Isogenic barcoded strains of Aspergillus fumigatus carrying the G54W or M220K

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1 AAC Accepted Manuscript Posted Online 28 September 2015 Antimicrob. Agents Chemother. doi: /aac Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 Fitness studies of azole resistant strains of A. fumigatus Isabel Valsecchi 1, Emilia Mellado 2, Rémi Beau 1, Shriya Raj 1 and Jean-Paul Latgé 1. 1 Unité des Aspergillus, Institut Pasteur Paris, France. 2 Mycology Reference Laboratory, Instituto de Salud Carlos III, Madrid, Spain Abstract Isogenic barcoded strains of Aspergillus fumigatus carrying the G54W or M220K mutations in Cyp51A were constructed. In vitro, the growth and conidiation capacities of the mutants were similar to the parental strain. Competition studies in absence of azoles showed that there was no adverse fitness cost for the azole resistant A. fumigatus strains in vitro or in vivo, when compared to the parental strain Introduction Azoles antifungal agents are widely deployed in medicine and agriculture for the treatment and prevention of fungal diseases. They target membrane ergosterol synthesis, by blocking a key biosynthetic enzyme cytochrome P450 lanosterol/eburitol C-14-alpha-demethylase, encoded by cyp51a (1). The emergence of azole resistant strains of A. fumigatus in hospitals has been repeatedly reported in recent years, specifically in those patients on long-term treatment with azoles, e.g. cystic fibrosis. However, azole resistant strains are now 1

2 being reported in patients who have not been previously exposed to azoles. Importantly, almost all mechanisms underlying resistance to triazoles described until now in A. fumigatus have primarily involved mutations in the cyp51a gene. The most common mutations found in patient isolates involve the G54 or M220 amino acids, or the presence of tandem repeats within the promoter region in combination with an L98 mutation (2). Nevertheless, the percentage of azole resistant A. fumigatus strains remains low in the population. In a recent large prospective multicenter international surveillance study, a total of 3,788 Aspergillus isolates were screened in 22 centers across 19 countries between 2009 and Resistance to azoles was detected in most centers with an overall prevalence of azole resistance of 3.2% (3). In several other national studies, the computed percentages were highly dependent on the local epidemiology, as well as the sample size of isolates tested for azole resistance (4-6). In contrast, the extensive use of azoles in agriculture led to the dramatic emergence of resistant phytopathogenic fungi, with a global invasion by (a) azole resistant(s) strain(s) within three to six years following the appearance of the first resistant strain (Fungicide Resistance Action Committee [FRAC]; ) (7). Therefore, it is necessary to understand the reasons underlying the limited spread of resistant strains in hospitals when compared to their rapid dispersal in agriculture. To this end, one of the first hypotheses explored is the occurrence of a fitness defect in the azole resistant strains when compared in competitive mixed models of growth and infection in vitro and in vivo, respectively (8). Indeed, it has been reported since 1976 that mutants of plant pathogenic fungi 2

3 46 47 exhibiting resistance to sterol biosynthesis inhibitors have reduced pathogenicity and fitness attributes (9-11) Generation and phenotypic characterization of isogenic resistant and sensitive strains In order to analyze the fitness of resistant strains, (i.e. the ability of a resistant strain to propagate and evolve competitively with sensitive strains within a given environment), it is necessary to use isogenic sensitive and resistant strains. Previous studies have shown that it is impossible to investigate the fitness cost of azole resistance in vivo using clinical isolates of multiple origins, and which may 57 carry different mutations that confer drug resistance (12,13). Therefore, we constructed strains in the CEA17_ΔakuB KU80 background that were isogenic to the parental strain, except for the G54W or M220K mutations within the cyp51a gene (14). Complemented strains were constructed by reintroducing the parental cyp51a gene. Strategies for replacement and complementation are shown in Suppl. Fig.1 A. Confirmation by Southern hybridization is shown in Suppl. Fig.1 B. The G54W and M220K mutants exhibited resistance to itraconazole and differential resistance to posaconazole (15), while the complemented strains reverted to parental sensitivity to the two azoles (Suppl. Fig.2). The G54W and M220K mutant strains showed growth and conidiation characteristics similar to the parental strain on different media (MM-pH 7.0, Sabouraud (Sab), and Malt 6% KCl) at 37 C and 50 C (Suppl. Fig.3). In addition, the 3

4 69 percentage and kinetics of conidial germination in Sab medium were similar for the 70 mutants and the parental strain. These results confirmed that the mutations 71 conferring resistance to azoles did not adversely impact fungal growth (16) Fitness analysis Since their biological traits were identical, fitness of each mutant and parental strain was investigated by growing them together in vitro and in vivo, in the absence of azoles in the experimental set up shown in Suppl. Fig.4. To estimate the relative growth of mutants and parental strain (indicated by fungal mass), quantitative PCR was performed using primers annealing the barcode sequences (Suppl. Fig.5). The genomic DNA extracted from the inoculation mixtures of either G54W or M220K and parental conidia were used as controls Fitness in vitro. A mixture containing equal amounts of parental Cyp51A-expressing and G54W (or M220K)-expressing conidia was spotted onto the center of a Petri dish containing RPMI ph 7.0 medium but lacking itraconazole, and incubated at 37 (Suppl. Fig.4 A,a). C. After three days, a circle of 0.6 cm was excised from the edge of the colony and conidia were resuspended in an aqueous solution of 0.05% Tween-20. Five μl of this mix was spotted onto a second RPMI plate and processed as before. Following the third such passage, three circles measuring 0.6 cm each were processed for the extraction of gdna. qpcr was performed using strain-specific 91 barcode primers (see above) to quantify the growth of strains. No significant 4

5 differences were observed in the Ct values between the mutants and parental strain (Fig.1 A). In an alternative experimental (Suppl. Fig.4 A,b), strains were further analyzed by co-inoculating malt slants with a parent:mutant ratio of 1:1 and grown for 15 days. Conidia were recovered and then plated onto MM in the absence or presence of 10 µg/ml itraconazole, in order to quantify the amount of total and azole resistant colonies. A suspension recovered from the sixth successive passage was used to estimate the ratio of strains by enumerating the CFUs. The ratio of resistant: sensitive strains following six passages was the same when compared with that determined for the original mixture (Fig.1 B). These data showed that the G54W or M220K point mutations in Cyp51A were not associated with any fitness cost in vitro Fitness in vivo. Seven-week-old OF1 male mice (Charles River Laboratory, L Arbresle, France) were immunosuppressed and infected via the intranasal route as described earlier (17). Each mouse was administered an inoculum of 5x10 7 conidia consisting of a 1:1 ratio of G54W and parental, or M220K and parental conidia. Three days following inoculation, mice were sacrificed, lungs extracted, and frozen in liquid nitrogen. The 110 lungs were processed similarly for all three replicates. qpcr was performed on total infected lung gdna (Supplementary data, Suppl. Fig.4 B) using strain-specific barcode primers. No significant differences were observed for Ct values between the parental and mutant strains (Fig.1 C). This result showed that the Cyp51A deletions undertaken were not associated with any fitness cost in vivo. 5

6 The molecular mechanisms of azole resistance in A. fumigatus are well characterized. The most common mechanism of azole resistance found in Aspergillus spp. involves modifications within the cyp51a encoded target site that leads to an impaired interaction between the azole and the target enzyme (18). The ability of the resulting resistant strains to persist within a given population, however, depends on their fitness capabilities (19). To this end, there are few studies that measure the impact of these mechanisms on A. fumigatus fitness in different environments. Most in vitro studies with various fungal pathogens established no direct relationship between the cost of developing drug resistance and changes in fitness, by measuring competitive growth of susceptible and resistant isolates (12,13,20,21). The direct relationship between the development of drug resistance and virulence has been indeed only demonstrated in very few yeast strains (22 23). To our knowledge, this is the first study wherein the fitness of azole resistant mutants of A. fumigatus has been evaluated with a mixture of isogenic strains in axenic media and in mice. Our data showed that the acquisition of azole resistance was not associated with any fitness penalty in A. fumigatus. A recent study (24) arrived at the same conclusion despite using non-isogenic strains. Isogenic A. fumigatus strains carrying different Cyp51A mutations exhibited no differences in virulence in the alternative host Galleria mellonella. However, this study did not look at competitive growth of strains within the host preventing a proper evaluation of the fitness cost (16). Features that favor the occurrence of drug resistant strains, such as a short biological cycle, abundant sporulation, and dispersal of spores over long distances 6

7 (7), are typically observed in A. fumigatus. However, like rust fungi, these are not sufficient to promote the expansion of resistant strains. Moreover, multidrug resistance, which seems to play a major role in spreading resistance acquisition among plant pathogens (25,26), has rarely been described for medically important pathogens (2,27). We could speculate that the normal soil habitat of this fungus might impose nutritive or antagonistic restrictions, which may lead to an increased susceptibility and consequent dilution of the resistant strain in the environmental population. In this study, we only analyzed two out of three clinically relevant mutations, i.e. G54 and M220, which are encountered as commonly as the TR34/L98H mutation in patients (2,3). In the recent van der Linden study comprising 28 patients, equal numbers of strains were found carrying one or the other mutation: 8 strains (G54/M220) and 9 strains (TR34L98H). Moreover, outcome was worse in those patients infected with G54/M220 mutant strains (71.1% had a fatal outcome) than those with TR34/L98H, where a fatal outcome was relatively lower (55%) While most of the triazole resistance mechanisms described for A. fumigatus primarily involve mutations in the Cyp51A, 47% of resistant isolates have unknown mechanisms of resistance (4). Therefore, complementary studies with mutants that overexpress Cyp51A due to the presence of tandems repeats in its promoter, or strains overexpressing azole efflux transporters (ABC and MFS) (2,3) are required, since azole resistance in clinical isolates carrying mutations at other loci may result in virulence defects (28,29). The fitness profiles of these resistant mutants could be influenced by complex changes in the expression of several genes that are independent of the target enzyme metabolism (19). 7

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13 274 Figure Legends Figure 1. Growth of mutant strains G54W (a) and M220K (b) in competition with the sensitive parental strain at a 1:1 ratio. (A) Competition was performed on RPMI-agar and growth was estimated with qpcr quantification of gdna after three passages; (B) Competition was performed on Malt agar; growth was estimated as CFU in the presence or absence of 10 µg/ml itraconazole after 6 successive cultures (6 th) and compared to the initial ratio (0) (C); qpcr quantification of growth (gdna) in mice estimated as in A. Statistical analysis was based on three replicates. 13

14 A C B a b a b a b Fig.1