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Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
Correspondence
Chia Y. Lee
clee2{at}uams.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; O'Riordan & Lee, 2004). Additionally, staphylococcal capsule was recently implicated as one of the few extracellular factors required for virulence in Caenorhabditis elegans (Bae et al., 2004).
The cap5 and cap8 operons required for the synthesis of CP5 and CP8 are allelic (Sau et al., 1997a). Twelve of the 16 genes are virtually identical between the cap5 and cap8 operons, which explains why CP5 and CP8 have almost identical trisaccharide-repeating units that differ only in the location of the O-acetylation and the position linking monosaccharides. Molecular characterization of the cap8 locus indicates that all 16 genes are transcribed as a large transcript from the primary promoter upstream of the first gene. Several internal promoters are also present but these are much weaker than the primary promoter (Sau et al., 1997b). A 10 bp inverted repeat located upstream of the –35 sequence of the primary cap8 promoter has been shown to be required for full expression of CP8, suggesting that a DNA-binding regulator is involved in the control of capsule production (Ouyang et al., 1999). The promoter regions between the cap5 and cap8 operons of several strains are also highly similar, suggesting that the two capsules are regulated similarly (Herbert et al., 2001; Sau et al., 1997a).
Several regulatory genes which globally control the expression of many virulence determinants of S. aureus have been identified. These regulators appear to form a complex network of regulation to coordinately control the virulence gene expression (for reviews see Bronner et al., 2004; Cheung et al., 2004; Novick, 2003). Previously, we showed that agr had a significant effect on capsule production (Luong et al., 2002). Recently, we found that mgrA, also known as norR (Truong-Bolduc et al., 2003) or rat (Ingavale et al., 2003), affected capsule production (Luong et al., 2003). In this study, we employed Tn551 mutagenesis to identify several putative regulatory loci that influenced CP5/8 production. We chose to focus on arl, a locus which has previously been shown to regulate autolysis and some secreted proteins (Fournier & Hooper, 2000; Fournier et al., 2001), and showed that the locus positively regulated CP5/8 expression at the transcriptional level mainly via an mgrA-dependent pathway.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. S. aureus strain RN4220 was used as the recipient for electroporation of the constructed plasmids. Transductions between S. aureus strains were carried out using bacteriophage 52A. S. aureus strains were cultivated on tryptic soy agar (Difco). Electroporation in S. aureus was carried out by the procedure of Kraemer & Iandolo (1990). Nitrocefin was purchased from Becton Dickinson Microbiology Section.
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. To identify Tn551 insertion sites, chromosomal DNA with Tn551 insertion was digested with Sau3A, ligated with T4 DNA ligase and amplified by inverse PCR using two inverse PCR primers (Tn917R and Rn917L; Table 2) as previously described (Luong et al., 2003). The amplified fragments were cloned into pGEMT-easy vector (Promega) and sequenced.
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) by reversing the orientation of tet (pT181) and ori (pE194) with respect to the rest of the plasmid. Plasmids pLL33 and pLL35 were constructed by ligating the blaZ reporter gene and the multiple cloning sites from pWN1819 and pSA3800, respectively, to pCL96. Plasmid pCL15 was derived from pSI-1 (Yansura & Henner, 1984) by removing the EcoRI site (end-filling), adding the ampicillin-resistance gene from pBluescript at the BamHI site (which also destroyed the BamHI site) and replacing the cloning site with the multiple cloning site of pUC18. To construct Pcap5 : : blaZ reporter fusions, a 627 bp fragment containing the cap5 promoter was amplified from Newman chromosomal DNA using primers cp8bla.f and cp8bla.r. To clone the mgrA gene under the control of Pspac in pCL15, a 570 bp fragment containing the mgrA gene without the promoter was amplified using primers mgr74 and mgr75 and cloned into the HindIII and XbaI sites of pCL15. The same method was used to clone an 820 bp fragment containing the arlR gene in pCL15 using primers arlR19 and arlR20. To clone the arlR gene for complementation, a PCR fragment was amplified from Newman chromosomal DNA using arlR17 and arlR18 and then cloned into pCL96 to form pTL3287.
Strain construction.
To construct the Newman 
arlR mutant, the upstream and downstream fragments of the target region to be replaced were amplified by PCR using two sets of primers (arlR3 and arlR4 for upstream fragment and arlR5 and arlR6 for downstream fragment) and sequence-verified. The two fragments were cloned into pCL52.2 and used for allele replacement as described previously (Lin et al., 1994). The mutant (CYL1164) with a 383 bp internal deletion of the arlR gene without any replacing antibiotic-resistance marker was obtained and verified by PCR.
The Newman mgr : : cat strain (CYL1050) was constructed by transducing mgrA : : cat from CYL1040 (Luong et al., 2003) to strain Newman. To construct the Newman mgrA strain (CYL1460), the plasmid used for the construction of CYL1040 was digested with ApaI to delete the cat gene. The resultant plasmid was transferred to CYL1050 for allele replacement as described by Lin et al. (1994) except that the clones were screened for the loss of chloramphenicol resistance. The resultant strain CYL1460 was confirmed by PCR with appropriate primers. To construct mgrA arlR double mutant CYL1206, the mgrA : : cat from CYL1040 was transduced to CYL1164.
RNA extraction and mRNA quantification.
These were carried out as described previously (Luong et al., 2002). For quantification of mRNA by real-time RT-PCR, gene-specific primers (SG16S1, SG16S2, SGhu1, SGhu2, SGarl1, SGarl2, SGmgrA1, SGmgrA2, SGcap8A1, SGcap8A2; Table 2
) were used to amplify 100–200 nucleotide fragments of the target genes (Table 2). DNase I-treated RNA was incubated with the SuperScript III platinum SYBR Green One-Step qRT-PCR master mix (Invitrogen) and the reaction was carried out using the ABI Prism 7300 Detection system (Applied Biosystems). No-template reactions were included as negative controls. A negative control without reverse transcriptase in the reaction was performed to exclude the possibility of DNA contamination. Standard curves were established to confirm that the primers amplified at the same rate. The 16S rRNA and/or the hu gene was used for normalization of the target mRNA. Relative expression levels were determined by using the protocol for the standard curve method (Applied Biosystems user bulletin no. 2).
Gel mobility shift assay.
The arlR gene was amplified by primers arlR1 and arlR2, cloned into the NdeI and BamHI sites of pET15b (Novagen) and used for purifying His6-ArlR fusion protein by nickel-affinity column chromatography following the manufacturer's directions (Novagen). A 138 bp fragment containing the promoter of the cap5 operon (from +4 to –135 with respect to the transcriptional start site) was amplified by PCR using primers cp8gs6 and cp8gs15, purified using a Qiagen PCR purification kit, end-labelled and used in gel mobility shift assay as previously described (Ouyang et al., 1999). Phosphorylation of His6-ArlR was carried out by incubating 2.3 µg (86.7 pmol) purified His6-ArlR at 37 °C for 2 h in 10 µl reaction buffer containing 50 mM Tris/HCl (pH 8.0), 10 mM MgCl2, 2 mM DTT and 30 mM acetyl phosphate. Successful phosphorylation was determined by detecting a shifted band in an 8 % non-denatured PAGE system.
Other tests.
-Lactamase activity was assayed by the nitrocefin method as described previously (Luong et al., 2002). CP5 production was quantified as described by Luong et al. (2003).
Statistical analysis.
Data from reporter gene fusion analyses were assessed using a paired Student's t test for comparing two samples. P values of <0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) but its use for monitoring the activity in isolated colonies had not been tested. We therefore transferred the plasmid into strains Becker, Newman and COL for testing XylE activity. We found that COL(pCL8074) exhibited moderate intensity of yellow colour, strain Becker(pCL8074) exhibited high intensity and Newman(pCL8074) exhibited low intensity. We chose strain COL for further transposon mutagenesis for screening potential regulatory genes because of the possibility of obtaining both classes of mutation, activator and repressor, in the same screen. Random transposition was carried out as described in Methods. Several putative mutants with reduced or enhanced yellow colour as compared to the wild-type were identified. Forty-four of these mutants were backcrossed by phage transduction to the parental strain COL and the phenotypes of 17 of them remained unchanged, indicating that the phenotypes are due to the Tn551 insertion and not to secondary mutations during mutagenesis. The chromosomal regions flanking Tn551 insertion sites in the 17 mutants identified above were amplified by inverse PCR, which yielded 12 different patterns according to the sizes of the amplified fragments. The fragments representing each of the 12 patterns were subjected to DNA sequencing. BLAST searches of the sequenced DNA fragment against the genomes of strains 8325 and N315 showed that the insertion sites in these mutants were within the coding region of arlR, sbcD, sbcC, clpC, N315 SA1288, N315 SA1708 and N315 SA0816. A single insertion site was found in each gene, except that there were six different insertion sites within sbcD gene. The insertions within sbcC, sbcD and clpC resulted in increased XylE activities, whereas insertions in other genes resulted in decreased XylE activities.
arlR is an activator for capsule production
To study whether the genes identified above are involved in capsule regulation, we chose to focus on arlR, the response regulator of the arlRS two-component regulatory system. The arlRS system has been shown to be involved in regulation of several virulence genes in S. aureus (Fournier & Hooper, 2000; Fournier et al., 2001). The two genes form an operon, and mutations in each gene have been shown to result in the same phenotype (Fournier et al., 2001). To further confirm that the arl locus is involved in the regulation of capsular production, we transduced the Tn551 insertion in the arlR gene (at between 66 and 67 bp from the translational start site) from strain COL to strain Newman, which produces type 5 capsule. The resultant strain (CYL815) produced much less capsule than the wild-type strain when the capsule was isolated from whole cell pellet or from the supernatant (Fig. 1a). We then constructed an arlR-deletion mutant (CYL1164) in which the 383 bp internal portion of the gene was deleted by allele replacement. As shown in Fig. 2, similar to the Tn551 mutant CYL815, the mutant CYL1164 produced an almost undetectable amount of CP5 as compared to the wild-type strain. To confirm that the mutant phenotype is due to the deletion of arlR, we complemented CYL1164 with pTL3287, carrying a PCR-amplified 739 bp DNA fragment containing the intact arlR gene from strain Newman. As shown in Fig. 2, the capsule production of the CYL1164(pTL3287) was restored to the wild-type level. Since the cloned fragment contained only the arlR gene without other ORFs, the results indicated that the mutation in arlR, not other secondary mutations or polarity, affects capsule production in strain Newman. These results suggested that the arlR gene, and thus the arl locus, is involved in the activation of capsule production.
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show that the amount of cap5 mRNA isolated from the mutant CYL815 was much less than that isolated from Newman. This result is similar to the assay of CP5 production described above, suggesting that arlR is an activator regulating CP5 production primarily at the transcriptional level.
To confirm the transcriptional regulation of cap5 expression by arlR and to determine whether arlR can also have an effect on CP5 production at the post-translational level, we constructed blaZ reporter gene fusions. The fusion plasmids were transduced into strain Newman and mutant strain CYL1164. As shown in Fig. 3, the Pcap5 activity in the arlR mutant was significantly less than that in the wild-type strain in both transcriptional and translational fusion assays at all time points. We also obtained similar results for BlaZ activities when the fusion plasmids were put into the Tn551 insertional mutants (data not shown). The fact that the BlaZ activities decreased in a similar pattern in both transcriptional and translational fusions suggests that the regulation of the cap5 genes by arlR is controlled at the transcriptional level. However, we noted that translational fusions had a tenfold higher activity than the transcriptional fusions (Fig. 3). This difference could be due to the difference in ribosome-binding site of the two constructs.
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) with increasing concentrations of purified His6-ArlR. We found a shifted band, which could be inhibited with 500-fold excess of unlabelled competitor DNA but not by an irrelevant DNA fragment (data not shown). However, the shifting could only be detected when the amount of ArlR was or exceeded 5 µM, suggesting that ArlR binds to the cap5 promoter region but only at a high concentration. Phosphorylation of ArlR with acetyl phosphate did not facilitate the binding (data not shown). Taking these results together, we concluded that ArlR might not regulate the capsule production by direct binding to the promoter region of the cap5 operon. Although the binding occurred under high concentrations of the protein, it was most likely not physiologically relevant.
arlR regulates cap5 genes primarily through mgrA
We have previously shown that mgrA is a major activator of cap5(8) gene expression (Luong et al., 2003, 2006). Since we showed above that arl most likely did not regulate the cap5 genes directly, we speculated that it might regulate capsule production through mgrA. To test this possibility, we compared the mgrA mRNA produced from wild-type strain Newman and an arl deletion mutant from 18 h cultures using real-time RT-PCR. We found that the mgrA mRNA was reduced 18-fold in the arlR mutant (data not shown), indicating that mgrA is positively regulated by the arl locus. On the other hand, the amount of arlR mRNA from an mgrA mutant was similar to that from the wild-type, indicating that mgrA has no significant effect on arl expression. These results suggest that arl may up-regulate cap5 through up-regulation of mgrA.
To further confirm that arl functions upstream of mgrA with respect to cap5 gene regulation, we measured the cap5 mRNA by real-time RT-PCR to determine whether overproducing MgrA can compensate cap5 expression in an arlR mutant and, conversely, whether overproducing ArlR can compensate cap5 expression in an mgrA mutant. The rationale for these experiments is that overproduction of MgrA, but not overproduction of ArlR, should restore the arl effect on capsule if arl is upstream of mgrA in the regulatory circuit. On the other hand, a reverse effect should be detected if arl acts downstream of mgrA. To this end, we cloned the mgrA or arlR gene under the control of the IPTG-inducible promoter Pspac in pCL15. The results in Fig. 4(a) show that MgrA overproduction was able to restore the cap5 mRNA in the arl mutant to the wild-type level, whereas the results in Fig. 4(b) show that ArlR overproduction did not restore the cap5 mRNA level in the mgrA mutant. These results support the contention that arl functions upstream of the mgrA.
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, for both transcriptional and translation fusions, mutations in mgrA or arlR substantially reduced the cap5 promoter activity, and the double mutation reduced the activity more than the single mutations. The additional reduction in cap5 promoter activities at various time points in the double mutant as compared to that of the mgrA single mutant are statistically significant, with P values (paired t test) ranging from 0.0005 to 0.0135 for the transcriptional blaZ assays (Fig. 5a) and from 0.0029 to 0.021 for the translational blaZ assays (Fig. 5b). This reduction of the cap5 promoter activity resulting from arl mutation in the mgrA mutant suggests that arl also regulates cap5 independently from mgrA. However, although the reduction is statistically significant, it is only moderate, suggesting that the mgrA-independent pathway(s) may only play a minor role in cap5 regulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Fournier et al., 2001), but how it regulates capsule has not been studied. Our results showed that arl positively controlled the production of capsule. These results are consistent with a recent gene profiling study showing that two of the cap5 genes were upregulated by arl (Liang et al., 2005) and with a study showing that a natural deletion in the arlRS locus of the sequenced N315 strain affected CP5 production (Cocchiaro et al., 2006). The arl locus is composed of two genes, arlS and arlR, encoding the sensor and regulator, respectively, of a typical prokaryotic two-component regulatory system in which the sensor senses an environmental cue(s) and relates the information to the response regulator, usually, by phosphorylation (Klumpp & Krieglstein, 2002). However, to date, no environmental cue has been reported to be mediated by ArlS. Several environmental factors, which include iron concentration, oxygen tension, carbon dioxide and growth media, have been reported to influence capsule production (Lee & Lee, 2006; O'Riordan & Lee, 2004). The environmental control of virulence gene expression in S. aureus is an understudied area. It would be of interest to determine if the signal of any of these environmental factors affecting capsule production is transduced through the arl system.
The expression of virulence genes in S. aureus is controlled by very complicated regulatory circuitry (Bronner et al., 2004; Cheung et al., 2004; Novick, 2003) in which various regulators form a complex network to coordinately regulate different virulence genes. With regard to capsule, we have previously shown that both agr and mgr positively regulate capsule production mostly at the transcriptional level, whereas sarA does not play a role in transcriptional regulation but may have a minor effect at the post-translational level (Luong et al., 2002, 2003). In this study, we found that arl played an activator role, exerting its effect at the transcriptional level, similar to that of agr and mgr. However, how arl regulates its target genes has not been studied. Since arl is a two-component system, it is likely that ArlR binds some target genes directly. Although our gel mobility shift experiments showed that both the unphosphorylated and phosphorylated forms of ArlR could bind to the cap5 promoter region, the concentrations of the protein at which the binding occurred were most likely too high to be within the physiological condition. Hence the binding may not be biologically significant. Our results, therefore, suggest that ArlR most likely does not regulate capsule production by direct binding to the cap5 promoter region.
As the gel-shift experiments indicated that arl probably regulates capsule through other regulators, to test this possibility we investigated whether arl affected mgrA expression. We found that mgrA expression was drastically reduced (18-fold) in an arlR deletion mutant, whereas mgrA mutation did not affect the expression of arlR, indicating that arl activates mgrA but not the opposite. We also showed that mgrA expressed from an inducible promoter could override the arl effect on cap5 genes but not vice versa, suggesting that arl functions upstream of mgrA in activating capsule. These results therefore strongly indicate that arlR activates cap5 genes through mgrA. Interestingly, further experiments using gel-shift assays did not find evidence of ArlR binding to the mgrA promoter or MgrA binding to the cap5 promoter (results not shown), suggesting that intermediary regulators exist upstream and downstream of mgrA in the arlRS-mgrA-cap5 regulatory pathway.
It should be noted here that our quantitative RT-PCR results showing that mgrA did not affect arl expression are inconsistent with those reported from Ingavale et al. (2003) and Manna et al. (2004). In these two previous reports, MgrA was first shown to activate arl expression by direct binding to the arl promoter region (Ingavale et al., 2003) but was later reported to indirectly repress arl through sarV (Manna et al., 2004). Because these two studies used the same strain (the sigB-deficient RN6390) and the results are contradictory, it is difficult to draw a firm conclusion as to how mgrA regulates arl based on the studies. On the other hand, since the sigB-positive strain Newman was used in our studies, it is conceivable that the discrepancy between our results and those from the two reports could be explained by strain difference.
In this study, besides arl, we also identified five additional potential regulatory loci affecting capsule gene expression. Our preliminary data showed that two of these newly identified regulators, sbcDC and clpC, were also involved in capsule gene regulation. In addition to mgrA and agr, recent reports showed that sae and sigB were also involved in capsule regulation (Steinhuber et al., 2003; Bischoff et al., 2004). Taken together, these results indicate that capsule is under the regulation of a surprisingly large number of regulators. One interesting question to ask would be why capsule regulation is regulated by so many regulators. Capsular polysaccharide is a large molecule that resides on the surface of the bacteria. Because of its size, capsule anchored on the cell surface is an important defence shield for the bacteria to avoid host phagocytosis by masking the C3b deposited on the bacterial cell wall from the phagocytes (Cunnion et al., 2003; Karakawa et al., 1988; Thakker et al., 1998). However, for the same reason, it can also mask other surface components required for staphylococcal pathogenesis. Indeed, it has been shown that capsule impedes the initial attachment of S. aureus to endothelial cells by masking the adhesins (Pohlmann-Dietze, 2000). Thus, the organism needs to regulate capsule production according to requirements at various stages of the infection process. S. aureus can cause various types of infection at various sites of the host; it is therefore not unreasonable to postulate that capsule is regulated differently in different infections. The need for the organism to fine-tune capsule production under different conditions may explain why capsule is regulated by several regulators. The impressive number of regulatory genes involved in capsule regulation further supports the notion that virulence genes in S. aureus are regulated by a complex regulatory network.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 30 May 2006;
revised 9 July 2006;
accepted 17 July 2006.
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T. T. Luong, M. G. Lei, and C. Y. Lee Staphylococcus aureus Rbf Activates Biofilm Formation In Vitro and Promotes Virulence in a Murine Foreign Body Infection Model Infect. Immun., January 1, 2009; 77(1): 335 - 340. [Abstract] [Full Text] [PDF]
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C. D. Majerczyk, M. R. Sadykov, T. T. Luong, C. Lee, G. A. Somerville, and A. L. Sonenshein Staphylococcus aureus CodY Negatively Regulates Virulence Gene Expression J. Bacteriol., April 1, 2008; 190(7): 2257 - 2265. [Abstract] [Full Text] [PDF]
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Z. Chen, T. T. Luong, and C. Y. Lee The sbcDC Locus Mediates Repression of Type 5 Capsule Production as Part of the SOS Response in Staphylococcus aureus J. Bacteriol., October 15, 2007; 189(20): 7343 - 7350. [Abstract] [Full Text] [PDF]
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S. Meier, C. Goerke, C. Wolz, K. Seidl, D. Homerova, B. Schulthess, J. Kormanec, B. Berger-Bachi, and M. Bischoff {sigma}B and the {sigma}B-Dependent arlRS and yabJ-spoVG Loci Affect Capsule Formation in Staphylococcus aureus Infect. Immun., September 1, 2007; 75(9): 4562 - 4571. [Abstract] [Full Text] [PDF]
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