Deborah A. Hogan

Medically important bacterial–fungal interactions

Whether it is in the setting of disease or in a healthy state, the human body contains a diverse range of microorganisms, including bacteria and fungi. The interactions between these taxonomically diverse microorganisms are highly dynamic and dependent on a multitude of microorganism and host factors. Human disease can develop from an imbalance between commensal bacteria and fungi or from invasion of particular host niches by opportunistic bacterial and fungal pathogens. This Review describes the clinical and molecular characteristics of bacterial–fungal interactions that are relevant to human disease.

Farnesol, a common sesquiterpene, inhibits PQS production in Pseudomonas aeruginosa.

Farnesol is a sesquiterpene produced by many organisms, including the fungus Candida albicans. Here, we report that the addition of farnesol to cultures of Pseudomonas aeruginosa, an opportunistic human bacterial pathogen, leads to decreased production of the Pseudomonas quinolone signal (PQS) and the PQS‐controlled virulence factor, pyocyanin. Within 15 min of farnesol addition, decreased transcript levels of pqsA, the first gene in the PQS biosynthetic operon, were observed. Transcript levels of pqsR (mvfR), which encodes the transcription factor that positively regulates pqsA, were unaffected. An Escherichia coli strain producing PqsR and containing the pqsA promoter fused to lacZ similarly showed that farnesol inhibited PQS‐stimulated transcription. Electrophoretic mobility shift assays showed that, like PQS, farnesol stimulated PqsR binding to the pqsA promoter at a previously characterized LysR binding site, suggesting that farnesol promoted a non‐productive interaction between PqsR and the pqsA promoter. Growth with C. albicans leads to decreased production of PQS and pyocyanin by P. aeruginosa, suggesting that the amount of farnesol produced by the fungus is sufficient to impact P. aeruginosa PQS signalling. Related isoprenoid compounds, but not other long‐chain alcohols, also inhibited PQS production at micromolar concen‐trations, suggesting that related compounds may participate in interspecies interactions with P. aeruginosa.

Talking to Themselves: Autoregulation and Quorum Sensing in Fungi

Extracellular autoinducing compounds in the supernatants of microbial cultures were first recognized for their roles in the induction of genetic competence in gram-positive bacteria ([17][1], [94][2]) and in the regulation of light production in marine vibrios ([60][3]). In 1994, this form of

Farnesol and dodecanol effects on the Candida albicans Ras1‐cAMP signalling pathway and the regulation of morphogenesis

Candida albicans hypha formation which has been stimulated via the Ras1‐cAMP‐Efg1 signalling cascade is inhibited by farnesol, a C. albicans autoregulatory factor, and small molecules such as dodecanol. In cultures containing farnesol or dodecanol, hypha formation was restored upon addition of dibutyryl‐cAMP. The CAI4‐Ras1G13V strain, which carries a dominant‐active variant of Ras1 and forms hyphae in the absence of inducing stimuli, grew as yeast in medium with farnesol or dodecanol; the heat shock sensitivity of the CAI4‐Ras1G13V strain was also suppressed by these compounds. Neither Pde1 nor Pde2 was necessary for the repression of hyphal growth by farnesol or dodecanol. Two transcripts, CTA1 and HSP12, which are at higher levels upon mutation of Ras1 or Cdc35, were increased in abundance in cells grown with farnesol or dodecanol. Microscopic analysis of strains carrying CTA1 and HWP1 promoter fusions grown with intermediate concentrations of farnesol or dodecanol indicated a link between cells with the increased expression of cAMP‐repressed genes and cells repressed for hypha formation. Because several cAMP‐controlled outputs are affected by farnesol and dodecanol, our findings suggest that these compounds impact activity of the Ras1‐Cdc35 pathway, thus leading to an alteration of C. albicans morphology.

Candida albicans Interactions with Bacteria in the Context of Human Health and Disease

Humans are colonized by diverse populations of bacteria and fungi when in a healthy state and in the settings of disease, and the interactions between these microbial populations can be beneficial or detrimental to the host [1]. Among these microbial populations, Candida albicans is the fungus most commonly detected in association with humans [2], and numerous studies have described C. albicans interactions with its bacterial neighbors [1]. Here, with a focus on C. albicans, we provide examples of how bacterial-fungal interactions can influence human health. In addition, we highlight studies that give insight into the molecular mechanisms that govern the physical associations, interspecies communication, and changes in microbial behavior and survival that occur when bacteria and fungi occupy the same sites.

Control of Candida albicans Metabolism and Biofilm Formation by Pseudomonas aeruginosa Phenazines

ABSTRACT Candida albicans has developmental programs that govern transitions between yeast and filamentous morphologies and between unattached and biofilm lifestyles. Here, we report that filamentation, intercellular adherence, and biofilm development were inhibited during interactions between Candida albicans and Pseudomonas aeruginosa through the action of P. aeruginosa-produced phenazines. While phenazines are toxic to C. albicans at millimolar concentrations, we found that lower concentrations of any of three different phenazines (pyocyanin, phenazine methosulfate, and phenazine-1-carboxylate) allowed growth but affected the development of C. albicans wrinkled colony biofilms and inhibited the fungal yeast-to-filament transition. Phenazines impaired C. albicans growth on nonfermentable carbon sources and led to increased production of fermentation products (ethanol, glycerol, and acetate) in glucose-containing medium, leading us to propose that phenazines specifically inhibited respiration. Methylene blue, another inhibitor of respiration, also prevented the formation of structured colony biofilms. The inhibition of filamentation and colony wrinkling was not solely due to lowered extracellular pH induced by fermentation. Compared to smooth, unstructured colonies, wrinkled colony biofilms had higher oxygen concentrations within the colony, and wrinkled regions of these colonies had higher levels of respiration. Together, our data suggest that the structure of the fungal biofilm promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by bacterial molecules such as phenazines or compounds with similar activities disrupts these pathways. These findings may suggest new ways to limit fungal biofilms in the context of disease. IMPORTANCE Many of the infections caused by Candida albicans, a major human opportunistic fungal pathogen, involve both morphological transitions and the formation of surface-associated biofilms. Through the study of C. albicans interactions with the bacterium Pseudomonas aeruginosa, which often coinfects with C. albicans, we have found that P. aeruginosa-produced phenazines modulate C. albicans metabolism and, through these metabolic effects, impact cellular morphology, cell-cell interactions, and biofilm formation. We suggest that the structure of C. albicans biofilms promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by phenazines inhibits biofilm development. Our findings not only provide insight into interactions between these species but also provide valuable insights into novel pathways that could lead to the development of new therapies to treat C. albicans infections. Many of the infections caused by Candida albicans, a major human opportunistic fungal pathogen, involve both morphological transitions and the formation of surface-associated biofilms. Through the study of C. albicans interactions with the bacterium Pseudomonas aeruginosa, which often coinfects with C. albicans, we have found that P. aeruginosa-produced phenazines modulate C. albicans metabolism and, through these metabolic effects, impact cellular morphology, cell-cell interactions, and biofilm formation. We suggest that the structure of C. albicans biofilms promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by phenazines inhibits biofilm development. Our findings not only provide insight into interactions between these species but also provide valuable insights into novel pathways that could lead to the development of new therapies to treat C. albicans infections.

Prevalence of Streptococci and Increased Polymicrobial Diversity Associated with Cystic Fibrosis Patient Stability

Diverse microbial communities chronically colonize the lungs of cystic fibrosis patients. Pyrosequencing of amplicons for hypervariable regions in the 16S rRNA gene generated taxonomic profiles of bacterial communities for sputum genomic DNA samples from 22 patients during a state of clinical stability (outpatients) and 13 patients during acute exacerbation (inpatients). We employed quantitative PCR (qPCR) to confirm the detection of Pseudomonas aeruginosa and Streptococcus by the pyrosequencing data and human oral microbe identification microarray (HOMIM) analysis to determine the species of the streptococci identified by pyrosequencing. We show that outpatient sputum samples have significantly higher bacterial diversity than inpatients, but maintenance treatment with tobramycin did not impact overall diversity. Contrary to the current dogma in the field that Pseudomonas aeruginosa is the dominant organism in the majority of cystic fibrosis patients, Pseudomonas constituted the predominant genera in only half the patient samples analyzed and reported here. The increased fractional representation of Streptococcus in the outpatient cohort relative to the inpatient cohort was the strongest predictor of clinically stable lung disease. The most prevalent streptococci included species typically associated with the oral cavity (Streptococcus salivarius and Streptococcus parasanguis) and the Streptococcus milleri group species. These species of Streptococcus may play an important role in increasing the diversity of the cystic fibrosis lung environment and promoting patient stability.

The Pseudomonas aeruginosa secreted protein PA2934 decreases apical membrane expression of the cystic fibrosis transmembrane conductance regulator.

ABSTRACT We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl− ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in α/β hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

Antifungal mechanisms by which a novel Pseudomonas aeruginosa phenazine toxin kills Candida albicans in biofilms

Pseudomonas aeruginosa produces several phenazines including the recently described 5‐methyl‐phenazine‐1‐carboxylic acid (5MPCA), which exhibits a novel antibiotic activity towards pathogenic fungi such as Candida albicans. Here we characterize the unique antifungal mechanisms of 5MPCA using its analogue phenazine methosulphate (PMS). Like 5MPCA, PMS induced fungal red pigmentation and killing. Mass spectrometry analyses demonstrated that PMS can be covalently modified by amino acids, a process that yields red derivatives. Furthermore, soluble proteins from C. albicans grown with either PMS or P. aeruginosa were also red and demonstrated absorbance and fluorescence spectra similar to that of PMS covalently linked to either amino acids or proteins in vitro, suggesting that 5MPCA modification by protein amine groups occurs in vivo. The red‐pigmented C. albicans soluble proteins were reduced by NADH and spontaneously oxidized by oxygen, a reaction that likely generates reactive oxygen species (ROS). Additional evidence indicated that ROS generation precedes 5MPCA‐induced fungal death. Reducing conditions greatly enhanced PMS uptake by C. albicans and killing. Since 5MPCA was more toxic than other phenazines that are not modified, such as pyocyanin, we propose that the covalent binding of 5MPCA promotes its accumulation in target cells and contributes to its antifungal activity in mixed‐species biofilms.

Identification of Two Gene Clusters and a Transcriptional Regulator Required for Pseudomonas aeruginosa Glycine Betaine Catabolism

Glycine betaine (GB), which occurs freely in the environment and is an intermediate in the catabolism of choline and carnitine, can serve as a sole source of carbon or nitrogen in Pseudomonas aeruginosa. Twelve mutants defective in growth on GB as the sole carbon source were identified through a genetic screen of a nonredundant PA14 transposon mutant library. Further growth experiments showed that strains with mutations in two genes, gbcA (PA5410) and gbcB (PA5411), were capable of growth on dimethylglycine (DMG), a catabolic product of GB, but not on GB itself. Subsequent nuclear magnetic resonance (NMR) experiments with 1,2-(13)C-labeled choline indicated that these genes are necessary for conversion of GB to DMG. Similar experiments showed that strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine. Mutant analyses and (13)C NMR studies also confirmed that the soxBDAG genes, predicted to encode a sarcosine oxidase, are required for sarcosine catabolism. Our screen also identified a predicted AraC family transcriptional regulator, encoded by gbdR (PA5380), that is required for growth on GB and DMG and for the induction of gbcA, gbcB, and dgcAB in response to GB or DMG. Mutants defective in the previously described gbt gene (PA3082) grew on GB with kinetics similar to those of the wild type in both the PAO1 and PA14 strain backgrounds. These studies provided important insight into both the mechanism and the regulation of the catabolism of GB in P. aeruginosa.