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.

Keywords: Candida albicans; Clavispora lusitaniae; Exophiala dermatitidis; Mrs4; cystic fibrosis; fungi; iron.

Fig 1

Non-synonymous SNPs in MRS4 were found in whole-genome sequence data from 12 to 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. Subjects A, B, and C had 139, 59, and 207 variable loci within each respective isolate population. (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 six transmembrane (TM) domains 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 were quantified by analysis of individual reads.

Fig 2

Mrs4^Q254* confers loss of function in C. lusitaniae. (A) An mrs4∆ mutant and mrs4∆ 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°C in YPD or YPD with 80 µM BPS iron chelator. Columns labeled with a are non-significantly different from each other and are significantly different from columns labeled with b and c. (C) Indicated strains were grown for 24 h at 37°C in YPD supplemented with 2.5 mM CoCl₂ (left) and 12.5 µM CdCl₂ (right). There were at least three replicates per sample. Indicated P-values are from a one-way analysis of variance (ANOVA) with Tukey’s post hoc correction. ns, not significant.

Fig 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 mrs4∆ derivatives that were then complemented with the MRS4^REF allele were grown in YPD supplemented with (A) 2.5 mM CoCl₂ 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°C. Indicated P-values are from one-way ANOVA with Tukey’s post hoc correction. ns, not significant.

Fig 4

Loss of Mrs4 function leads to increased expression of iron acquisition genes. (A) Design of RNA-seq sample preparation. Sextuplicate cultures of B_L01 mrs4∆ 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 80 µM BPS or vehicle prior to RNA isolation. Gene expression heatmaps indicate 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. Gene names in parentheses indicate predicted orthologs in C. albicans. When more than one C. lusitaniae gene is predicted to be most similar to the same C. albicans gene (FRE1 and CCC2), the alleles are differentiated with lowercase letters.

Fig 5

Decreased Mrs4 function increases ferric reductase activity and intracellular iron content. (A) B_L01-derived strains mrs4∆, mrs4∆::MRS4^REF, and mrs4∆::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 represents greater levels of ferric iron reduction. Inclusion of 10 mM of FeCl₃ (+Fe) as a competitor eliminates TTC reduction. (B) B_L01-derived strains mrs4∆, mrs4∆::MRS4^REF, mrs4∆::MRS4^Q254*, and hap43∆ were serially diluted from 1 OD and spotted on YPD plates, then allowed to grow for 24 h at 37°C, and then analyzed as in panel A. (C) C. lusitaniae DH2383 and its mrs4∆ 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 mrs4∆ 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 inductively coupled plasma mass spectrometry (ICP-MS). Data represent the averages of three technical replicates for two experiments done on separate days. Indicated P-values are from Student’s t-tests.

Fig 6

An Mrs4 loss-of-function subpopulation also emerged in E. dermatitidis during a chronic CF lung infection. (A) Two alleles of MRS4 were found in E. dermatitidis isolates from a single chronic CF lung infection. Of the 23 isolates sequenced, 7 genomes encoded the reference Mrs440E (E.d. REF), which is identical to previously sequenced E. dermatitidis strains, and 16 isolates encoded an Mrs4^40G variant (E40G). (B) C. lusitaniae B_L01 mrs4∆ strains complemented with the two E. dermatitidis MRS4 alleles grown in YPD with 80 µM BPS for 24 h. C. lusitaniae B_L01 mrs4∆ strains 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 correction. ns, not significant.