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 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 was much higher in strains with Mrs4 loss of function variants. Parallel studies found that a subpopulation of a CF-associated Exophiala dermatiditis infection also had a non-synonymous loss of function 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.

Figure 1.. Non-synonymous SNPs in MRS4 were found in whole genome sequence data from 12–20 C. lusitaniae isolates from each of three subjects with chronic CF infections.

A) Analysis of loci that were heterogeneous in three C. lusitaniae populations in three separate individuals (Subjects A, B, and C) found that only CLUG_02526 (MRS4) had subpopulations with non-synonymous substitutions in all three infections. B) Two MRS4 alleles were detected in each population. “REF” indicates the MRS4 sequence in environmental and acute infection isolates of C. lusitaniae. C) The MRS4 sequence encodes a barrel-structure iron transporter on the inner mitochondrial membrane; the protein is 318 amino acids long and comprised of six transmembrane alpha-helices denoted by light blue bars. Each mutation is predicted to disrupt or truncate one of these transmembrane domains (see SUSPECT analysis, Fig S3). D) Pooled sequencing was performed on isolates from bronchoalveolar lavage fluid taken from specific lobes of subjects A and B. The relative abundances of MRS4 alleles was quantified by analysis of individual reads.

Figure 2.

Mrs4^Q254* confers loss of function in C. lusitaniae. A) An mrs4Δ mutant and mrs4Δ mutants complemented with MRS4^Q254* or the MRS4^REF were constructed in the B_L01 clinical isolate background B) Strains were assessed for growth in a 96-well plate after 24 h at 37° in YPD or YPD with 80 μM BPS iron chelator. Columns labelled with a are non-significantly different from each other, and are significantly different from columns labelled with b and c. C) Indicated strains were grown for 24 h at 37° in YPD supplemented with 2.5 mM CoCl₂ (left) and 12.5μM CdCl₂ (right). There were at least three replicates per sample. Indicated p-values are from a one-way ANOVA with Tukey’s post-hoc correction, ns, not significant.

Figure 3.. MRS4 mutations in each clinical population demonstrate LOF phenotypes.

Representative parent isolates of each mutation from Subject B (B_L04, MRS4^A147D), Subject A (A_U05, MRS4^A235V), and Subject C, (C_M06, MRS4^G138V) and their mrs4Δ derivatives that were then complemented with the MRS4^REF allele, were grown in YPD supplemented with A) 2.5 mM CoCl₃ and B) 12.5 μM CdCl₂. Data represent the endpoint OD600 measured by a Synergy Neo2 plate reader after 24 h of growth at 37°. Indicated p-values are from one-way ANOVA with Tukey’s post-hoc , ns, not significant.

Figure 4.. Lack of MRS4 growth phenotypes in complex and minimal media with glycerol and glucose.

Cells from exponential phase cultures of B_L01, its mrs4Δ derivative, and mrs4Δ mutants complemented with MRS4^Q254* or the MRS4^REF were inoculated into a 96-well plate and grown 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. OD₆₀₀ was measured over time using a Synergy Neo2 plate reader.

Figure 5.. Loss of Mrs4 function leads to increased expression of iron acquisition genes.

A) Design of RNA-seq sample preparation. Sextuplicate cultures of B_L01 mrs4Δ complemented with either REF or Q254* MRS4 alleles were grown overnight, then sub-cultured into YPD and grown for 5 h. Cultures were grown for an additional hour with either 80μM BPS or vehicle prior to RNA isolation. Gene expression heatmaps of differentially expressed genes (P < 0.05 and a log₂ fold-change ≥|1|) in a comparison between B) the B_L01::MRS4^REF strain grown in YPD (iron replete) or YPD with BPS (iron deplete) C) B_L01::MRS4^REF and B_L01::MRS4^Q254* grown in YPD with BPS, and D) B_L01::MRS4^REF and B_L01::MRS4^Q254* grown in YPD.

Figure 6.. Decreased Mrs4 function increases ferric reductase activity and intracellular iron content.

A) B_L01 derived strains mrs4Δ, mrs4Δ::MRS4^REF, mrs4Δ::MRS4^Q254* were spotted on YNB-glycerol plates. Plates were incubated for 24 h at 37°C. Each plate was overlayed with a 10 ml solution of 1 mg/ml tetrazolium chloride (TTC) and incubated for 5 min prior to imaging. Red pigmentation indicated represents greater levels of ferric iron reduction. Inclusion of 10 mM of FeCl₃ (+Fe) as a competitor eliminates TTC reduction. 24 B) B_L01 derived strains mrs4Δ, mrs4Δ::MRS4^REF, mrs4Δ::MRS4^Q254* and hap43Δ were serially diluted from 1 OD and spotted on YPD plates, then allowed to grow for 24 hours at 37° C then analyzed as in panel A. C) C. lusitaniae DH2383 and its mrs4Δ mutant and C. albicans SC5314 with single and double knockouts of mrs4 were analyzed for surface ferric reductase activity on YNB-glycerol. D) B_L01 mrs4Δ strains complemented with MRS4^REF and MRS4^Q254* alleles were grown in YPD or YPD + 80 μM BPS as outlined in Fig. 5A. Whole cell iron was quantified using ICP-MS. Data represents the averages of three technical replicates for two experiments done on separate days. Indicated p-values are from Student’s t-tests.

Figure 7.. An Mrs4 loss-of-function subpopulation also emerged in Exophiala dermatiditis during a chronic CF lung infection.

A) Two alleles of MRS4 were found in E. dermatitidis isolates from in a single chronic CF lung infection. Of the 23 isolates sequenced, seven genomes encoded the reference Mrs4^40E (E.d. REF), which is identical to previously sequenced E. dermitiditis strains, and sixteen isolates encoded an Mrs4^40Gvariant (E40G). B) C. lusitaniae B_L01 mrs4Δ strains complemented with the two E. dermatitidis MRS4 alleles grown in YPD with 80 μM BPS for 24 h. C. lusitaniae B_L01 mrs4Δ expressing functional MRS4^REF or MRS4^Q254* were included for comparison. Indicated p-value are from a one-way ANOVA with Tukey’s post-hoc , ns, not significant.