Previous Article | Next Article 
Infection and Immunity, August 2001, p. 4742-4748, Vol. 69, No. 8
Microbiology and Tumorbiology Center,
Karolinska Institutet, S-17177 Stockholm, Sweden
Received 5 February 2001/Returned for modification 14 March
2001/Accepted 16 May 2001
Data have been presented indicating that Staphylococcus
aureus cell surface protein can be degraded by extracellular
proteases produced by the same bacterium. We have found that in
sarA mutant cells, which produce high amounts of four major
extracellular proteases (staphylococcal serine protease [V8 protease]
[SspA], cysteine protease [SspB], aureolysin [metalloprotease]
[Aur], and staphopain [Scp]), the levels of cell-bound
fibronectin-binding proteins (FnBPs) and protein A were very low
compared to those of wild-type cells, in spite of unaltered or
increased transcription of the corresponding genes. Cultivation of
sarA mutant cells in the presence of the global protease
inhibitor Staphylococcus aureus
produces several cell surface proteins which bind specifically to
different host extracellular matrix proteins and plasma proteins
(12, 13, 32). For many of the cell surface proteins a role
in colonization and virulence has been demonstrated in animal models of
infection (17, 23, 27, 33). Two highly homologous
fibronectin-binding proteins (FnBPA and FnBPB), encoded by
fnbA and fnbB, have been characterized (14,
21, 25, 41) and shown to be involved in adherence to damaged
heart valves (23) and to promote internalization of
S. aureus by epithelial cells (9). Although
S. aureus is primarily considered to be an extracellular
pathogen, the intracellular niche could promote long-term colonization
and maintenance of chronic infections.
Protein A (Spa), which binds immunoglobulin G (IgG) by the Fc segment,
is a major surface protein present in virtually all strains of S. aureus (10, 11). Strains of S. aureus with
a high content of Spa are more resistant to phagocytosis by human neutrophils in vitro than strains with less Spa (34).
Reduced virulence of a spa mutant compared to that of the
corresponding wild type was demonstrated in a mouse intraperitoneal
infection (31).
We have recently shown that transcription of the fnbA and
fnbB genes is negatively regulated by agr and by
an agr-independent mechanism that restricts fnb
mRNA synthesis to the early exponential phase of growth
(38). A similar temporal control of fnb
transcription was also found in another strain of S. aureus
(Newman) (43). However, only fnbA appeared to
be regulated by agr in this strain. It was also found that
fnbA, but not fnbB, was positively regulated by
sarA. As for fnbA and fnbB,
transcription of spa is negatively regulated by
agr (20). However, unlike for fnbA,
transcription of spa is negatively controlled by
sarA (3, 42).
Data from recent studies indicate that both FnBPs and protein A may be
degraded by extracellular proteases (3, 26, 42). Four
major extracellular proteases are produced by S. aureus
(1): staphylococcal serine protease (V8 protease) (SspA),
a metalloprotease named aureolysin (Aur), a cysteine protease (Scp)
named staphopain (18), and a second cysteine protease
(SspB) encoded within the same operon as SspA (2, 36). All
four proteases appear to be synthesized as preproenzymes, which are
proteolytically cleaved to generate the mature enzymes. In the case of
the serine protease the proform is enzymatically inactive and needs to
be cleaved by aureolysin to become active (8). The proform
of SspB that appeared to possess enzyme activity seems to be processed
by SspA (36). Which enzymes are involved in the processing
of aureolysin and staphopain remains to be determined.
The synthesis of extracellular proteases is positively regulated by
agr and negatively regulated by sarA (2,
20) in such a way that protease production takes place mainly
during the postexponential phase of growth, when synthesis of cell
surface proteins has ceased. Because of the sensitivity of FnBPs and a
limited number of unidentified cell surface proteins to degradation by
staphylococcal serine protease, it has been suggested that this enzyme
participates in the transition of S. aureus cells from an
adhesive to an invasive phenotype (26). However, since
there are four major proteases which are all regulated in the same way
and which are involved in the maturation of each other, we decided to
analyze which enzyme(s) is involved in the degradation of FnBPs and
protein A in growing cultures of S. aureus. By studying the
effects of different protease inhibitors and protease knockout mutants,
we have come to the conclusion that staphylococcal serine protease (V8
protease) is the most important for degrading FnBPs and protein A.
Bacterial strains and plasmids and cultivation conditions.
The bacterial strains and plasmids used in this study are listed in
Table 1. S. aureus strains
were precultured in tryptic soy broth for 16 to 18 h. Cells from 2 ml of preculture were inoculated into 100 ml of brain heart infusion
(BHI) broth in a 1-liter baffled flask to an initial optical density at
600 nm (OD600) of 0.25 to 0.3 and were incubated on a
rotary shaker (180 rpm) at 37°C. To test the effect of protease
inhibitors, parallel overnight cultures of the sarA mutant
strain 11D2 were transferred to 50 ml of BHI broth (OD600
of 0.1) with or without 0.4 U of the universal protease inhibitor
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.8.4742-4748.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Decreased Amounts of Cell Wall-Associated Protein A
and Fibronectin-Binding Proteins in Staphylococcus aureus
sarA Mutants due to Up-Regulation of Extracellular
Proteases
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2-macroglobulin resulted in a 16-fold increase
in cell-bound FnBPs, indicating that extracellular proteases were
responsible for the decreased amounts of FnBPs in sarA
mutant cells. The protease inhibitor E64 had no effect on the level of
FnBPs, indicating that cysteine proteases were not involved.
Inactivation of either ssp or aur in the
prototype S. aureus strain 8325-4 resulted in a threefold increase in the amount of cell-bound FnBPs. Inactivation of the same
protease genes in a sarA mutant of 8325-4 resulted in a 10- to 20-fold increase in cell-bound protein A. As the serine protease requires aureolysin to be activated, it can thus be concluded that the
serine protease is the most important protease in the release of
cell-bound FnBPs and protein A.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2-macroglobulin (Boehringer Mannheim) ml
1
or 10 µM cysteine protease-specific inhibitor E64
[L-trans-epoxysuccinyl-leucylamido-(4-guanidino) butane] (Sigma). Samples of cell lysates and supernatants were taken
at indicated time points, and cell-associated FnBPs were analyzed by
Western blotting.
TABLE 1.
Bacterial strains and plasmids used in this study
Northern blot analysis.
Total S. aureus RNA was
prepared by extraction of lysostaphin-treated cells with hot phenol as
described previously (20). Northern blot analysis of
fnb mRNA and RNAIII, using digoxigenin-labeled antisense RNA
probes, was performed as described earlier (38). Serine
protease (sspA), aureolysin (aur), and staphopain
(scp) mRNAs were analyzed using 32P-labeled
probes as described previously (28). DNA fragments of 889 bp (ssp), 1,197 bp (aur), and 407 bp
(scp), encompassing the beginning of the structural genes,
were generated by PCR using S. aureus 8325-4 chromosomal DNA
as the template. PCR fragments were radiolabeled to a specific activity
of 1 × 108 to 5 × 108 cpm
µg1 with [-32P]dCTP using a random
primer labeling kit (Boehringer Mannheim Biochemica). Northern blot
images were processed and quantitated using the PhosphorImager 445SI
and Image QuanT software (Molecular Dynamics).
Construction of plasmids and protease mutants by allelic
replacement.
The plasmids were constructed in Escherichia
coli DH5, and molecular biology techniques and recombinant DNA
manipulations were done as previously described (37).
Plasmid DNA was extracted using the Qiagen plasmid mini-kit. E. coli transformants were selected on Luria-Bertani (Difco) plates
containing 100 µg of ampicillin ml1.
11 (29) was
performed in order to obtain a double crossover and an allelic
replacement of the metalloprotease gene. The aur mutation in
AK1 was verified by PCR and by Southern blotting using an
aur-specific probe. The lack of metalloprotease in AK1 was
verified by Western blotting (see below).
Strain AK2 (ssp allelic replacement mutant) was constructed
similarly to AK1. PCR fragments (244 bp [nucleotides 7 to 251] and
332 bp [nucleotides 2693 to 3025], GeneBank accession no. 309515)
flanking the ssp operon were generated using forward and reverse primers with the same restriction sites as in the construction of pAK1. The PCR fragments were inserted at either side of the ermB casette in pKT4 to generate plasmid pAK2, which was
then transferred into S. aureus RN4220 by electroporation.
Recombinants were selected on erythromycin plates and lincomycin plates
as described above. Restriction mapping and PCR analysis of one
transformant (AK20) using primers internal to the ssp operon
and flanking primers in the erythromycin cassette confirmed the
integration of pAK2 in the ssp operon. The ssp
mutation of AK20 was transduced in S. aureus 8325-4 using
phage 11 (29) to obtain a double crossover and
replacement of the ssp operon by the ermB
cassette. Erythromycin-resistant clones were grown on casein agar
plates, and colonies with small zones of proteolysis were checked for
allelic replacement by PCR and Southern blotting. The loss of SspA in
one clone, AK2, was verified by Western blotting (see below).
Strains AK3 (sarA aur double mutant) and AK5 (sarA
ssp double mutant) were constructed by transfer of the
sarA::km mutation from PC1839 into AK1
and AK2 using the transducing phage 11 as described before. The
mutations were confirmed by PCR.
Western blotting of cell wall proteins and secreted proteins. Cell-associated FnBPs and protein A were released by lysostaphin treatment of equal numbers of bacterial cells (1.2 OD600 units) as described previously (20). Released proteins were separated in sodium dodecyl sulfate (SDS)-7.5% polyacrylamide gels (24) and transferred to polyvinylidene difluoride-based membranes (Immobilon-P; Millipore) using a Bio-Rad Mini Trans-Blot electrophoretic transfer cell as recommended by the supplier. Monoclonal antibodies (goat) against the fibronectin-binding D domains of FnBPA and FnBPB from S. aureus (a gift from L. K. Rantamäki, University of Helsinki, Finland) were used and were detected with horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibodies (DAKO A/S, Glostrup, Denmark). Protein A was detected by Western ligand blotting using IgG2a from mouse (monoclonal antibody to human CD14; Nordic BioSite Inc.). IgG2a bound to protein A was detected using HRP-conjugated sheep anti-mouse antibodies (Amersham Life Science). For Western blot analysis of the metalloprotease and the serine protease, culture supernatants corresponding to a bacterial density of 0.12 OD600 units were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride-based membranes as described above. Polyclonal rabbit anti-metalloprotease and -serine protease antibodies (19) were used and detected with HRP-conjugated sheep anti-rabbit antibodies (Amersham Life Science).
Quantitation of FnBPs and protein A on Western blots was made by the Personal Densitometer SI and Image QuanT software (Molecular Dynamics). Preliminary sequence data for the S. aureus strain COL were obtained from The Institute for Genomic Research website at http://www.tigr.org and for S. aureus strain 8325 were obtained from the University of Oklahoma Genome sequencing project website at http://www.genome.ou.edu/staph.html.| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Analysis of fnb transcripts and cell-associated FnBPs in S. aureus DB (wild type) and the sarA mutant 11D2. It has been reported that S. aureus sarA mutant cells bind less fibronectin than the corresponding parental cells (4, 7, 16, 43). This could be explained at least in part by the recent observation that transcription of fnbA, but not fnbB, was down-regulated in a sarA mutant (43). However, the decreased fibronectin binding in the sarA mutant could also be the result of increased production of extracellular proteases (2).
Inactivation of sarA in the clinical S. aureus strain DB had little effect on the maximum concentration of fnb mRNA, although the peak level was reached after 0.5 h (OD = 0.35) in the mutant compared to 1 h (OD = 0.7) in the wild-type strain (Fig. 1). There was no significant difference in growth rate between the strains. It should be pointed out that we used a probe that recognized both fnbA and fnbB mRNA. In accordance with the transcription analysis, synthesis of cell wall-associated FnBPs was restricted to the first hour of growth in both strains (Fig. 2A). However, in spite of roughly equal levels of fnb mRNAs, the amounts of FnBPs were 6- to 10-fold lower in sarA mutant cells than in wild-type cells (Fig. 2A). Full-length FnBPs appear on SDS-PAGE as bands with molecular masses of approximately 200 kDa (14, 15, 21). Control experiments using the fnbA knockout mutant DU5881 (Fig. 2A), together with previous experiments with the fnbA fnbB double mutant DU5883 (38), identified the largest band as FnBPA and the slightly smaller band as FnBPB. The amounts of FnBPA and FnBPB were equally reduced in the sarA mutant and the wild-type strain. The intensity of several smaller protein bands previously shown to represent degradation products (38) was also reduced in the sarA mutant. As the antiserum is specific for the fibronectin-binding domains of FnBPA and FnBPB, these results are consistent with the reduced binding of fibronectin to sarA mutant cells (6, 43).
|
|
2-macroglobulin, a universal protease inhibitor that
inhibits the activity of all major staphylococcal proteases (26,
35; unpublished results), or the cysteine protease inhibitor E64, which inhibits the staphylococcal cysteine proteases (18, 35, 36). At least 16 times more cell-associated FnBPs were found
on cells grown in the presence of 2-macroglobulin than on cells grown in the absence of inhibitor (Fig. 2B). The addition of
the inhibitor had no effect on bacterial growth rate (data not shown).
No increase in the amount of FnBPs could be seen when the
sarA mutant was cultivated in the presence of E64 (data not shown), indicating that cysteine proteases are not involved in the
degradation of FnBPs. The amount of FnBPs on sarA mutant
cells grown in the presence of 2-macroglobulin was
roughly the same as that on wild-type cells grown without protease
inhibitor (Fig. 2A), which is in agreement with the roughly equal
levels of fnb mRNAs in these strains (Fig. 1).
Effect of ssp and aur knockout mutations on
FnBP levels.
Our results indicate that serine protease and
aureolysin are the major players in the degradation of FnBPs. Specific
knockout mutants were therefore constructed. Because of difficulties in genetically manipulating strains DB and 11D2, the mutations were made
in the prototype S. aureus strain 8325-4, which produces high amounts of proteases and possesses relatively low levels of cell
wall-associated FnBPs. The serine protease gene (sspA) is
part of an operon containing two additional open reading frames, one
(sspB) coding for cysteine protease and the other
(sspC) coding for a 12-kDa cytoplasmic protein with unknown
function (2, 36). The aureolysin gene, on the other hand,
seems to be mono-cistronic. The ssp operon and the
metalloprotease gene, aur, were inactivated by allelic
replacement as described in Materials and Methods. Mutants AK1
(aur) and AK2 (ssp) showed dramatically reduced
zones of proteolysis on casein agar plates (Fig.
3). Inactivation of either protease gene
resulted in the same (three- to fivefold, in three different
experiments) increase in the amount of cell wall-associated FnBPs as
for the wild-type strain and a slower disappearance of FnBPs from the
cell surface (Fig. 4). Inactivation of
ssp and aur in the sarA mutant PC1839
gave similar but less-pronounced results (data not shown). As the
serine protease is produced as an inactive proenzyme which is activated
through cleavage by the metalloprotease (8), inactivation
of aur also leads to the loss of serine protease activity.
As inhibition of cysteine proteases had no effect on FnBP levels and as
the increase in FnBPs was roughly the same in AK1 and AK2, it can be
concluded that the serine protease is the most important protease for
the degradation of FnBPs.
|
|
Effect of serine protease and metalloprotease on protein A.
Previous studies in our laboratory have shown that the increased
transcription of spa (protein A) in the sarA
mutant PC1839 was not accompanied by a corresponding increase in
protein A production (42). To determine whether this was
due to degradation of protein A by extracellular proteases, the
aur and ssp knockout mutations were transferred
by phage transduction to the sarA mutant PC1839 to form
strain AK3 (aur sarA mutant) and AK5 (ssp sarA
mutant), respectively. Mutations were verified by PCR using primers
specific for the inserted ermB gene and the flanking DNA
regions. Compared to the parental strain PC1839, AK5 had a slightly
smaller zone of proteolysis that was less dense (Fig. 3). Strain AK3,
on the other hand, showed no zone of proteolysis. Both mutants showed a
10- to 20-fold increase in cell wall-associated protein A compared to
the level in the parental strain PC1839 (Fig.
5). All immunoglobulin-binding bands seen
in Fig. 5 represent protein A as they were absent in a spa
knockout mutant (data not shown). Inactivation of ssp or aur in strain 8325-4 resulted in comparable increases in
protein A (data not shown). Since inactivation of the ssp
operon (sspA and sspB) resulted in roughly the
same increase in protein A as inactivation of aur did, it
can be concluded that SspA is the most important enzyme in the
degradation of protein A.
|
Transcription of ssp and hld (RNAIII) in DB
(wild type) and 11D2 (sarA).
As the serine protease
appears to be responsible for the degradation of FnBPs, transcription
of sspA in S. aureus strains DB and 11D2 was
analyzed. Northern blot analysis confirmed that transcription of the
ssp operon was up-regulated in the sarA mutant strain 11D2 compared to that in wild-type strain DB (Fig.
6A). In mutant cells the level of
ssp transcript increased dramatically during the
postexponential phase of growth (2 to 3 h). However, significant
amounts of ssp transcript were seen at time zero, suggesting
that SspA was produced during the early exponential phase of growth at
the same time that FnBPs were produced (Fig. 1). Increased protease
production by the sarA mutant 11D2 was also indicated by
large zones of precipitation around bacteria grown on casein agar
plates (Fig. 3).
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
In the present study we have demonstrated that the decreased expression of FnBPs in 11D2 (sar mutant) cells compared to that of the wild-type cells (DB) was due to increased proteolytic degradation of FnBPs rather than to decreased transcription of the fnb genes. We also found that the decreased amount of protein A in sarA mutant cells was the result of increased protease production. The major protease responsible for the degradation of FnBPs and protein A appeared to be the staphylococcal serine protease (V8).
Contrary to the present results, it was recently shown that transcription of fnbA, but not fnbB, was down-regulated in a sarA mutant (43). In strain Newman, used in a previous study (43), FnBPA appeared to be the dominating FnBP, while in strains DB and 8325-4, which we used, roughly equal amounts of FnBPA and FnBPB were produced (Fig. 2A). In strain 8325-4, FnBPA and FnBPB also seemed to contribute equally to the adherence of S. aureus cells to fibronectin-coated surfaces (15). The different relative levels of FnBPA and FnBPB and the different transcription patterns of fnbA and fnbB with respect to their regulation by sarA may be explained by differences between the fnb promoter sequences (16, 43). Strain-dependent differences may, however, also be due to variations in the levels of expression of sarA and other regulators (e.g., agr). Expression of both fnbA and fnbB was negatively controlled by agr in strain 8325-4 (38), while only fnbA appeared to be regulated by agr in strain Newman (43).
The observation that cultivation of S. aureus in the presence of E64 did not significantly affect the amount of cell wall-associated FnBPs strongly suggests that cysteine proteases (SspB and Scp) were not involved in the degradation of FnBPs. The increase in FnBPs seen in the ssp knockout mutant was therefore most likely due to the loss of SspA activity. This was supported by the observation that deletion of the metalloprotease that is required for the activation of SspA resulted in the same increase in FnBPs as the ssp deletion and by the finding that SspA alone can degrade cell wall-associated FnBPs (26). However, a nonpolar sspA mutation in strain SP6391 (derived from 8325-4) did not enhance fibronectin binding, suggesting that the degradation of FnBPs might be the result of the combined activity of two or more proteases (36). However, this discrepancy could also be explained by the observation that binding of soluble fibronectin is not proportional to the amount of cell wall-associated FnBPs. A 16-fold increase in FnBPs resulted in only a twofold increase in fibronectin binding (38). A similar discrepancy between the level of cell-bound FnBPs and binding of soluble fibronectin can also be deduced from data reported previously (43).
FnBPs, protein A, and other cell surface proteins are covalently linked to the cell wall peptidoglycan (40). The release of FnBPs and protein A from the cell wall could therefore also be due to increased autolysin activity. In sarA mutants both proteases and autolysin activity are up-regulated, suggesting that the decreased levels of FnBPs and protein A might be the combined result of increased proteolysis and cell wall turnover. However, the increased levels of protein A in the ssp sarA and aur sarA double mutants compared to that in the sarA mutant (Fig. 5) strongly indicate that proteolytic degradation is the most important factor.
Previous reports clearly indicated that the production of FnBPs is inhibited by agr and stimulated by sarA (38, 43), while the production of proteases is inhibited by sarA and stimulated by agr (2, 42). This means that the production of serine protease would generally be down-regulated when fnbA and fnbB are expressed. Under conditions where no extracellular proteases are produced, cell wall-associated FnBPs appeared to be very stable and disappeared from the bacterial surface at a rate that was proportional to the cell doubling time, meaning that complete down-regulation of the fibronectin-binding phenotype would take several generations. It therefore seems reasonable that FnBPs can be actively released from the bacterial surface soon after de novo synthesis has been turned off. Depending on the regulation of sarA and agr expression and the relative levels of sarA and RNAIII, the amount of FnBPs on the bacterial surface can be modulated to meet the specific requirements during the course of infection.
In the case of protein A the situation seems more complicated, as sarA represses transcription of both spa and ssp. Thus, increased protein A production, as a result of down-regulation of sarA, will be counteracted by the concomitant increase in protease production. However, as agr is also positively regulated by sarA (7), down-regulation of sarA might result in a decreased level of RNAIII, which in turn results in decreased protease production and therefore less degradation of protein A. This is consistent with the observation that an agr sarA double mutant produced higher amounts of protein A than a sarA mutant (42).
In our study we demonstrated that cell wall-associated FnBPs and protein A can be released through the activity of the staphylococcal serine protease. The relevance of this in adaptation to various environmental conditions during the infection process remains to be determined.
| |
ACKNOWLEDGMENTS |
|---|
We thank Agneta Wahlquist and Lena Norénius for skillful technical assistance. We are also grateful to A. Cheung for providing us with strains DB and 11D2.
This work was supported by grant 4513 from the Swedish Medical Research Council.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Microbiology and Tumorbiology Center (MTC), Box 280, Karolinska Institutet, S-17177 Stockholm, Sweden. Phone: 46(8)7287172. Fax: 46(8)342651. E-mail: Staffan.Arvidson{at}mtc.ki.se.
Editor: E. I. Tuomanen
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Arvidson, S. 2000. Extracellular enzymes, p. 379-385. In V. A. Fischetti, R. P. Novick, J. J. Ferretti, D. A. Portnoy, and J. I. Rood (ed.), Gram-positive pathogens. ASM Press, Washington, D.C. |
| 2. |
Chan, P. F., and S. J. Foster.
1998.
Role of SarA in virulence determinant production and environmental signal transduction in Staphylococcus aureus.
J. Bacteriol.
180:6232-6241 |
| 3. | Cheung, A. L., K. Eberhardt, and J. H. Heinrichs. 1997. Regulation of protein A synthesis by the sar and agr loci of Staphylococcus aureus. Infect. Immun. 65:2243-2249[Abstract]. |
| 4. |
Cheung, A. L.,
K. J. Eberhardt,
E. Chung,
M. R. Yeaman,
P. M. Sullam,
M. Ramos, and A. S. Bayer.
1994.
Diminished virulence of a sarlagr mutant of Staphylococcus aureus in the rabbit model of endocarditis.
J. Clin. Investig.
94:1815-1822.
|
| 5. |
Cheung, A. L., and V. A. Fischetti.
1988.
Variation in the expression of cell wall proteins of Staphylococcus aureus grown on solid and liquid media.
Infect. Immun.
56:1061-1065 |
| 6. |
Cheung, A. L.,
J. M. Koomey,
C. A. Butler,
S. J. Projan, and V. A. Fischetti.
1992.
Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr.
Proc. Natl. Acad. Sci. USA
89:6462-6466 |
| 7. |
Cheung, A. L., and P. Ying.
1994.
Regulation of alpha- and beta-hemolysins by the sar locus of Staphylococcus aureus.
J. Bacteriol.
176:580-585 |
| 8. |
Drapeau, G. R.
1978.
Role of metalloprotease in activation of the precursor of staphylococcal protease.
J. Bacteriol.
136:607-613 |
| 9. |
Dziewanowska, K.,
J. M. Patti,
C. F. Deobald,
K. W. Bayles,
W. R. Trumble, and G. A. Bohach.
1999.
Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells.
Infect. Immun.
67:4673-4678 |
| 10. | Forsgren, A., V. Ghetie, R. Lindmark, and J. Sjoquist. 1983. Protein A and its exploitation, p. 429-480. In C. S. F. Easmon, and C. Adlam (ed.), Staphylococci and staphylococcal infections, vol. 2. Academic Press Inc. Ltd., London, United Kingdom. |
| 11. |
Forsgren, A., and J. Sjoquist.
1966.
Protein A from S. aureus. I. Pseudo-immune reaction with human gamma-globulin.
J. Immunol.
97:822-827 |
| 12. | Foster, T. J., and M. Höök. 1998. Surface protein adhesins of Staphylococcus aureus. Trends Microbiol. 6:484-488[CrossRef][Medline]. |
| 13. | Foster, T. J., and D. McDevitt. 1994. Surface-associated proteins of Staphylococcus aureus: their possible roles in virulence. FEMS Microbiol. Lett. 118:199-205[CrossRef][Medline]. |
| 14. |
Froman, G.,
L. M. Switalski,
P. Speziale, and M. Hook.
1987.
Isolation and characterization of a fibronectin receptor from Staphylococcus aureus.
J. Biol. Chem.
262:6564-6571 |
| 15. | Greene, C., D. McDevitt, P. Francois, P. E. Vaudaux, D. P. Lew, and T. J. Foster. 1995. Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes. Mol. Microbiol. 17:1143-1152[CrossRef][Medline]. |
| 16. |
Heinrichs, J. H.,
M. G. Bayer, and A. L. Cheung.
1996.
Characterization of the sar locus and its interaction with agr in Staphylococcus aureus.
J. Bacteriol.
178:418-423 |
| 17. | Heinz, S. A., T. Schenning, A. Heimahl, and J.-I. Flock. 1996. Collagen binding of Staphylococcus aureus is a virulence factor in experimental endocarditis. J. Infect. Dis. 174:83-88[Medline]. |
| 18. | Hofmann, B., D. Schomburg, and H. J. Hecht. 1993. Crystal structure of a thiol proteinase from Staphylococcus aureus V-8 in the E-64 inhibitor complex. Acta Crystallogr. 49(Suppl.):102. |
| 19. | Janzon, L., and S. Arvidson. 1990. The role of the delta-lysin gene (hld) in regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus. EMBO J. 9:1391-1399[Medline]. |
| 20. | Janzon, L., S. Löfdahl, and S. Arvidson. 1986. Evidence for a coordinate transcriptional control of alpha-toxin and protein A in Staphylococcus aureus. FEMS Microbiol. Lett. 33:193-198. |
| 21. | Jonsson, K., C. Signas, H. P. Muller, and M. Lindberg. 1991. Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene. Eur. J. Biochem. 202:1041-1048[Medline]. |
| 22. | Kreiswirth, B., S. Löfdahl, M. J. Betly, M. O'Reilly, M. P. Schleivert, M. S. Bergdoll, and R. P. Novick. 1983. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305:709-712[CrossRef][Medline]. |
| 23. |
Kuypers, J. M., and R. A. Proctor.
1989.
Reduced adherence to traumatized rat heart valves by low-fibronectin-binding mutant of Staphylococcus aureus.
Infect. Immun.
57:2306-2312 |
| 24. | Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[CrossRef][Medline]. |
| 25. |
McGavin, M. J.,
G. Raucci,
S. Gurusiddappa, and M. Hook.
1991.
Fibronectin binding determinants of the Staphylococcus aureus fibronectin receptor.
J. Biol. Chem.
266:8343-8347 |
| 26. | McGavin, M. J., C. Zahradka, R. Kelly, and J. E. Scott. 1997. Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease. Infect. Immun. 65:2621-2628[Abstract]. |
| 27. | Moreillon, P., J. M. Entenza, P. Francioli, D. McDevitt, T. J. Foster, P. Francois, and P. Vaudaux. 1995. Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis. Infect. Immun. 63:4738-4743[Abstract]. |
| 28. | Morfeldt, E., L. Janzon, S. Arvidson, and S. Lofdahl. 1988. Cloning of a chromosomal locus (exp) which regulates the expression of several exoprotein genes in Staphylococcus aureus. Mol. Gen. Genet. 211:435-440[CrossRef][Medline]. |
| 29. | Novick, R. P. 1991. Genetic systems in staphylococci. Methods Enzymol. 204:587-636[Medline]. |
| 30. | Novick, R. P. 1967. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology 33:155-166[CrossRef][Medline]. |
| 31. |
Patel, A. H.,
P. Nowlan,
E. D. Weavers, and T. Foster.
1987.
Virulence of protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement.
Infect. Immun.
55:3103-3110 |
| 32. | Patti, J. M., B. L. Allen, M. J. McGavin, and M. Hook. 1994. MSCRAMM-mediated adherence of microorganisms to host tissues. Annu. Rev. Microbiol. 48:585-617[Medline]. |
| 33. |
Patti, J. M.,
T. Bremell,
D. Krajewska-Pietrasik,
A. Abdelnour,
A. Tarkowski,
C. Ryden, and M. Hook.
1994.
The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis.
Infect. Immun.
62:152-161 |
| 34. | Peterson, P. K., J. Verhoef, L. D. Sabath, and P. G. Quie. 1977. Effect of protein A on staphylococcal opsonization. Infect. Immun. 55:3103-3110. |
| 35. |
Potempa, J.,
A. Dubin,
G. Korzus, and J. Travis.
1988.
Degradation of elastin by a cysteine proteinase from Staphylococcus aureus.
J. Biol. Chem.
263:2664-2667 |
| 36. |
Rice, K.,
R. Peralta,
D. Bast,
J. de Azavedo, and M. J. McGavin.
2001.
Description of staphylococcus serine protease (ssp) operon in Staphylococcus aureus and nonpolar inactivation of sspA-encoded serine protease.
Infect. Immun.
69:159-169 |
| 37. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 38. |
Saravia-Otten, P.,
H. P. Muller, and S. Arvidson.
1997.
Transcription of Staphylococcus aureus fibronectin binding protein genes is negatively regulated by agr and an agr-independent mechanism.
J. Bacteriol.
179:5259-5263 |
| 39. | Schenk, S., and R. A. Laddaga. 1992. Improved methods for electroporation of Staphylococcus aureus. FEMS Microbiol. Lett. 94:133-138[CrossRef]. |
| 40. |
Schneewind, O.,
A. Fowler, and K. F. Faull.
1995.
Structure of the cell wall anchor of surface proteins in Staphylococcus aureus.
Science
268:103-106 |
| 41. |
Signas, C.,
G. Raucci,
K. Jonsson,
P. E. Lindgren,
G. M. Anantharamaiah,
M. Hook, and M. Lindberg.
1989.
Nucleotide sequence of the gene for a fibronectin-binding protein from Staphylococcus aureus: use of this peptide sequence in the synthesis of biologically active peptides.
Proc. Natl. Acad. Sci. USA
86:699-703 |
| 42. | Tegmark, K., A. Karlsson, and S. Arvidson. 2000. Identification and characterization of SarH1, a new global regulator of virulence gene expression in Staphylococcus aureus. Mol. Microbiol. 37:398-409[CrossRef][Medline]. |
| 43. | Wolz, C., P. Pöhlmann-Dietze, A. Steinhuber, Y.-T. Chien, A. W. Manna, W. van Wamell, and A. L. Cheung. 2000. agr-independent regulation of fibronectin-binding protein(s) by the regulatory locus sar in Staphylococcus aureus. Mol. Microbiol. 36:230-243[CrossRef][Medline]. |
Infection and Immunity, August 2001, p. 4742-4748, Vol. 69, No. 8
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.8.4742-4748.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Weiss, E. C., Spencer, H. J., Daily, S. J., Weiss, B. D., Smeltzer, M. S.
(2009). Impact of sarA on Antibiotic Susceptibility of Staphylococcus aureus in a Catheter-Associated In Vitro Model of Biofilm Formation. Antimicrob. Agents Chemother.
53: 2475-2482
[Abstract] [Full Text] -
Lauderdale, K. J., Boles, B. R., Cheung, A. L., Horswill, A. R.
(2009). Interconnections between Sigma B, agr, and Proteolytic Activity in Staphylococcus aureus Biofilm Maturation. Infect. Immun.
77: 1623-1635
[Abstract] [Full Text] -
O'Neill, E., Pozzi, C., Houston, P., Humphreys, H., Robinson, D. A., Loughman, A., Foster, T. J., O'Gara, J. P.
(2008). A Novel Staphylococcus aureus Biofilm Phenotype Mediated by the Fibronectin-Binding Proteins, FnBPA and FnBPB. J. Bacteriol.
190: 3835-3850
[Abstract] [Full Text] -
Whiston, E. A., Sugi, N., Kamradt, M. C., Sack, C., Heimer, S. R., Engelbert, M., Wawrousek, E. F., Gilmore, M. S., Ksander, B. R., Gregory, M. S.
(2008). {alpha}B-Crystallin Protects Retinal Tissue during Staphylococcus aureus- Induced Endophthalmitis. Infect. Immun.
76: 1781-1790
[Abstract] [Full Text] -
Adhikari, R. P., Arvidson, S., Novick, R. P.
(2007). A Nonsense Mutation in agrA Accounts for the Defect in agr Expression and the Avirulence of Staphylococcus aureus 8325-4 traP::kan. Infect. Immun.
75: 4534-4540
[Abstract] [Full Text] -
Maiques, E., Ubeda, C., Tormo, M. A., Ferrer, M. D., Lasa, I., Novick, R. P., Penades, J. R.
(2007). Role of Staphylococcal Phage and SaPI Integrase in Intra- and Interspecies SaPI Transfer. J. Bacteriol.
189: 5608-5616
[Abstract] [Full Text] -
O'Neill, E., Pozzi, C., Houston, P., Smyth, D., Humphreys, H., Robinson, D. A., O'Gara, J. P.
(2007). Association between Methicillin Susceptibility and Biofilm Regulation in Staphylococcus aureus Isolates from Device-Related Infections. J. Clin. Microbiol.
45: 1379-1388
[Abstract] [Full Text] -
Oscarsson, J., Kanth, A., Tegmark-Wisell, K., Arvidson, S.
(2006). SarA Is a Repressor of hla ({alpha}-Hemolysin) Transcription in Staphylococcus aureus: Its Apparent Role as an Activator of hla in the Prototype Strain NCTC 8325 Depends on Reduced Expression of sarS. J. Bacteriol.
188: 8526-8533
[Abstract] [Full Text] -
Cassat, J., Dunman, P. M., Murphy, E., Projan, S. J., Beenken, K. E., Palm, K. J., Yang, S.-J., Rice, K. C., Bayles, K. W., Smeltzer, M. S.
(2006). Transcriptional profiling of a Staphylococcus aureus clinical isolate and its isogenic agr and sarA mutants reveals global differences in comparison to the laboratory strain RN6390.. Microbiology
152: 3075-3090
[Abstract] [Full Text] -
Renzoni, A., Barras, C., Francois, P., Charbonnier, Y., Huggler, E., Garzoni, C., Kelley, W. L., Majcherczyk, P., Schrenzel, J., Lew, D. P., Vaudaux, P.
(2006). Transcriptomic and Functional Analysis of an Autolysis-Deficient, Teicoplanin-Resistant Derivative of Methicillin-Resistant Staphylococcus aureus.. Antimicrob. Agents Chemother.
50: 3048-3061
[Abstract] [Full Text] -
Shaw, L. N., Aish, J., Davenport, J. E., Brown, M. C., Lithgow, J. K., Simmonite, K., Crossley, H., Travis, J., Potempa, J., Foster, S. J.
(2006). Investigations into {sigma}B-Modulated Regulatory Pathways Governing Extracellular Virulence Determinant Production in Staphylococcus aureus.. J. Bacteriol.
188: 6070-6080
[Abstract] [Full Text] -
Sambanthamoorthy, K., Smeltzer, M. S., Elasri, M. O.
(2006). Identification and characterization of msa (SA1233), a gene involved in expression of SarA and several virulence factors in Staphylococcus aureus.. Microbiology
152: 2559-2572
[Abstract] [Full Text] -
Roberts, C., Anderson, K. L., Murphy, E., Projan, S. J., Mounts, W., Hurlburt, B., Smeltzer, M., Overbeek, R., Disz, T., Dunman, P. M.
(2006). Characterizing the Effect of the Staphylococcus aureus Virulence Factor Regulator, SarA, on Log-Phase mRNA Half-Lives.. J. Bacteriol.
188: 2593-2603
[Abstract] [Full Text] -
Frees, D., Sorensen, K., Ingmer, H.
(2005). Global Virulence Regulation in Staphylococcus aureus: Pinpointing the Roles of ClpP and ClpX in the sar/agr Regulatory Network. Infect. Immun.
73: 8100-8108
[Abstract] [Full Text] -
Trotonda, M. P., Manna, A. C., Cheung, A. L., Lasa, I., Penades, J. R.
(2005). SarA Positively Controls Bap-Dependent Biofilm Formation in Staphylococcus aureus. J. Bacteriol.
187: 5790-5798
[Abstract] [Full Text] -
Liang, X., Zheng, L., Landwehr, C., Lunsford, D., Holmes, D., Ji, Y.
(2005). Global Regulation of Gene Expression by ArlRS, a Two-Component Signal Transduction Regulatory System of Staphylococcus aureus. J. Bacteriol.
187: 5486-5492
[Abstract] [Full Text] -
Resch, A., Rosenstein, R., Nerz, C., Gotz, F.
(2005). Differential Gene Expression Profiling of Staphylococcus aureus Cultivated under Biofilm and Planktonic Conditions. Appl. Environ. Microbiol.
71: 2663-2676
[Abstract] [Full Text] -
Tormo, M. A., Marti, M., Valle, J., Manna, A. C., Cheung, A. L., Lasa, I., Penades, J. R.
(2005). SarA Is an Essential Positive Regulator of Staphylococcus epidermidis Biofilm Development. J. Bacteriol.
187: 2348-2356
[Abstract] [Full Text] -
Li, D., Renzoni, A., Estoppey, T., Bisognano, C., Francois, P., Kelley, W. L., Lew, D. P., Schrenzel, J., Vaudaux, P.
(2005). Induction of Fibronectin Adhesins in Quinolone-Resistant Staphylococcus aureus by Subinhibitory Levels of Ciprofloxacin or by Sigma B Transcription Factor Activity Is Mediated by Two Separate Pathways. Antimicrob. Agents Chemother.
49: 916-924
[Abstract] [Full Text] -
Shaw, L. N., Golonka, E., Szmyd, G., Foster, S. J., Travis, J., Potempa, J.
(2005). Cytoplasmic Control of Premature Activation of a Secreted Protease Zymogen: Deletion of Staphostatin B (SspC) in Staphylococcus aureus 8325-4 Yields a Profound Pleiotropic Phenotype. J. Bacteriol.
187: 1751-1762
[Abstract] [Full Text] -
Entenza, J.-M., Moreillon, P., Senn, M. M., Kormanec, J., Dunman, P. M., Berger-Bachi, B., Projan, S., Bischoff, M.
(2005). Role of {sigma}B in the Expression of Staphylococcus aureus Cell Wall Adhesins ClfA and FnbA and Contribution to Infectivity in a Rat Model of Experimental Endocarditis. Infect. Immun.
73: 990-998
[Abstract] [Full Text] -
Bisognano, C., Kelley, W. L., Estoppey, T., Francois, P., Schrenzel, J., Li, D., Lew, D. P., Hooper, D. C., Cheung, A. L., Vaudaux, P.
(2004). A RecA-LexA-dependent Pathway Mediates Ciprofloxacin-induced Fibronectin Binding in Staphylococcus aureus. J. Biol. Chem.
279: 9064-9071
[Abstract] [Full Text] -
Xiong, Y.-Q., Bayer, A. S., Yeaman, M. R., van Wamel, W., Manna, A. C., Cheung, A. L.
(2004). Impacts of sarA and agr in Staphylococcus aureus Strain Newman on Fibronectin-Binding Protein A Gene Expression and Fibronectin Adherence Capacity In Vitro and in Experimental Infective Endocarditis. Infect. Immun.
72: 1832-1836
[Abstract] [Full Text] -
Shaw, L., Golonka, E., Potempa, J., Foster, S. J.
(2004). The role and regulation of the extracellular proteases of Staphylococcus aureus. Microbiology
150: 217-228
[Abstract] [Full Text] -
Sterba, K. M., Mackintosh, S. G., Blevins, J. S., Hurlburt, B. K., Smeltzer, M. S.
(2003). Characterization of Staphylococcus aureus SarA Binding Sites. J. Bacteriol.
185: 4410-4417
[Abstract] [Full Text] -
Beenken, K. E., Blevins, J. S., Smeltzer, M. S.
(2003). Mutation of sarA in Staphylococcus aureus Limits Biofilm Formation. Infect. Immun.
71: 4206-4211
[Abstract] [Full Text] -
Said-Salim, B., Dunman, P. M., McAleese, F. M., Macapagal, D., Murphy, E., McNamara, P. J., Arvidson, S., Foster, T. J., Projan, S. J., Kreiswirth, B. N.
(2003). Global Regulation of Staphylococcus aureus Genes by Rot. J. Bacteriol.
185: 610-619
[Abstract] [Full Text] -
Massimi, I., Park, E., Rice, K., Muller-Esterl, W., Sauder, D., McGavin, M. J.
(2002). Identification of a Novel Maturation Mechanism and Restricted Substrate Specificity for the SspB Cysteine Protease of Staphylococcus aureus. J. Biol. Chem.
277: 41770-41777
[Abstract] [Full Text] -
Cordwell, S. J., Larsen, M. R., Cole, R. T., Walsh, B. J.
(2002). Comparative proteomics of Staphylococcus aureus and the response of methicillin-resistant and methicillin-sensitive strains to Triton X-100. Microbiology
148: 2765-2781
[Abstract] [Full Text] -
Karlsson, A., Arvidson, S.
(2002). Variation in Extracellular Protease Production among Clinical Isolates of Staphylococcus aureus Due to Different Levels of Expression of the Protease Repressor sarA. Infect. Immun.
70: 4239-4246
[Abstract] [Full Text] -
Blevins, J. S., Beenken, K. E., Elasri, M. O., Hurlburt, B. K., Smeltzer, M. S.
(2002). Strain-Dependent Differences in the Regulatory Roles of sarA and agr in Staphylococcus aureus. Infect. Immun.
70: 470-480
[Abstract] [Full Text] -
Mongodin, E., Bajolet, O., Cutrona, J., Bonnet, N., Dupuit, F., Puchelle, E., Bentzmann, S. d.
(2002). Fibronectin-Binding Proteins of Staphylococcus aureus Are Involved in Adherence to Human Airway Epithelium. Infect. Immun.
70: 620-630
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»