Loss of LasR function leads to decreased repression of Pseudomonas aeruginosa PhoB activity at physiological phosphate concentrations.

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.

How P. aeruginosa cells with diverse stator composition collectively swarm.

Swarming 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.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

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.

Pseudomonas aeruginosa transcriptome analysis of metal restriction in ex vivo cystic fibrosis sputum.

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.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in Cystic Fibrosis.

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 lung, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC 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 and noteworthy: The experiments in this report identify a novel mechanim whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet haves in OMVs secreted by P. aeruginiosa , 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. Graphical abstract: The anti-inflammatory effect of tobramycin mediated by 5' tRNA-fMet halves secreted in P. aeruginosa OMVs. (A) P. aeruginosa colonizes the CF lungs and secrets OMVs. OMVs diffuse through the mucus layer overlying bronchial epithelial cells and induce IL-8 secretion, which recruits neutrophils that causes lung damage. ( B ) Tobramycin increases 5' tRNA-fMet halves in OMVs secreted by P. aeruginosa . 5' tRNA-fMet halves are delivered into host cells after OMVs fuse with lipid rafts in CF-HBEC and down-regulate protein expression of MAPK10, IKBKG, and EP300, which suppresses IL-8 secretion and neutrophils in the lungs. A reduction in neutrophils in CF BALF is predicted to improve lung function and decrease lung damage.

Citrate cross-feeding by Pseudomonas aeruginosa supports lasR mutant fitness.

Cross-feeding of metabolites between subpopulations can affect cell phenotypes and population-level behaviors. In chronic Pseudomonas aeruginosa lung infections, subpopulations with loss-of-function (LOF) mutations in the lasR gene are common. LasR, a transcription factor often described for its role in virulence factor expression, also impacts metabolism, which, in turn, affects interactions between LasR+ and LasR- genotypes. Prior transcriptomic analyses suggested that citrate, a metabolite secreted by many cell types, induces virulence factor production when both genotypes are together. An unbiased analysis of the intracellular metabolome revealed broad differences including higher levels of citrate in lasR LOF mutants. Citrate consumption by LasR- strains required the CbrAB two-component system, which relieves carbon catabolite repression and is elevated in lasR LOF mutants. Within mixed communities, the citrate-responsive two-component system TctED and its gene targets OpdH (porin) and TctABC (citrate transporter) that are predicted to be under catabolite repression control were induced and required for enhanced RhlR/I-dependent signaling, pyocyanin production, and fitness of LasR- strains. Citrate uptake by LasR- strains markedly increased pyocyanin production in co-culture with Staphylococcus aureus, which also secretes citrate and frequently co-infects with P. aeruginosa. This citrate-induced restoration of virulence factor production by LasR- strains in communities with diverse species or genotypes may offer an explanation for the contrast observed between the markedly deficient virulence factor production of LasR- strains in monocultures and their association with the most severe forms of cystic fibrosis lung infections. These studies highlight the impact of secreted metabolites in mixed microbial communities.IMPORTANCECross-feeding of metabolites can change community composition, structure, and function. Here, we unravel a cross-feeding mechanism between frequently co-observed isolate genotypes in chronic Pseudomonas aeruginosa lung infections. We illustrate an example of how clonally derived diversity in a microbial communication system enables intra- and inter-species cross-feeding. Citrate, a metabolite released by many cells including P. aeruginosa and Staphylococcus aureus, was differentially consumed between genotypes. Since these two pathogens frequently co-occur in the most severe cystic fibrosis lung infections, the cross-feeding-induced virulence factor expression and fitness described here between diverse genotypes exemplify how co-occurrence can facilitate the development of worse disease outcomes.

Analysis of Pseudomonas aeruginosa transcription in an ex vivo cystic fibrosis sputum model identifies metal restriction as a gene expression stimulus.

Chronic Pseudomonas aeruginosa lung infections are a distinctive feature of cystic fibrosis (CF) pathology, that challenge adults with CF even with the advent of highly effective modulator therapies. Characterizing P. aeruginosa transcription in the CF lung and identifying factors that drive gene expression could yield novel strategies to eradicate infection or otherwise improve outcomes. To complement published P. aeruginosa gene expression studies in laboratory culture models designed to model the CF lung environment, we employed an ex vivo sputum model in which laboratory strain PAO1 was incubated in sputum from different CF donors. As part of the analysis, we compared PAO1 gene expression in this "spike-in" sputum model to that for P. aeruginosa grown in artificial sputum medium (ASM). Analyses focused on genes that were differentially expressed between sputum and ASM and genes that were most highly expressed in sputum. We present a new approach that used sets of genes with correlated expression, identified by the gene expression analysis tool eADAGE, to analyze the differential activity of pathways in P. aeruginosa grown in CF sputum from different individuals. A key characteristic of P. aeruginosa grown in expectorated CF sputum was related to zinc and iron acquisition, but this signal varied by donor sputum. In addition, a significant correlation between P. aeruginosa expression of the H1-type VI secretion system and corrector use by the sputum donor was observed. These methods may be broadly useful in looking for variable signals across clinical samples.

Bacterial Outer Membrane Vesicles and Immune Modulation of the Host.

This article reviews the role of outer membrane vesicles (OMVs) in mediating the interaction between Gram-negative bacteria and their human hosts. OMVs are produced by a diverse range of Gram-negative bacteria during infection and play a critical role in facilitating host-pathogen interactions without requiring direct cell-to-cell contact. This article describes the mechanisms by which OMVs are formed and subsequently interact with host cells, leading to the transport of microbial protein virulence factors and short interfering RNAs (sRNA) to their host targets, exerting their immunomodulatory effects by targeting specific host signaling pathways. Specifically, this review highlights mechanisms by which OMVs facilitate chronic infection through epigenetic modification of the host immune response. Finally, this review identifies critical knowledge gaps in the field and offers potential avenues for future OMV research, specifically regarding rigor and reproducibility in OMV isolation and characterization methods.

Citrate cross-feeding between Pseudomonas aerguinosa genotypes supports lasR mutant fitness.

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 circumventing 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.

Citrate cross-feeding between Pseudomonas aerguinosa genotypes supports lasR mutant fitness.

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 circumventing 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.

Comparative effects of CFTR modulators on phagocytic, metabolic and inflammatory profiles of CF and nonCF macrophages.

Macrophage dysfunction has been well-described in Cystic Fibrosis (CF) and may contribute to bacterial persistence in the lung. Whether CF macrophage dysfunction is related directly to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in macrophages or an indirect consequence of chronic inflammation and mucostasis is a subject of ongoing debate. CFTR modulators that restore CFTR function in epithelial cells improve global CF monocyte inflammatory responses but their direct effects on macrophages are less well understood. To address this knowledge gap, we measured phagocytosis, metabolism, and cytokine expression in response to a classical CF pathogen, Pseudomonas aeruginosa in monocyte-derived macrophages (MDM) isolated from CF F508del homozygous subjects and nonCF controls. Unexpectedly, we found that CFTR modulators enhanced phagocytosis in both CF and nonCF cohorts. CFTR triple modulators also inhibited MDM mitochondrial function, consistent with MDM activation. In contrast to studies in humans where CFTR modulators decreased serum inflammatory cytokine levels, modulators did not alter cytokine secretion in our system. Our studies therefore suggest modulator induced metabolic effects may promote bacterial clearance in both CF and nonCF monocyte-derived macrophages.

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.

In host evolution of Exophiala dermatitidis in cystic fibrosis lung micro-environment.

Individuals with cystic fibrosis (CF) are susceptible to chronic lung infections that lead to inflammation and irreversible lung damage. While most respiratory infections that occur in CF are caused by bacteria, some are dominated by fungi such as the slow-growing black yeast Exophiala dermatitidis. Here, we analyze isolates of E. dermatitidis cultured from two samples, collected from a single subject 2 years apart. One isolate genome was sequenced using long-read Nanopore technology as an in-population reference to use in comparative single nucleotide polymorphism and insertion-deletion variant analyses of 23 isolates. We then used population genomics and phylo-genomics to compare the isolates to each other as well as the reference genome strain E. dermatitidis NIH/UT8656. Within the CF lung population, three E. dermatitidis clades were detected, each with varying mutation rates. Overall, the isolates were highly similar suggesting that they were recently diverged. All isolates were MAT 1-1, which was consistent with their high relatedness and the absence of evidence for mating or recombination between isolates. Phylogenetic analysis grouped sets of isolates into clades that contained isolates from both early and late time points indicating there are multiple persistent lineages. Functional assessment of variants unique to each clade identified alleles in genes that encode transporters, cytochrome P450 oxidoreductases, iron acquisition, and DNA repair processes. Consistent with the genomic heterogeneity, isolates showed some stable phenotype heterogeneity in melanin production, subtle differences in antifungal minimum inhibitory concentrations, and growth on different substrates. The persistent population heterogeneity identified in lung-derived isolates is an important factor to consider in the study of chronic fungal infections, and the analysis of changes in fungal pathogens over time may provide important insights into the physiology of black yeasts and other slow-growing fungi in vivo.

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa.

Lung infections caused by antibiotic-resistant strains of Pseudomonas aeruginosa are difficult to eradicate in immunocompromised hosts such as those with cystic fibrosis. We previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AECs) deliver microRNA let-7b-5p to P. aeruginosa to suppress biofilm formation and increase sensitivity to beta-lactam antibiotics. In this study, we show that EVs secreted by AECs transfer multiple distinct short RNA fragments to P. aeruginosa that are predicted to target the three subunits of the fluoroquinolone efflux pump MexHI-OpmD, thus increasing antibiotic sensitivity. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%), and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S ribosomal RNA (rRNA) external transcribed spacer that was transferred to P. aeruginosa by EVs reduced planktonic growth of P. aeruginosa in the presence of ciprofloxacin, reduced the minimum inhibitory concentration of P. aeruginosa for ciprofloxacin by over 50%, and significantly reduced protein levels of both MexH and OpmD. In conclusion, an rRNA fragment secreted by AECs in EVs that targets the fluoroquinolone efflux pump MexHI-OpmD downregulated these proteins and increased the ciprofloxacin sensitivity of P. aeruginosa. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with ciprofloxacin-resistant P. aeruginosa infections.NEW & NOTEWORTHY Human RNA fragments transported in extracellular vesicles interfere with Pseudomonas aeruginosa drug efflux pumps. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

How individual P. aeruginosa cells with diverse stator distributions collectively form a heterogeneous macroscopic swarming population.

Swarming 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 WT strain, with MotAB stators produced ∼40-fold more than MotCD stators. However, recruitment of MotCD stators in free swimming cells requires higher liquid viscosities, while MotAB stators are readily recruited at low viscosities. Importantly, we find that cells with MotCD stators are ∼10x more likely to have an active motor compared to cells without, so wild-type, WT, populations are intrinsically heterogeneous and not reducible to MotAB-dominant or MotCD-dominant behavior. 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 recruitment. 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. Importance: After extensive study, it is now known that there exist multifactorial influences on swarming motility in P. aeruginosa , but it is not clear precisely why stator selection in the flagellum motor is so important or how this process is collectively initiated or arrested. Here, we show that for P. aeruginosa PA14, MotAB stators are produced ∼40-fold more than MotCD stators, but recruitment of MotCD over MotAB stators requires higher liquid viscosities. 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 active ∼10x more often than MotAB. What emerges from this complex landscape of stator recruitment 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 they potentially relate to surface sensing circuitry.

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.

Community composition shapes microbial-specific phenotypes in a cystic fibrosis polymicrobial model system.

Interspecies interactions can drive the emergence of unexpected microbial phenotypes that are not observed when studying monocultures. The cystic fibrosis (CF) lung consists of a complex environment where microbes, living as polymicrobial biofilm-like communities, are associated with negative clinical outcomes for persons with CF (pwCF). However, the current lack of in vitro models integrating the microbial diversity observed in the CF airway hampers our understanding of why polymicrobial communities are recalcitrant to therapy in this disease. Here, integrating computational approaches informed by clinical data, we built a mixed community of clinical relevance to the CF lung composed of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica. We developed and validated this model biofilm community with multiple isolates of these four genera. When challenged with tobramycin, a front-line antimicrobial used to treat pwCF, the microorganisms in the polymicrobial community show altered sensitivity to this antibiotic compared to monospecies biofilms. We observed that wild-type P. aeruginosa is sensitized to tobramycin in a mixed community versus monoculture, and this observation holds across a range of community relative abundances. We also report that LasR loss-of-function, a variant frequently detected in the CF airway, drives tolerance of P. aeruginosa to tobramycin specifically in the mixed community. Our data suggest that the molecular basis of this community-specific recalcitrance to tobramycin for the P. aeruginosa lasR mutant is increased production of phenazines. Our work supports the importance of studying a clinically relevant model of polymicrobial biofilms to understand community-specific traits relevant to infections.

Computationally Efficient Assembly of Pseudomonas aeruginosa Gene Expression Compendia.

Thousands of Pseudomonas aeruginosa RNA sequencing (RNA-seq) gene expression profiles are publicly available via the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). In this work, the transcriptional profiles from hundreds of studies performed by over 75 research groups were reanalyzed in aggregate to create a powerful tool for hypothesis generation and testing. Raw sequence data were uniformly processed using the Salmon pseudoaligner, and this read mapping method was validated by comparison to a direct alignment method. We developed filtering criteria to exclude samples with aberrant levels of housekeeping gene expression or an unexpected number of genes with no reported values and normalized the filtered compendia using the ratio-of-medians method. The filtering and normalization steps greatly improved gene expression correlations for genes within the same operon or regulon across the 2,333 samples. Since the RNA-seq data were generated using diverse strains, we report the effects of mapping samples to noncognate reference genomes by separately analyzing all samples mapped to cDNA reference genomes for strains PAO1 and PA14, two divergent strains that were used to generate most of the samples. Finally, we developed an algorithm to incorporate new data as they are deposited into the SRA. Our processing and quality control methods provide a scalable framework for taking advantage of the troves of biological information hibernating in the depths of microbial gene expression data and yield useful tools for P. aeruginosa RNA-seq data to be leveraged for diverse research goals. IMPORTANCE Pseudomonas aeruginosa is a causative agent of a wide range of infections, including chronic infections associated with cystic fibrosis. These P. aeruginosa infections are difficult to treat and often have negative outcomes. To aid in the study of this problematic pathogen, we mapped, filtered for quality, and normalized thousands of P. aeruginosa RNA-seq gene expression profiles that were publicly available via the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). The resulting compendia facilitate analyses across experiments, strains, and conditions. Ultimately, the workflow that we present could be applied to analyses of other microbial species.

Compendium-Wide Analysis of Pseudomonas aeruginosa Core and Accessory Genes Reveals Transcriptional Patterns across Strains PAO1 and PA14.

Pseudomonas aeruginosa is an opportunistic pathogen that causes difficult-to-treat infections. Two well-studied divergent P. aeruginosa strain types, PAO1 and PA14, have significant genomic heterogeneity, including diverse accessory genes present in only some strains. Genome content comparisons find core genes that are conserved across both PAO1 and PA14 strains and accessory genes that are present in only a subset of PAO1 and PA14 strains. Here, we use recently assembled transcriptome compendia of publicly available P. aeruginosa RNA sequencing (RNA-seq) samples to create two smaller compendia consisting of only strain PAO1 or strain PA14 samples with each aligned to their cognate reference genome. We confirmed strain annotations and identified other samples for inclusion by assessing each sample's median expression of PAO1-only or PA14-only accessory genes. We then compared the patterns of core gene expression in each strain. To do so, we developed a method by which we analyzed genes in terms of which genes showed similar expression patterns across strain types. We found that some core genes had consistent correlated expression patterns across both compendia, while others were less stable in an interstrain comparison. For each accessory gene, we also determined core genes with correlated expression patterns. We found that stable core genes had fewer coexpressed neighbors that were accessory genes. Overall, this approach for analyzing expression patterns across strain types can be extended to other groups of genes, like phage genes, or applied for analyzing patterns beyond groups of strains, such as samples with different traits, to reveal a deeper understanding of regulation. IMPORTANCE Pseudomonas aeruginosa is a ubiquitous pathogen. There is much diversity among P. aeruginosa strains, including two divergent but well-studied strains, PAO1 and PA14. Understanding how these different strain-level traits manifest is important for identifying targets that regulate different traits of interest. With the availability of thousands of PAO1 and PA14 samples, we created two strain-specific RNA-seq compendia where each one contains hundreds of samples from PAO1 or PA14 strains and used them to compare the expression patterns of core genes that are conserved in both strain types and to determine which core genes have expression patterns that are similar to those of accessory genes that are unique to one strain or the other using an approach that we developed. We found a subset of core genes with different transcriptional patterns across PAO1 and PA14 strains and identified those core genes with expression patterns similar to those of strain-specific accessory genes.

SOPHIE: Generative Neural Networks Separate Common and Specific Transcriptional Responses.

Genome-wide transcriptome profiling identifies genes that are prone to differential expression (DE) across contexts, as well as genes with changes specific to the experimental manipulation. Distinguishing genes that are specifically changed in a context of interest from common differentially expressed genes (DEGs) allows more efficient prediction of which genes are specific to a given biological process under scrutiny. Currently, common DEGs or pathways can only be identified through the laborious manual curation of experiments, an inordinately time-consuming endeavor. Here we pioneer an approach, Specific cOntext Pattern Highlighting In Expression data (SOPHIE), for distinguishing between common and specific transcriptional patterns using a generative neural network to create a background set of experiments from which a null distribution of gene and pathway changes can be generated. We apply SOPHIE to diverse datasets including those from human, human cancer, and bacterial pathogen Pseudomonas aeruginosa. SOPHIE identifies common DEGs in concordance with previously described, manually and systematically determined common DEGs. Further molecular validation indicates that SOPHIE detects highly specific but low-magnitude biologically relevant transcriptional changes. SOPHIE's measure of specificity can complement log2 fold change values generated from traditional DE analyses. For example, by filtering the set of DEGs, one can identify genes that are specifically relevant to the experimental condition of interest. Consequently, these results can inform future research directions. All scripts used in these analyses are available at https://github.com/greenelab/generic-expression-patterns. Users can access https://github.com/greenelab/sophie to run SOPHIE on their own data.