Bacterial outer membrane vesicle (OMV)-mediated delivery of proteins to host cells is an important mechanism of host-pathogen communication. Emerging evidence suggests that OMVs contain ...differentially packaged short RNAs (sRNAs) with the potential to target host mRNA function and/or stability. In this study, we used RNA-Seq to characterize differentially packaged sRNAs in Pseudomonas aeruginosa OMVs, and to show transfer of OMV sRNAs to human airway cells. We selected one sRNA for further study based on its stable secondary structure and predicted mRNA targets. Our candidate sRNA (sRNA52320), a fragment of a P. aeruginosa methionine tRNA, was abundant in OMVs and reduced LPS-induced as well as OMV-induced IL-8 secretion by cultured primary human airway epithelial cells. We also showed that sRNA52320 attenuated OMV-induced KC cytokine secretion and neutrophil infiltration in mouse lung. Collectively, these findings are consistent with the hypothesis that sRNA52320 in OMVs is a novel mechanism of host-pathogen interaction whereby P. aeruginosa reduces the host immune response.
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.
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.
Pseudomonas aeruginosa and Candida albicans are opportunistic pathogens whose interactions involve the secreted products ethanol and phenazines. Here, we describe the role of ethanol in mixed-species ...co-cultures by dual-seq analyses. P. aeruginosa and C. albicans transcriptomes were assessed after growth in mono-culture or co-culture with either ethanol-producing C. albicans or a C. albicans mutant lacking the primary ethanol dehydrogenase, Adh1. Analysis of the RNA-Seq data using KEGG pathway enrichment and eADAGE methods revealed several P. aeruginosa responses to C. albicans-produced ethanol including the induction of a non-canonical low-phosphate response regulated by PhoB. C. albicans wild type, but not C. albicans adh1DELTA/DELTA, induces P. aeruginosa production of 5-methyl-phenazine-1-carboxylic acid (5-MPCA), which forms a red derivative within fungal cells and exhibits antifungal activity. Here, we show that C. albicans adh1DELTA/DELTA no longer activates P. aeruginosa PhoB and PhoB-regulated phosphatase activity, that exogenous ethanol complements this defect, and that ethanol is sufficient to activate PhoB in single-species P. aeruginosa cultures at permissive phosphate levels. The intersection of ethanol and phosphate in co-culture is inversely reflected in C. albicans; C. albicans adh1DELTA/DELTA had increased expression of genes regulated by Pho4, the C. albicans transcription factor that responds to low phosphate, and Pho4-dependent phosphatase activity. Together, these results show that C. albicans-produced ethanol stimulates P. aeruginosa PhoB activity and 5-MPCA-mediated antagonism, and that both responses are dependent on local phosphate concentrations. Further, our data suggest that phosphate scavenging by one species improves phosphate access for the other, thus highlighting the complex dynamics at play in microbial communities.
Microbial cells do not live in isolation in their environment, but rather they communicate with each other using chemical signals. This sophisticated mode of cell-to-cell signalling, known as quorum ...sensing, was first discovered in bacteria, and coordinates the behaviour of microbial population behaviour in a cell-density-dependent manner. More recently, these mechanisms have been described in eukaryotes, particularly in fungi, where they regulate processes such as pathogenesis, morphological differentiation, secondary metabolite production and biofilm formation. In this manuscript, we review the information available to date on these processes in yeast, dimorphic fungi and filamentous fungi. We analyse the diverse chemical 'languages' used by different groups of fungi, their possible cross-talk and interkingdom interactions with other organisms. We discuss the existence of these mechanisms in multicellular organisms, the ecophysiological role of QS in fungal colonisation and the potential applications of these mechanisms in biotechnology and pathogenesis.
Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of ...Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.
Phenazines are redox-active small molecules that play significant roles in the interactions between pseudomonads and diverse eukaryotes, including fungi. When Pseudomonas aeruginosa and Candida ...albicans were cocultured on solid medium, a red pigmentation developed that was dependent on P. aeruginosa phenazine biosynthetic genes. Through a genetic screen in combination with biochemical experiments, it was found that a P. aeruginosa-produced precursor to pyocyanin, proposed to be 5-methyl-phenazinium-1-carboxylate (5MPCA), was necessary for the formation of the red pigmentation. The 5MPCA-derived pigment was found to accumulate exclusively within fungal cells, where it retained the ability to be reversibly oxidized and reduced, and its detection correlated with decreased fungal viability. Pyocyanin was not required for pigment formation or fungal killing. Spectral analyses showed that the partially purified pigment from within the fungus differed from aeruginosins A and B, two red phenazine derivatives formed late in P. aeruginosa cultures. The red pigment isolated from C. albicans that had been cocultured with P. aeruginosa was heterogeneous and difficult to release from fungal cells, suggesting its modification within the fungus. These findings suggest that intracellular targeting of some phenazines may contribute to their toxicity and that this strategy could be useful in developing new antifungals.
Individuals with cystic fibrosis (CF) often acquire chronic lung infections that lead to irreversible damage. We sought to examine regional variation in the microbial communities in the lungs of ...individuals with mild-to-moderate CF lung disease, to examine the relationship between the local microbiota and local damage, and to determine the relationships between microbiota in samples taken directly from the lung and the microbiota in spontaneously expectorated sputum. In this initial study, nine stable, adult CF patients with an FEV1>50% underwent regional sampling of different lobes of the right lung by bronchoalveolar lavage (BAL) and protected brush (PB) sampling of mucus plugs. Sputum samples were obtained from six of the nine subjects immediately prior to the procedure. Microbial community analysis was performed on DNA extracted from these samples and the extent of damage in each lobe was quantified from a recent CT scan. The extent of damage observed in regions of the right lung did not correlate with specific microbial genera, levels of community diversity or composition, or bacterial genome copies per ml of BAL fluid. In all subjects, BAL fluid from different regions of the lung contained similar microbial communities. In eight out of nine subjects, PB samples from different regions of the lung were also similar in microbial community composition, and were similar to microbial communities in BAL fluid from the same lobe. Microbial communities in PB samples were more diverse than those in BAL samples, suggesting enrichment of some taxa in mucus plugs. To our knowledge, this study is the first to examine the microbiota in different regions of the CF lung in clinically stable individuals with mild-to-moderate CF-related lung disease.
Microbial interactions are increasingly recognized as an integral part of microbial physiology. Cell–cell communication mediated by quorum sensing and metabolite exchange is a formative element of ...microbial interactions. However, loss-of-function mutations in quorum-sensing components are common across diverse species. Furthermore, quorum sensing is modulated by small molecules and environmental conditions that may be altered in the presence of other microbial species. Recent evidence highlights how strain heterogeneity impacts microbial interactions. There is great potential for microbial interactions to act as selective pressures that influence the emergence of common mutations in quorum-sensing genes across the bacterial and fungal domains.
Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental ...factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate different environmental and intracellular signals, including metabolic status, to decide the fate of cellular morphology.
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