1 Institute of Medical Microbiology and Hygiene, Building 43, University of Saarland, D-66421 Homburg/Saar, Germany
2 Institute of Molecular Biology, Center of Excellence for Molecular Medicine, Slovak Academy of Sciences, 845 51 Bratislava, Slovak Republic
3 Institute of Medical Microbiology and Hygiene, University of Tübingen, D-72074 Tübingen, Germany
4 Institute of Molecular Infection Biology, University of Würzburg, D-97070 Würzburg, Germany
5 Institute of Infection, Immunity and Inflammation, University of Nottingham, Nottingham NG7 2RD, UK
6 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Sutton Bonington LE12 5RD, UK
Correspondence
Niamh Harraghy
bhnhar{at}uniklinik-saarland.de
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ABSTRACT |
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INTRODUCTION |
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S. aureus produces two types of adhesin (reviewed by Navarre & Schneewind, 1999). One set has a characteristic LPXTG motif that anchors the adhesin to the staphylococcal cell surface (Mazmanian et al., 1999
). The members of this family of adhesins are called MSCRAMM molecules, and they include protein A, the fibronectin-binding proteins (FnBPs), clumping factors A and B (ClfA and B), and more recently described molecules, such as IsdA, (also known as FrpA and StbA; Wiltshire & Foster, 2001
; Mazmanian et al., 2002
; Morrissey et al., 2002
; Taylor & Heinrichs, 2002
), Bsp (Tung et al., 2000
) and HarA (Dryla et al., 2003
). Many of these proteins have been implicated as bridging molecules between the bacterium and the host cell (e.g. Sinha et al., 1999
; Hartleib et al., 2000
; Massey et al., 2001
). Members of the second set of adhesins are noncovalently anchored to the cell surface, and include the fibrinogen-binding protein (Efb; Palma et al., 2001
), coagulase (Boden & Flock, 1989
), and Eap and Emp (discussed below).
In recent years it has emerged that staphylococcal adhesins may have additional, diverse functions (e.g. Chavakis et al., 2002; Bjerketorp et al., 2004
; Heilmann et al., 2005
), and that bacteria may alter expression of these molecules in response to changing environmental conditions (e.g. Clarke et al., 2004
). Moreover, a number of studies have implicated staphylococcal adhesins as being important in the host response to infection (Jahreis et al., 1995
, 2000
; Miyamoto et al., 2001
; Chavakis et al., 2002
; Lee et al., 2002
). Our work focuses on two of these adhesins, Eap and Emp. These proteins were initially identified by their ability to bind to various extracellular matrix proteins (Boden & Flock, 1992
; Jönsson et al., 1995
; Palma et al., 1999
; Hussain et al., 2001b
). Eap has been shown to be involved in the adherence to and invasion of eukaryotic cells by S. aureus (Hussain et al., 2002
; Kreikemeyer et al., 2002
; Haggar et al., 2003
). Moreover, Eap has emerged as being important in modulation of the immune response to infection by interfering with neutrophil recruitment (Chavakis et al., 2002
), as well as inhibiting the delayed-type hypersensitivity response, and inducing T-cell death (Lee et al., 2002
). It also appears to be important in chronic infections (Lee et al., 2002
). Recently, a novel function for Eap as a potent inhibitor of angiogenesis has been described (Sobke et al., 2004
). The importance of Emp during infection is not yet known, but, like Eap, it also binds to fibronectin, fibrinogen and vitronectin (Hussain et al., 2001b
).
For both Eap and Emp, little is known about the environmental conditions and regulators that affect their expression. The expression of staphylococcal virulence genes is controlled by a complex regulatory network; for a more comprehensive overview of virulence gene regulation in S. aureus, the reader is referred to a number of excellent review articles (Arvidson & Tegmark, 2001; Cheung & Zhang, 2002
; Cheung et al., 2002
, 2004
; Novick, 2003
), while a short review of the regulators that are the focus of this study is given below.
agr (accessory gene regulator) was one of the first recognized global regulators of staphylococcal gene expression (Rescei et al., 1986; Janzon et al., 1989
). The agr system is a quorum sensing one, and consists of an operon of four genes. The first is a membrane-associated protease (agrB). The second, agrD, produces the pre-pro-peptide that is subsequently modified by AgrB and secreted as a peptide thiolactone consisting of 79 amino acid residues, in which the central cysteine residue is covalently linked to the C-terminal amino acid carboxylate forming a cyclic thioester. This functions as the quorum-sensing pheromone, which is recognized by a membrane-associated sensor kinase (agrC), which in turn activates the cytoplasmic regulator (agrA) (Ji et al., 1995
; Morfeldt et al., 1996
; Lina et al., 1998
; Zhang et al., 2002
). These genes form the transcript RNAII, and are under the control of the P2 promoter (Novick et al., 1993
, 1995
; Ji et al., 1995
). A second, divergent transcript, RNAIII, is the effector molecule of the agr system, and is under the control of the P3 promoter (Janzon & Arvidson, 1990
). This molecule is synthesized in response to the environmental concentration of the octapeptide (Ji et al., 1995
). Expression of RNAIII is temporal, with maximal expression occurring in the transition from the post-exponential to the stationary phase. It has been postulated that this temporal expression is responsible for the repression of cell surface proteins, and the enhanced expression of exoproteins during the later stages of growth (Kornblum et al., 1990
).
The sar (staphylococcal accessory regulator) locus was identified by Cheung et al. (1992), and encodes a single DNA-binding protein, SarA. However, upstream of sarA are three distinct promoters that produce three distinct transcripts (sarA, sarB and sarC), with the sarA and sarB transcripts preferentially expressed during the exponential phase, and maximal expression of the sarC transcript occurring during the late stationary phase (Bayer et al., 1996
; Manna et al., 1998
; Blevins et al., 2002
). All of the transcripts terminate at the same stemloop structure (Bayer et al., 1996
), thereby resulting in constitutive production of SarA (Blevins et al., 1999
). In the past few years, analyses of the staphylococcal genome sequence have revealed the presence of a large number of SarA homologues, termed the SarA protein family (reviewed by Cheung et al., 2002
, 2004
). These homologues appear to be involved in the control of sarA expression.
SarA is believed to regulate RNAIII expression by binding to the P2 (and, to a lesser extent, P3) promoter of the agr system, resulting in enhanced transcription of RNAII and correspondingly RNAIII (Heinrichs et al., 1996; Cheung et al., 1997
; Chien & Cheung, 1998
; Chien et al., 1998
). SarA does not mediate its effects only through agr. For example, the FnBPs are regulated by agr, but also by SarA, in an agr-independent manner (Saravia-Otten et al., 1997
; Wolz et al., 2000
). An agr mutation has no obvious effect on sarA transcription (Cheung et al., 1997
; Horsburgh et al., 2002
).
Another staphylococcal regulator recently subjected to microarray analysis is SigmaB (B) (Bischoff et al., 2004
).
B was initially identified in Bacillus subtilis as being an important regulator of the general stress and heat-shock response. The homologous operon in S. aureus has been mapped, and found to consist of a four-gene operon, consisting of rsbU (required for
B activation), rsbV (the anti-anti-sigma factor), rsbW (the anti-sigma factor which regulates
B activity post-translationally) and sigB (Wu et al., 1996
; Kullik & Giachino, 1997
; Miyazaki et al., 1999
, Giachino et al., 2001
; Palma & Cheung, 2001
). During the course of their study on the sigB operon, Kullik & Giachino (1997)
noticed that there was an 11 bp deletion in the rsbU gene in derivatives of 8325. This effectively rendered these strains
B-negative. Unfortunately, the 8325 lineage had up to this time been used extensively in gene regulation studies and in vivo models of infection. However, since then, several rsbU+ strains in the 8325 background have been constructed (Giachino et al., 2001
; Horsburgh et al., 2002
).
B is also involved in the regulation of other regulators. It is well known that the sarC transcript is
B dependent (Deora et al., 1997
; Manna et al., 1998
; Gertz et al., 2000
). However, there are conflicting reports as to whether
B influences the levels of SarA (Gertz et al., 2000
; Bischoff et al., 2001
; Horsburgh et al., 2002
).
B is also involved in the regulation of agr, but in this case it has been reported to have a negative effect (Bischoff et al., 2001
; Horsburgh et al., 2002
).
sae (S. aureus exoprotein expression) has emerged as an important regulator of virulence gene expression. This regulator was initially identified following isolation of a mutant that was defective in the production of several exoproteins (Giraudo et al., 1994, 1999
). The sae locus is composed of four ORFs, two of which encode a classical two-component system, which is now known to be more complex (Novick & Jiang, 2003
; Steinhuber et al., 2003
). A sae mutation has no effect on expression of agr and sarA (Giraudo et al., 1997
). However, sae is activated by agr, at least in some strains, by an unknown mechanism (Giraudo et al., 2003
; Novick & Jiang, 2003
; Goerke et al., 2005
). The importance of sae as a virulence factor in vivo has been demonstrated in several animal models (Rampone et al., 1996
; Benton et al., 2004
; Goerke et al., 2005
). Furthermore, it has been shown that sae is essential for virulence-gene expression in vivo (Goerke et al., 2001
, 2005
).
The interactions of the various regulatory loci are only partially understood, and have been analysed mainly in relation to the coordinate expression of selected extracellular or cell-bound proteins. Little is known about the regulation of the adhesins that are non-covalently attached to the cell wall. Given the importance of Eap and, possibly, Emp as virulence factors of S. aureus, understanding the regulation of these genes may give additional information to the overall picture of staphylococcal virulence, as well as contributing to our understanding of the staphylococcal global regulatory network. In this study we investigate the regulation of eap and emp by mapping their transcription start sites, and describe the contribution of the global regulators agr, sarA and sae to the regulation of eap and emp.
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METHODS |
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Luminescence assay.
For assaying eap expression, cells were diluted 1 : 100 in MBB and MBB plus 0·5 % glucose, both supplemented with 5 µg chloramphenicol ml1, and 200 µl of the cell suspension was transferred to wells of a 96-well clear-bottom white plate (Greiner). All samples were assayed in triplicate. The plate was then incubated with shaking at 37 °C in a Victor 2 multilabel reader. Optical density and luminescence readings were taken every 30 min. To normalize the data, the luminescence values were divided by the corresponding OD595 value.
Isolation of RNA and S1-nuclease mapping.
At pre-determined time points, the cell suspension of S. aureus Newman was immediately poured into a 50 ml Falcon tube containing about 15 ml crushed ice that was prechilled to 80 °C, and total RNA was prepared essentially as recently described in Kormanec (2001). High-resolution S1 nuclease mapping was performed according to Kormanec (2001)
. Samples (40 µg) of RNA were hybridized to approximately 0·02 pmol of a suitable DNA probe labelled at one 5' end with [
-32P]ATP (approx. 3x106 c.p.m. per pmol probe). The probes used were prepared by PCR amplification from the corresponding plasmids as follows: probe EMP was a 1100 bp DNA fragment prepared by PCR amplification from the plasmid pEmp1 using the 5' end-labelled reverse primer emp1 from the emp coding region, and the direct universal primer 48; probe EAP was a 600 bp DNA fragment prepared by PCR amplification from the plasmid pEAP1 using the 5' end-labelled reverse primer eap1 from the eap coding region, and the direct universal primer 47 (Table 2
). Oligonucleotides were labelled at their 5' ends with [
-32P]ATP (166·5 TBq mmol1; ICN Biochemicals) and T4 polynucleotide kinase (New England Biolabs). The protected DNA fragments were analysed on DNA sequencing gels, together with G+A and T+C sequencing ladders derived from the end-labelled fragments (Maxam & Gilbert, 1980
). Before assigning the transcription start point (TSP), 1·5 nt were subtracted from the length of the protected fragment to account for the difference in the 3' ends resulting from the S1-nuclease digestion, and the chemical sequencing reactions. All mapping experiments were done twice with independent sets of RNA with similar results.
Primer-extension analysis.
A 50 µg quantity of total RNA was dissolved in 60 µl hybridization buffer (40 mM PIPES, pH 6·4, 1 mM EDTA, 0·4 M NaCl, 80 %, v/v, formamide) at 65 °C, denatured together with 0·5 pmol of the 32P-labelled oligonucleotide primer (emp1 or eap1) for 5 min at 95 °C, and annealed for 4 h at 45 °C. DNA samples were ethanol-precipitated, dissolved in 9 µl water, and the following components were added: 0·75 µl RNasin (Promega), 3 µl 5x AMV-RT buffer (Promega), 0·75 µl 5 mM each of dATP, dGTP, dTTP and dCTP, and 0·75 µl actinomycin (4 mg ml1), and the mixture was incubated for 2 min at 42 °C. The primer extension was initiated by adding 1·3 µl (26 U) AMV-RT (Finnzymes), and incubated for 2 h at 42 °C. The reaction was terminated with 25 µl RNase mix [100 µg ml1 DNase-free RNase A, 30 µg ml1 sonicated salmon sperm DNA, TE buffer (10 mM Tris/HCl, 1 mM EDTA), pH 8)] and incubated for 30 min at 37 °C. After addition of 20 µl 1 M NaCl, the mixture was extracted with alkaline phenol/chloroform, and DNA was precipitated with ethanol. The pellet was dissolved in 5 µl loading buffer (80 %, v/v, formamide, 10 mM NaOH, 1 mM EDTA, 0·05 % xylene cyanol, 0·05 % bromophenol blue), heated for 2 min at 95 °C, and an aliquot was loaded on a 6 % denaturing gel, and separated together with the G+A and T+C sequencing ladders derived from the corresponding end-labelled fragments (EMP, EAP) (Maxam & Gilbert, 1980).
RNA isolation for real-time PCR.
Cells were grown as described above, and at the pre-determined time points, 0·8 ml culture was removed and centrifuged at 5000 g for 5 min at 4 °C. RNA was prepared with the Nucleospin RNA II kit (Macherey and Nagel), with the following modifications. Following centrifugation, the cells were resuspended in 100 µl TE buffer (pH 8), and then 500 µl of the supplied buffer R1 plus 5 µl -mercaptoethanol (Roth) was added. This suspension was transferred to a Lysing Matrix B tube (Q-biogene), and spun for 20 s at a speed of 6·5 m s1 in a Fastprep (Thermo Savant). After centrifugation at 14 000 g (MiniSpin; Eppendorf), the supernatant was collected for further centrifugation. The supernatant was then transferred to the gDNA shredding column supplied with the kit, and the manufacturer's protocol was then followed. All samples were treated with the supplied DNase I. The absence of contaminating genomic DNA was confirmed by real-time PCR using primers for gyrB, as described below.
Real-time PCR.
cDNA was prepared using the High Capacity cDNA Kit (Applied Biosystems), according to the manufacturer's instructions. Primers for real-time PCR were designed using Primer Express software (Applied Biosystems). Prior to analysis of the cDNA samples, a number of optimization reactions were performed. For optimizing the primer concentration, real-time PCR with primer concentrations ranging from 50 to 900 nM was performed. The optimal primer concentrations were as follows: eap (300 nM, forward and reverse), emp (100 nM, forward and reverse), sae (50 nM forward, 100 nM reverse) and gyrB (300 nM, forward and reverse). To check the efficiency of the PCR, standard curves were made using serial dilutions of cDNA. Real-time PCR was performed on an Applied Biosystems 7000 instrument using the SYBR Green Mastermix (Applied Biosystems), according to the manufacturer's instructions. gyrB was used as an internal control, and the gene-specific transcripts were expressed as the n-fold difference relative to gyrB, using the formula 2CT, where CT is the threshold cycle value.
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RESULTS |
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Global regulators affecting expression of eap and emp
In order to identify the main regulator of eap and emp, we investigated the contribution of a number of glucose-repressed and important regulators of virulence-gene expression in S. aureus to the regulation of eap and emp. This was performed using real-time PCR in defined regulatory mutants. The initial experiments, showing enhanced expression of eap and emp in Newman compared with the 8325 derivatives SH1000 and 8325-4, suggest that B is not essential for expression of eap and emp, since neither gene showed enhanced expression in SH1000 (rsbU+) when compared with 8325-4 (rsbU). This was in spite of the fact that the level of
B activity in SH1000 was similar to Newman, as assayed by real-time PCR, using expression of asp23 as an indicator of
B activity (data not shown). The expression of eap and emp was also examined in agr, sarA and sae mutants (ALC355, ALC637 and AS3, respectively). In both the agr and sarA mutants, expression of eap and emp was reduced three- to fivefold at the 3 h time point (Fig. 3
). However, in the sae mutant, expression of both genes was severely repressed, which was particularly evident at the 3 h time point (Fig. 3
), thereby demonstrating that sae is essential for expression of eap and emp.
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DISCUSSION |
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Mapping the TSPs of eap and emp revealed that both genes are transcribed from a single promoter. This was an important point to establish, since while there are no genes in the immediate vicinity of eap, there are two genes encoding staphylococcal adhesins immediately upstream of emp (Kuroda et al., 2001). The translation start codon of emp is only 353 bp downstream of the vwb stop codon, raising a possibility of co-transcription. However, previous studies (Bjerketorp et al., 2002
) based on sequence analysis of this region have suggested that these genes are not linked, and we can now show that emp does indeed have its own promoter.
In order to study the regulation of eap and emp, we used two different reporter-gene assays. Initial experiments showed that expression of both the eap and emp constructs in the 8325 background was very low. We therefore decided to transfer the constructs to strain Newman, a strain that has previously been shown to produce considerably more Eap than other strains (Hussain et al., 2001a). In Newman, enhanced expression of both eap and emp reporters was obtained. Moreover, using real-time PCR we could confirm that Newman does indeed have much higher levels of expression of eap and emp compared with SH1000 and 8325-4 (data not shown), thereby confirming the reporter-gene assay data. Taken together, these data showed that the differential expression of eap and emp in strains Newman and SH1000 and 8325-4 was not due to differences in the promoter sequences (since the 8325-4 promoter of both genes was used for the reporter assays in all strains), or to the proteins (e.g. as a consequence of enhanced protein stability), and therefore must be due to differential expression of (a) key regulator(s). The difference in
B activity between strains Newman and 8325-4 is unlikely to be responsible for the observed differences in expression of eap and emp, since SH1000 (8325-4 rsbU+) did not show increased expression of either gene compared with 8325-4 (rsbU). These findings are also consistent with the recently published microarray data (Bischoff et al., 2004
) showing that
B is not essential for expression of either gene. However, since sarA is partly under
B control (Bischoff et al., 2001
), one should not exclude the possibility that
B is functioning either directly or indirectly at the post-transcriptional level (N. Harraghy, unpublished observation).
We next examined expression of eap and emp in agr and sarA mutants. In these mutants, expression of both eap and emp was reduced three- to fivefold (Fig. 3). For eap, these findings are in agreement with previous transcriptional profiling of agr- and sarA-regulated genes in S. aureus (Dunman et al., 2001
), where it was shown that eap is upregulated by both agr and sarA. However, although emp did not emerge in this study as being under control of either regulator, our data indicate that it is also under control of both agr and sarA.
Although agr and sarA were shown to be involved in the regulation of eap and emp, neither gene was completely repressed in the agr and
sarA strains. Our observation that both eap and emp were repressed in the presence of glucose, and their enhanced expression in strain Newman compared with the 8325 derivatives, taken together with the recently published data of Steinhuber et al. (2003)
and Novick & Jiang (2003)
, pointed to sae as being a key regulator of eap and emp. Steinhuber et al. (2003)
noted that expression of sae was reduced in ISP479C (an 8325 derivative) compared with Newman, an observation that we could confirm in our 8325 derivatives, SH1000 and 8325-4 (Fig. 4
). Novick & Jiang (2003)
and Weinrick et al. (2004)
reported that sae expression is repressed in the presence of glucose as a consequence of changes in the pH that occur when bacteria are grown in such a medium. We could also confirm that there were marked differences in the pH of MBB containing glucose compared with MBB without glucose, and that in the glucose-containing medium, expression of sae was repressed. The recent transcriptome analysis of genes induced under mild acidic conditions, such as those seen when bacteria are grown in the presence of glucose, has revealed a wide range of genes, including eap and emp, that are affected by this change in pH (Weinrick et al., 2004
). This leads to the possibility that changes in pH, such as those that S. aureus would encounter following arrival at a new niche, or invasion of eukaryotic cells, may alter expression of key regulators, thereby altering expression of virulence factors, and hence aid the adaptation of S. aureus to changing environmental conditions. It is also possible that sae is responding to signals other than pH, as exemplified by our observation that the temporal pattern of sae expression was dependent on the growth medium used.
From the data presented in this paper, it is clear that sae is essential for transcription of both eap and emp. However, the exact mechanism of sae control is still not known. The interactions between the various global regulators of S. aureus are highly complex, and often paradoxical. For example, Novick & Jiang (2003) have suggested that sae is both dependent and independent of agr, and both agr-dependent and -independent regulation of genes has been described (e.g. Saravia-Otten et al., 1997
; Blevins et al., 1999
; Wolz et al., 2000
). Since repression of eap and emp in the agr mutant is modest in comparison with that seen in the sae mutant, this may be indicative of a decrease in sae expression as a result of the agr mutation. Alternatively, it may indicate that RNAIII interacts directly with the eap and emp promoters, and that regulation of eap and emp by sae is independent of agr.
Repression of eap and emp in the sarA mutant may be a direct consequence of a decrease in agr expression, considering that the eap transcript levels are very similar in both the agr and sarA backgrounds. The additional repression of emp seen in the sarA mutant as compared with the agr mutants suggests that, in addition to any effect via agr, SarA may act directly on the sae or emp promoters. However, it is also possible that the decrease in expression of both genes is a result of SarA acting independently of agr. It should also be noted that although in strain Newman eap and emp appear to be under similar regulatory control, the expression of eap is about 10-fold higher than that of emp. This suggests the interaction of additional transcription factors with the eap and emp promoters. Characterization of the eap and emp promoters, and the factors binding to them, is currently under investigation in our laboratories.
In conclusion, we have identified sae as an important regulator of the staphylococcal adhesins Eap and Emp, which is capable of modulating their expression under different environmental conditions. It may well be that sae is a crucial regulator in the adaptation of S. aureus to changing environments, which consequently may contribute to the intracellular survival of S. aureus (e.g. altering virulence-gene expression in response to changing pH), or may orchestrate complex interactions with the eukaryotic immune system via expression of these secreted adhesins.
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ACKNOWLEDGEMENTS |
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Received 19 January 2005;
revised 28 February 2005;
accepted 3 March 2005.
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