Ambrose L. Cheung

SarA, a global regulator of virulence determinants in Staphylococcus aureus, binds to a conserved motif essential for sar-dependent gene regulation.

The expression of many virulence determinants inStaphylococcus aureus including α-hemolysin-, protein A-, and fibronectin-binding proteins is controlled by global regulatory loci such as sar and agr. In addition to controlling target gene expression via agr(e.g. α-hemolysin), the sar locus can also regulate target gene transcription via agr-independent mechanisms. In particular, we have found that SarA, the major regulatory protein encoded within sar, binds to a conserved sequence, homologous to the SarA-binding site on the agrpromoter, upstream of the −35 promoter boxes of several target genes including hla (α-hemolysin gene), spa(protein A gene), fnb (fibronectin-binding protein genes), and sec (enterotoxin C gene). Deletion of the SarA recognition motif in the promoter regions of agr andhla in shuttle plasmids rendered the transcription of these genes undetectable in agr and hla mutants, respectively. Likewise, the transcription activity of spa(a gene normally repressed by sar), as measured by a XylE reporter fusion assay, became derepressed in a wild type strain containing a shuttle plasmid in which the SarA recognition site had been deleted from the spa promoter region. However, DNase I footprinting assays demonstrated that the SarA-binding region on thespa and hla promoter is more extensive than the predicted consensus sequence, thus raising the possibility that the consensus sequence is an activation site within a larger binding region. Because the sar and agr regulate an assortment of virulence factors in S. aureus, we propose, based on our data, a unifying hypothesis for virulence gene activation in S. aureus whereby SarA is a regulatory protein that binds to its consensus SarA recognition motif to activate (e.g. hla) or repress (e.g. spa) the transcription of sar target genes, thus accounting for both agr-dependent andagr-independent mode of regulation.

The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level

Abstract Agr and sar are known regulatory loci of Staphylococcus aureus that control the production of several extracellular and cell-wall-associated proteins. A pleiotropic insertional mutation in S. aureus, designated sae, that leads to the production of drastically diminished levels of α- and β-hemolysins and coagulase and slightly reduced levels of protein A has been described. The study of the expression of the genes coding for these exoproteins in the sae::Tn551 mutant (carried out in this work by Northern blot analyses) revealed that the genes for α- and β-hemolysins (hla and hlb) and coagulase (coa) are not transcribed and that the gene for protein A (spa) is transcribed at a somewhat reduced level. These results indicate that the sae locus regulates these exoprotein genes at the transcriptional level. Northern blot analyses also show that the sae mutation does not affect the expression of agr or sar regulatory loci. An sae::Tn551agr::tetM double mutant has been phenotypically characterized as producing reduced or null levels of α-, β-, and δ-hemolysins, coagulase, and high levels of protein A. Northern blot analyses carried out in this work with the double mutant revealed that hla, hlb, hld, and coa genes are not transcribed, while spa is transcribed at high levels. The fact that coa is not expressed in the sae agr mutant, as in the sae parental strain, while spa is expressed at the high levels characteristic of the agr parental strain, suggests that sae and agr interact in a complex way in the control of the expression of the genes of several exoproteins.

Dynamics of Neutrophil Infiltration during Cutaneous Wound Healing and Infection Using Fluorescence Imaging

Neutrophil influx is an early inflammatory response that is essential for the clearance of bacteria and cellular debris during cutaneous wounding. A non-invasive real-time fluorescence imaging technique was developed to examine the kinetics of enhanced green fluorescence protein-polymorphonuclear leukocyte (EGFP-PMN) influx within a wound. We hypothesized that infection or systemic availability would directly regulate the dynamics of EGFP-PMN recruitment and the efficiency of wound closure. Neutrophil recruitment increased dramatically over the first 24 hours from 10(6) at 4 hours up to a maximum of 5 x 10(6) EGFP-PMNs at 18 hours. A high rate of EGFP-PMN turnover was evidenced by approximately 80% decrease in EGFP signal within 6 hours. In response to wound colonization by Staphylococcus aureus or injection of GM-CSF, systemic PMNs increased twofold above saline control. This correlated with an increase in EGFP-PMN recruitment up to approximately 10(7) within the wound. Despite this effect by these distinct inflammatory drivers, wound closure occurred at a rate similar to the saline-treated control group. In summary, a non-invasive fluorescence-based imaging approach combined with genetic labeling of neutrophils provides a dynamic inner view of inflammation and the kinetics of neutrophil infiltration into the wounded skin over extended durations.

Agr-independent regulation of fibronectin-binding protein(s) by the regulatory locus sar in Staphylococcus aureus.

Fibronectin‐binding proteins (FnBPs) are thought to be important for the attachment of Staphylococcus aureus during infection. The regulation of the genes fnbA and fnbB by the global regulatory loci sar and agr was examined using site‐specific regulatory mutants of S. aureus strain Newman. The results from binding assays using both aqueous and solid‐phase fibronectin as well as ligand blotting with biotinylated fibronectin showed that the expression of FnBPA is enhanced in the agr mutant but inhibited in the sar mutant and the sar–agr double mutant. The same regulatory pattern was observed in Northern blot analysis using fnbA‐specific probes. The introduction of sar on a multicopy plasmid increased the already enhanced fnbA transcription of the agr mutant. FnBPB was not detectable by ligand blotting and the fnbB promoter activity in promoter fusion assays was not affected by either sar or agr. The sequence encompassing ORF3 located upstream of sarA was found to be essential for the activation of fnbA transcription. We hypothesize that this sequence may modulate SarA expression and/or activity on the post‐transcriptional level. Gel shift assays demonstrated that SarA binds to the fnbA promoter fragments, probably as a dimer. DNase I footprinting assays with SarA revealed a protected area of 102 bp upstream of fnbA.

SarA level is a determinant of agr activation in Staphylococcus aureus.

The control of virulence determinant expression in Staphylococcus aureus is a complex process involving global regulatory loci such as sar and agr. The sar locus consists of a 372 bp sarA open reading frame (ORF) preceded by a triple promoter region interspersed with two putative smaller ORFs (ORF3 and ORF4). The triple promoter system yields three overlapping sar transcripts (sarA, sarC and sarB of 0.56, 0.8 and 1.2 kb respectively). We have recently shown that the SarA protein binds to the agr promoter region to stimulate the transcription of RNAII and RNAIII, two major transcripts encoded within the agr locus. To assess the role of the region upstream of sarA in agr expression, we evaluated the contribution of ORF3 and ORF4 to SarA protein expression and to agr activation by introducing nonsense mutations into the respective ORFs. Northern analysis of sar mutant clones containing these mutations carried on a shuttle plasmid revealed that all three sar‐related transcripts are present. Using anti‐SarA monoclonal antibodies with defined epitopes in a competitive ELISA to determine the SarA protein level, we found that the introduction of a stop codon in ORF3 on a shuttle plasmid carrying the intact sar locus in a sar mutant led to a significant decrease in SarA protein level compared with the non‐mutated control. The effect of a nonsense mutation in ORF4 on SarA levels is much less. Likewise, an analogous sar mutant clone with a deletion in ORF3 also displayed a lower SarA level than its intact counterpart. The reduction in SarA expression correlated with a lower level of agr activation in the corresponding sar mutant clone. These data suggest that ORF3, and to a lesser degree ORF4, may affect agr expression by modulating SarA protein expression.

Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial cells.

The propensity of Staphylococcus aureus to cause acute endovascular infections during transient bacteremia is poorly understood. To examine the events leading to the attachment of staphylococci to endothelium, adherence assays were developed to study the role of blood factors in the mediation of staphylococcal adherence to cultured human umbilical vein endothelium in vitro. Results indicate that the preferential attachment of S. aureus to endothelial cells is mediated by fibrinogen adsorbed from plasma onto the endothelial surface. The binding is apparently specific because it could be blocked by goat anti-human fibrinogen antibody in a dose-dependent fashion while nonimmune goat IgG, mouse MAb against AG-1 (a platelet antigen found on the endothelial cell surface), nonspecific mouse MAb and rabbit antibodies to human vitronectin and fibronectin were not inhibitory. Our data also indicate that fibrinogen is a necessary but not the only blood constituent in the mediation of binding of S. aureus to endothelium. This was supported by the finding that fibrinogen alone, at a concentration equivalent to that in plasma, did not potentiate staphylococcal adherence as much as plasma while afibrinogenemic plasma reconstituted with fibrinogen did. Because fibrinogen is known to bind to endothelial cells, our data is consistent with the hypothesis that fibrinogen and additional plasma factor(s), possibly activated during inflammation, promote staphylococcal adherence to endothelium. In addition, the role of the fibrinogen binding receptor of S. aureus as an adherence factor to native endothelium is also suggested.

A method to detect proteinase activity using unprocessed X-ray films

Routine assays to detect proteinases in biological samples are generally tedious and time-consuming. To expedite the recognition of proteinases, we have developed an assay utilizing the gelatin on the surface of an unprocessed Kodak X-Omat AR film as the proteolytic substrate. A positive reaction is indicated by a clear zone on the film after it has been rinsed with running water. This proteinase assay has been found to be inexpensive, rapid, and simple. Besides its ease of use, this assay has been found to be quantitatively reproducible with a well-defined endpoint. More importantly, this assay method is applicable to a variety of proteolytic enzymes under diverse pH (5-8.5) and salt conditions (up to 5 M NaCl) and has a sensitivity similar to that of azocoll. Since the assay does not require sophisticated equipment, it is useful as a general laboratory procedure.

The role of fibrinogen in mediating staphylococcal adherence to fibers

The use of tampons and surgical gauze pads and colonization with Staphylococcus aureus have been established as risk factors for the development of toxic shock syndrome. To elucidate the role of blood factors in the mediation of staphylococcal adherence to fibers used in tampons and surgical packing, an adherence assay with cotton fibers was developed. Results demonstrated that cotton disks precoated with fibrinogen in the presence of human serum albumin bound a significant percentage of the inoculum for both staphylococcal strains tested when compared to human serum albumin controls. Likewise, fibers pretreated with plasma or defibrinated blood containing a small amount of fibrin revealed comparable staphylococcal adherence to that of fibrinogen. In contrast, fibers pretreated with serum, fibronectin, or vitronectin did not exhibit significant augmentation in staphylococcal attachment in comparison to human serum albumin controls. The attachment of staphylococci to fibrinogen and/or fibrin appeared to be specific and is blocked by goat anti-human fibrinogen antibody, but not fibronectin, vitronectin, or nonimmune goat IgG. Thus, our data indicate that fibrinogen/fibrin is the dominant blood component in the mediation of staphylococcal adherence to fibers used in tampons and surgical gauze pads.

Role of surface proteins in staphylococcal adherence to fibers in vitro.

To study the role of surface proteins in the adherence of Staphylococcus aureus to fibers that are used in tampon and surgical gauze pad manufacture, we have developed an adherence assay with S. aureus cells and cotton and rayon fibers. Results suggest that staphylococcal adherence is dependent on both the substrate and the material used to coat these fibers. Scanning electron micrographs supported the adherence results and revealed more cells on the surface of cotton than rayon fibers. Treatment of staphylococcal cells with proteolytic enzymes significantly reduced binding to pure cotton and detergent-treated cotton fibers. Immunoblot analysis of cell wall proteins suggested that surface proteins in the mol wt range of 120-220 kD were involved in the adherence of S. aureus to cotton fibers. Although the adherence of S. aureus to cotton fibers alone appeared to be mediated through surface charge or hydrophobic interactions, bacterial binding to fibers which have been pretreated with defibrinated blood appeared to be more specific and independent of the surface constituents of the fibers. The results of these studies implicate staphylococcal surface proteins in the adherence of S. aureus to commercially available tampon fibers and surgical gauze pads.

Are the structures of SarA and SarR similar

Abstract In Staphylococcus aureus , the production of virulence determinants including hemolysin is controlled by global regulators such as SarA. Recently the crystal structures of SarA and SarR, a SarA homolog and a member of the SarA family of proteins, were solved. A motif found in SarR is similar to that found in the winged-helix protein family, and it is possible that the SarA family of proteins uses DNA bending to regulate gene transcription.