ABSTRACT
Nontypeable Haemophilus influenzae (NTHi) is an extremely common human pathobiont that persists on the airway mucosal surface within biofilm communities, and our previous work has shown that NTHi biofilm maturation is coordinated by the production and uptake of autoinducer 2 (AI-2) quorum signals. To directly test roles for AI-2 in maturation and maintenance of NTHi biofilms, we generated an NTHi 86-028NP mutant in which luxS transcription was under the control of the xylA promoter (NTHi 86-028NP luxS xylA::luxS), rendering AI-2 production inducible by xylose. Comparison of biofilms under inducing and noninducing conditions revealed a biofilm defect in the absence of xylose, whereas biofilm maturation increased following xylose induction. The removal of xylose resulted in the interruption of luxS expression and biofilm dispersal. Measurement of luxS transcript levels by real-time reverse transcription-PCR (RT-PCR) showed that luxS expression peaked as biofilms matured and waned before dispersal. Transcript profiling revealed significant changes following the induction of luxS, including increased transcript levels for a predicted family 8 glycosyltransferase (NTHI1750; designated gstA); this result was confirmed by real-time RT-PCR. An isogenic NTHi 86-028NP gstA mutant had a biofilm defect, including decreased levels of sialylated matrix and significantly altered biofilm structure. In experimental chinchilla infections, we observed a significant decrease in the number of bacteria in the biofilm population (but not in effusions) for NTHi 86-028NP gstA compared to the parental strain. Therefore, we conclude that AI-2 promotes NTHi biofilm maturation and the maintenance of biofilm integrity, due at least in part to the expression of a probable glycosyltransferase that is potentially involved in the synthesis of the biofilm matrix.
Subject(s)
Bacterial Proteins/metabolism , Biofilms/growth & development , Carrier Proteins/metabolism , Glycosyltransferases/metabolism , Haemophilus influenzae/metabolism , Homoserine/analogs & derivatives , Lactones/metabolism , Animals , Bacterial Proteins/genetics , Carbon-Sulfur Lyases/genetics , Carrier Proteins/genetics , Chinchilla/microbiology , Gene Expression Profiling , Glycosyltransferases/genetics , Haemophilus Infections/microbiology , Haemophilus influenzae/genetics , Homoserine/genetics , Homoserine/metabolism , Intracellular Signaling Peptides and Proteins , Mutation , Otitis Media/microbiology , Real-Time Polymerase Chain Reaction , Transcription, Genetic , Xylose/metabolismABSTRACT
BACKGROUND: Vasodilator-stimulated phosphoprotein (VASP) mediates focal adhesion, actin filament binding and polymerization in a variety of cells, thereby inhibiting cell movement. Phosphorylation of VASP via cAMP and cGMP dependent protein kinases releases this "brake" on cell motility. Thus, phosphorylation of VASP may be necessary for epithelial cell repair of damage from allergen-induced inflammation. Two hypotheses were examined: (1) injury from segmental allergen challenge increases VASP phosphorylation in airway epithelium in asthmatic but not nonasthmatic normal subjects, (2) regular in vivo beta2-agonist use increases VASP phosphorylation in asthmatic epithelium, altering cell adhesion. METHODS: Bronchial epithelium was obtained from asthmatic and non-asthmatic normal subjects before and after segmental allergen challenge, and after regularly inhaled albuterol, in three separate protocols. VASP phosphorylation was examined in Western blots of epithelial samples. DNA was obtained for beta2-adrenergic receptor haplotype determination. RESULTS: Although VASP phosphorylation increased, it was not significantly greater after allergen challenge in asthmatics or normals. However, VASP phosphorylation in epithelium of nonasthmatic normal subjects was double that observed in asthmatic subjects, both at baseline and after challenge. Regularly inhaled albuterol significantly increased VASP phosphorylation in asthmatic subjects in both unchallenged and antigen challenged lung segment epithelium. There was also a significant increase in epithelial cells in the bronchoalveolar lavage of the unchallenged lung segment after regular inhalation of albuterol but not of placebo. The haplotypes of the beta2-adrenergic receptor did not appear to associate with increased or decreased phosphorylation of VASP. CONCLUSION: Decreased VASP phosphorylation was observed in epithelial cells of asthmatics compared to nonasthmatic normals, despite response to beta-agonist. The decreased phosphorylation does not appear to be associated with a particular beta2-adrenergic receptor haplotype. The observed decrease in VASP phosphorylation suggests greater inhibition of actin reorganization which is necessary for altering attachment and migration required during epithelial repair.
Subject(s)
Adrenergic beta-Agonists/administration & dosage , Albuterol/administration & dosage , Asthma/metabolism , Cell Adhesion Molecules/metabolism , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Receptors, Adrenergic, beta/metabolism , Asthma/classification , Bronchodilator Agents/administration & dosage , Humans , Phosphorylation/drug effectsABSTRACT
The nucleotide sequences, genetic organization, and distribution of plasmids pEU30 (30,314 bp) and pEL60 (60,145 bp) from the plant pathogen Erwinia amylovora are described. The newly characterized pEU30 and pEL60 plasmids inhabited strains isolated in the western United States and Lebanon, respectively. The gene content of pEU30 resembled plasmids found in plant-associated bacteria, while that of pEL60 was most similar to IncL/M plasmids inhabiting enteric bacteria.
