ABSTRACT
The clinical utility of fluoroquinolones (FQs) for the treatment of Pseudomonas aeruginosa (PA) and other serious Gram-negative infections is currently decreasing due to the rapid emergence of resistance. Because previous studies have shown that efflux is a common mechanism contributing to FQ resistance in PA, one suggested approach to extend the longevity of this class of drugs is combination therapy with an efflux pump inhibitor (EPI). In order to determine the viability of this approach, it is necessary to understand the relative contribution of efflux- vs. target-mediated mechanisms of FQ resistance in the clinic. A set of 26 recent PA clinical isolates were characterized for antibiotic resistance profiles, efflux pump expression, topoisomerase mutations, and FQ susceptibility with and without an EPI. The contribution of OprM to the overall antibiotic resistance was assessed in a subset of these strains. Our results suggest that the co-administration of an EPI with FQs or other antibiotics currently in use would not be sufficient to combat the complexity of resistance mechanisms now present in many clinical isolates.
Subject(s)
Fluoroquinolones/pharmacology , Mutation , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Drug Resistance, Bacterial , Humans , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/metabolismABSTRACT
Understanding the structural biology of type IV pili, fibres responsible for the virulent attachment and motility of numerous bacterial pathogens, requires a detailed understanding of the three-dimensional structure and chemistry of the constituent pilin subunit. X-ray crystallographic refinement of Neisseria gonorrhoeae pilin against diffraction data to 2.6 A resolution, coupled with mass spectrometry of peptide fragments, reveals phosphoserine at residue 68. Phosphoserine is exposed on the surface of the modelled type IV pilus at the interface of neighbouring pilin molecules. The site-specific mutation of serine 68 to alanine showed that the loss of the phosphorylation alters the morphology of fibres examined by electron microscopy without a notable effect on adhesion, transformation, piliation or twitching motility. The structural and chemical characterization of protein phosphoserine in type IV pilin subunits is an important indication that this modification, key to numerous regulatory aspects of eukaryotic cell biology, exists in the virulence factor proteins of bacterial pathogens. These O-linked phosphate modifications, unusual in prokaryotes, thus merit study for possible roles in pilus biogenesis and modulation of pilin chemistry for optimal in vivo function.
Subject(s)
Membrane Proteins/chemistry , Neisseria gonorrhoeae/chemistry , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Disaccharides/chemistry , Escherichia coli/chemistry , Fimbriae Proteins , Immunoblotting , Mass Spectrometry , Membrane Proteins/ultrastructure , Microscopy, Electron , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phenotype , Phosphates/chemistry , Phosphorylation , Phosphoserine/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship , Transformation, GeneticABSTRACT
Secretins are a large family of proteins associated with membrane translocation of macromolecular complexes, and a subset of this family, termed PilQ proteins, is required for type IV pilus biogenesis. We analysed the status of PlIQ expression in Neisseria meningitidis (Mc) and found that PlIQ mutants were non-piliated and deficient in the expression of pilus-associated phenotypes. Sequence analysis of the 5' portion of the pilQ ORF of the serogroup B Mc strain 44/76 showed the presence of seven copies of a repetitive sequence element, in contrast to the situation in N. gonorrhoeae (Gc) strains, which carry either two or three copies of the repeat. The derived amino acid sequence of the consensus nucleotide repeat was an octapeptide PAKQQAAA, designated as the small basic repeat (SBR). This gene segment was studied in more detail in a collection of 52 Mc strains of diverse origin by screening for variability in the size of the PCR-generated DNA fragments spanning the SBRs. These strains were found to harbour from four to seven copies of the repetitive element. No association between the number of copies and the serogroup, geographic origin or multilocus genotype of the strains was evident. The presence of polymorphic repeat elements in Mc PilQ is unprecedented within the secretin family. To address the potential function of the repeat containing domain, Mc strains were constructed so as to express chimeric PilQ molecules in which the number of SBR repeats was increased or in which the repeat containing domain was replaced in toto by the corresponding region of the Pseudomonas aeruginosa (Pa) PilQ protein. Although the strain expressing PilQ with an increased number of SBRs was identical to the parent strain in pilus phenotypes, a strain expressing PilQ with the equivalent Pa domain had an eightfold reduction in pilus expression level. The findings suggest that the repeat containing domain of PilQ influences Mc pilus expression quantitatively but not qualitatively.
