Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

In Pseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanr derivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanr strains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections

Candida albicans Ethanol Stimulates Pseudomonas aeruginosa WspR-Controlled Biofilm Formation as Part of a Cyclic Relationship Involving Phenazines

In chronic infections, pathogens are often in the presence of other microbial species. For example, Pseudomonas aeruginosa is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF) and co-infections with Candida albicans are common. Here, we show that P. aeruginosa biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus C. albicans. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic- di-GMP (c-di-GMP), and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted P. aeruginosa colonization of CF airway epithelial cells. P. aeruginosa production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by C. albicans. Using a C.albicans adh1/adh1 mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of P. aeruginosa phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both P. aeruginosa and C. albicans has been associated with worse outcomes in cystic fibrosis

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

<i>Candida albicans</i> Ethanol Stimulates <i>Pseudomonas aeruginosa</i> WspR-Controlled Biofilm Formation as Part of a Cyclic Relationship Involving Phenazines

<div><p>In chronic infections, pathogens are often in the presence of other microbial species. For example, <i>Pseudomonas aeruginosa</i> is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF) and co-infections with <i>Candida albicans</i> are common. Here, we show that <i>P. aeruginosa</i> biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus <i>C. albicans</i>. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP), and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted <i>P. aeruginosa</i> colonization of CF airway epithelial cells. <i>P. aeruginosa</i> production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by <i>C. albicans</i>. Using a <i>C. albicans adh1</i>/<i>adh1</i> mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of <i>P. aeruginosa</i> phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both <i>P. aeruginosa</i> and <i>C. albicans</i> has been associated with worse outcomes in cystic fibrosis.</p></div

<i>P. aeruginosa</i> Δ<i>wspR</i> shows loss of swarm repression in the presence of ethanol.

<p><i>P. aeruginosa</i> strain PA14 WT, Δ<i>wspR</i>, and Δ<i>wspR</i> strains containing either plasmid-borne <i>wspR</i> or the empty vector were analyzed on swarm medium with and without 1% ethanol (EtOH) and with 0.02% arabinose (to induce <i>wspR</i> expression in the complemented strain). Images are representative of at least 5 experiments for each strain.</p

<i>C. albicans</i> promotes <i>P. aeruginosa</i> strain PAO1 WT biofilm formation on airway epithelial cells in part through ethanol production.

<p><i>P. aeruginosa</i> PAO1 WT was cultured with a monolayer of ΔF508 CFTR-CFBE cells alone or with <i>C. albicans</i> CAF2 (reference strain), the <i>C. albicans adh1/adh1</i> mutant (<i>adh1</i>), and its complemented derivative, <i>adh1/adh1+ADH1</i> (<i>adh1-R</i>). Data are combined from three independent experiments with 3–5 technical replicates per experiment, (* represents a statistically significant difference (p<0.05) between indicated strains). Error bars represent one standard deviation.</p

Ethanol leads to higher levels of PCN crystal formation and 5MPCA derivatives.

<p><b>A</b>. <i>P. aeruginosa</i> strain PA14 wild type (WT) was grown on medium without and with 1% ethanol. With ethanol, PCN crystals form and the colony has a yellowish color likely attributed to reduced PCN. <b>B</b>. <i>P. aeruginosa</i> strain PA14 WT was cultured on lawns of <i>C. albicans</i> CAF2 (WT reference strain), the <i>C. albicans adh1/adh1</i> mutant, and its complemented derivative (<i>adh1/adh1+ADH1</i>); the PA14 Δ<i>phz</i> mutant defective in phenazine production was plated on the <i>C. albicans</i> CAF2 for comparison.</p

Ethanol acts through the Wsp system.

<p><b>A</b>. Attachment of <i>P. aeruginosa</i> strain PAO1 WT, Δ<i>wspR</i> and Δ<i>wspA</i> mutants to the bottom of a polystyrene dish during growth in M63 medium with glucose and casamino acids, and with vehicle (Control) or with 1% ethanol for 6 hours. <b>B</b>. The number of cells per field for each condition was enumerated. Images and data are representative of results from more than three separate experiments. Significance determination was based on an ordinary one-way ANOVA followed by Sidak's multiple comparisons test for each intrastrain comparison; ***, <i>P</i><0.001. <b>C</b>. Δ<i>wspR</i> and Δ<i>wspFR</i> strains expressing WspR-E253A-YFP were grown without and with 1% EtOH, and the number of cells with fluorescent clusters were counted out of a total of approximately 100 cells examined per condition across two experiments.</p

Ethanol increases c-di-GMP levels in <i>P. aeruginosa</i> strain PA14 WT but not in a Δ<i>wspR</i> mutant.

<p>c-di-GMP levels from cultures grown on swarm plates without (black) or with 1% ethanol (grey) were measured by LC-MS. Error bars represent one standard deviation (*,p<0.05, N = 5); ns, not significant).</p

Our proposed model for the impacts of fungally-produced ethanol on <i>P. aeruginosa</i> behaviors.

<p>Our previous work has shown that <i>P. aeruginosa</i> phenazines increase fungal ethanol production. Here, we show that ethanol stimulates the Wsp system, leading to a WspR-dependent increase in c-di-GMP levels and a concomitant increase in Pel production and biofilm formation on plastic and on airway epithelial cells. In addition, ethanol altered phenazine production by promoting 5MPCA release and the accumulation of PCN.</p