bis(3--5-)-cyclic-diguanylic-acid and Cystic-Fibrosis

The treatment of lung infection in the context of cystic fibrosis (CF) is limited by a biofilm mode of growth of pathogenic organisms. When compared to planktonically grown bacteria, bacterial biofilms can survive extremely high levels of antimicrobials. Within the lung, bacterial biofilms are aggregates of microorganisms suspended in a matrix of self-secreted proteins within the sputum. These structures offer both physical protection from antibiotics as well as a heterogeneous population of metabolically and phenotypically distinct bacteria. The bacteria themselves and the components of the extracellular matrix, in addition to the signaling pathways that direct their behaviour, are all potential targets for therapeutic intervention discussed in this review. This review touches on the successes and failures of current anti-biofilm strategies, before looking at emerging therapies and the mechanisms by which it is hoped they will overcome current limitations.

Topics: Alginates; Anti-Bacterial Agents; Bacteriophages; Biofilms; Cell Communication; Cyclic GMP; Cystic Fibrosis; Humans; Iron; Lung Diseases; Quorum Sensing

The Gram-negative pathogen Pseudomonas aeruginosa is found ubiquitously within the environment and is recognised as an opportunistic human pathogen that commonly infects burn wounds and immunocompromised individuals, or patients suffering from the autosomal recessive disorder cystic fibrosis (CF). During chronic infection, P. aeruginosa is thought to form structured aggregates known as biofilms characterised by a self-produced matrix which encases the bacteria, protecting them from antimicrobial attack and the host immune response. In many cases, antibiotics are ineffective at eradicating P. aeruginosa from chronically infected CF airways. Cyclic-di-GMP has been identified as a key regulator of biofilm formation; however, the way in which its effector proteins elicit a change in biofilm formation remains unclear. Identifying regulators of biofilm formation is a key theme of current research and understanding the factors that activate biofilm formation may help to expose potential new drug targets that slow the onset of chronic infection. This minireview outlines the contribution made by exopolysaccharides to biofilm formation, and describes the current understanding of biofilm regulation in P. aeruginosa with a particular focus on CF airway-associated infections.

Topics: Alginates; Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cyclic GMP; Cystic Fibrosis; Drug Resistance, Bacterial; Escherichia coli Proteins; Glucuronic Acid; Hexuronic Acids; Humans; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial; Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common pathogen that colonizes the lungs of patients with cystic fibrosis. Isolates from sputum are typically all derived from the same strain of bacterium but show extensive phenotypic heterogeneity. One of these variants is the so-called small colony variant, which also shows increased ability to form a biofilm and is frequently resistant to multiple antibiotics. The presence of small colony variants in the sputum of patients with cystic fibrosis is associated with a worse clinical condition. The underlying mechanism responsible for generation of the small colony phenotype remains unclear, but a final common pathway would appear to be elevation of intracellular levels of cyclic di-GMP. This phenotypic variant is thus not just a laboratory curiosity, but a significant bacterial adaptation that favors survival within the lung of patients with cystic fibrosis and contributes to the pulmonary damage caused by P. aeruginosa.

Topics: Anti-Bacterial Agents; Biofilms; Chronic Disease; Cyclic GMP; Cystic Fibrosis; Drug Resistance, Bacterial; Humans; Lung; Phenotype; Pseudomonas aeruginosa; Pseudomonas Infections

A subpopulation of small-colony variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolates obtained from colonized cystic fibrosis lungs. Since most SCVs have until now been isolated from clinical samples, it remains unclear how widespread the ability of P. aeruginosa strains to develop this phenotype is and what the genetic mechanism(s) behind the emergence of SCVs are according to the origin of the isolate. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to spontaneously adopt an SCV-like smaller alternative morphotype distinguishable from that of the ancestral parent strain under laboratory culture conditions. We found that all the P. aeruginosa strains tested could adopt an SCV phenotype, regardless of their origin. Whole-genome sequencing of SCVs obtained from clinical and environmental sources revealed single mutations exclusively in two distinct c-di-GMP signaling pathways, the Wsp and YfiBNR pathways. We conclude that the ability to switch to an SCV phenotype is a conserved feature of P. aeruginosa and results from the acquisition of a stable genetic mutation, regardless of the origin of the strain.

Topics: Cyclic GMP; Cystic Fibrosis; Humans; Mutation; Phenotype; Pseudomonas aeruginosa; Pseudomonas Infections

How diversity evolves and persists in biofilms is essential for understanding much of microbial life, including the uncertain dynamics of chronic infections. We developed a biofilm model enabling long-term selection for daily adherence to and dispersal from a plastic bead in a test tube. Focusing on a pathogen of the cystic fibrosis lung, Burkholderia cenocepacia, we sequenced clones and metagenomes to unravel the mutations and evolutionary forces responsible for adaptation and diversification of a single biofilm community during 1,050 generations of selection. The mutational patterns revealed recurrent evolution of biofilm specialists from generalist types and multiple adaptive alleles at relatively few loci. Fitness assays also demonstrated strong interference competition among contending mutants that preserved genetic diversity. Metagenomes from five other independently evolved biofilm lineages revealed extraordinary mutational parallelism that outlined common routes of adaptation, a subset of which was found, surprisingly, in a planktonic population. These mutations in turn were surprisingly well represented among mutations that evolved in cystic fibrosis isolates of both Burkholderia and Pseudomonas. These convergent pathways included altered metabolism of cyclic diguanosine monophosphate, polysaccharide production, tricarboxylic acid cycle enzymes, global transcription, and iron scavenging. Evolution in chronic infections therefore may be driven by mutations in relatively few pathways also favored during laboratory selection, creating hope that experimental evolution may illuminate the ecology and selective dynamics of chronic infections and improve treatment strategies.

Topics: Bacterial Adhesion; Base Sequence; Biofilms; Burkholderia cenocepacia; Burkholderia Infections; Chronic Disease; Cyclic GMP; Cystic Fibrosis; Directed Molecular Evolution; DNA, Bacterial; Ecosystem; Genome, Bacterial; Humans; Lung Diseases; Mannose; Metagenome; Mutation; Opportunistic Infections; Phylogeny; Selection, Genetic

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.

Topics: Bacterial Proteins; Cell Line, Tumor; Chromatography, Thin Layer; Cyclic GMP; Cystic Fibrosis; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Humans; Lung; Oligonucleotide Array Sequence Analysis; Polysaccharides, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction