Examination of cyp 51 A and cyp 51 B expression level of the first Polish azole resistant clinical Aspergillus fumigatus isolate *

Aspergillus fumigatus is one of the most prevalent airborne fungal pathogens causing infections worldwide. Most A. fumigatus strains are susceptible to azoles, which are administered as the first line therapeutics. However, during last decade the acquired resistance to triazoles by these species has been described. There is a number of publications concerning the examination of clinical A. fumigatus strains from different countries, however there has been no report from Poland. Here, we describe for the first time, an examination of cyp51A and cyp51B expression level of 11 clinical A. fumigatus strains isolated during 2007–2014 period from the collection of Medical University in Wrocław. Their susceptibility to itraconazole, voriconazole and posaconazole has been examined. The MIC values of triazoles for one of the examined isolates were respectively: > 8 mg/L for itraconazole, 2 mg/L for voriconazole and 0.5 mg/L for posaconazole. The cyp51A gene with its promoter region of all isolates was sequenced. It was found that the resistant isolate harbors the TR34/L98H mutation in the cyp51A gene and when cultured on media supplemented with voriconazole exhibits overexpression of both, cyp51A and cyp51B genes. The level of cyp51A gene expression was about 50 times higher than cyp51B.


INTRODUCTION
Aspergillus (A.) fumigatus is a saprophytic, cosmopolitan fungus present in the soil, rotting plant debris, damp walls or in-house dust (Soubani et al., 2002).It plays an important role in the carbon and nitrogen cycle in the environment.However, it is also recorded as the etiological agent of aspergillosis.Modern diagnostics and application of antifungals improved the outcome of aspergillosis treatment, however, mortality of immunocompromised patients with invasive aspergillosis reaches 40-90% (Shapiro et al., 2011).Among all potential antifungal agents for treatment of A. fumigatus infections, antibiotics from the group of azoles, polyenes and echinocandins, are being administered.Because of the broad spectrum, relatively low toxic azoles are the first-line therapeuticals.(Valiante et al., 2015) The activity of azoles is based on inhibition of lanosterol 14-α-demethylase (Odds et al., 2003).Azoles are a group of antifungal compounds which are applied in clinical medicine and in agriculture (Verweij et al., 2009).In 1997, the first itraconazole re-sistant A. fumigatus strains were identified in the Netherlands (Denning et al., 1997).The number of azole resistant strains still increases for e.g. in the Netherlands from 2% in 2000 to 8% in 2009.In Great Britain the highest increase was observed -5% in 2004, 14% in 2008 and 20% in 2009.However, the published data are probably underestimated due to the lack of routine testing of susceptibility of A. fumigatus isolates (Perlin et al., 2015).Most of the azole resistant strains harbor the TR 34 /L98H mutation in the cyp51A gene, which is related to azole resistance (Mellado et al., 2007).Another suggested mechanism of acquiring the azole resistance is connected to the expression level of the cyp51 genes (Shapiro et al., 2011;van Ingen et al., 2015).In this paper we present the results of examination of cyp51A and cyp51B genes expression level of 11 clinical A. fumigatus isolates grown on Sabouraud agar and Sabouraud agar supplemented with voriconazole.

Isolates.
Eleven clinical A. fumigatus isolates from the collection of Wrocław Medical University were included in this study.Minimal Inhibitory Concentration (MIC) was assessed for itraconazole (ITR), voriconazole (VOR) and posaconazole (POS) using broth microdilution methods, according to European Committee for Antimicrobial Susceptibility Testing E.Def 9.1 (Rodriguez-Tudela et al., 2008).One of the tested isolates (no.55) has been characterized as VOR and POS intermediate susceptible (MIC for VOR: 2 mg/L and POS: 0.25 mg/L) and resistant to itraconazole (MIC for ITR: 32 mg/L).
A PCR-based assay was applied to screen for the presence of the 34-bp tandem repeat in the promoter region of the cyp51A gene as it was described elsewhere (Bromley et al., 2014).2x Master Mix HighGC (A&A Biotechnology) was applied for all of the PCR assays performed.
Examination of cyp51A and cyp51B expression level.All isolates were grown on Sabouraud agar plates for 72-96 h at room temperature (with no antimycotics and supplemented with 1 mg/L of voriconazole, respectively).Mycelia from the whole surface of the plate (diameter 900 mm) were placed in 2 ml tubes with zirconia/silica beads (A&A Biotechnology) and 400 µl of de-ionized water was added.The tubes were placed in a bead beater and shaken for 2 min at 2300 rpm.Lysates were then transferred into 1.5 ml tubes.The next steps of RNA extraction were performed by means of Total RNA Mini kit (A&A Biotechnology).Thus obtained RNA was treated by a DNase and further purified with Clean up RNA Concentrator (A&A Biotechnology).The concentration was determined using the NanoDrop1000 (Thermo Scientific).cDNA synthesis was carried out by means of TranScriba kit (A&A Biotechnology) with an oligo(dT)18 solution, 1 µg RNA and the rest of components from the kit.
Quantitative analysis of expression of the cyp51A, cyp51B (Buied et al., 2013) and reference β-tubulin (Glass N.L., 1995) genes was performed by real-time PCR with a LightCycler Nano PCR Real-Time System (Roche).Real-time PCR was performed in 20 µl reaction volume containing the following reagents: 10 µl RealTime 2 X PCR Master Mix SYBR (A&A Biotechnology), 1 µl each primer solution (10 mM), 1 µl of total cDNA sample and distilled water.All reactions were performed in triplicates.The experiments were also repeated in triplicate for each specimen.Quantitative analysis of the level of expression of the investigated genes was carried out using the R = 2 -ΔΔCq formula, also known as the Livak's method (Livak et al., 2001).The azole resistant A. fumigatus strain from Centraalbureau voor Schimmelcultures (accession number CBS 133436) was applied as a calibrator.

Species identification and analysis of cyp51A sequence
The results of rRNA fragment and β-tubulin gene sequencing confirmed classical identification of the examined isolates as A. fumigatus.Amplification of the promoter region of cyp51A gene revealed presence of a 34 bp tandem repeat.Sequencing of cyp51A gene confirmed point mutation in the azole resistant isolate (no.55) resulting in L98H substitution.

Examination of cyp51A and cyp51B expression level
The expression level of the cyp51A gene of 9/11 A. fumigatus isolates grown on Sabouraud agar with voriconazole was higher when compared to isolates cultured without any azoles.Two isolates cultured in the presence of voriconazole exhibited a lower expression level of the cyp51A gene.However, the dramatic increase from R = 0.354 to R = 2036 was detected in the azole resistant isolate no.55 harboring the TR 34 /L98H mutation (in comparison, the highest increase of azole susceptible isolate reached R = 1.777 from R = 0.008).Similar results were obtained for examination of the expression level of the cyp51B gene.We observed a change of R = 0.044 for the resistant isolate grown without access to voriconazole, to R = 42.942 for the same isolate cultured in the presence of voriconazole.However, the increase in expression level of cyp51B was lower when compared to an increase in cyp51A gene expression (Table 1).

DISSCUSSION
The relation between the occurrence of the TR 34 / L98H mutation and resistance to azoles is well documented (Diaz-Guerra et al., 2003;Howard et al., 2011;Mellado et al., 2007;Snelders et al., 2011).We report here a great difference in expression of cyp51A and cy-p51B genes of the azole resistant A. fumigatus isolate harboring the TR 34 /L98H mutation when grown on Sabouraud agar and Sabouraud agar supplemented with 1 mg/L voriconazole respectively.Our findings indirectly confirm the hypothesis of azole resistance induction by the presence of azoles in the environment (Snelders et al., 2009, Snelders et al., 2012, Stensvold et al., 2012, Verweji et al., 2009), as the elevated expression level of cyp51 genes can be the reason for azole resistance occurrence.However, further examination of other resistance mechanisms is necessary, not only due to a dramatic increase of expression level of cyp51A gene in isolate no.55, but also as most likely the azole resistance is a result of combination of different factors.Beside tandem repeats in the promoter region of the cyp51A gene and a point mutation in this gene, some transcription factors can also be involved in upregulation of cyp51A.For example, the SrbA protein is a regulating factor from the SREBP family (sterol regulator element binding protein), which is involved in the sterol biosynthesis.It is possible that SrbA can interfere with the cyp51A gene and influence its activity (Blosser et al., 2011).Other mechanisms may also induce azole resistance -for e.g. a mutation in the transcription factor HapE which binds the CCAAT sequence (Wei et al., 2015), overexpression of MFS (major facilitator superfamily) transporters (Shapiro et al., 2011) or overexpression of ABC transporters (ATP binding cassette superfamily).

Financial support
This work was funded by the National Science Centre grant no 2013/11/B/NZ7/04935.