Biomedical subjects

A L Cheung

Publications and source records attributed to A L Cheung.

At least 37 records · Page 2Linked to original sources

PubMed2000

Clinical relevance of HPV 16/18 testing methods in cervical squamous cell carcinoma.

Three different in situ hybridization (ISH) methods were compared for their clinical relevance and suitability in detecting human papillomavirus (HPV) 16/18 in 55 cases of squamous cell carcinoma (SCC) of the uterine cervix. After the initial biopsy, surgery, and/or radiation therapy, patients were followed for 5 to 8 years. A biotinylated cDNA probe for HPV 16/18 was applied to serial sections in combination with conventional streptavidin-biotin-peroxidase ISH (a widely applied routine procedure), streptavidin-Nanogold-silver ISH, and tyramide-signal amplified (TSA) streptavidin-Nanogold-gold ISH. The TSA principle is also known as catalyzed reporter deposition and is, apart from in situ PCR, probably today's most sensitive technique for detecting papillomavirus infection by microscopic means. Nearly 65.5% of the cases showed specific HPV 16/18 detection with TSA ISH, whereas 43.6% were positive with streptavidin-Nanogold-silver-ISH, and only 40.0% with peroxidase-based ISH. Statistical analyses comparing early and advanced stages in both HPV-positive and -negative groups revealed a significantly better outcome for early disease patients; statistical significance was most pronounced with TSA ISH. In a subgroup of patients who had received radiation therapy without prior surgery (n = 35), those with advanced disease were significantly less likely to have HPV 16/18 infection than those with early disease. A significantly better overall survival was observed in those women with HPV 16/18-positive carcinomas who had undergone surgery before radiation therapy (seen with all three methods). We conclude that TSA, in addition to being the most sensitive HPV in situ method applied in this study, gave the most significant and clinically relevant statistical results.

Adult↗

PubMed1999

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 in Staphylococcus aureus including alpha-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. alpha-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 agr promoter, upstream of the -35 promoter boxes of several target genes including hla (alpha-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 and hla 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 the spa 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 and agr-independent mode of regulation.

Bacterial Proteins↗

PubMed1999

Downregulation and abnormal expression of E-cadherin and beta-catenin in nasopharyngeal carcinoma: close association with advanced disease stage and lymph node metastasis.

Nasopharyngeal carcinoma (NPC) is predominantly of the undifferentiated histological subtype. Histological differentiation is of limited prognostic significance in NPC. Recent studies have suggested that downregulation of the cadherin-catenin cell adhesion complex may play a crucial role in the initial stage of cancer invasion and metastasis and is associated with poor prognosis in human cancers. Expression of E-cadherin has not been reported previously in NPC, and its prognostic value in NPC is unknown. The purpose of this study was to examine the expression pattern of E-cadherin and its associated partner, beta-catenin, in NPC and their possible applications as prognostic markers to predict the clinical outcome of NPC. Expression of the E-cadherin and beta-catenin was examined by immunohistochemical methods in 74 cases of primary NPC and 17 of their corresponding lymph node metastases. Normal nasopharyngeal epithelium showed strong and homogeneous immunocytochemical staining of E-cadherin and beta-catenin at the cell membranes and intercellular junctions. In contrast, primary NPC showed variable and heterogeneous staining patterns of E-cadherin and beta-catenin. Loss of membranous E-cadherin expression was significantly associated with advanced stages of diseases (P<.001). Eighty percent to ninety percent of NPC in stages IV and V (Ho's staging), respectively, showed a reduced (<35%) membranous staining of E-cadherin compared with normal nasopharyngeal epithelium. Expression of beta-catenin also was downregulated in advanced NPC. Ninety percent to one hundred percent of NPC in stages IV and V (Ho's staging) expressed a reduction (<35%) of imnmunocytochemical staining of beta-catenin. The expression pattern of beta-catenin staining was strongly associated with the expression of E-cadherin (P<.001). Unlike E-cadherin, nuclear staining of beta-catenin expression was observed in some of the primary NPC and lymph node metastasis. Reduced expression of E-cadherin and beta-catenin expression was associated with a shorter survival of NPC patients (P<.001). In advanced NPC patients (stages IV and V), a significant difference in survival was observed in tumors with higher or lower levels of E-cadherin expression (P=.0224, log-rank test). These observations suggests that expression of E-cadherin and beta-catenin may have prognostic values in NPC patients.

Adult↗

PubMed1999

Hyperproduction of alpha-hemolysin in a sigB mutant is associated with elevated SarA expression in Staphylococcus aureus.

To evaluate the role of SigB in modulating the expression of virulence determinants in Staphylococcus aureus, we constructed a sigB mutant of RN6390, a prototypic S. aureus strain. The mutation in the sigB gene was confirmed by the absence of the SigB protein in the mutant on an immunoblot as well as the failure of the mutant to activate sigmaB-dependent promoters (e.g., the sarC promoter) of S. aureus. Phenotypic analysis indicated that both alpha-hemolysin level and fibrinogen-binding capacity were up-regulated in the mutant strain compared with the parental strain. The increase in fibrinogen-binding capacity correlated with enhanced expression of clumping factor and coagulase on immunoblots. The effect of the sigB mutation on the enhanced expression of the alpha-hemolysin gene (hla) was primarily transcriptional. Upon complementation with a plasmid containing the sigB gene, hla expression returned to near parental levels in the mutant. Detailed immunoblot analysis as well as a competitive enzyme-linked immunosorbent assay of the cell extract of the sigB mutant with anti-SarA monoclonal antibody 1D1 revealed that the expression of SarA was higher in the mutant than in the parental control. Despite an elevated SarA level, the transcription of RNAII and RNAIII of the agr locus remained unaltered in the sigB mutant. Because of a lack of perturbation in agr, we hypothesize that inactivation of sigB leads to increased expression of SarA which, in turn, modulates target genes via an agr-independent but SarA-dependent pathway.

Animals↗

PubMed1999

Detection of human papillomavirus in cervical carcinoma: comparison of peroxidase, Nanogold, and catalyzed reporter deposition (CARD)-Nanogold in situ hybridization.

We compared three in situ hybridization (ISH) methods for their applicability and sensitivity in detecting human papillomavirus (HPV) in 61 cases (1 Grade 1, 18 Grade 2, 42 Grade 3) of routinely processed squamous cell cervical carcinoma. A commercially available biotinylated probe for HPV-16/18 was applied to serial sections and detected by conventional streptavidin-biotin-peroxidase ISH, streptavidin-Nanogold-silver ISH, and catalyzed reporter deposition (CARD)-Nanogold-gold ISH. The latter method involved signal amplification by peroxidase-catalyzed deposition of biotinylated tyramides at the hybridization sites, followed by detection of accumulated biotin by streptavidin-Nanogold made visible by autometallography. The HPV-16/18 detection rates for the three methods were 39.3, 44.3, and 65.6%, respectively. In all of the three ISH methods, a punctate staining pattern (single or multiple intranuclear spots of variable size), presumably indicating viral integration, was highly predominant among the positive cases. Two of the cases identified as positive by streptavidin-biotinperoxidase ISH were rated negative with streptavidin-Nanogold-silver ISH, whereas six cases that were clearly negative with streptavidin-biotinperoxidase ISH became positively stained with streptavidin-Nanogold ISH. All of these discordant cases were positive by the highly sensitive CARD-Nanogold-gold ISH. In addition, the high detection sensitivity of CARD-Nanogold-gold ISH was confirmed by its ability to detect single copies of HPV-16 in SiHa cells. In general, we found that the intense black reaction product from Nanogold autometallography gave superior contrast to that obtained with the peroxidase system. After tyramide signal amplification, the staining was so clearly visible that preparations could be readily screened under low magnification. Our findings precisely demonstrated the need for improved sensitivity in the in situ detection of HPV. The CARD-Nanogold-gold technology looks promising as a highly sensitive method for routine ISH in molecular pathology.

Carcinoma, Squamous Cell↗

PubMed1998

Molecular interactions between two global regulators, sar and agr, in Staphylococcus aureus.

The expression of many virulence determinants in Staphylococcus aureus is controlled by regulatory loci such as agr and sar. We have previously shown that the SarA protein is required for optimal transcription of RNAII and RNAIII in the agr locus. To define the specific molecular interaction, we overexpressed SarA as a glutathione S-transferase (GST) fusion protein by cloning the 372-base pair (bp) sarA gene into the vector. The purified GST-SarA as well as cleaved SarA were able to bind specifically to the P2, P3, and the combined P2-P3 promoter fragments of agr in gel shift assays. Using monoclonal antibodies to SarA, we found that SarA is a part of the retarded protein-DNA complex as evidenced by the formation of a supershifted band. The SarA binding site on the agr promoter, mapped by DNase I footprinting assay, covered a 29-bp region between the P2 and P3 promoters devoid of any direct repeats. A synthetic 45-bp fragment encompassing the 29-bp sequence also bound the SarA protein in band shift assays. Serial in-frame deletion analysis of sarA revealed that, with the exception of 15 residues in the N terminus, almost all of SarA (residues 16-124) is essential for agr binding activity. Northern analysis confirmed that only the sar mutant clone containing a truncated sarA gene with a 15-residue deletion in the N terminus (SarA16-124) could activate agr transcription to a level approaching that of the full-length counterpart (SarA1-124). Taken together, these data indicated that SarA is a DNA-binding protein with binding specificity to the P2 and P3 interpromoter region of agr, thereby activating RNAII and RNAIII transcription.

Bacterial Proteins↗

PubMed1998

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.

Animals↗

PubMed1998

Selective activation of sar promoters with the use of green fluorescent protein transcriptional fusions as the detection system in the rabbit endocarditis model.

The global regulatory locus sar is composed of three overlapping transcripts initiated from a triple-promoter system (designated P1, P3, and P2). To explore if the individual sar promoters are differentially expressed in vitro and in vivo, we constructed a shuttle plasmid (pALC1434) containing a promoterless gfpUV gene (a gfp derivative [Clontech]) preceded by a polylinker region. Recombinant shuttle vectors containing individual sar promoters upstream of the gfpUV reporter gene were then introduced into Staphylococcus aureus RN6390. Northern and immunoblot analysis revealed that P1 is stronger than the P2 and P3 promoters in vitro. Additionally, the levels of the gfpUV transcript driven by individual sar promoters also correlated with the growth cycle dependency of these promoters in liquid cultures, thus suggesting the utility of pALC1434 as a vehicle for reporter fusion. Using the rabbit endocarditis model, we examined the expression of these three GFPUV fusions in vivo by fluorescence microscopy of infected cardiac vegetations 24 h after initial intravenous challenge. Similar to the in vitro findings, P1 was activated both in the center and on the surface of the vegetations. In contrast, the P3 promoter was silent both in vivo and in vitro as determined by fluorescence microscopy. Remarkably, P2 was silent in vitro but became highly activated in vivo. In particular, the sar P2 promoter was activated on the surface of the vegetation but not in the center of the lesion. These data imply that in vivo promoter activation of sar differed from that observed in vitro. Moreover, the individual sar promoters may be differentially expressed in different areas within the same anatomic niche, presumably reflecting the microbial physiological response to distinct host microenvironments. As the sar locus controls the synthesis of both extracellular and cell wall virulence determinants, these promoter-gfpUV constructs should be useful to characterize many aspects of S. aureus gene regulation in vivo.

Animals↗

PubMed1998

Characterization of a sar homolog of Staphylococcus epidermidis.

Coagulase-negative staphylococci are common nosocomial pathogens. A regulatory element, designated sar, partially controls exoprotein synthesis in coagulase-positive Staphylococcus aureus by modulating the expression of another regulatory locus, called agr. We report here the cloning of a sar homolog in S. epidermidis. The major open reading frame within sar in S. epidermidis is highly homologous (84%) to the S. aureus SarA protein. Primer extension studies revealed three sar transcripts (0.64, 0.76, and 0.85 kb) initiated from three distinct promoters. The interpromoter region in S. epidermidis differs from its S. aureus counterpart, possibly suggesting target gene differences and a disparate pattern for sar activation. Remarkably, the S. epidermidis sar homolog interacts with an agr promoter fragment of S. aureus in gel shift assays. Additionally, S. epidermidis sar fragments could restore hemolysin production in an S. aureus sar mutant. As typical virulence determinants controlled by sar in S. aureus are not present in S. epidermidis, an examination of functional and structural similarities and divergence of sar in staphylococci will be of major interest.

Amino Acid Sequence↗

PubMed1998

Factors affecting the collagen binding capacity of Staphylococcus aureus.

To determine whether the ability of Staphylococcus aureus to bind collagen involves an adhesin other than the collagen adhesin encoded by cna, we examined the collagen binding capacity (CBC) of 32 strains of S. aureus. With only two exceptions, a high CBC corresponded with the presence of cna. Both exceptions involved cna-positive strains with a low CBC. The first was a single strain (ACH5) that encoded but did not express cna. The second were the mucoid strains Smith diffuse and M, both of which encoded and expressed cna but bound only minimal amounts of collagen. Analysis of capsule mutants suggests that the reduced CBC observed in the mucoid strains was due to masking of the collagen adhesin on the cell surface and that this masking effect is restricted to heavily encapsulated strains. Differences in the CBC of the remaining cna-positive strains were correlated to variations in the level of cna transcription and were independent of the number of B domain repeats in the cna gene. In all cna-positive strains other than ACH5, cna transcription was temporally regulated, with cna mRNA levels being highest in cells taken from exponentially growing cultures and falling to almost undetectable levels as cultures entered the post-exponential growth phase. The CBC was also highest with cells taken from exponentially growing cultures. Mutation of agr resulted in a slight increase in cna transcription and a corresponding increase in CBC during the exponential growth phase but did not affect the temporal pattern of cna transcription. Mutation of sar resulted in a more dramatic increase in CBC and a delay in the post-exponential-phase repression of cna transcription. Mutation of both sar and agr had an additive effect on both CBC and cna transcription. We conclude that (i) cna encodes the primary collagen-binding adhesin in S. aureus, (ii) sar is the primary regulatory element controlling expression of cna, and (iii) the regulatory effects of sar and agr on cna transcription are independent of the interaction between sar and agr.

Adhesins, Bacterial↗

PubMed1998

Transcriptional analysis of different promoters in the sar locus in Staphylococcus aureus.

The expression of extracellular virulence determinants in Staphylococcus aureus is controlled by a 510-nucleotide RNA molecule (RNAIII) which is a part of the agr system. The agr operon, which encodes a multicomponent signal transduction system, is partially under the influence of an unlinked regulatory locus called sar. The sar locus is composed of three overlapping transcripts, designated sarA (0.56 kb), sarC (0.8 kb), and sarB (1.2 kb), originating from the P1, P3, and P2 promoters, respectively. In this study, we analyzed the differential expression of these promoters by using transcriptional fusion with the xylE reporter gene to study the activation of the sar locus. The data confirm the existence of three independent promoters with different promoter activities. Maximal promoter activity was observed with the combined fusion of P2-P3-P1 promoters. Expression studies with a sigB mutant revealed that the P3 promoter is SigB dependent. Analysis of these transcriptional fusions in a sarA mutant and in complemented strains with each of the sar transcriptional units revealed that the sar locus is autoregulatory, with SarA acting as a positive regulator. From various transcriptional fusion studies of the upstream region of the P1 promoter, we have localized a 34-bp sequence which seems to play a role in down-modulating P1 transcription. Using heparin-Sepharose and DNA-specific columns, we partially purified a 12-kDa protein, possibly a repressor, which binds to the promoter regions upstream of P2 and P1 and which also binds to the 34-bp sequence. These data indicated that the regulation of the sar locus is complex and may involve the sar gene product(s) and other regulatory protein(s).

Base Sequence↗

PubMed1997

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

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 alpha- and beta-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 alpha- and beta-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::Tn551 agr::tetM double mutant has been phenotypically characterized as producing reduced or null levels of alpha-, beta-, and delta-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.

Bacterial Proteins↗

PubMed1997

Hyperproduction of alpha-toxin by Staphylococcus aureus results in paradoxically reduced virulence in experimental endocarditis: a host defense role for platelet microbicidal proteins.

Staphylococcal alpha-toxin targets several cell types which are important components of cardiac vegetations in endocarditis, including platelets, erythrocytes, and endothelial cells. We evaluated the in vivo role of Staphylococcus aureus alpha-toxin in experimental endocarditis by using isogenic strains differing in the capacity to produce functional alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU1090(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1090 with a single copy of the hla gene cloned into the chromosomal lipase locus). In vitro, wild-type alpha-toxin (from parental strain 8325-4) extensively lysed both erythrocytes and platelets. In contrast, mutant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic filters or compounds, and recoverable from anionic filter membranes by hypertonic saline elution. These characteristics are consistent with those of cationic platelet microbicidal proteins (PMPs). Reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis confirmed the presence of three distinct PMPs (1, 2, and 3) in platelet lysates. In experimental endocarditis, the two variant staphylococcal strains producing either minimal alpha-toxin or nonlytic alpha-toxin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibited significantly lower virulence in vivo than the parental strain (decreased intravegetation staphylococcal densities). Paradoxically, the two variant staphylococcal strains producing alpha-toxin at supraparental levels in vitro [strains DU1090(p1212) and DU1090(pCL84::hla)] also exhibited significantly decreased induction rates and intravegetation staphylococcal densities in experimental endocarditis versus the parental strain. The reduced in vivo virulence of the latter variant staphylococcal strains could not be explained by differences in bacteremic clearance or initial adherence to sterile vegetations (compared to the parental strain). These findings suggest that the reduced virulence exhibited by the variant staphylococcal strains in this model was related to pathogenetic events subsequent to bacterial adherence to the damaged endocardium. Excess intravegetation secretion of alpha-toxin, leading to increased PMP release (secondary to either increased platelet secretion or lysis), may well explain the reduced virulence observed in experimental endocarditis.

Animals↗

PubMed1997

Staphylococcal accessory regulator (sar) in conjunction with agr contributes to Staphylococcus aureus virulence in endophthalmitis.

Previous studies showed that an agr mutant strain of Staphylococcus aureus was partially attenuated in virulence compared to a parental strain in experimental endophthalmitis. The purpose of this study was to determine whether the sar locus, either alone or through interactions with agr, contributes to the regulation of virulence in S. aureus endophthalmitis. Experimental endophthalmitis was established by the midvitreous injection of approximately 30 CFU of S. aureus RN6390 or the isogenic attenuated strains RN6911 (agr mutant), ALC136 (sar mutant), and ALC135 (agr sar double mutant). Unexpectedly, the rate of reduction in electroretinographic B-wave amplitude in eyes infected with strain ALC136 (sar mutant) was not significantly different from the parental strain on postinfection day (PID) 5 (10% retention). In contrast, ALC135 (agr sar double mutant)-infected eyes retained 73% of preoperative B-wave amplitude on PID 5. Therefore, unlike agr, a mutation in the sar locus alone does not alter the overall virulence of wild-type S. aureus in experimental endophthalmitis. However, the combined effect of insertional mutations in both the sar and agr global regulators leads to near-complete attenuation of virulence.

Bacterial Proteins↗

PubMed1997

Regulation of protein A synthesis by the sar and agr loci of Staphylococcus aureus.

The synthesis of protein A in Staphylococcus aureus is regulated by global regulatory loci such as sar and agr. Phenotypic data indicate that both sar and agr suppress protein A synthesis; like agr, sar also regulates protein A production at the transcriptional level. To determine the genetic requirement of sar in protein A suppression, we transformed shuttle plasmids containing various sar fragments into a sar mutant. Our results indicated that the 560-bp sarA transcript, or, more probably, the SarA protein (13.5 kDa), is sufficient for suppressing protein A gene transcription when introduced on a multicopy plasmid or as a single copy in the chromosome. Immunoblot analysis with a chicken anti-protein A antibody also confirmed the reduction in protein A expression in these sar mutant clones. Complementation studies revealed that the transcription of the protein A gene can be suppressed in a sar mutant background by a plasmid containing RNAIII. Surprisingly, in agr deletion mutant clones and in clones derived from the agr-sar double mutant, protein A gene transcription can also be suppressed by plasmids containing the sarA transcript plus additional upstream sequence but not the sarA transcript alone. These data suggest that the sar locus can down-modulate protein A gene transcription via both RNAIII-dependent and RNAIII-independent pathways. Consistent with the hypothesis of an RNAIII-independent pathway is an additional genetic requirement for protein A suppression in the agr deletion mutant RN6911 as well as the isogenic double sar-agr mutant, whereas in the sar mutant background, the sarA transcript encoding the SarA protein alone is sufficient. These data suggested that both sar and agr are coregulators of protein A synthesis in S. aureus.

Base Sequence↗

PubMed1997

Phenotypic resistance to thrombin-induced platelet microbicidal protein in vitro is correlated with enhanced virulence in experimental endocarditis due to Staphylococcus aureus.

Thrombin-induced platelet microbicidal protein (tPMP) is secreted by rabbit platelets following thrombin stimulation, and it kills common endovascular pathogens in vitro, including Staphylococcus aureus. Therefore, pathogens which exhibit tPMP resistance in vitro possess a potential survival advantage in vivo at sites of endovascular damage. We generated an isogenic S. aureus strain pair, differing in tPMP susceptibility, by transposon (Tn551) mutagenesis of a tPMP-susceptible (tPMPs) parental strain (ISP479) to derive a stably tPMP-resistant (tPMPr) strain, ISP479R. ISP479 and ISP479R were equivalent in vitro in the following phenotypes: biotyping, antiobiograms, platelet adherence and aggregation, growth kinetics, cell wall-associated protein A expression, and fibrinogen binding. Genotypic comparisons of chromosomal DNA of strains ISP479 and ISP479R following restriction endonuclease digestion revealed indistinguishable pulsed-field gel electrophoretic patterns. The genotype exhibited by strain ISP479R was linked to the tPMP-resistant phenotype, as it was transducible into the initially tPMP-susceptible parental strain, ISP479. Southern hybridization verified the presence of a single copy of Tn551 in the same chromosomal restriction site of both ISP479R and tPMPr transductants of ISP479. The correlation of in vitro tPMP susceptibility phenotypes with the ability to induce experimental endocarditis (a prototypical endovascular infection) was evaluated. Despite equivalent rates of endocarditis induction, animals infected with strain ISP479R achieved significantly higher vegetation bacterial densities over a 7-day post-challenge period than did animals infected with strain ISP479. These data suggest that tPMPr microbial strains have a selective advantage in experimental staphylococcal endocarditis. Furthermore, the major impact of tPMP resistance upon endocarditis pathogenesis appears to involve a postvalvular adherence event(s), most probably by facilitating bacterial proliferation within vegetations.

Animals↗

PubMed1997

sar Genetic determinants necessary for transcription of RNAII and RNAIII in the agr locus of Staphylococcus aureus.

The temporal expression of most virulence factors in Staphylococcus aureus is regulated by pleiotropic loci such as agr and sar. We have previously shown that the sar locus affects hemolysin production because it is required for agr transcription. To delineate the sar genetic determinant required for agr transcription, single copies of fragments from the sar locus, encompassing the individual sar transcripts (sarA, sarC, and sarB), were introduced into a sar mutant via the integration vector pCL84. Although a DNA fragment encompassing the sarA transcript plus a 189-bp upstream region was sufficient for agr expression, complementation analysis revealed that the sarB transcript was the most effective in augmenting agr transcription as determined by RNAII and RNAIII transcription and gel retardation assays with the P2 and P3 promoters of agr. As the region upstream of the sarA transcript encodes a 39-amino-acid open reading frame, ORF3, it is possible that posttranslational cooperation between the sarA gene product and ORF3 may be necessary for optimal agr expression. Deletion studies demonstrated that an intact sarA gene is essential for agr transcription. However, mutagenesis and in vitro translation studies revealed that unlike the agr locus, the required element is the SarA protein and not the RNA molecule. Taken together, these results indicate that the sarA-encoded protein, possibly in conjunction with peptides encoded in the upstream region, regulates hemolysin production by controlling agr P2 and P3 transcription.

Amino Acid Sequence↗

PubMed1996

Role of the staphylococcal accessory gene regulator (sar) in septic arthritis.

Staphylococcus aureus arthritis is a highly erosive disease in which both host and bacterial factors are of importance for its induction and progression. At the transcriptional level, three known loci act in regulating production of exoproteins and expression of cell wall structures. The aim of our study was to assess the role of the sar locus as a virulence determinant in the pathogenesis of septic arthritis. A recently established murine model of hematogenously spread S. aureus arthritis was employed. S. aureus strains, isogenic for the sar locus, were inoculated intravenously into NMRI mice, and the clinical, bacteriological, serological, and histopathological progression of the disease was studied. Within 1 week after inoculation of bacteria, the frequency of arthritis was 79% in the group of mice inoculated with the sar+ strain, whereas the corresponding frequency in sar mutants was 21% (P < 0.01). Mice inoculated with the sar+ staphylococcal strain exhibited a more pronounced T- and B-lymphocyte activation than those inoculated with the sar mutant, evidenced by splenomegaly, polyclonal B-cell activation, and high serum levels of interleukin 6 and gamma interferon. Also, infection with sar+ staphylococci induced a pronounced weight loss. To assess the relationship between clinical signs and spread of bacteria, we analyzed the homing pattern and persistence of S. aureus in host tissues. Kidneys and joints from sar+-inoculated subjects displayed a higher degree of bacterial persistence than other organs. Our results suggest that molecules controlled by the sar locus are important virulence determinants in the induction and progression of septic arthritis.

Animals↗