Pseudomonas aeruginosa infections in adults with cystic fibrosis (CF) are comprised of heterogeneous populations, most often tracing ancestry back to a single recent common ancestor. What is not clear is the physical spatial structure within the lung infection population, its stability over time, and whether this physical structure leads to different evolutionary trajectories in different adaptive environments. To compare the P. aeruginosa populations across a single lung, we performed whole genome sequence analyses of 450 isolates recovered from lavage samples of the three different lobes of the right lung from a person with mild-to-moderate CF lung disease at three time points over the course of ~1.5 years. We found that isolates fell into five distinct phylogenetic lineages with evidence for repeated translocation of isolates from different lineages across lobes and loss-of-function mutations in lasR and mucA were present in all 450 isolates. The well-resolved phylogenetic analyses revealed a structured population in which we find the coexistence of a slowly evolving lineage and more rapidly evolving lineages. There is also support for numerous migration events. Furthermore, strong evidence for parallel adaptive mutations in multiple genes revealed distinct evolutionary paths affecting mucoid phenotypes and genetic variation in antibiotic resistance-associated pathways across coexisting populations within a single individual over time. These results provide an example of within-host evolution leading to microheterogeneity that may be useful to consider in future study of infection metapopulations dynamics over the course of chronic infection.IMPORTANCEIndividuals with cystic fibrosis (CF) commonly have chronic lung infections that contain clonally derived Pseudomonas aeruginosa populations with genotypic and phenotypic diversity. This study describes a substantial data set containing 450 isolates from different lobes of the right lung across three time points from an individual with mild-to-moderate CF lung disease. Some regional enrichment for specific lineages with parallel mutations among individual lobes of the lung was observed, but longitudinal analysis also demonstrated that compartmentalization is not strictly maintained and that isolates migrate between lobes of the lung over time. Perspectives on within lung evolution will be important for understanding the pathogen populations in chronic respiratory infections in CF and other diseases.

ABSTRACT Innate immune signaling plays a key role in host response to infection, yet the pattern recognition receptors that detect non-model gut-associated yeasts remain poorly defined. Here, we investigated macrophage sensing of Debaryomyces hansenii , a food-derived yeast that we found to be enriched within intestinal ulcers of Crohn disease (CD) patients. Using a cell surface receptor antibody screen of bone marrow-derived macrophages infected with a CD patient isolate of D. hansenii , we showed that D. hansenii -induced macrophage activation characterized by increased expression of co-stimulatory molecules, MHC-II, and pattern recognition receptors, including the C-type lectin receptor Dectin-1. Antibody blockade experiments showed both Dectin-1 and complement receptor 3 subunit CD11b were required for phagocytosis of D. hansenii , while Dectin-1 was uniquely required for production of the pro-inflammatory cytokine tumor necrosis factor (Tnf). CRISPR-Cas9-mediated deletion of Dectin-1 phenocopied antibody neutralization effects on phagocytosis. Furthermore, deletion of Dectin-1 or its downstream signaling adaptor molecule Card9 resulted in reduced Tnf secretion in response to D. hansenii . Dectin-1-mediated uptake of D. hansenii was observed in primary bone marrow-derived macrophage and dendritic cells, as well as across the spectrum of macrophage polarization states. Together, these findings define the role of Dectin-1-Card9 signaling axis in innate immune cell sensing of D. hansenii . These findings support the emerging relevance of innate immune recognition of a yeast in Crohn disease pathogenesis.

ABSTRACT Strains of food-derived microbes can become facultative pathogens in susceptible human hosts. Surprisingly, we previously isolated Debaryomyces hansenii , a yeast common in fermented foods, from Crohn disease (CD) ulcers, raising questions about its strain-specific traits that influence host interactions. Here, we further developed the genetic tractability of D. hansenii and identified a single adhesin, Hil1, as a major determinant of colony morphology, biofilm formation, and immune targeting in CD patients. We used Agrobacterium tumefaciens -mediated transformation to perform a forward genetic screen in a food-derived reference strain. We isolated mutants that converted from a wrinkled, biofilm-forming phenotype to a smooth, non-adherent phenotype characteristic of CD patient isolates. Mapping of multiple insertion sites showed a disrupted subtelomeric Hyr/Iff-like adhesin gene, herein referred to as HIL1 . CRISPR-Cas9-mediated deletion of HIL1 recapitulated the mutant phenotype, demonstrating that HIL1 was necessary for biofilm formation and high cell-surface hydrophobicity phenotypes. To contextualize these findings, we performed comparative genomics on a D. hansenii strain collection to assess allelic variation in the number of HIL1 tandem repeats. Longer alleles in food strains correlated with increased biofilm formation, while CD-isolated strains contained shorter HIL1 alleles and reduced binding to surfaces. Serology profiling showed that HIL1 was a direct antigenic target of circulating immunoglobulin G (IgG) in CD patients. Together, these results suggest Hil1 is a key, strain-variable adhesin shaping fungal surface properties and host immune recognition. This work establishes D. hansenii as a genetically tractable system and shows how adhesin polymorphisms may influence fungal behavior in food and disease contexts. IMPORTANCE Debaryomyces hansenii is a yeast that is common in food and is generally recognized as safe for human consumption, though recently it has been identified within diseased regions of the intestine in Crohn disease patients. A current need is to determine the genetic and phenotypic differences between safe food isolates and isolates from human Crohn disease patient ulcers. Here, we used a loss-of-function genetic screen and identified HIL1 , an adhesin that we found mediates cellular adhesion in many food strains but not in patient strains. We identified circulating HIL1 -reactive antibodies in patients with Crohn disease, indicating that food strains can be a target of host immune responses through Hil1.

Cystic fibrosis (CF) is characterized by chronic muco-obstructive lung disease and infection. People with CF (pwCF) are often colonized with Pseudomonas aeruginosa for years to decades, allowing for evolutionary adaptation. In chronic P. aeruginosa lung isolates from pwCF, the quorum-sensing regulator LasR is frequently nonfunctional; however, the factors enabling lasR loss-of-function (LOF) mutant selection are incompletely understood. We hypothesized that LOF mutations in lasR could allow P. aeruginosa to resist the selective pressure of phagocytosis. We found that in multiple strain backgrounds, LasR LOF decreased phagocytosis by both model THP-1 and primary monocyte-derived macrophages, and lasR complementation increased phagocytosis in mutant strains. While exogenous administration of the quorum-sensing autoinducer 3-oxo-C12-homoserine-lactone, which is made by an enzyme regulated by LasR activity, inhibited phagocytosis and mitochondrial respiration, the phagocytosis resistance seen with lasR mutants appears to be bacterial cell intrinsic rather than due to secreted factors. Finally, we found that lasR LOF mutations altered the inflammatory profile upon infection of CF macrophages, with a shift from IL-1 family cytokine expression toward canonical inflammatory markers, including IL-6 and TNFα. Collectively, these data provide a potential explanation for both the prevalence of lasR mutants in the CF lung as well as their association with worse outcomes.IMPORTANCECystic fibrosis (CF) is a genetically inherited disease that leads to chronic lung infections. Pseudomonas aeruginosa is often implicated in the worsening of lung disease, and it evolves in the lung over time to resist eradication. One of the most commonly disrupted genes in P. aeruginosa isolates from chronically infected CF lungs is lasR, which encodes a transcription factor that regulates multiple virulence factors. What contributes to the apparent fitness of lasR mutants in the CF lung is not well known. Our study shows that lasR loss-of-function mutants resist phagocytosis by macrophages, one of the fundamental mechanisms of clearance by the immune system. We identify mechanisms promoting resistance to phagocytosis and explore the downstream consequences on inflammatory responses. Understanding why lasR mutations arise could inform strategies to eradicate them from the CF lung and improve outcomes.

While the Pseudomonas aeruginosa LasR transcription factor plays a role in quorum sensing (QS) across phylogenetically-distinct lineages, isolates with loss-of-function mutations in lasR (LasR- strains) are commonly found in diverse settings including infections where they are associated with worse clinical outcomes. In LasR- strains, the transcription factor RhlR, which is controlled by LasR, can be alternately activated in low inorganic phosphate (Pi) concentrations via the two-component system PhoR-PhoB. Here, we demonstrate a new link between LasR and PhoB in which the absence of LasR increases PhoB activity at physiological Pi concentrations and raises the Pi concentration necessary for PhoB inhibition. PhoB activity was also less repressed by Pi in mutants lacking different QS regulators (RhlR and PqsR) and in mutants lacking genes required for the production of QS-regulated phenazines suggesting that decreased phenazine production was one reason for decreased PhoB repression by Pi in LasR- strains. In addition, the CbrA-CbrB two-component system, which is elevated in LasR- strains, was necessary for reduced PhoB repression by Pi and a Δcrc mutant, which lacks the CbrA-CbrB-controlled translational repressor, activated PhoB at higher Pi concentrations than the wild type. The ΔlasR mutant had a PhoB-dependent growth advantage in a medium with no added Pi and increased virulence-determinant gene expression in a medium with physiological Pi, in part through reactivation of QS. This work suggests PhoB activity may contribute to the virulence of LasR- P. aeruginosa and subsequent clinical outcomes.

Pseudomonas aeruginosa, which causes many types of infections, often has loss-of-function mutations in the gene encoding LasR, a transcription factor involved in quorum sensing. Here, we report that LasR- strains are more sensitive to the electrophile methylglyoxal (MGO), which is produced by many cells, including those involved in immune defense. MGO, due to its high reactivity, must be rapidly detoxified to prevent the formation of damaging adducts on macromolecules. GloA3 is a GSH-dependent MGO glyoxalase involved in MGO detoxification, and we found that intracellular GSH levels were lower in ∆lasR strains, as measured by multiple methods. Furthermore, we show that exogenous GSH and GloA3 overexpression both enhance GloA3-dependent MGO resistance, particularly in a ∆lasR mutant. Previous reports suggest that one factor that contributes to the selection for LasR- strains is a growth advantage resulting from increased activity of the CbrAB two-component system, which lowers the Crc-mediated translational repression of many RNAs. Genetic data presented here suggest that low Crc activity leads to increased MGO sensitivity in LasR- strains but not through effects on GSH or GloA3. Together, these data suggest that LasR- strains are more sensitive to MGO due to multiple factors, including lower intracellular levels of GSH and decreased Crc activity, and that these factors may represent fitness trade-offs for LasR- strains.IMPORTANCEMethylglyoxal (MGO) is a highly reactive metabolite detected in various disease states, including those involving Pseudomonas aeruginosa. P. aeruginosa requires the glutathione-dependent glyoxalase enzyme GloA3 for MGO resistance. This study reveals that P. aeruginosa strains with mutations in the gene encoding the transcription factor LasR, commonly found in clinical isolates, are more sensitive to MGO due to lower intracellular glutathione levels and high activity of the CbrAB-Crc regulatory pathway. Thus, sensitivity to MGO and other electrophiles may represent a trade-off for P. aeruginosa in infections.

Pseudomonas aeruginosa causes acute and chronic infections such as those that occur in the lungs of people with cystic fibrosis (CF). In infection environments, oxygen (O 2 ) concentrations are often low. The transcription factor Anr responds to low O 2 by upregulating genes necessary for P. aeruginosa fitness in microoxic and anoxic conditions. Anr regulates dnr , a gene encoding a transcriptional regulator that promotes the expression of genes required for using nitrate as an alternative electron acceptor during denitrification. In CF sputum, transcripts involved in denitrification are highly expressed. While Dnr is necessary for the anoxic growth of P. aeruginosa in CF sputum and artificial sputum media (ASMi), the contribution of denitrification to P. aeruginosa fitness in oxic conditions has not been well described. Here we show that P. aeruginosa requires dnr for fitness in ASMi and the requirement for dnr is abolished when nitrate is excluded from the media. Additionally, we show that P. aeruginosa consumes nitrate in lysogeny broth (LB) under microoxic conditions. Furthermore, strains without a functioning quorum sensing regulator LasR, which leads to elevated Anr activity, consume nitrate in LB even in normoxia. There was no growth advantage for P. aeruginosa when nitrate was present at concentrations from 100 µM to 1600 µM. However, P. aeruginosa consumption of nitrate in oxic conditions created a requirement for Dnr and Dnr-regulated NorCB likely due to the need to detoxify nitric oxide. These studies suggest that Anr- and Dnr-regulated processes may impact P. aeruginosa physiology in many common culture conditions. Importance:Pseudomonas aeruginosa is an opportunistic pathogen commonly isolated from low-oxygen environments such as the lungs of people with cystic fibrosis. While the importance of P. aeruginosa energy generation by denitrification is clear in anoxic environments, the effects of denitrification in oxic cultures is not clear. Here, we show that nitrate is consumed even in oxic environments and while it does not appear to stimulate growth, it does impact fitness. Further, we report that two regulators that are best known for their roles in anoxic conditions also contribute to P. aeruginosa fitness in commonly- used laboratory media in presence of oxygen.

Infections caused by the emerging pathogenic yeast Clavispora (Candida) lusitaniae can be difficult to manage due to multi-drug resistance. Resistance to the frontline antifungal fluconazole (FLZ) in Candida spp. is commonly acquired through gain-of-function (GOF) mutations in the gene encoding the transcription factor Mrr1. These activated Mrr1 variants enhance FLZ efflux via upregulation of the multi-drug transporter gene MDR1. Recently, it was reported that, unlike in the well-studied Candida albicans species, C. lusitaniae and Candida parapsilosis with activated Mrr1 also have high expression of CDR1, which encodes another multi-drug transporter with overlapping but distinct transported substrate profiles and Cdr1-dependent FLZ resistance. To better understand the mechanisms of Mrr1 regulation of MDR1 and CDR1, and other co-regulated genes, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analysis of Mrr1 binding sites. Mrr1 bound the promoter regions of MDR1 and CDR1, as well as FLU1, which encodes another transporter capable of FLZ efflux. Mdr1 and Cdr1 independently contributed to the decreased susceptibility of the MRR1GOF strains against diverse clinical azoles and other antifungals, including 5-flucytosine. A consensus motif, CGGAGWTAR, enriched in Mrr1-bound C. lusitaniae DNA was also conserved upstream of MDR1 and CDR1 across species, including C. albicans. CUT&RUN and RNA-seq data were used to define the Mrr1 regulon, which includes genes involved in transport, stress response, and metabolism. Activated and inducible Mrr1 bound similar regions in the promoters of Mrr1 regulon genes. Our studies provide new evolutionary insights into the coordinated regulation of multi-drug transporters and potential mechanism(s) that aid secondary resistance acquisition in emerging Candida. IMPORTANCE Understanding antifungal resistance in emerging Candida pathogens is essential to managing treatment failures and guiding the development of new therapeutic strategies. Like other Candida species, the environmental opportunistic fungal pathogen Clavispora (Candida) lusitaniae can acquire resistance to the antifungal fluconazole by overexpression of the multi-drug efflux pump Mdr1 through gain-of-function (GOF) mutations in the gene encoding the transcription factor Mrr1. Here, we show that C. lusitaniae Mrr1 also directly regulates CDR1, another major multi-drug transporter gene, along with MDR1. In strains with activated Mrr1, upregulation of MDR1 and CDR1 protects against diverse antifungals, potentially aiding the rise of other resistance mutations. Mrr1 also regulates several stress response and metabolism genes, thereby providing new perspectives into the physiology of drug-resistant strains. The identification of an Mrr1 binding motif that is conserved across strains and species will advance future efforts to understand multi-drug resistance across Candida species.

In the opportunistic pathogen Candida albicans, hyphal growth and virulence factor expression are regulated by environmental and chemical cues. Farnesol is a secreted autoregulatory molecule that represses filamentation. It is derived from farnesyl pyrophosphate (FPP), an ergosterol biosynthesis pathway intermediate. Although Dpp1, Dpp2, and Dpp3 were proposed to synthesize farnesol, a mutant lacking all three had only a modest reduction in farnesol. To identify other farnesol biosynthesis genes, we employed transcription factor mutants that underproduced or overproduced farnesol in a screen and analyzed their transcriptomes. CWH8 was the only transcript correlated with farnesol production. Cwh8 is a lipid phosphatase known to recycle dolichyl pyrophosphate during glycosylation of cell wall proteins. The cwh8ΔΔ mutant had a > 99% reduction in farnesol compared to the parent strain and was hyperfilamentous in embedded conditions. It had ~900 fold lower levels of farnesyl phosphate, the product of FPP dephosphorylation, suggesting Cwh8 acts directly on FPP. Complementation of the C. albicans cwh8ΔΔ mutant with CWH8 from C. albicans restored cell wall integrity and farnesol production. However, complementation with CWH8 from Clavispora lusitaniae, a species lacking farnesol signaling, restored cell wall integrity but failed to rescue farnesol production. This indicates that while Cwh8’s role in maintaining cell wall integrity is conserved, differences in substrate specificity or interactions with species-specific cofactors underlie its involvement in farnesol biosynthesis in C. albicans. Finally, fluconazole was fungicidal for cwh8ΔΔ rather than fungistatic. These studies demonstrate how an extracellular signaling system can arise and be co-opted to promote fungal fitness in complex environments.IMPORTANCEFarnesol secretion distinguishes the human opportunistic pathogen Candida albicans from non-secreting, non-pathogenic yeasts. Despite 20 years of research, surprisingly little is known about how farnesol is synthesized and regulated. Using transcriptomic profiles from mutants with altered farnesol production, we identified CWH8 as a critical enzyme in this process. CWH8 null mutants produced no farnesol. Our novel assay measuring cellular farnesyl pyrophosphate (FPP) and farnesyl phosphate (FP) showed that Cwh8 converts FPP to FP in the first step of farnesol biosynthesis, in addition to its established role in recycling dolichyl pyrophosphate. Farnesol-secreting fungi had huge metabolic pools of FP, while nonsecretors had small pools. Furthermore, expressing CWH8 from Clavispora lusitaniae, a non-farnesol-secreting species, failed to restore farnesol production in C. albicans. This suggests that changes in Cwh8 enzyme specificity drove the evolution of farnesol as a signaling molecule and virulence factor in C. albicans.

Pseudomonas aeruginosa causes acute and chronic infections, such as those that occur in the lungs of people with cystic fibrosis (CF). In infection environments, oxygen (O2) concentrations are often low. The transcription factor Anr (anaerobic regulation of arginine deiminase and nitrate reduction) responds to low O2 by upregulating genes necessary for P. aeruginosa fitness in microoxic and anoxic conditions. Anr regulates Dnr (dissimilative nitrate respiration regulator), a gene encoding a transcriptional regulator that promotes the expression of genes required for using nitrate as an alternative electron acceptor during denitrification. In CF sputum, transcripts involved in denitrification are highly expressed. While Dnr is necessary for the anoxic growth of P. aeruginosa in CF sputum and artificial sputum media (ASMi), the contribution of denitrification to P. aeruginosa fitness in oxic conditions has not been well described. Here, we show that P. aeruginosa requires dnr for fitness in ASMi, and the requirement for dnr is abolished when nitrate is excluded from the media. Additionally, we show that P. aeruginosa consumes nitrate in lysogeny broth (LB) under microoxic conditions. Furthermore, strains without a functioning quorum sensing regulator LasR, which leads to elevated Anr activity, consume nitrate in LB even in normoxia. There was no growth advantage for P. aeruginosa when nitrate was present at concentrations from 100 to 1,600 µM. However, P. aeruginosa consumption of nitrate in oxic conditions created a requirement for Dnr and Dnr-regulated NorCB, likely due to the need to detoxify nitric oxide. These studies suggest that Anr- and Dnr-regulated processes may impact P. aeruginosa physiology in many common culture conditions.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen commonly isolated from low-oxygen environments such as the lungs of people with cystic fibrosis. While the importance of P. aeruginosa energy generation by denitrification is clear in anoxic environments, the effects of denitrification in oxic cultures are not well understood. Here, we show that nitrate is consumed in microoxic environments and, in some strains, in normoxic environments. While nitrate does not appear to stimulate microoxic growth rate or yield, it does impact physiology. We show that the regulators Anr (anaerobic regulation of arginine deiminase and nitrate reduction) and Dnr (dissimilative nitrate respiration regulator), which are best known for their roles in anoxic conditions, contribute to P. aeruginosa fitness in common laboratory media in the presence of oxygen.

Across the tree of life, clonal populations—from cancer to chronic bacterial infections — frequently give rise to subpopulations with different metabolic phenotypes. Metabolic exchange or cross-feeding between subpopulations can have profound effects on both cell phenotypes and population-level behavior. In Pseudomonas aeruginosa, subpopulations with loss-of-function mutations in the lasR gene are common. Though LasR is often described for its role in density-dependent virulence factor expression, interactions between genotypes suggest potential metabolic differences. The specific metabolic pathways and regulatory genetics enabling such interactions were previously undescribed. Here, we performed an unbiased metabolomics analysis that revealed broad differences in intracellular metabolomes, including higher levels of intracellular citrate in LasR- strains. We found that while both strains secreted citrate, only LasR- strains, consumed citrate in rich media. Elevated activity of the CbrAB two component system which relieves carbon catabolite repression enabled citrate uptake. Within mixed genotype communities, we found that the citrate responsive two component system TctED and its gene targets OpdH (porin) and TctABC (transporter) required for citrate uptake were induced and required for enhanced RhlR signalling and virulence factor expression in LasR- strains. Enhanced citrate uptake by LasR- strains eliminates differences in RhlR activity between LasR+ and LasR- strains thereby circum-venting the sensitivity of LasR- strains to quorum sensing controlled exoproducts. Citrate cross feeding also induces pyocyanin production in LasR- strains co-cultured with Staphylococcus aureus, another species known to secrete biologically-active concentrations of citrate. Metabolite cross feeding may play unrecognized roles in competitive fitness and virulence outcomes when different cell types are together. IMPORTANCE Cross-feeding can change community composition, structure and function. Though cross-feeding has predominantly focused on interactions between species, here we unravel a cross-feeding mechanism between frequently co-observed isolate genotypes of Pseudomonas aeruginosa. Here we illustrate an example of how such clonally-derived metabolic diversity enables intraspecies cross-feeding. Citrate, a metabolite released by many cells including P. aeruginosa, was differentially consumed between genotypes, and this cross-feeding induced virulence factor expression and fitness in genotypes associated with worse disease.

ABSTRACTSwarming is a macroscopic phenomenon in which surface bacteria organize into a motile population. The flagellar motor that drives swarming in Pseudomonas aeruginosa is powered by stators MotAB and MotCD. Deletion of the MotCD stator eliminates swarming, whereas deletion of the MotAB stator enhances swarming. Interestingly, we measured a strongly asymmetric stator availability in the wild-type (WT) strain, with MotAB stators produced at an approximately 40-fold higher level than MotCD stators. However, utilization of MotCD stators in free swimming cells requires higher liquid viscosities, while MotAB stators are readily utilized at low viscosities. Importantly, we find that cells with MotCD stators are ~10× more likely to have an active motor compared to cells uses the MotAB stators. The spectrum of motility intermittency can either cooperatively shut down or promote flagellum motility in WT populations. In P. aeruginosa, transition from a static solid-like biofilm to a dynamic liquid-like swarm is not achieved at a single critical value of flagellum torque or stator fraction but is collectively controlled by diverse combinations of flagellum activities and motor intermittencies via dynamic stator utilization. Experimental and computational results indicate that the initiation or arrest of flagellum-driven swarming motility does not occur from individual fitness or motility performance but rather related to concepts from the “jamming transition” in active granular matter.IMPORTANCEIt is now known that there exist multifactorial influences on swarming motility for P. aeruginosa, but it is not clear precisely why stator selection in the flagellum motor is so important. We show differential production and utilization of the stators. Moreover, we find the unanticipated result that the two motor configurations have significantly different motor intermittencies: the fraction of flagellum-active cells in a population on average with MotCD is active ~10× more often than with MotAB. What emerges from this complex landscape of stator utilization and resultant motor output is an intrinsically heterogeneous population of motile cells. We show how consequences of stator recruitment led to swarming motility and how the stators potentially relate to surface sensing circuitry.

The electrophile methylglyoxal (MG) is produced by microorganisms and host cells through central metabolic pathways. MG is a highly reactive electrophile, so it must be rapidly detoxified to prevent damaging modifications to macromolecules. Pseudomonas aeruginosa , a pathogen of concern due to its ability develop multidrug resistance, causes many types of infections that have been associated with elevated MG levels, including cystic fibrosis (CF). P. aeruginosa isolates commonly have mutations that lead to LasR loss-of-function (LasR-) and we found that lasR mutations confer sensitivity to MG in multiple strain backgrounds. LasR- strains have increased activity of the CbrAB two-component system which represses Crc regulation of metabolism. Here, we show that higher CbrAB activity and low Crc activity renders cells sensitive to MG. We found that P. aeruginosa LasR- strains are more sensitive to MG and have lower intracellular reduced glutathione (GSH) compared to their LasR+ comparators. Consistent with published reports, mutants lacking gloA3 , which encodes a MG-glyoxalase, and mutants lacking GSH biosynthesis enzymes ( gshA or gshB ) were sensitive to MG. Exogenous GSH rescued MG sensitivity in LasR- strains and gshA or gshB mutants, but not in a gloA3 mutant strain. We propose that low GSH levels in LasR- strains contribute to increased sensitivity to MG and H2O2.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

Chronic Pseudomonas aeruginosa lung infections are a feature of cystic fibrosis (CF) that many patients experience even with the advent of highly effective modulator therapies. Identifying factors that impact P. aeruginosa in the CF lung could yield novel strategies to eradicate infection or otherwise improve outcomes. To complement published P. aeruginosa studies using laboratory models or RNA isolated from sputum, we analyzed transcripts of strain PAO1 after incubation in sputum from different CF donors prior to RNA extraction. We compared PAO1 gene expression in this "spike-in" sputum model to that for P. aeruginosa grown in synthetic cystic fibrosis sputum medium to determine key genes, which are among the most differentially expressed or most highly expressed. Using the key genes, gene sets with correlated expression were determined using the gene expression analysis tool eADAGE. Gene sets were used to analyze the activity of specific pathways in P. aeruginosa grown in sputum from different individuals. Gene sets that we found to be more active in sputum showed similar activation in published data that included P. aeruginosa RNA isolated from sputum relative to corresponding in vitro reference cultures. In the ex vivo samples, P. aeruginosa had increased levels of genes related to zinc and iron acquisition which were suppressed by metal amendment of sputum. We also found a significant correlation between expression of the H1-type VI secretion system and CFTR corrector use by the sputum donor. An ex vivo sputum model or synthetic sputum medium formulation that imposes metal restriction may enhance future CF-related studies.IMPORTANCEIdentifying the gene expression programs used by Pseudomonas aeruginosa to colonize the lungs of people with cystic fibrosis (CF) will illuminate new therapeutic strategies. To capture these transcriptional programs, we cultured the common P. aeruginosa laboratory strain PAO1 in expectorated sputum from CF patient donors. Through bioinformatic analysis, we defined sets of genes that are more transcriptionally active in real CF sputum compared to a synthetic cystic fibrosis sputum medium. Many of the most differentially active gene sets contained genes related to metal acquisition, suggesting that these gene sets play an active role in scavenging for metals in the CF lung environment which may be inadequately represented in some models. Future studies of P. aeruginosa transcript abundance in CF may benefit from the use of an expectorated sputum model or media supplemented with factors that induce metal restriction.

To manage and treat chronic fungal diseases effectively, we require an improved understanding of their complexity. There is an increasing appreciation that chronic infection populations are often heterogeneous due to diversification and drift, even within a single microbial species. Genetically diverse populations can contribute to persistence and resistance to treatment by maintaining cells with different phenotypes capable of thriving in these dynamic environments. In chronic infections, fungal pathogens undergo prolonged challenges that can drive trait selection to convergent adapted states through restricted access to critical nutrients, assault by immune effectors, competition with other species, and antifungal drugs. This review first highlights the various genetic and epigenetic mechanisms that promote diversity in pathogenic fungal populations and provide an additional barrier to assessing the actual heterogeneity of fungal infections. We then review existing studies of evolution and genetic heterogeneity in fungal populations from lung infections associated with the genetic disease cystic fibrosis. We conclude with a discussion of open research questions that, once answered, may aid in diagnosing and treating chronic fungal infections.

Many bacteria have hemerythrin (Hr) proteins that bind O-2, including Pseudomonas aeruginosa, in which microoxia-induced Hr (Mhr) provide fitness advantages under microoxic conditions. Mhr has a 23 amino-acid extension at its C-terminus relative to a well-characterized Hr from Methylococcus capsulatus, and similar extensions are also found in Hrs from other bacteria. The last 11 amino acids of this extended, C-terminal tail are highly conserved in gammaproteobacteria and predicted to form a helix with positively charged and hydrophobic faces. In cellular fractionation assays, wild-type (WT) Mhr was found in both membrane and cytosolic fractions, while a Mhr(W143*) variant lacking the last 11 residues was largely in the cytosol and did not complement Mhr function in competition assays. Mhr(L112Y), a variant that has a much longer-lived O-2-bound form, was fully functional and had a similar localization pattern to that of WT Mhr. Both Mhr(W143*) and Mhr(L112Y) had secondary structures, stabilities, and O-2-binding kinetics similar to those of WT Mhr. Fluorescence studies revealed that the C-terminal tail, and particularly the fragment corresponding to its last 11 residues, was sufficient and necessary for association with lipid vesicles. Molecular dynamics simulations and subsequent cellular analysis of Mhr variants have demonstrated that conserved, positively charged residues in the tail are important for Mhr interactions with negatively charged membranes and the contribution of this protein to competitive fitness. Together, these data suggest that peripheral interactions of Mhr with membranes are guided by the C-terminal tail and are independent of O-2-binding.

Fungi and bacteria coexist in many polymicrobial communities, yet the molecular basis of their interactions remains poorly understood. Here, we show that the fungus Candida albicans sequesters essential magnesium ions from the bacterium Pseudomonas aeruginosa. To counteract fungal Mg2+ sequestration, P. aeruginosa expresses the Mg2+ transporter MgtA when Mg2+ levels are low. Thus, loss of MgtA specifically impairs P. aeruginosa in co-culture with C. albicans, but fitness can be restored by supplementing Mg2+. Using a panel of fungi and bacteria, we show that Mg2+ sequestration is a general mechanism of fungal antagonism against gram-negative bacteria. Mg2+ limitation enhances bacterial resistance to polymyxin antibiotics like colistin, which target gram-negative bacterial membranes. Indeed, experimental evolution reveals that P. aeruginosa evolves C. albicans-dependent colistin resistance via non-canonical means; antifungal treatment renders resistant bacteria colistin-sensitive. Our work suggests that fungal-bacterial competition could profoundly impact polymicrobial infection treatment with antibiotics of last resort.