ABSTRACT
Microbial pathogenicity in Staphylococcus aureus is a complex process involving a number of virulence genes that are regulated by global regulatory systems including sar and agr. To evaluate the roles of these two loci in virulence, we constructed sar-/agr- mutants of strains RN6390 and RN450 and compared their phenotypic profiles to the corresponding single sar- and agr- mutants and parents. The secretion of all hemolysins was absent in the sar-/agr- mutants while residual beta-hemolysin activity remained in single agr- mutants. The fibronectin binding capacity was significantly diminished in both single sar- mutants and double mutants when compared with parents while the reduction in fibrinogen binding capacity in the double mutants was modest. In the rabbit endocarditis model, there was a significant decrease in both infectivity rates and intravegetation bacterial densities with the double mutant as compared to the parent (RN6390) at 10(3)-10(6) CFU inocula despite comparable levels of early bacteremia among various challenge groups. Notably, fewer bacteria in the double mutant group adhered to valvular vegetations at 30 min after challenge (10(6) CFU) than the parent group. These studies suggest that both the sar and agr loci are involved in initial valvular adherence, intravegetation persistence and multiplication of S. aureus in endocarditis.
Subject(s)
Endocarditis, Bacterial/microbiology , Genes, Bacterial , Staphylococcus aureus/pathogenicity , Animals , Bacterial Adhesion , Disease Models, Animal , Mutation , Phenotype , Rabbits , Staphylococcus aureus/genetics , VirulenceABSTRACT
The roles of fimB and fimE in the phase-variable expression of type 1 fimbriae in Escherichia coli were examined. A method was developed to study the effects of fimB and fimE on both recombination of the fim invertible element and fimbrial expression. The method used an allelic exchange procedure consisting of two steps. The first step, construction of intermediate strains, deleted fimB and fimE. This step locked the invertible element in either the on or the off orientation. The second step of the exchange procedure introduced either wild-type or mutant alleles of fimB and/or fimE into the chromosome of the intermediate strains. Analysis of the resulting strains supported the current, plasmid-based model of recombination. Unexpectedly, strains in which the invertible element was locked in the on orientation (either by mutation of both fimB and fimE or, in a control strain, by mutation of the left inverted repeat sequence of the invertible element) continued to exhibit phase-variable expression of type 1 fimbriae. A strain in which fimA was transcribed from the tac promoter continued to exhibit phase-variable fimbrial expression, suggesting that inversion-independent phase variation cannot be explained by variable transcription initiation of fimA.
Subject(s)
Bacterial Proteins/biosynthesis , Chromosome Inversion , DNA, Bacterial/genetics , DNA-Binding Proteins/biosynthesis , Escherichia coli Proteins , Escherichia coli/genetics , Escherichia coli/physiology , Fimbriae Proteins , Fimbriae, Bacterial/physiology , Genes, Bacterial , Integrases , Alleles , Bacterial Proteins/genetics , Base Sequence , Cloning, Molecular , DNA, Bacterial/metabolism , DNA, Recombinant/metabolism , DNA-Binding Proteins/genetics , Escherichia coli/growth & development , Escherichia coli/ultrastructure , Fimbriae, Bacterial/ultrastructure , Gene Expression , Microscopy, Electron , Molecular Sequence Data , Mutagenesis, Site-Directed , Transcription, GeneticABSTRACT
The phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a short DNA element. This element (switch) acts in cis to control transcription of fimA, the major fimbrial subunit gene. Thus, fimA is transcribed when the switch is in one orientation (the on orientation) but not the other (the off orientation). The fim inversion requires either fimB (on-to-off or off-to-on inversion) or fimE (on-to-off inversion only), as well as integration host factor, and is also influenced by the abundant DNA-binding protein H-NS. Here we report that an additional gene, lrp, a factor known to influence the expression of both Pap and K99 fimbriae, is also required for normal activity of the fim switch. The frequencies of both fimB-promoted and fimE-promoted inversions, and consequently the phase variation of type 1 fimbriation, are lower in lrp mutants. Lrp affects slightly the transcription of both fimB (which is increased) and fimE (which is decreased). We believe that these alterations in fimB and fimE transcription alone are unlikely to account for the sharp reduction in switching found in lrp mutants.
