DNA-binding domain were also the genetic cause of CMC, which involved a gain of STAT1 function due to impaired dephosphorylation in the coiled-coil domain mutations.

In this case, direct sequence analysis of STAT1 exons indicated presence of c.1162A > G, which then resulted in p.K388E in exon 14. As previously described [10, 22], K388E was a GOF mutation in the DNA-binding domain. The patients showed an early onset and received different systemic or topical anti-fungal treatments for more than 10 years, especially azoles. Long-term azoles exposure may lead to generation of C. albicans resistant strains. Therefore, we speculated that the azoles-resistant C. albicans isolated from this case was induced by long-term exposure of anti-fungal drugs.

Azole drugs can target the ergosterol bio-synthetic pathway. Ergosterol is a crucial component of membrane of the fungal cell. Interruption of its synthesis allows accumulation of 14 α-methyl sterols, which is encoded by ERG11 in Candida spp. The function of 14 α-methyl sterols is to alter the membrane stability, permeability, and the action of membrane-bound enzymes [23, 24]. Additionally, inhibition of ERG3, a Δ^5,6-desaturase could lead to a depletion of ergosterol and accumulation of 14a-methylfecosterol, which allowed continuous growth in the presence of azole despite altered membrane composition [25]. Also, ERG11 over-expression confers azole resistance, which is more common among azole resistant isolates of C. albicans [26]. Over-expression of efflux pumps, encoded by genes of the ATP-binding cassette (ABC) super-family or the major facilitator super-family (MFS), is the most common cause for drug resistance. Such process could decrease the intracellular drug concentration by increasing target abundance. Hence, more drugs are required to inhibit the activity of pathogens, which then results in reduction of drug susceptibility [27].

To further investigate the resistance mechanism of C. albicans in this case, we sequenced ERG11 and ERG3. In total, 14 nucleotide substitutions were detected in ERG11 and ERG3, among which 13 nucleotide substitutions were synonymous mutations and 1 was missense mutations of ERG11 resulted in amino acid changes (p.E266D). In the previous study, such mutation was not reported to be related to azole resistance in C. albicans [28]. Additionally, RT-PCR was performed to detect the expression of CDR1, CDR2 and ERG11 mRNA between CA-R and the type strain SC5314. The relative expression of CDR1 and CDR2 rather than ERG11 in multiple azole resistant strains was obviously higher than that of SC5314 strain. These suggested that the main cause for azoles-resistant CA-R may be related to the over-expression of CDR1 and CDR2.

Several aspects have been reported to affect the expression of the azole target and/or drug pumps, including mutations in the drug target enzyme and efflux pumps over-expression, loss of heterozygosity, increased chromosomal copy number, aneuploidy, as well as the isochromosome [29]. These constituted the main reasons of azole resistance of C. albicans together with the biofilm formation [30]. They can occur in a single set or concurrently, which can produce additive effects or lead to cross-resistance among azoles. In this study, we only focused on the determination of drug efflux pump genes and target enzyme sequence. In future, additional studies are required to investigate whether there are other causes of drug resistance.

With the increasing understanding on CMC mutation mechanism, strategies based on the defects in STAT1 GOF mutations may serve as a candidate for treating CMC, such as decreasing hyperphosphorylation of STAT1 and restoring Th17 function by blocking inhibitory mechanisms [31]. However, the efficacy of these treatment options has not been well confirmed. Therefore, long-term systemic application of anti-fungal drugs is often preferred in clinical settings. Clinically, within a safe range, these patients were usually given a higher therapeutic dose of the drugs. After the symptoms were controlled, it was gradually reduced and finally the maintenance treatment was performed at the smallest controllable dose. Drug exposure in the form of prophylaxis, repeated, or long-term therapy is associated with the emergence of resistance [30]. At present, there are no system reports about anti-fungal drugs susceptibility of pathogen in CMC patients, who were receiving long-term oral anti-fungal treatment. Therefore, further investigations are still needed. Over-expression of the efflux pump was mainly responsible for the drug resistance. Efflux pump over-expression will reduces the entry of the drug into the pathogens, thereby reducing the effect of the drug. However, in presence of increased extracellular drug concentration, it may still be effective [30]. Although the pathogen in this case is resistant to the multiple azoles, the anti-fungal drug is still selected after informing the patient and his family of the currently feasible treatment options. Fortunately, the symptoms showed remission after administration of itraconazole in this case.

There are some limitations in this study. Firstly, there was only one case. Although the number of studies on gene mutation induced CMC showed increase, there are still rare cases in clinical settings. Secondly, we only investigated the over-expression of target genes encoding the enzyme and the mutation of target genes. We did not investigate the roles of other drug-resistant mechanisms.

In conclusion, we reported a case of CMC with multiple azole-resistant C. albicans, and investigated the possible mechanism of the drug resistance. The expression of CDR1 and CDR2 gene was up-regulated. For special patients, such as CMC or other immunodeficiency patients, drug resistance monitoring is very necessary.