Repression of the Staphylococcus aureus Accessory Gene Regulator in Serum and In Vivo

Subgenomic DNA microarrays were employed to evaluate the expression of the accessory gene regulator (agr locus) as well as multiple virulence-associated genes in Staphylococcus aureus. Gene expression was examined during growth of S. aureus in vitro in standard laboratory medium and rabbit serum and in vivo in subcutaneous chambers implanted in either nonimmune rabbits or rabbits immunized with staphylococcal enterotoxin B. Expression of RNAIII, the effector molecule of the agr locus, was dramatically repressed in serum and in vivo, despite the increased expression of secreted virulence factors sufficient to cause toxic shock syndrome (TSS) in the animals. Statistical analysis and clustering of virulence genes based on their expression profiles in the various experimental conditions demonstrated no positive correlation between the expression of agr and any staphylococcal virulence factors examined. Disruption of the agr locus had only a minimal effect on the expression in vivo of the virulence factors examined. An effect of immunization on the expression of agr and virulence factors was also observed. These results suggest that agr activation is not necessary for development of staphylococcal TSS and that regulatory circuits responding to the in vivo environment override agr activity.     

Population studies of methicillin-resistant and -sensitive Staphylococcus aureus strains reveal a lack of variability in the agrD gene, encoding a staphylococcal autoinducer peptide

The virulence of Staphylococcus aureus is controlled by the accessory gene regulator (agr) system, including an extracellular inducer encoded by agrD. Variable agr PCR restriction fragment length polymorphism (RFLP) patterns of unique S. aureus strains (n = 192) were determined for a region comprising agrD and parts of the neighboring agrC and agrB genes. Twelve unique RFLP patterns were identified among S. aureus strains in general; these patterns were further specified by sequencing. All sequences could be catalogued in the three current agr groups. A major proportion of the S. aureus strains belong to agr group 1, whereas only 6% of the methicillin-susceptible S. aureus strains and 5% of the methicillin-resistant S. aureus strains belong to agr groups 2 and 3, respectively. The homology between groups varied from 75 to 80%, and within groups it varied from 96 to 100%. Different levels of sequence variability were observed in the different agr genes. agr-related bacterial interference among colonizing S. aureus strains in the noses of persistent and intermittent human carriers was studied. S. aureus strains belonging to different agr groups were encountered in the same individual. This may suggest that the activity of the agrD gene product does not define colonization dynamics, which is further substantiated by the rarity of agr group 2 and 3 strains.     

RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus

Cell-density-dependent gene regulation by quorum-sensing systems has a crucial function in bacterial physiology and pathogenesis. We demonstrate here that the Staphylococcus aureus agr quorum-sensing regulon is divided into (1) control of metabolism and PSM cytolysin genes, which occurs independently of the small regulatory RNA RNAIII, and (2) RNAIII-dependent control of additional virulence genes. Remarkably, PSM expression was regulated by direct binding of the AgrA response regulator. Our findings suggest that quorum-sensing regulation of PSMs was established before wide-ranging control of virulence was added to the agr regulon, which likely occurred by development of the RNAIII-encoding region around the gene encoding the PSM delta-toxin. Moreover, the agr regulon in the community-associated methicillin-resistant S. aureus MW2 considerably differed from that previously determined using laboratory strains. By establishing a two-level model of quorum-sensing target gene regulation in S. aureus, our study gives important insight into the evolution of virulence control in this leading human pathogen.     

Cross-Strain Quorum Sensing Inhibition by Staphylococcus aureus. Part 1: A Spatially Homogeneous Model

Staphylococcus aureus uses the agr quorum sensing (QS) system to regulate reciprocally colonisation and virulence factor production. S. aureus strains can be divided into four agr groups: those within a specific agr group activate the QS systems of strains belonging to the same group, while inhibiting agr expression in strains of other groups. Furthermore, agr homologues exist in many more species of gram-positive bacteria, raising the likelihood of cross-species interference. In principle, a non-pathogenic strain of S. aureus or other species of bacteria employing agr could be engineered to inhibit the QS systems of pathogenic strains using agr, thus down-regulating their production of virulence factors. We present three models of the agr operon belonging to strains competing for dominance, each comprising one of the three possible phosphorylation cascades governing the two component system (TCS) of the agr system. Bifurcation analyses clarify the aspects of QS most crucial in determining the efficacy of using a non-pathogenic strain for therapeutic purposes if the target TCS cascade is known and illustrate the qualitative and quantitative differences which occur as a result of mechanistic differences between the models. We highlight those results that, in concert with appropriate experimental data, would be most useful in ascertaining whether or not a classical TCS is in operation in a particular strain if this information is unknown.     

A molecular phylogeny of enteric bacteria and implications for a bacterial species concept

A molecular phylogeny for seven taxa of enteric bacteria (Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia plymuthica) was made from multiple isolates per taxa taken from a collection of environmental enteric bacteria. Sequences from five housekeeping genes (gapA, groEL, gyrA, ompA, and pgi) and the 16S rRNA gene were used to infer individual gene trees and were concatenated to infer a composite molecular phylogeny for the species. The isolates from each taxa formed tight species clusters in the individual gene trees, suggesting the existence of 'genotypic' clusters that correspond to traditional species designations. These sequence data and the resulting gene trees and consensus tree provide the first data set with which to assess the utility of the recently proposed core genome hypothesis (CGH). The CGH provides a genetically based approach to applying the biological species concept to bacteria.     

A novel DNA microarray for rapid diagnosis of enteropathogenic bacteria in stool specimens of patients with diarrhea

A microarray technique for the detection and identification of enteropathogenic bacteria at the species and subspecies levels was developed in this study, and the target bacteria included pathogenic Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella enterica, Campylobacter jejuni, Shigellae, Yersinia enterocolitica, and Listeria monocytogenes. The virulence gene of each pathogen was chosen as the amplification target, labeled with a fluorescence dye by multiplex polymerase chain reaction (PCR), and hybridized to the specific virulence gene probes that had been immobilized on a microchip. Stool specimens from 34 patients with diarrhea were tested in this study. Five were positive for multiple genera. Nested PCRs and sequencing were used to amplify and identify the related genes, which were found to share 95.8% to 100% of the nucleotide identity with the corresponding regions in the Genbank database. Real-time PCR was used to determine the number of gene copies to determine the sensitivity of this technique, which was shown to be 58 copies/microl. The results indicated that the microarray technique which targets multiple virulence genes of enteropathogenic bacteria at the species and subspecies levels is an attractive diagnostic tool for rapidly and simultaneously identifying multiple enteropathogenic pathogens in clinical practice, especially in patients with infectious diarrhea.     

Escherichia coli molecular phylogeny using the incongruence length difference test

Molecular phylogeny of the species Escherichia coli using the E. coli reference (ECOR) collection strains has been hampered by (1) the absence of rooting in the commonly used phenogram obtained from multilocus enzyme electrophoresis (MLEE) data and (2) the existence of recombination events between strains that scramble phylogenetic trees reconstructed from the nucleotide sequences of genes. We attempted to determine the phylogeny for E. coli based on the ECOR strain data by extracting from GenBank the nucleotide sequences of 11 chromosomal structural and 2 plasmid genes for which the Salmonella enterica homologous gene sequences were available. For each of the 13 DNA data sets studied, incongruence with a nonnucleotide whole-genome data set including MLEE, random amplified polymorphic DNA, and rrn restriction fragment length polymorphism data was measured using the incongruence length difference (ILD) test of Farris et al. As previously reported, the incongruence observed between the gnd and plasmid gene data and the whole-genome data was multiple, indicating numerous horizontal transfer and/or recombination events. In five cases, the incongruence detected by the ILD test was punctual, and the donor group was identified. Congruence was not rejected for the remaining data sets. The strains responsible for incongruences with the whole-genome data set were removed, leading to a "prior-agreement" approach, i.e., the determination of a phylogeny for E. coli based on several genes, excluding (1) the genes with multiple incongruences with the whole genome data, (2) the strains responsible for punctual incongruences, and (3) the genes incongruent with each other. The obtained phylogeny shows that the most basal group of E. coli strains is the B2 group rather than the A group, as generally thought. The D group then emerges as the sister group of the rest. Finally, the A and B1 groups are sister groups. Interestingly, the most primitive taxon within E. coli in terms of branching pattern, i.e., the B2 group, includes highly virulent extraintestinal strains with derived characters (extraintestinal virulence determinants) occurring on its own branch.      

Evaluation of Staphylococcus aureus virulence factors using a silkworm model

Previous studies have indicated that the silkworm model is useful for identifying virulence genes of Staphylococcus aureus, a human pathogenic bacterium. Here we examined the scope of S.?aureus virulence factors that can be evaluated using the silkworm model. Gene-disrupted mutants of the agr locus, arlS gene and saeS gene, which regulate the expression of cell surface adhesins and hemolysins, exhibited attenuated virulence in silkworms. Mutants of the hla gene encoding α-hemolysin, the hlb gene encoding β-hemolysin, and the psmα and psmβ operons encoding cytolysins, however, showed virulence in silkworms indistinguishable from that of the parent strain. Thus, these S.?aureus cytolysins are not required for virulence in silkworms. In contrast, the gene-disrupted mutants of clfB, fnbB and sdrC, which encode cell-wall-anchored proteins, attenuated S.?aureus virulence in silkworms. In addition, the mutant of the srtA gene encoding sortase A, which anchors cell-wall proteins, showed attenuated virulence in silkworms. These findings suggest that the silkworm model can be used to evaluate S.?aureus cell-wall proteins and regulatory proteins as virulence factors.     

Assignment of Staphylococcus aureus isolates to clonal complexes based on microarray analysis and pattern recognition

A DNA microarray was designed for the rapid genotyping of Staphylococcus aureus. It covers 185 distinct genes and about 300 alleles thereof, including species-specific controls, accessory gene regulator (agr) alleles, genes encoding virulence factors, and microbial surface components recognizing adhesive matrix molecules, capsule type-specific genes, as well as resistance determinants. It was used to examine 100 clinical isolates and reference strains. Relationships of leukocidin and ssl/set (staphylococcal superantigen-like or exotoxin-like) genes were reviewed considering these experimental results as well as published sequences. A good correlation of overall hybridization pattern and multilocus sequence typing was found. Analysis of hybridization profiles thus allowed not only to assess virulence and drug resistance, but also to assign isolates to strains and to clonal complexes. Hybridization data were used to construct a split network tree and to analyse relationships between strains. Allelic variations of a number of genes indicate a division of S. aureus into three major branches that are not in accordance to agr group or capsule-type affiliations. Additionally, there are some isolated lineages, such as ST75, ST93, or ST152. These strains produce aberrant hybridization profiles, indicating that only a part of the gene pool of S. aureus has been investigated yet.