Mrs4 loss of function in fungi during adaptation to the cystic fibrosis lung

ABSTRACT The genetic disease cystic fibrosis (CF) frequently leads to chronic lung infections by bacteria and fungi. We identified three individuals with CF with persistent lung infections dominated by Clavispora (Candida) lusitaniae. Whole-genome sequencing analysis of multiple isolates from each infection found evidence for selection for mutants in the gene MRS4 in all three distinct lung-associated populations. In each population, we found one or two unfixed, non-synonymous mutations in MRS4 relative to the reference allele found in multiple environmental and clinical isolates including the type strain. Genetic and phenotypic analyses found that all evolved alleles led to loss of function (LOF) of Mrs4, a mitochondrial iron transporter. RNA-seq analyses found that Mrs4 variants with decreased activity led to increased expression of genes involved in iron acquisition mechanisms in both low iron and replete iron conditions. Furthermore, surface iron reductase activity and intracellular iron were much higher in strains with Mrs4 LOF variants. Parallel studies found that a subpopulation of a CF-associated Exophiala dermatitidis infection also had a non-synonymous LOF mutation in MRS4. Together, these data suggest that MRS4 mutations may be beneficial during chronic CF lung infections in diverse fungi, perhaps, for the purposes of adaptation to an iron-restricted environment with chronic infections. IMPORTANCE The identification of MRS4 mutations in Clavispora (Candida) lusitaniae and Exophiala dermatitidis in individuals with cystic fibrosis (CF) highlights a possible adaptive mechanism for fungi during chronic CF lung infections. The findings of this study suggest that loss of function of the mitochondrial iron transporter Mrs4 can lead to increased activity of iron acquisition mechanisms, which may be advantageous for fungi in iron-restricted environments during chronic infections. This study provides valuable information for researchers working toward a better understanding of the pathogenesis of chronic lung infections and more effective therapies to treat them.

) differ only by the number of synonymous mutations indicated in the matrix.

Figure S3
. Non-synonymous SNPs occur in predicted high-severity impact regions.SuSPect (Yates, Filippis, Kelley, & Sternberg, 2014) mutational analysis heatmaps indicate that the predicted severity of each possible particular amino acid substitution at a given position and the average impact of all possible amino acid substitutions on a scale from 0 (least severe) to 100 (most severe).Higher areas of impact coincide with predicted transmembrane alpha helix regions (see Fig. 1A).C. lusitaniae MRS4 mutations are highlighted in red with their predicted impact score in parentheses (except for the Q254* mutation).The predicted effects of the E. dermatiditis MRS4 substitution, which leads to an E40G substitution, is also shown.For full citation, see Supplemental References.for 24 h at 37˚ in YP medium supplemented with A) 2% glucose or B) 2% glycerol or in YNB defined medium without amino acids supplemented with C) 2% glucose or D) 2% glycerol.OD 600 was measured over time using a Synergy Neo2 plate reader.The exponential phase growth rates were not significantly different across the three strains in any of the conditions in three independent experiments.E) An mrs4∆ strain, constructed in DH2383, which has MRS4 REF sequence, and the DH2383 parent strain were spotted in a dilution series on YPD plates supplemented with cobalt, cadmium, and hydrogen peroxide as described in the Materials and Methods for 48 h before imaging.F) B_L01 parent, mrs4∆, and complemented strains with the MRS4 REF or native MRS Q254* allele were grown in a 96-well plate format in YPD containing 1 mM H2O2 for 24 h at 37˚.Final yield was measured as absorbance at 600 nm on a Synergy Neo2 plate reader.Table S1: Loci with heterogeneity in encoded amino acid sequences within more than one CF-associated C. lusitaniae population.Whole genome SNP tables were compiled from the sequencing of chronic infection isolates from each individual with CF and compared against each other.Genes which had acquired unfixed mutations (multiple alleles within the population) in more than one population are listed below.MRS4 is the only gene which had unfixed mutations in all three independent populations, and is not listed here among genes which had unfixed mutations associated with two independent populations.C. albicans gene names or their alias are denoted next to their predicted C. lusitaniae ortholog, which was determined by Candida Genome Database.

Figure S2 .
Figure S2.The MRS4 sequences of environmental, reference, and acute infection isolates of C. lusitaniae differ by only a small number of synonymous SNPs.Comparison of synonymous SNPs in the MRS4 sequences encoding the "reference" amino acid sequence.MRS4 alleles of C. lusitaniae clinical strains (DH2383 and ATCC 42740) and environmental isolates (see TableS3) differ only by the number of synonymous mutations indicated in the matrix.

Figure S4 .Figure S6 .
Figure S4.Restriction of mrs4 growth by chelator in defined minimal media.B_L01 mrs4∆ complemented with MRS4 REF and MRS4 Q254* alleles, and SC5314 parent and mrs4∆/∆ derivative were inoculated into Yeast Nitrogen Base, 2% glucose with and without 5 µM bathophenanthroline, sensitivity to low iron is observed in MRS4 defective or knockout strains in C. lusitaniae and C. albicans respectively.Indicated p-values are from Student's T-Test with Holms-Sidak correction.

Figure S7 .Figure S8 .
Figure S7.Quantitative RT-PCR analysis of HAP43 and FTR1 in the absence and presence of Mrs4 activity in iron-chelated conditions.Transcripts encoding the iron-regulating transcription factor Hap43 and high-affinity iron transporter Ftr1 were measured in B_L01 mrs4∆ strains complemented with MRS4 REF or native MRS Q254* in cells grown in YPD for 6 h, or in YPD for 5 h followed by 1 h growth after the addition of 80µM BPS.

Table S2 : Mrs4 loss of function does not alter secondary carbon metabolite secretion in C. lusitaniae.
B_L01 derivatives B_L01::MRS4 REF and B_L01::MRS4 Q254* , and C. albicans SC5314 with single and double knockouts of MRS4, were cultured overnight and sub-cultured into YNB minimal media with 100mM glucose as a primary carbon source.Supernatants were collected and analyzed by HPLC for consumption of glucose, and production of major secondary metabolites.Molar carbon ratios were calculated for consumed glucose and citrate to produced acetate, ethanol, and glycerol, relative to the base media.