Biomedical subjects

Ambrose L Cheung

Publications and source records attributed to Ambrose L Cheung.

27 records · Page 2Linked to original sources

PubMed2003

A recA-LexA-dependent pathway mediates ciprofloxacin-induced fibronectin binding in Staphylococcus aureus.

Subinhibitory concentrations of ciprofloxacin (CPX) raise the fibronectin-mediated attachment of fluoroquinolone-resistant Staphylococcus aureus by selectively inducing fnbB coding for one of two fibronectin-binding proteins: FnBPB. To identify candidate regulatory pathway(s) linking drug exposure to up-regulation of fnbB, we disrupted the global response regulators agr, sarA, and recA in the highly quinolone-resistant strain RA1. Whereas agr and sarA mutants of RA1 exposed to CPX still displayed increased adhesion to fibronectin, the CPX-triggered response was abolished in the uvs-568 recA mutant, but was restored following complementation with wild type recA. Steady-state levels of recA and fnbB, but not fnbA, mRNA were co-coordinately increased >3-fold in CPX-exposed strain RA1. Electrophoretic mobility shift assays revealed specific binding of purified S. aureus SOS-repressor LexA to recA and fnbB, but not to fnbA or rpoB promoters. DNase I footprint analysis showed LexA binding overlapping the core promoter elements in fnbB. We conclude that activation of recA and derepression of lexA-regulated genes by CPX may represent a response to drug-induced damage that results in a novel induction of a virulence factor leading to increased bacterial tissue adherence.

Anti-Infective Agents↗

PubMed2003

Fibrinogen depletion attenuates Staphyloccocus aureus infection by preventing density-dependent virulence gene up-regulation.

Staphylococcus aureus undergoes a density-dependent conversion in phenotype from tissue-adhering to tissue-damaging and phagocyte-evading that is mediated in part by the quorum-sensing operon, agr, and its effector, RNAIII. Contributions of host factors to this mechanism for regulating virulence have not been studied. We hypothesized that fibrinogen, as a component of the inflammatory response, could create spatially constrained microenvironments around bacteria that increase density independently of bacterial numbers and thus potentiate quorum-sensing-dependent virulence gene expression. Here we show that transient fibrinogen depletion significantly reduces the bacterial burden and the consequential morbidity and mortality during experimental infection with wild-type S. aureus, but not with bacteria that lack expression of the quorum-sensing operon, agr. In addition, it inhibits in vivo activation of the promoter for the agr effector, RNAIII, and downstream targets of RNAIII, including alpha hemolysin and capsule production. Moreover, both in vitro and in vivo, the mechanism for promoting this phenotypic switch in virulence involves clumping of the bacteria, demonstrating that S. aureus responds to fibrinogen-mediated bacterial clumping by enhancing density-dependent virulence gene expression. These data demonstrate that down-modulation of specific inflammatory components of the host that augment bacterial quorum sensing can be a strategy for enhancing host defense against infection.

Afibrinogenemia↗

PubMed2003

sarU, a sarA homolog, is repressed by SarT and regulates virulence genes in Staphylococcus aureus.

In searching the Staphylococcus aureus genome, we previously identified sarT, a homolog of sarA, which encodes a repressor for alpha-hemolysin synthesis. Adjacent but transcribed divergently to sarT is sarU, which encodes a 247-residue polypeptide, almost twice the length of SarA. Sequence alignment disclosed that SarU, like SarS, which is another SarA homolog, could be envisioned as a molecule with two halves, with each half being homologous to SarA. SarU, as a member of the SarA family proteins, disclosed conservation of basic residues within the helix-turn-helix motif and within the beta hairpin loop, two putative DNA binding domains within this protein family. The transcription of sarU is increased in a sarT mutant. Gel shift and transcriptional fusion studies revealed that SarT can bind to the sarU promoter region, probably acting as a repressor for sarU transcription. The expression of RNAII and RNAIII of agr is decreased in a sarU mutant. As RNAIII expression is up-regulated in a sarT mutant, we hypothesize that sarT may down regulate agr RNAIII expression by repressing sarU, a positive activator of agr expression. We propose that, in addition to the quorum sensing effect of the autoinducing peptide of agr, the sarT-sarU pathway may represent a secondary amplification loop whereby the expression of agr (e.g., those found in vivo) might repress sarT, leading to increased expression of sarU. Elevated sarU expression would result in additional amplification of the original agr signal.

Amino Acid Sequence↗

PubMed2003

SarT influences sarS expression in Staphylococcus aureus.

Staphylococcus aureus is a gram-positive pathogen that is capable of expressing a variety of virulence proteins in response to environmental signals. Virulence protein expression in S. aureus is controlled by a network of regulatory loci including sarA and agr. The sarA/agr network is associated with the expression of cell wall-associated adhesins during exponential growth and the expression of secreted enzymes and toxins in the transition to post-exponential growth. A number of sarA homologs, including sarT and sarS, have been identified in the S. aureus genome. Previous studies have shown that sarA influences expression of both sarT and sarS in the global regulatory network. SarS has been shown to bind to the spa promoter to induce expression of protein A. SarT, one of the SarA homologs that represses hla expression and is repressible by SarA and agr, was found to induce sarS expression in this report. Northern blot analysis of sarS and spa expression in S. aureus RN6390, and the isogenic sarT, sarT sarA, and sarT agr mutants showed that while sarA regulated spa expression directly, the agr locus used sarT as an intermediary to regulate sarS, thus leading to spa repression in agr-activated cells. Gel shift and footprinting analysis showed that SarT binds to the sarS promoter, indicating that the interaction of the sarT gene product with the upstream region of sarS is likely direct. Induction of sarS and spa by SarT in agr(+) strains was confirmed by a tetracycline-inducible system to titrate sarT expression.

Antigens, Bacterial↗

PubMed2003

Crystal structure of the SarS protein from Staphylococcus aureus.

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sarA and agr). One of these determinants, protein A (spa), is activated by sarS, which encodes a 250-residue DNA-binding protein. Genetic analysis indicated that the agr locus likely mediates spa repression by suppressing the transcription of sarS. Contrary to SarA and SarR, which require homodimer formation for proper function, SarS is unusual within the SarA protein family in that it contains two homologous halves, with each half sharing sequence similarity to SarA and SarR. Here we report the 2.2 A resolution X-ray crystal structure of the SarS protein. SarS has folds similar to those of SarR and, quite plausibly, the native SarA structure. Two typical winged-helix DNA-binding domains are connected by a well-ordered loop. The interactions between the two domains are extensive and conserved. The putative DNA-binding surface is highly positively charged. In contrast, negatively charged patches are located opposite to the DNA-binding surface. Furthermore, sequence alignment and structural comparison revealed that MarR has folds similar to those of SarR and SarS. Members of the MarR protein family have previously been implicated in the negative regulation of an efflux pump involved in multiple antibiotic resistance in many gram-negative species. We propose that MarR also belongs to the winged-helix protein family and has a similar mode of DNA binding as SarR and SarS and possibly the entire SarA protein family member. Based on the structural differences of SarR, SarS, and MarR, we further classified these winged-helix proteins to three subfamilies, SarA, SarS, and MarR. Finally, a possible transcription regulation mechanism is proposed.

Bacterial Proteins↗

PubMed2003

Salicylic acid attenuates virulence in endovascular infections by targeting global regulatory pathways in Staphylococcus aureus.

Aspirin has been previously shown to reduce the in vivo virulence of Staphylococcus aureus in experimental endocarditis, through antiplatelet and antimicrobial mechanisms. In the present study, salicylic acid, the major in vivo metabolite of aspirin, mitigated two important virulence phenotypes in both clinical and laboratory S. aureus strains: alpha-hemolysin secretion and fibronectin binding in vitro. In addition, salicylic acid reduced the expression of the alpha-hemolysin gene promoter, hla, and the fibronectin gene promoter, fnbA. Transcriptional analysis, fluorometry, and flow cytometry revealed evidence of salicylic acid-mediated activation of the stress-response gene sigB. Expression of the sigB-repressible global regulon sarA and the global regulon agr were also mitigated by salicylic acid, corresponding to the reduced expression of the hla and fnbA genes in vitro. Studies in experimental endocarditis confirmed the key roles of both sarA and sigB in mediating the antistaphylococcal effects of salicylic acid in vivo. Therefore, aspirin has the potential to be an adjuvant therapeutic agent against endovascular infections that result from S. aureus, by downmodulating key staphylococcal global regulons and structural genes in vivo, thus abrogating relevant virulence phenotypes.

Animals↗

PubMed2002

Activation and transcriptional interaction between agr RNAII and RNAIII in Staphylococcus aureus in vitro and in an experimental endocarditis model.

This study compared the promoter activation profiles of the 2 major transcripts of the Staphylococcus aureus global regulon, agr (RNAII and RNAIII). In vitro, RNAIII activation temporally followed RNAII activation and was absent in agr mutants. In experimental endocarditis, maximal RNAII activation in vegetations occurred early, followed by progressive increases in RNAIII activation (P<.05; 2 vs. 48 h); this paralleled significant increases in vegetation bacterial densities over time (P<.05; 2 and 6 vs. 48 h). At 48 h of infection, S. aureus densities in vegetations were significantly higher than those in kidney or spleen tissue (P<.05), paralleling a significantly greater RNAIII activation profile in vegetations than in the latter tissues (P<.05). Of importance, RNAIII activation was observed in vegetations in 2 agr mutants. These data demonstrate that RNAIII activation in vivo is time and cell density dependent, may be tissue specific, and can occur through RNAII-dependent and -independent mechanisms.

Animals↗

PubMed2002

Global regulation of virulence determinants in Staphylococcus aureus by the SarA protein family.

In S. aureus, the production of virulence determinants such as cell wall adhesins and exotoxins during the growth cycle is controlled by global regulators such as SarA and agr. Genomic scan reveals 16 two-component regulatory systems (e.g. agr and sae) as well as a family of SarA homologs in S. aureus. We call the SarA homologs the SarA protein family. Many of the members in this protein family are either small basic proteins (<153 residues) or two-domain proteins in which a single domain shares sequence similarity to each of the small basic proteins. Recent crystal structures of SarR and SarA reveal dimeric structures for these proteins. Because of its structure and unique mode of DNA binding, SarR, and possibly other SarA family members, may belong to a new functional class of the winged-helix family, accommodating long stretch of DNA with bending points. Based on sequence homology, we hypothesize that the SarA protein family may entail homologous structures with similar DNA-binding motifs but divergent activation domains. An understanding of how these regulators interact with each other in vivo and how they sense environmental signals to control virulence gene expression (e.g. alpha-hemolysin) will be important to our eventual goal of disrupting the regulatory network.

Amino Acid Sequence↗

PubMed2002

Regulation of Staphylococcus aureus type 5 capsular polysaccharides by agr and sarA in vitro and in an experimental endocarditis model.

The expression of antiphagocytic polysaccharide capsules is an important pathogenetic step in establishing Staphylococcus aureus infections. Using a green fluorescent protein reporter gene (gfp) system, we examined the expression and genetic regulation of the cap5 promoter (capsular polysaccharide 5 genes) by two major global regulators of S. aureus (agr and sarA) in vitro and in a rabbit endocarditis model. In vitro, cap5 expression substantially increased during the post-exponential phase in parental, as well assarA mutant constructs. However, cap5 expression was greatly reduced in agr and agr/sarA double mutants. In the endocarditis model, the extent of cap5 expression in vegetations infected with the parental strain was substantially higher than that observed with the agr/sarA double mutants (P<0.05). Similar trends were noted in renal, but not splenic abscesses. Collectively, these data suggest that agr positively regulates cap5 expression both in vitro and in vivo, while the contribution of sarA to cap5 regulation, although modest, is readily discerned in vivo in agr minus background. In addition, the regulation ofcap5 expression by these global regulators may vary in distinct anatomic niches in vivo.

Animals↗