Introduction Echinocandins are cyclic hexapeptides N-linked to a fatty acyl side chain inhibiting the ß-1,3-d-glucan synthase, which is responsible for biosynthesis of the major cell wall biopolymer. Studies reveal the presence of occasional strains from highly susceptible species that display uncharacteristically high MIC values. One form of an adaptive increased resistance is the Eagle effect or paradoxical growth effect which was originally described for a C. tropicalis strain growing in the presence of Cilofungin and later observed in different Candida spp. cultivated in the presence of echinocandins We compared the effect of echinocandins on C. albicans and C. dubliniensis and demonstrated that caspofungin and micafungin, but not anidulafungin induced a paradoxical growth effect in both species (Fleischhacker et al 2008). In order to analyze this effect in greater detail, we examined the effect of echinocandins on the expression of a panel of 26 genes in both Candida spp. Methods We used 4 strains of C. albicans, i.e. ATCC 90028, SC5314, Kor3 and Stil 1( the latter two clinical isolates from our strain collection) and 4 C. dubliniensis strains, i.e. CBS8500, Fr 12, Hun 3 and Rud 9 (the latter three clinical isolates from our strain collection). Strains were grown in 50-mL culture tubes containing 20 mL RPMI 1640 medium with/ without Anidulafungin (0.25μg/ml), Caspofungin (0.125μg/ml and 8μg/ml) and Micafungin (0.125μg/ml and 64μg/ml) respectively. They were incubated for 48 h on a rotary shaker (123 r.p.m.) at 37o C.The isolated RNA (FastRNA Pro Green kit) was DNAse digested and reverse transcribed. The expression level of 26 taget genes was determined by quantitative real-time PCR (Platinum® SYBR® Green qPCR SuperMix UDG kit, LC 480 machine) using URA 3 and TEF 1 as reference genes. Results The results of our analysis are summarized in Table 1 (only the data which were reproducible in two independent experiments were taken into account).We observed a complex gene expression pattern in both Candida spp. The echinocandins have a stronger effect on C. dubliniensis than on the C. albicans strains. A common feature in both species is the up-regulation of the Chs 8 gene. The genes associated with the chitin biosynthesis pathway (Chs3, Chs1, Chs8 and Chs2) are stronger affected in C. dubliniensis than in C. albicans. In C. dubliniensis all 4 afore mentioned genes are upregulated at least in two strains by all three echinocandins while in C. albicans the regulation of the Chs3 gene is not altered in any of the strains and an upregulation of the CHS2 gene is seen in one strain only. In C. dubliniensis there are 9 genes upregulated in at least one strain (i.e. Fks1, Chs7, Crz1, Chs4, Chiti, Rho1, Phr1, Phr2 and Mkc1), while in C. albicans there are only 4 genes whose regulation is affected by all echinocandins (i.e. Chs1, Chs5, Alg6 and Fks2). The regulation of eight other genes (Gsl1, Chs4, Chs6, Rho1, Phr1, Phr2, Mkc1 and Cwt1) is affected only by one or two echinocandins and this up-regulation is seen only in a few C. albicans strains. Interestingly in both species the reference strains (ATCC 90028 and SC 5314 in C. albicans, CBS 8500 in C. dubliniensis) do not differ in their response to the echinocandin treatment from the clinical isolates (Kor3 and Stil1 in C. albicans, Hun3, Fr12 and Rud9 in C. dubliniensis). In C. albicans a downregulation of the Fks1, Alg7, Crz1, Alg8 and Mkc1 genes is seen, whereas in C. dubliniensis the genes encoding for Chs6, Alg2, Alg8 and Alg 6 are downregulated. Conclusions The structural similarity of the three echinocandins and the overall ß-1,3-glucan synthase inhibition led to the assumption that the effects induced would be similar in different Candida spp. This in mind and the observation that the echinocandin induced Eagle effect and trailing was different in C. albicans and C. dubliniensis (Fleischhacker et al 2008) we decided to analyze the expression pattern of a set 26 genes belonging to the ß-1,3-D-glucan biosynthesis pathway and genes encoding for the cell wall chitin biosynthetic process. We observed an upregulation of the genes encoding for Chs3, Chs1, Chs8 and Chs2 in agreement with data observed by Kaneko et al (2010), while Stevens et al. (2005) concluded from their observations that the paradoxical growth is not related to an up-regulation of ß-1,3-glucan synthase activity in the presence of drug and Bizerra et al (2010) observed a decreased quantity of β-1,3-glucan content in C. albicans, C. tropicalis, C. orthopsilosis and C. parapsilosis, Our observation that C. albicans and C. dubliniensis responded differently to the echinocandins (Fleischhacker et al 2008) was also confirmed by Chamilos et al. (2007). The clinical relevance of echinocandin induced paradoxical effects remains uncertain and so far this seems to be an effect which is only observed in vitro. Nevertheless the peak concentrations seen in serum of patients treated with caspofungin ( 8 μg/ml, Shields et al 2011) reach al level able to induce an eagle effect in vitro. The same holds for mice which were given intraperitoneal doses of up to 80 mg/kg who reached levels of 60 μg/ml and 53 μg/mL, for anidulafungin and micafungin respectively (Andes et al 2008a, 2008b). We conclude from our experiments that the different echinocandins induce a complex gene expression pattern which is substantially different in C. albicans from C. dubliniensis. It might be that the effects we have seen in the panel of 26 analyzed genes are the basis for the different responses (i.e. paradoxical growth effect and trailing) of the two species to anidulafungin, caspofungin, and micafungin, respectively.

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