Dataset Information

Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene.

ABSTRACT: A 5.9-kb DNA fragment was cloned from Pseudomonas aeruginosa PA103 by its ability to functionally complement a fur mutation in Escherichia coli. A fur null mutant E. coli strain that contains multiple copies of the 5.9-kb DNA fragment produces a 15-kDa protein which cross-reacts with a polyclonal anti-E. coli Fur serum. Sequencing of a subclone of the 5.9-kb DNA fragment identified an open reading frame predicted to encode a protein 53% identical to E. coli Fur and 49% identical to Vibrio cholerae Fur and Yersinia pestis Fur. While there is extensive homology among these Fur proteins, Fur from P. aeruginosa differs markedly at its carboxy terminus from all of the other Fur proteins. It has been proposed that this region is a metal-binding domain in E. coli Fur. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants of strain PA103 that produce altered Fur proteins. These manganese-resistant Fur mutants constitutively produce siderophores and exotoxin A when grown in concentrations of iron that normally repress their production. A multicopy plasmid carrying the P. aeruginosa fur gene restores manganese susceptibility and wild-type regulation of exotoxin A and siderophore production in these Fur mutants.

SUBMITTER: Prince RW

PROVIDER: S-EPMC204560 | biostudies-other | 1993 May

REPOSITORIES: biostudies-other

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Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene.

Prince R W RW Cox C D CD Vasil M L ML

Journal of bacteriology 19930501 9

A 5.9-kb DNA fragment was cloned from Pseudomonas aeruginosa PA103 by its ability to functionally complement a fur mutation in Escherichia coli. A fur null mutant E. coli strain that contains multiple copies of the 5.9-kb DNA fragment produces a 15-kDa protein which cross-reacts with a polyclonal anti-E. coli Fur serum. Sequencing of a subclone of the 5.9-kb DNA fragment identified an open reading frame predicted to encode a protein 53% identical to E. coli Fur and 49% identical to Vibrio choler ...[more]

PMID: 8478325

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Project description:Cystic fibrosis (CF) is a genetic disease that causes patients to accumulate thick, dehydrated mucus in the lung and develop chronic, polymicrobial infections due to reduced mucociliary clearance. These chronic polymicrobial infections and subsequent decline in lung function are significant factors in the morbidity and mortality of CF. Pseudomonas aeruginosa and Streptococcus spp. are among the most prevalent organisms in the CF lung; the presence of P. aeruginosa correlates with lung function decline, and the Streptococcus milleri group (SMG), a subgroup of the viridans streptococci, is associated with exacerbations in patients with CF. Here we characterized the interspecies interactions that occur between these two genera. We demonstrated that multiple P. aeruginosa laboratory strains and clinical CF isolates promote the growth of multiple SMG strains and oral streptococci in an in vitro coculture system. We investigated the mechanism by which P. aeruginosa enhances growth of streptococci by screening for mutants of P. aeruginosa PA14 that are unable to enhance Streptococcus growth, and we identified the P. aeruginosapqsL::TnM mutant, which failed to promote growth of Streptococcus constellatus and S. sanguinis Characterization of the P. aeruginosa ΔpqsL mutant revealed that this strain cannot promote Streptococcus growth. Our genetic data and growth studies support a model whereby the P. aeruginosa ΔpqsL mutant overproduces siderophores and thus likely outcompetes Streptococcus sanguinis for limited iron. We propose a model whereby competition for iron represents one important means of interaction between P. aeruginosa and Streptococcus spp.IMPORTANCE Cystic fibrosis (CF) lung infections are increasingly recognized for their polymicrobial nature. These polymicrobial infections may alter the biology of the organisms involved in CF-related infections, leading to changes in growth, virulence, and/or antibiotic tolerance, and could thereby affect patient health and response to treatment. In this study, we demonstrate interactions between P. aeruginosa and streptococci using a coculture model and show that one interaction between these microbes is likely competition for iron. Thus, these data indicate that one CF pathogen may influence the growth of another, and they add to our limited knowledge of polymicrobial interactions in the CF airway.

Project description:The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

Dataset Information

Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene.

Publications

Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene.

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