ABSTRACT
The CFTR gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF). The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with Pseudomonas aeruginosa causing lethal bacterial infections in CF, is not known. Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells. The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased P. aeruginosa adherence were corrected by incubating CF respiratory epithelial cells with weak bases. Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN. These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Glycoproteins , Lung/metabolism , Membrane Proteins , Pseudomonas aeruginosa/metabolism , Acids , Bacterial Adhesion , Cell Line , Glycosylation , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Membrane Glycoproteins/metabolism , Proton-Translocating ATPases/antagonists & inhibitors , Pseudomonas aeruginosa/physiology , Recombinant Fusion Proteins/metabolism , Sialyltransferases/metabolism , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , trans-Golgi Network/metabolismABSTRACT
Pseudomonas aeruginosa is a significant threat to human health as it is frequently recalcitrant to conventional antibacterial therapy. This ubiquitous gram-negative bacterium is notorious for its nutritional and ecological flexibility and its resistance to both antibiotic treatments and sanitary measures. These properties contribute to its prominence as a leading source of opportunistic nosocomial (hospital acquired) and a less appreciated, but significant cause of community acquired infections. P. aeruginosa remains a considerable problem for patients with burns, neutropenic individuals, and cystic fibrosis patients (CF). In this review, we will address the current issues in P. aeruginosa infections in CF. A major emphasis will be placed on the factors predisposing CF patients to colonization with P. aeruginosa.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/complications , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/pathogenicity , Respiratory Tract Infections/microbiology , Animals , Bacterial Adhesion , Biofilms/growth & development , Cystic Fibrosis/metabolism , Cystic Fibrosis/microbiology , Disease Models, Animal , Humans , Inflammation/immunology , Mice , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Pseudomonas Infections/complications , Pseudomonas Infections/metabolism , Pseudomonas aeruginosa/physiology , Respiratory Mucosa/enzymology , Respiratory Mucosa/microbiology , Respiratory Tract Infections/complications , Respiratory Tract Infections/metabolism , Sialyltransferases/metabolismSubject(s)
Cystic Fibrosis/microbiology , Pseudomonas aeruginosa/physiology , Pseudomonas aeruginosa/pathogenicity , Alginates/metabolism , Animals , Biofilms/growth & development , Blood Bactericidal Activity , Carbohydrate Dehydrogenases/genetics , Cell Line , Cystic Fibrosis/complications , Disease Models, Animal , Glucuronic Acid , Hexuronic Acids , Humans , Mice , Mutation , Opportunistic Infections/etiology , Opportunistic Infections/microbiology , Pseudomonas aeruginosa/genetics , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , Respiratory Tract Infections/etiology , Respiratory Tract Infections/microbiologyABSTRACT
BACKGROUND: Many beta lactams are well absorbed by the small intestine, although the reasons for this are poorly understood. AIMS: To characterise the uptake of penicillin G into human small intestinal brush border membrane vesicles (BBMV) and to compare the uptake characteristics to those of rabbit BBMV. METHODS AND RESULTS: Uptake of penicillin G was studied in human BBMV. Penicillin G was actively transported into the lumen of BBMV via an H+ dependent, Na+ independent uptake system. The carrier mediated process was saturable and adhered to Michaelis-Menten kinetics (Vmax 52 nmol penicillin G per mg protein per 30 seconds, Km 13.9 mM). These results are similar to those previously reported in rabbit BBMV. CONCLUSIONS: It is suggested that penicillin G can be used as a model molecule for peptide and beta lactam transport studies as it is cheap and readily available in isotopically labelled form. Furthermore, rabbit BBMV may be used as an acceptable substitute for human BBMV for the study of penicillin transport.
Subject(s)
Intestinal Absorption , Jejunum/metabolism , Penicillin G/pharmacokinetics , Penicillins/pharmacokinetics , Animals , Biological Transport, Active , Culture Techniques , Humans , Jejunum/ultrastructure , Microvilli/metabolism , Osmolar Concentration , RabbitsABSTRACT
Uptake of penicillin-G has been studied in rabbit intestinal brush-border membrane vesicles (BBMV). Penicillin-G was transported into the lumen of BBMV via an H+-dependent, Na+-independent uptake system. This was a saturable carrier-mediated process, which adhered to Michaelis-Menten kinetics, having a pH optimum of 4.5 and resulting in a net-negative charge transfer. Vmax was 59 nmol penicillin-G (mg protein)-1 (30s)-1 and Km 22.7 mM. Ampicillin, penicillin-V, cefadroxil, cephalexin, cephalothin, cephradine, L-carnosine, glycyl-L-alanine, glycyl-L-tyrosine and glycylglycylglycine inhibited the uptake of penicillin-G. However, glycylsarcosine stimulated uptake by 92%. Countertransport experiments suggested that this effect took place at the active site of the transporter. Penicillin-G uptake appeared to be mediated via a common transport system shared by penicillins, cephalosporins and peptides.
Subject(s)
Intestine, Small/metabolism , Microvilli/metabolism , Penicillin G/pharmacokinetics , Penicillins/pharmacokinetics , Amino Acid Sequence , Amino Acids/pharmacology , Animals , Biological Transport/drug effects , Electrochemistry , Hydrogen-Ion Concentration , In Vitro Techniques , Intestine, Small/ultrastructure , Kinetics , Lactams/pharmacology , Male , Membrane Potentials , Molecular Sequence Data , Oligopeptides/pharmacology , RabbitsABSTRACT
A bacterial expression vector containing a segment of the v-abl gene from Abelson murine leukemia virus (A-MuLV) was constructed such that the gag region of v-abl was replaced by a sequence encoding the IgG-binding domain of the S. aureus protein A. pabl HP, a fusion protein encoded by this vector was rapidly purified to near homogeneity by affinity chromatography on IgG-Affigel and Mono Q FPLC. The Km of the pabl HP kinase for ATP varied with [Val5]-angiotensin II concentration and was 21.2 microM at saturating concentrations of [Val5]-angiotensin II. The Km for [Val5]-angiotensin II at saturating concentrations of ATP was 3.8 mM. The turnover number, at 20 degrees C, was 62 mumol min-1 mumol-1. Initial rate studies support a ternary complex kinetic mechanism for phosphoryl transferase. The substrate specificity of the pabl HP kinase was further characterized using synthetic peptides. This expression system, which enables the rapid purification of recombinant v-abl kinase is suitable for the comparative enzymological study of mutant v-abl enzymes generated by site-directed mutagenesis.
