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

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.

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

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.

GAUGE-Annotated Microbial Transcriptomic Data Facilitate Parallel Mining and High-Throughput Reanalysis To Form Data-Driven Hypotheses.

The NCBI Gene Expression Omnibus (GEO) provides tools to query and download transcriptomic data. However, less than 4% of microbial experiments include the sample group annotations required to assess differential gene expression for high-throughput reanalysis, and data deposited after 2014 universally lack these annotations. Our algorithm GAUGE (general annotation using text/data group ensembles) automatically annotates GEO microbial data sets, including microarray and RNA sequencing studies, increasing the percentage of data sets amenable to analysis from 4% to 33%. Eighty-nine percent of GAUGE-annotated studies matched group assignments generated by human curators. To demonstrate how GAUGE annotation can lead to scientific insight, we created GAPE (GAUGE-annotated Pseudomonas aeruginosa and Escherichia coli transcriptomic compendia for reanalysis), a Shiny Web interface to analyze 73 GAUGE-annotated P. aeruginosa studies, three times more than previously available. GAPE analysis revealed that PA3923, a gene of unknown function, was frequently differentially expressed in more than 50% of studies and significantly coregulated with genes involved in biofilm formation. Follow-up wet-bench experiments demonstrate that PA3923 mutants are indeed defective in biofilm formation, consistent with predictions facilitated by GAUGE and GAPE. We anticipate that GAUGE and GAPE, which we have made freely available, will make publicly available microbial transcriptomic data easier to reuse and lead to new data-driven hypotheses.IMPORTANCE GEO archives transcriptomic data from over 5,800 microbial experiments and allows researchers to answer questions not directly addressed in published papers. However, less than 4% of the microbial data sets include the sample group annotations required for high-throughput reanalysis. This limitation blocks a considerable amount of microbial transcriptomic data from being reused easily. Here, we demonstrate that the GAUGE algorithm could make 33% of microbial data accessible to parallel mining and reanalysis. GAUGE annotations increase statistical power and, thereby, make consistent patterns of differential gene expression easier to identify. In addition, we developed GAPE (GAUGE-annotated Pseudomonas aeruginosa and Escherichia coli transcriptomic compendia for reanalysis), a Shiny Web interface that performs parallel analyses on P. aeruginosa and E. coli compendia. Source code for GAUGE and GAPE is freely available and can be repurposed to create compendia for other bacterial species.

Balancing Positive and Negative Selection: In Vivo Evolution of Candida lusitaniae MRR1.

The evolution of pathogens in response to selective pressures present during chronic infections can influence their persistence and virulence and the outcomes of antimicrobial therapy. Because subpopulations within an infection can be spatially separated and the host environment can fluctuate, an appreciation of the pathways under selection may be most easily revealed through the analysis of numerous isolates from single infections. Here, we continued our analysis of a set of clonally derived Clavispora (Candida) lusitaniae isolates from a single chronic lung infection with a striking enrichment in the number of alleles of MRR1 Genetic and genomic analyses found evidence for repeated acquisition of gain-of-function mutations that conferred constitutive Mrr1 activity. In the same population, there were multiple alleles with both gain-of-function mutations and secondary suppressor mutations that either attenuated or abolished the constitutive activity, suggesting the presence of counteracting selective pressures. Our studies demonstrated trade-offs between high Mrr1 activity, which confers resistance to the antifungal fluconazole, host factors, and bacterial products through its regulation of MDR1, and resistance to hydrogen peroxide, a reactive oxygen species produced in the neutrophilic environment associated with this infection. This inverse correlation between high Mrr1 activity and hydrogen peroxide resistance was observed in multiple Candida species and in serially collected populations from this individual over 3 years. These data lead us to propose that dynamic or variable selective pressures can be reflected in population genomics and that these dynamics can complicate the drug resistance profile of the population.IMPORTANCE Understanding microbial evolution within patients is critical for managing chronic infections and understanding host-pathogen interactions. Here, our analysis of multiple MRR1 alleles in isolates from a single Clavispora (Candida) lusitaniae infection revealed the selection for both high and low Mrr1 activity. Our studies reveal trade-offs between high Mrr1 activity, which confers resistance to the commonly used antifungal fluconazole, host antimicrobial peptides, and bacterial products, and resistance to hydrogen peroxide. This work suggests that spatial or temporal differences within chronic infections can support a large amount of dynamic and parallel evolution and that Mrr1 activity is under both positive and negative selective pressure to balance different traits that are important for microbial survival.

Debaryomyces is enriched in Crohn's disease intestinal tissue and impairs healing in mice.

Alterations of the mycobiota composition associated with Crohn's disease (CD) are challenging to link to defining elements of pathophysiology, such as poor injury repair. Using culture-dependent and -independent methods, we discovered that Debaryomyces hansenii preferentially localized to and was abundant within incompletely healed intestinal wounds of mice and inflamed mucosal tissues of CD human subjects. D. hansenii cultures from injured mice and inflamed CD tissues impaired colonic healing when introduced into injured conventionally raised or gnotobiotic mice. We reisolated D. hansenii from injured areas of these mice, fulfilling Koch's postulates. Mechanistically, D. hansenii impaired mucosal healing through the myeloid cell-specific type 1 interferon-CCL5 axis. Taken together, we have identified a fungus that inhabits inflamed CD tissue and can lead to dysregulated mucosal healing.

Mrr1 regulation of methylglyoxal catabolism and methylglyoxal-induced fluconazole resistance in Candida lusitaniae.

Transcription factor Mrr1, best known for its regulation of Candida azole resistance genes such as MDR1, regulates other genes that are poorly characterized. Among the other Mrr1-regulated genes are putative methylglyoxal reductases. Methylglyoxal (MG) is a toxic metabolite that is elevated in diabetes, uremia, and sepsis, which are diseases that increase the risk for candidiasis, and MG serves as a regulatory signal in diverse organisms. Our studies in Clavispora lusitaniae, also known as Candida lusitaniae, showed that Mrr1 regulates expression of two paralogous MG reductases, MGD1 and MGD2, and that both participate in MG resistance and MG catabolism. Exogenous MG increased Mrr1-dependent expression of MGD1 and MGD2 as well as expression of MDR1, which encodes an efflux pump that exports fluconazole. MG improved growth in the presence of fluconazole and this was largely Mrr1-dependent with contributions from a secondary transcription factor, Cap1. Increased fluconazole resistance was also observed in mutants lacking Glo1, a Mrr1-independent MG catabolic enzyme. Isolates from other Candida species displayed heterogeneity in MG resistance and MG stimulation of azole resistance. We propose endogenous and host-derived MG can induce MDR1 and other Mrr1-regulated genes causing increased drug resistance, which may contribute to some instances of fungal treatment failure.

Evolution of drug resistance in an antifungal-naive chronic Candida lusitaniae infection.

Management of the limited number of antimicrobials currently available requires the identification of infections that contain drug-resistant isolates and the discovery of factors that promote the evolution of drug resistance. Here, we report a single fungal infection in which we have identified numerous subpopulations that differ in their alleles of a single gene that impacts drug resistance. The diversity at this locus was markedly greater than the reported heterogeneity of alleles conferring antibiotic resistance in bacterial infections. Analysis of genomes from hundreds of Clavispora (Candida) lusitaniae isolates, through individual and pooled isolate sequencing, from a single individual with cystic fibrosis revealed at least 25 nonsynonymous mutations in MRR1, which encodes a transcription factor capable of inducing fluconazole (FLZ) resistance in Candida species. Isolates with high-activity Mrr1 variants were resistant to FLZ due to elevated expression of the MDR1-encoded efflux pump. We found that high Mrr1-regulated Mdr1 activity protected against host and bacterial factors, suggesting drug resistance can be selected for indirectly and perhaps explaining the Mrr1 heterogeneity in this individual who had no prior azole exposure. Regional analysis of C. lusitaniae populations from the upper and lower lobes of the right lung suggested intermingling of subpopulations throughout. Our retrospective characterization of sputum and lung populations by pooled sequencing found that alleles that confer FLZ resistance were a minority in each pool, possibly explaining why they were undetected before unsuccessful FLZ therapy. New susceptibility testing regimes may detect problematical drug-resistant subpopulations in heterogeneous single-species infections.

Use of RNA-Protein Complexes for Genome Editing in Non-albicans Candida Species.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome modification systems have greatly facilitated the genetic analysis of fungal pathogens. In CRISPR-Cas9 genome editing methods designed for use in Candida albicans, DNAs that encode the necessary components are expressed in the target cells. Unfortunately, expression constructs that work efficiently in C. albicans are not necessarily expressed well in other pathogenic species within the genus Candida or the related genus Clavispora. To circumvent the need for species-specific expression constructs, we implemented an expression-free CRISPR genome editing system and demonstrated its successful use in three different non-albicans Candida species: Candida (Clavispora) lusitaniae, Candida glabrata, and Candida auris. In CRISPR-Cas9-mediated genome editing methods, a targeted double-stranded DNA break can be repaired by homologous recombination to a template designed by the investigator. In this protocol, the DNA cleavage is induced upon transformation of purified Cas9 protein in complex with gene-specific and scaffold RNAs, referred to as RNA-protein complexes (RNPs). In all three species, the use of RNPs increased both the number of transformants and the percentage of transformants in which the target gene was successfully replaced with a selectable marker. We constructed mutants defective in known or putative catalase genes in C. lusitaniae, C. glabrata, and C. auris and demonstrated that, in all three species, mutants were more susceptible to hydrogen peroxide than the parental strain. This method, which circumvents the need for expression of CRISPR-Cas9 components, may be broadly useful in the study of diverse Candida species and emergent pathogens for which there are limited genetic tools. IMPORTANCE Existing CRISPR-Cas9 genome modification systems for use in Candida albicans, which rely on constructs to endogenously express the Cas9 protein and guide RNA, do not work efficiently in other Candida species due to inefficient promoter activity. Here, we present an expression-free method that uses RNA-protein complexes and demonstrate its use in three Candida species known for their drug resistance profiles. We propose that this system will aid the genetic analysis of fungi that lack established genetic systems.

A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles.

Bacterial outer membrane vesicle (OMV)-mediated delivery of proteins to host cells is an important mechanism of host-pathogen communication. Emerging evidence suggests that OMVs contain differentially packaged short RNAs (sRNAs) with the potential to target host mRNA function and/or stability. In this study, we used RNA-Seq to characterize differentially packaged sRNAs in Pseudomonas aeruginosa OMVs, and to show transfer of OMV sRNAs to human airway cells. We selected one sRNA for further study based on its stable secondary structure and predicted mRNA targets. Our candidate sRNA (sRNA52320), a fragment of a P. aeruginosa methionine tRNA, was abundant in OMVs and reduced LPS-induced as well as OMV-induced IL-8 secretion by cultured primary human airway epithelial cells. We also showed that sRNA52320 attenuated OMV-induced KC cytokine secretion and neutrophil infiltration in mouse lung. Collectively, these findings are consistent with the hypothesis that sRNA52320 in OMVs is a novel mechanism of host-pathogen interaction whereby P. aeruginosa reduces the host immune response.

Mitochondrial Activity and Cyr1 Are Key Regulators of Ras1 Activation of C. albicans Virulence Pathways.

Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate different environmental and intracellular signals, including metabolic status, to decide the fate of cellular morphology.