Spontaneous phenotypic suppression of GacA-defective Vibrio fischeri is achieved via mutation of csrA and ihfA

Background: Symbiosis defective GacA-mutant derivatives of Vibrio fischeri are growth impaired thereby creating a selective advantage for growth-enhanced spontaneous suppressors. Suppressors were isolated and characterized for effects of the mutations on gacA-mutant defects of growth, siderophore activity and luminescence. The mutations were identified by targeted and whole genome sequencing. Results: Most mutations that restored multiple phenotypes were non-null mutations that mapped to conserved domains in or altered expression of CsrA, a post-transcriptional regulator that mediates GacA effects in a number of bacterial species. These represent an array of unique mutations compared to those that have been described previously. Different substitutions at the same amino acid residue were identified allowing comparisons of effects such as at the R6 residue, which conferred relative differences in luminescence and siderophore levels. The screen revealed residues not previously identified as critical for function including a single native alanine. Most csrA mutations enhanced luminescence more than siderophore activity, which was especially evident for mutations predicted to reduce the amount of CsrA. Although CsrA mutations compensate for many known GacA mutant defects, not all CsrA suppressors restore symbiotic colonization. Phenotypes of a suppressor allele of ihfA that encodes one subunit of the integration host factor (IHF) heteroduplex indicated the protein represses siderophore and activates luminescence in a GacA-independent manner. Conclusions: In addition to its established role in regulation of central metabolism, the CsrA regulator represses luminescence and siderophore as an intermediate of the GacA regulatory hierachy. Siderophore regulation was less sensitive to stoichiometry of CsrA consistent with higher affinity for the targets of this trait. The lack of CsrA null-mutant recovery implied these mutations do not enhance fitness of gacA mutants and alluded to this gene being conditionally essential. This study also suggests a role for IHF in the GacA-CsrB-CsrA regulatory cascade by potentially assisting with the binding of repressors of siderohphore and activators of luminescence. As many phosphorelay proteins reduce fitness when mutated, the documented instability used in this screen also highlights a potentially universal and underappreciated problem that, if not identified and strategically avoided, could introduce confounding variability during experimental study of these regulatory pathways

Pouring Salt on a Wound: Pseudomonas aeruginosa Virulence Factors Alter Na+ and Cl− Flux in the Lung

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen with multiple niches in the human body, including the lung. P. aeruginosa infections are particularly damaging or fatal for patients with ventilator-associated pneumonia, chronic obstructive pulmonary disease, and cystic fibrosis (CF). To establish an infection, P. aeruginosa relies on a suite of virulence factors, including lipopolysaccharide, phospholipases, exoproteases, phenazines, outer membrane vesicles, type III secreted effectors, flagella, and pili. These factors not only damage the epithelial cell lining but also induce changes in cell physiology and function such as cell shape, membrane permeability, and protein synthesis. While such virulence factors are important in initial infection, many become dysregulated or nonfunctional during the course of chronic infection. Recent work on the virulence factors alkaline protease (AprA) and CF transmembrane conductance regulator inhibitory factor (Cif) show that P. aeruginosa also perturbs epithelial ion transport and osmosis, which may be important for the long-term survival of this microbe in the lung. Here we discuss the literature regarding host physiology-altering virulence factors with a focus on Cif and AprA and their potential roles in chronic infection and immune evasion

Synthesis of Bioinspired Carbohydrate Amphiphiles that Promote and Inhibit Biofilms

The synthesis and characterization of a new class of bioinspired carbohydrate amphiphiles that modulate Pseudomonas aeruginosa biofilm formation are reported. The carbohydrate head is an enantiopure poly-amido-saccharide (PAS) prepared by a controlled anionic polymerization of β-lactam monomers derived from either glucose or galactose. The supramolecular assemblies formed by PAS amphiphiles are investigated in solution using fluorescence assays and dynamic light scattering. Dried samples are investigated using X-ray, infrared spectroscopy, and transmission electron microscopy. Additionally, the amphiphiles are evaluated for their ability to modulate biofilm formation by the Gram-negative bacterium Pseudomonas aeruginosa. Remarkably, from a library of eight amphiphiles, we identify a structure that promotes biofilm formation and two structures that inhibit biofilm formation. Using biological assays and electron microscopy, we relate the chemical structure of the amphiphiles to the observed activity. Materials that modulate the formation of biofilms by bacteria are important both as research tools for microbiologists to study the process of biofilm formation and for their potential to provide new drug candidates for treating biofilm-associated infections

Epoxide-Mediated CifR Repression of cif Gene Expression Utilizes Two Binding Sites in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro, and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees

Spontaneous phenotypic suppression of GacA-defective Vibrio fischeri is achieved via mutation of csrA and ihfA

Background: Symbiosis defective GacA-mutant derivatives of Vibrio fischeri are growth impaired thereby creating a selective advantage for growth-enhanced spontaneous suppressors. Suppressors were isolated and characterized for effects of the mutations on gacA-mutant defects of growth, siderophore activity and luminescence. The mutations were identified by targeted and whole genome sequencing. Results: Most mutations that restored multiple phenotypes were non-null mutations that mapped to conserved domains in or altered expression of CsrA, a post-transcriptional regulator that mediates GacA effects in a number of bacterial species. These represent an array of unique mutations compared to those that have been described previously. Different substitutions at the same amino acid residue were identified allowing comparisons of effects such as at the R6 residue, which conferred relative differences in luminescence and siderophore levels. The screen revealed residues not previously identified as critical for function including a single native alanine. Most csrA mutations enhanced luminescence more than siderophore activity, which was especially evident for mutations predicted to reduce the amount of CsrA. Although CsrA mutations compensate for many known GacA mutant defects, not all CsrA suppressors restore symbiotic colonization. Phenotypes of a suppressor allele of ihfA that encodes one subunit of the integration host factor (IHF) heteroduplex indicated the protein represses siderophore and activates luminescence in a GacA-independent manner. Conclusions: In addition to its established role in regulation of central metabolism, the CsrA regulator represses luminescence and siderophore as an intermediate of the GacA regulatory hierachy. Siderophore regulation was less sensitive to stoichiometry of CsrA consistent with higher affinity for the targets of this trait. The lack of CsrA null-mutant recovery implied these mutations do not enhance fitness of gacA mutants and alluded to this gene being conditionally essential. This study also suggests a role for IHF in the GacA-CsrB-CsrA regulatory cascade by potentially assisting with the binding of repressors of siderohphore and activators of luminescence. As many phosphorelay proteins reduce fitness when mutated, the documented instability used in this screen also highlights a potentially universal and underappreciated problem that, if not identified and strategically avoided, could introduce confounding variability during experimental study of these regulatory pathways. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0509-2) contains supplementary material, which is available to authorized users

Evidence for Direct Control of Virulence and Defense Gene Circuits by the Pseudomonas aeruginosa Quorum Sensing Regulator, MvfR

Pseudomonas aeruginosa defies eradication by antibiotics and is responsible for acute and chronic human infections due to a wide variety of virulence factors. Currently, it is believed that MvfR (PqsR) controls the expression of many of these factors indirectly via the pqs and phnAB operons. Here we provide strong evidence that MvfR may also bind and directly regulate the expression of additional 35 loci across the P. aeruginosa genome, including major regulators and virulence factors, such as the quorum sensing (QS) regulators lasR and rhlR, and genes involved in protein secretion, translation, and response to oxidative stress. We show that these anti-oxidant systems, AhpC-F, AhpB-TrxB2 and Dps, are critical for P. aeruginosa survival to reactive oxygen species and antibiotic tolerance. Considering that MvfR regulated compounds generate reactive oxygen species, this indicates a tightly regulated QS self-defense anti-poisoning system. These findings also challenge the current hierarchical regulation model of P. aeruginosa QS systems by revealing new interconnections between them that suggest a circular model. Moreover, they uncover a novel role for MvfR in self-defense that favors antibiotic tolerance and cell survival, further demonstrating MvfR as a highly desirable anti-virulence target