Overproduction of Type 8 Capsular Polysaccharide Augments ...

INFECTION AND IMMUNITY, July 2002, p. 3389–3395 0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.7.3389–3395.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 70, No. 7

Overproduction of Type 8 Capsular Polysaccharide Augments Staphylococcus aureus Virulence Thanh T. Luong and Chia Y. Lee* Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160 Received 5 December 2001/Returned for modification 6 March 2002/Accepted 25 March 2002

Type 8 capsular polysaccharide (CP8) is the most prevalent capsule type in clinical isolates of Staphylococcus aureus. However, its role in virulence has not been clearly defined. CP8 strains such as strain Becker produce a small amount of capsule on their surface in vitro. In contrast, CP1 strains such as strain M produce a large amount of capsule, which has been shown to be an important antiphagocytic virulence factor. The cap8 and cap1 operons, required for the synthesis of CP8 and CP1, respectively, have been cloned and sequenced. To test whether CP8 contributes to the pathogenesis of S. aureus, we replaced the weak native promoter of the cap8 operon in strain Becker with the strong constitutive promoter of the cap1 operon of strain M. The resultant strain, CYL770, synthesized cap8-specific mRNA at a level about sevenfold higher than that in the parent strain. Remarkably, the CYL770 strain produced about 80-fold more CP8. In a mouse infection model of bacteremia, the CP8-overproducing strain persisted longer in the bloodstream, the liver, and the spleen in mice than the parent strain. In addition, strain CYL770 was more resistant to ospsonophagocytosis in vitro by human polymorphonuclear leukocytes. These results indicate that CP8 is an antiphagocytic virulence factor of S. aureus.

linked genes. Recently, we identified two more closely linked cap1 genes (16a). The cap1 genes form an operon that is transcribed by a strong promoter to produce a long transcript (19). The cap5 and cap8 gene clusters, required for the synthesis of CP5 and CP8, respectively, have also been cloned and sequenced (22). The cap5 and cap8 operons are allelic, whereas the cap1 locus is located at a different location in a staphylococcal chromosome cassette element (16a, 22). Twelve of the 16 genes in the cap5 and cap8 operons share a high degree of identity, reflecting the highly similar nature of the repeating units of CP5 and CP8. Like the cap1 genes, all 16 genes of the cap8 locus are transcribed as a large transcript from a major promoter upstream of the first gene, cap8A. Although several internal promoters within the cap1 and cap8 gene clusters have also been identified by genetic complementation and reporter gene fusion studies, these internal promoters are much weaker than the respective primary promoters (22, 24). The production of CP5 and CP8 has been shown to be regulated by various environmental cues, such as iron concentration, carbon dioxide, and in vivo growth, etc. (6, 14). 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 regulation (20). Recently, we showed that the expression of the cap8 genes was under the control of agr and sarA, the two most-studied global regulators in S. aureus (16a). On the other hand, studies on the transcription of cap1 genes suggest that the primary cap1 promoter is constitutively expressed. Gene fusion studies carried out with the native host strains revealed that the primary cap1 promoter is a very strong promoter, whereas the primary cap8 promoter is a much weaker one (19, 24). Since the role of CP8, and to a lesser extent that of CP5, in staphylococcal virulence is not fully defined, we sought a ge-

For many human pathogens, capsular polysaccharides (CPs) play an important role in bacterial evasion of host immune surveillance, thereby conferring virulence to the pathogens. In Staphylococcus aureus, more than 90% of the strains produce CPs. Among the 11 serotypes of CP identified to date, type 1 CP (CP1), CP2, CP5, and CP8, have been chemically characterized. The repeating units of CP5 and CP8 are almost identical except for the linkages between the amino sugars and the position of the O acetylation. Type 1 and type 2 strains produce a large quantity of capsule. These capsules have been shown to play an important role in virulence. However, type 1 and type 2 strains are rarely isolated clinically. In fact, ⬃53 and ⬃22% of clinical isolates produce CP8 and CP5, respectively. Type 5 and type 8 strains, on the other hand, produce a much smaller amount of capsule than type 1 or type 2 strains in vitro. The small amount of CP5 and CP8 produced by these strains has hampered efforts to define the role of these capsules in virulence. Indeed, several reports concluded that CP5 and CP8 did not confer virulence to S. aureus (see reference 13 for a review). However, recent studies using specific animal models and growth conditions to enhance CP production have shown that CP5 does play a role in the pathogenesis of S. aureus, most probably by evading bacterial uptake and killing by phagocytes (1, 18, 26). Similar experiments, however, have not been employed to assess the role of CP8 in virulence. The cap1gene cluster, which is required for the synthesis of CP1, has been cloned and sequenced (12, 15). Initial sequencing showed that the cap1 locus was composed of 13 closely * Corresponding author. Mailing address: Department of Microbiology, Molecular Genetics and Immunology, Room 3025, WHW, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. Phone: (913) 588-7156. Fax: (913) 588-7295. E-mail: [email protected]. 3389

3390

LUONG AND LEE

INFECT. IMMUN.

FIG. 1. Construction of CYL770. The plasmid pCL8142, constructed as described in the text, was used to replace the 141-bp region containing the cap8 promoter in the chromosome of strain Becker. The initial step of plasmid integration by homologous recombination was performed at 43°C with selection for tetracycline resistance. Excision of the integrated plasmid and subsequent curing of the excised plasmid were carried out at 30°C without selection. The sequence at the junction of cap1 promoter and the cap8A gene, with the cap8 sequences in boldface, is shown below the chromosome of the resultant strain, CYL770. Dashed lines indicate regions of homology.

netic approach to overexpress CP8 for assessing its role in virulence. In this study, we replaced the cap8 primary promoter with that of the cap1 operon. We showed that the CP8-overexpressing strain was more resistant to opsonophagocytosis by human polymorphonuclear leukocytes (PMNs) and persisted longer in the blood and tissues of mice than the isogenic wild-type strain in a murine bacteremia model. MATERIALS AND METHODS Strains, plasmids, and growth conditions. S. aureus Becker, a CP8-producing strain containing the cap8 locus (23), was used as the parent strain for constructing the CP8-overproducing strain. S. aureus M, a CP1-producing strain containing the cap1 locus, was used as the source for the cap1 promoter (12). S. aureus RN4220 (11) was used as the recipient in electroporations of the constructed plasmids. Escherichia coli strain XL1-Blue was used as a host strain for plasmid constructions. S. aureus strains were cultivated in Trypticase soy medium (Difco Laboratories, Detroit, Mich.). E. coli strains were cultivated in Luria-Bertani medium (Difco Laboratories). Electroporation in S. aureus was carried out by the procedure of Kraemer and Iandolo (10). Transduction was carried out as described previously, using bacteriophage 52A (12). Plasmid vector pCL52.1 has been previously described (15). DNA manipulations. Standard DNA manipulations were performed as described by Sambrook et al. (21). Plasmid DNA was purified with a plasmid purification kit (Qiagen, Inc., Chatsworth, Calif.). Rapid small-scale plasmid DNA purification from E. coli was done by the method of Holmes and Quigley (8). Bulk chromosomal DNA from S. aureus was purified with a chromosomal DNA purification kit (Promega, Madison, Wis.). PCR amplification was carried out with the Advantage cDNA PCR kit (Clontech, Palo Alto, Calif.). The transfer of DNA to nitrocellulose membranes was by the method of Southern (25). Construction of the CP8-overexpressing strain CYL770. Figure 1 outlines the strategy for the construction of the CP8-overexpressing strain CYL770. We first constructed the plasmid pCL8142, which contained an insert of the 5⬘ control

region of the cap8 operon in which the cap8 promoter was replaced by the cap1 promoter, as shown in Fig. 1. The DNA fragment containing the 5⬘ portion of the cap8 operon under the control of the cap1 promoter (i.e., Pcap1::cap8) was constructed by the two-step overlapping PCR technique as described by Higuchi (7). The sequences of the primers used are given in Table 1. In the first step, two PCR fragments were obtained: a 250-bp fragment containing the cap1 promoter was amplified by using primers Ppa1fNcoI and Ppa1r, and an 852-bp fragment containing the cap8A gene and a partial cap8B gene was amplified by using primers Ppa8af7 and Ppa8ar1. In the second step, the two PCR fragments from the first step were annealed (Ppa1r and Ppa8af7 are complementary to each other) and used as a template for a second PCR amplification by Ppa1fNcoI (with an NcoI adaptor) and Ppa8ar1 (with a BamHI adaptor). The resultant 1,069-bp NcoI-BamHI fragment, which contains the cap1 promoter fused to the cap8 operon at the ATG start codon of cap8A, was cloned into the pGEM-T vector (Promega) and verified by sequencing. This fragment was then joined at the NcoI site with the 779-bp fragment containing the upstream sequence of the cap8 promoter obtained by PCR amplification using the primers Ppa8f8 (with an EcoRI adaptor) and Ppa8r2 (with an NcoI adaptor). Finally, the resulting 1,839-bp fragment was recloned into the BamHI and EcoRI sites of the temperature-sensitive E. coli-S. aureus shuttle vector pCL52.1 to generate the plasmid pCL8142, which was electroporated into strain RN4220 and subsequently transduced to strain Becker at 30°C. Strain Becker(pCL8142) was then used for constructing the Pcap1::cap8 strain CYL770 by temperature shift experiments as described before (15). The resultant strain was verified by PCR amplification (not shown). Mouse infection studies. CD-1 female mice, 7 to 8 weeks old, were obtained from Harlan (Indianapolis, Ind.). Groups of five mice were inoculated intraperitoneally with ⬃2 ⫻ 106 CFU/mouse in 0.2 ml of phosphate-buffered-saline. Blood samples were collected in heparinized tubes from the tail veins of separate groups of animals at various time points up to 4 h after inoculation. Bacterial counts were obtained by the plate count method on Trypticase soy agar. After 24 and 48 h, groups of five mice were sacrificed and tissue from the liver and spleen was collected, weighed, minced, diluted with phosphate-buffered saline, and plated on agar plates for bacterial counts.

S. AUREUS CAPSULE AUGMENTS VIRULENCE

VOL. 70, 2002

3391

TABLE 1. Primers used in this study Sequencea

Primer

Ppa8r2..................................................................................................................5⬘CCA TGG CTC TAA AGT AGT AAT ATT TTG3⬘ Ppa8f8 ..................................................................................................................5⬘GAA TTC GAG TCT ACA AGC GAT TAA A3⬘ Ppa8ar1................................................................................................................5⬘CCG GGA TCC AGA AGT AAC CAA CAA GCG3⬘ Ppa8af7 ................................................................................................................5⬘AAA TAG GAG GTT TAT ATG GAA AGT ACA TTA GAA TTA3⬘ Ppa1fNcoI ...........................................................................................................5⬘CGG CCA TGG CCA CAG TAT AAA TTA TAT CAG3⬘ Ppa1r....................................................................................................................5⬘TTCTAA TGT ACT TTC CAT ATA AAC CTC CTA TTT TCC3⬘ a Italic indicates a complementary pair. Restriction sites are underlined, as follows: for Ppa8r2, NcoI; for Ppa8f8, EcoRI; for Ppa8ar1, BamHI; and for Ppa1fNcoI, NcoI.

In vitro phagocytosis assay. The in vitro opsonophagocytosis assay was performed essentially as described by Kawakawa et al. (9). Briefly, human PMNs were obtained by resolving 8 to 10 ml of fresh blood collected from healthy adult volunteers in Mono-Poly resolving medium (ICN Biomedicals, Inc., Aurora, Ohio) according to the manufacturer’s instruction. The PMNs were resuspended in RPMI with 5% heat-inactivated fetal bovine serum and adjusted to 107 cells/ml. Human serum used as the complement source was prepared from blood collected from healthy adults by centrifugation of the clotted blood. Antibodies present in the serum were then absorbed by S. aureus Becker for 24 h. Bacterial cells used for the tests were prepared by pelleting and resuspending overnight bacterial cultures in RPMI plus 5% fetal bovine serum. The final mixture for the assay consisted of 3 ⫻ 106 PMNs, 20% serum, and 1 ⫻ 106 bacterial cells in a total volume of 0.5 ml. Control experiments without PMNs or complement were also performed. All tubes were rotated slowly at 37°C, and samples were plated out at the 45-, 60-, 90-, and 120-min time points. Other tests. Northern slot blotting was performed as described previously (16). The XylE enzymatic assay was carried out essentially as described by Zulowski et al. (28). To quantitate the production of CP8, the optical density at 660 nm (OD660) was measured from 18-h cultures grown in Trypticase soy broth before centrifugation. The cell-associated CP8 was isolated by autoclaving the cell pellet as described before (16). The CP8 released to the medium was treated with proteinase K (Sigma, St. Louis, Mo.) at 50 ␮g/ml for 1 h at 37°C, followed by heat inactivation at 75°C for 10 min. Serial dilutions of equivalent amounts of crude CP8 preparations (i.e., extracted from equivalent amounts of cells according to OD660) were loaded onto a nitrocellulose membrane in a dot blot apparatus. The CP8 was detected by the immunological detection method as described previously by using a CP8-specific rabbit antiserum (16) kindly provided by Ali Fattom, Nabi, Rockville, Md. Statistical analyses. Data from animal infection studies and opsonophagocytic assays were assessed with a two-tailed unpaired Student t test for comparisons between means. P values of ⬍0.05 were considered to be statistically significant.

RESULTS Construction and characterization of strain CYL770. To obtain a CP8-overexpressing strain, S. aureus Becker was genetically manipulated so that the cap8 operon in the chromosome could be transcribed by a strong constitutive promoter. To this end, a DNA fragment containing the 5⬘ control region of the cap8 operon was manipulated so that the original cap8 promoter was replaced with the cap1 promoter from S. aureus M. The engineered fragment was cloned into the temperaturesensitive plasmid pCL52.1 to form pCL8142 as described in Materials and Methods. Before proceeding to allele replacement, to ensure that the engineered DNA fragment containing the Pcap1::cap8 fusion was functional and was stronger than the natural cap8 promoter, we recloned the insert from pCL8142 into the xylE reporter vector pSL24. The resultant plasmid, pCL8123, was transferred to strain Becker, and the promoter activity of Becker(pCL8123) was compared with that of Becker containing pCL8074 (the natural cap8 promoter fused with the xylE reporter in pSL24 [24]). The results from three independent measurements showed that the XylE activities of Becker(pCL8123) and Becker(pCL8074) were 11.70 ⫾

0.72 and 1.75 ⫾ 0.27 mU/mg of protein, respectively. Thus, the Pcap1::cap8 fusion was about sevenfold higher in promoter strength than the natural cap8 promoter, indicating that the cap1 promoter is functional in the strain Becker genetic background. The plasmid pCL8142 was then used to construct strain CYL770 by employing the allele replacement technique. To assess the transcription of cap8 genes under the control of cap1 promoter in strain CYL770, we performed Northern slot blotting. The results showed that the level of the cap8 transcript from CYL770 was about four- to eightfold higher than that from the wild-type strain Becker (an example is shown in Fig. 2A). This increase was comparable to that measured by the plasmid reporter gene fusion method as described above. To compare the amounts of CP8 produced by the wild type and CYL770, both cell-associated and released CP8 from liquidgrown cultures were quantitated by the immuno-dot blotting method. Three independent measurements were carried out. An example of one of the experiments was shown in Fig. 2B. Comparing to the wild-type strain, strain CYL770 produced about 80-fold more CP8 in both the cell-associated and released forms. It is noteworthy that the amount of released CP8 was about 100 times larger than that of cell-associated CP8 (Fig. 2B) (note that the amount of CP8 loaded in the supernatants was about 14 times less than that in the pellets). This ratio between the cell-associated and released forms of CP8 was similar to that in an earlier report (14). The overproduction of CP8 in CYL770 could also be demonstrated on solid agar plates. As shown in Fig. 2C, CYL770 had a larger colony size and more mucoid appearance on Trypticase soy agar plates than the wild type. Together, these results indicate that the engineered strain CYL770 indeed constitutively produces much more CP8 than the wild-type strain in vitro. Virulence studies. To test whether overproduction of CP8 affected the virulence of the bacteria, we employed a murine bacteremia model by injecting groups of five mice intraperitoneally with either strain Becker or CYL770. The actual inocula were 2.17 ⫻ 106 and 1.87 ⫻ 106 CFU for strains Becker and CYL770, respectively. The number of bacteria in the blood was assayed at various time points up to 4 h as described in Materials and Methods. The results in Fig. 3 show that there was about a 1-log-unit difference between the wild type and CYL770 at 10 min after inoculation. These results were consistent with those reported by Thakker et al. (26), suggesting that the wild-type strain with a small amount of capsule was cleared from the blood much more rapidly than the capsuleoverproducing strain. In addition, we found that there were statistically significantly higher concentrations of the CYL770

3392

LUONG AND LEE

INFECT. IMMUN.

FIG. 3. Bacterial counts from blood samples from five mice at various time points after injection. The values are means of log10 CFU per milliliter of blood sample. Error bars represent standard errors of the means.

FIG. 2. Comparison of strains Becker and CYL770. (A) Expression of cap8-specific mRNA. Total RNA was purified from overnight cultures. Samples, twofold serial diluted, were loaded onto nitrocellulose membranes and probed with the cap8D gene (top panel). A duplicate set of samples was probed with the S. aureus 16S rRNA gene (bottom panel) to indicate equal loading of the samples. (B) Analysis of CP8 production. Equivalent amounts of samples adjusted according to OD660 were taken from overnight cultures. Samples, threefold serial diluted (dil), were loaded onto nitrocellulose membranes and incubated with rabbit anti-CP8 specific antibody. Twofold serial dilutions

strain than of the wild-type strain in the blood at all time points (P values ranged from 0.001 to 0.042), except for the 3-h time point. These results suggest that there were more bacterial cells of CYL770 than of the wild-type Becker strain disseminated from the peritoneal cavity to the bloodstream. The slight increase in the number of CYL770 cells from the time of inoculation to 2 h postinjection also suggested that the CP8 overproducer might have multiplied in the bloodstream to some extent before it was eventually cleared from the bloodstream. In contrast, no such increase in number was observed for the wild-type strain. Groups of five mice were also sacrificed at 24 and 48 h to test bacterial dissemination and clearance in tissues (Fig. 4). At 24 h after inoculation, strain CYL770 had significantly higher bacterial loads than the wild type in both liver (P ⫽ 0.0479) and spleen (P ⫽ 0.0159). After 48 h of inoculation, almost no wild-type bacteria were recovered from liver, whereas about 6 CFU of CYL770 per mg of tissue were recovered, although no statistical differences were found between the two groups (P ⫽ 0.0873). From the spleen tissue at 48 h, more CYL770 than Becker bacterial cells were recovered (P ⫽ 0.0426). These results suggest that the CP8-overproducing CYL770 is more resistant to clearing from mouse tissues. The remaining 40 mice, of which half were inoculated with Becker and half were inoculated with CYL770, were observed for up to 7 days. We found that none of them showed signs of sickness, and all of these mice survived at day 7 after inocula-

of CP8 standard with an initial amount of 200 ng were loaded as controls. The CP8 from cell pellets was 14-fold more concentrated than that from the supernatants according to the OD660 of the cultures. (C) Colony morphology, showing the size and appearance of the strains after incubation at 30°C for about 40 h.

VOL. 70, 2002


3393

FIG. 5. Bacterial counts from in vitro phagocytosis assays. The values are means of log10 CFU per milliliter of reaction mixture. Results represent averages from at least three independent experiments. Error bars indicate standard errors of the means.

FIG. 4. Bacterial counts from spleen and liver tissues from five mice at 24 and 48 h after injection. The values are means of CFU per milligram of tissue sample. Error bars represent standard errors of the means.

tion. These results showed that the inoculum of about 2 ⫻ 106 CFU was below the lethal dosage even for the CP8-overproduing strain CYL770, suggesting that the 50% lethal dose of CYL770 is much higher than that of the wild type. In vitro phagocytosis assay. The results described above indicate that the large amount of capsule produced from S. aureus Becker conferred protection for the bacteria from clearance in the bloodstream and tissues. One mechanism that capsule contributes to bacterial virulence is resistance to opsonophagocytic killing by PMNs. To test whether the overproduced CP8 in strain CYL770 can provide this mechanism of protection for the bacteria from PMNs, we compared strain CYL770 and the wild-type strain Becker by in vitro phagocytosis assays. Bacteria were incubated with PMNs and normal human sera as the complement source at 37°C for various lengths of time. Since normal human sera from human volunteers may contain antibodies to S. aureus cell wall components such as CP8, we extensively absorbed the sera with strain Becker before the assays to preclude possible interference from anti-CP8 antibodies. As shown in Fig. 5, the number of cells of the wild-type strain was reduced from 6.6 to 5.2 log10 units after incubation with human PMNs for 45 min, and the number remained at about the same level for up to 2 h of incubation. In contrast, the number of cells of strain CYL770 was reduced from 6.6 to 5.9, 5.8, 6.0, and 6.1 log10 units at 45, 60, 90, and 120 min, respectively, after incubation with PMNs. The differences between the two strains were statistically significant (P ⱕ 0.0098) at all time points except 45 min (P ⫽ 0.0644) as analyzed by Student’s t test. In the control experi-

ments in which either PMNs or sera were excluded from the incubation mixture, the bacterial counts for both strains increased steadily to about 7 log10 units at 120 min under the same incubation conditions (results not shown). Thus, these results showed that strain CYL770 was more resistant to opsonophagocytosis by human PMNs than the wild-type strain, indicating that CP8 contribute to S. aureus virulence by increasing the antiphagocytic activity of the bacteria. DISCUSSION CP5- and CP8-producing strains of S. aureus do not produce a large amount of capsule when cultivated in broth media in vitro. It is thus difficult to assess the role of these capsules in virulence, because a suitable in vivo animal model has not been developed for detecting such a small difference in capsule production between the wild type and the capsule-deficient mutants. As a result, several early studies had erroneously concluded that CP5 and CP8 were not virulence factors (13). However, growth of CP5 strains on solid agar media has been shown to increase CP5 production about 100-fold over that in liquid-grown cultures (26). This property had prompted researchers to reexamine the functional role of CP5 in virulence by using CP5 strains grown in agar plates as the inocula in several animal models of infection, including bacteremia and septic arthritis (1, 18, 26). These studies demonstrated that the increase in CP5 production from agar-grown cultures correlated with increased virulence of the strains. Likewise, CP8 production from strain Becker was shown to be enhanced more than 300-fold from cultures grown in solid agar media compared to those grown in liquid broth. However, the drastic increase in CP8 production was not shown to have an effect on virulence in a mouse lethality model (14). Animal models with higher sensitivity than the mouse lethality model, such as those used for testing virulence of agar-grown CP5 strains, have not

3394

LUONG AND LEE

been used to assess the virulence of agar-grown CP8 strains. In this study, we took a molecular genetic approach to alter the promoter strength of the cap8 operon in the chromosome to achieve high production of capsule for assessing the role of CP8 in S. aureus pathogenesis. Our data showed that overproduction of CP8 augmented the virulence of S. aureus in a murine bacteremia model in which the bacteria were inoculated intraperitoneally and were assayed for their ability to disseminate to and survive in the bloodstream and tissues. Our results suggest that CP8 helps the bacteria to escape the host immune surveillance as the bacteria disseminate into the bloodstream and tissues. We also showed that the CP8-overproducing strain was more resistant to opsonophagocytic killing by human PMNs in the presence of complement than the wild-type strain, indicating that the overproduced CP8 contributes to the virulence of the bacteria by evading phagocytosis. Thus, our results showed that like CP1 and CP5, CP8 was also an antiphagocytic virulence factor of S. aureus. In this study, we chose the cap1 promoter for replacing the cap8 promoter to increase the CP8 production of strain Becker, because we had shown that the cap1 promoter is a strong promoter responsible for the high production of CP1 in S. aureus M (19). Furthermore, the cap1 promoter is constitutive, thereby avoiding the need for induction with exogenous inducers during the in vivo testing of virulence. Previously, we showed that the strength of the cap1 promoter was about 60- to 80-fold higher than that of the cap8 promoter when the tests were carried out in the respective natural hosts by using xylE reporter fusion (19, 24). However, the results of the xylE fusion experiments in this study showed that upon fusing to the cap8 operon, the cap1 promoter exhibited only about a sevenfold increase in activity over the native cap8 promoter. The Pcap1::cap8 in the chromosome of CYL770 also showed a similar degree of increase in promoter activity over the wild type as measured by Northern slot blotting (Fig. 2A). This much-less-than-expected increase could be due to differences in genetic background between strains M and Becker. It is also possible that a negative cis-acting regulatory element is located downstream of the cap8 promoter within the coding region of the 5⬘ end of the cap8 operon. Examples of a cis-acting element within the coding region have been reported for other bacteria (3, 4, 17). Indeed, our preliminary study showed that deletions of this region increased the Pcap1::cap8 promoter activity. However, further studies are required to confirm such a possibility. Although the promoter activity assayed by xylE reporter fusion and by Northern blotting showed only about a 7-fold increase in CYL770, the capsule quantification results showed about an 80-fold increase in CP8 production in liquid cultures (Fig. 2B). Attempts to estimate the production of CP8 of CYL770 on agar plates failed because the cells severely clumped together, which resulted in difficulty in accurately measuring the CP8 production. The reason for the discrepancy in increase of the promoter activity and that of the CP8 production from CYL770 is unknown. CP8 is a complex carbohydrate whose synthesis requires a number of enzymes in a series of reactions. It is possible that one or more enzymes involved in the biosynthetic pathway are rate limiting and that their enzymatic activities could be greatly increased once the concentration of the enzymes is above a certain threshold level, which could lead to high-level production of CP8.

INFECT. IMMUN.

Although overproduction of CP8 could be achieved using the agar growth method, similar to that used for virulence studies of CP5, we believe that the molecular genetic approach that we employed in this study to achieve CP8 overproduction had additional advantages. First, since strain CYL770 produced a high quantity of CP8 independent of growth conditions, we were able to perform the experiments by growing the isogenic pair of strains under the exact same growth conditions. Growing the control and mutant strains under the same conditions was essential for preventing possible effects on the virulence tests from other cellular components whose expression could be affected under different growth conditions. For example, different cell surface proteins have been observed in S. aureus grown on solid versus liquid media (2, 26). Second, since the cap1 promoter is a constitutive promoter (19), strain CYL770 is likely to produce CP8 at a constant amount throughout the in vivo tests. In contrast, capsule produced by agar-grown cultures would not be consistently maintained due to varied environmental factors in vitro and in vivo, which would likely complicate the analyses. Type 1 strains such as S. aureus M produce a large amount of capsule. It has been shown that the thick capsule is able to shield the deposited C3b complement on the cell wall from recognition by the phagocytes, thereby rendering the cells resistant to phagocytosis (27). Most recently, the degree of deposition of C3b and iC3b has been shown to be correlated with the amount of capsule produced in CP5 strain Reynolds and in CP1 strains (5). Since our data showed that CP8 was antiphagocytic, it is most likely that CP8 also affected C3b and iC3b deposition on the cell wall of S. aureus cells. Thus, it appears that all staphylococcal capsules may contribute to the virulence of S. aureus by the same antiphagocytic mechanism. In this regard, one would expect that if a type 8 strain produced CP8 to the same extent as the CP1 produced by type 1 strains, the CP8 strain would be as virulent as the type 1 strains. However, an injection of 2 ⫻ 106 CFU of strain CYL770 was not lethal to mice whereas the 50% lethal doses of CP1 strains such as M or SA1 were about 4 ⫻ 104 CFU, even though CYL770 produced about 80-fold more CP8 than Becker whereas strain M was estimated to produce about 10- to 60fold more capsule than strain Becker (14). In view of this, it is possible that differences in biological properties between CP1 and CP8 may contribute to such a disparity. However, it is also possible that the amount of CP1 produced by type 1 strains has been underestimated. On this note, we found that although the colonies of CYL770 were more mucoid than those of Becker (Fig. 2C), the degree of mucoidy was much less than that of strain M. If the degree of colony mucoidy is an indication of the amount of capsule, this would suggest that type 1 strains might produce much more CP1 than the CP8 produced from CYL770. ACKNOWLEDGMENTS We thank Kelly Bush for excellent technical supports and Shu Ouyang for assisting in construction of pCL8142. This work was supported by grant AI37027 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Bhasin, N., A. Albus, F. Michon, P. J. Livolsi, J.-S. Park, and J. C. Lee. 1998. Identification of a gene essential for O-acetylation of the Staphylococcus aureus type 5 capsular polysaccharide. Mol. Microbiol. 27:9–21.

VOL. 70, 2002 2. 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. 3. Chugani, S. A., M. R. Parsek, and A. M. Chakrabarty. 1998. Transcriptional repression mediated by LysR-type regulator CatR bound at multiple binding sites. J. Bacteriol. 180:2367–2372. 4. Cowan, J. M., M. L. Urbanowski, M. Talmi, and G. V. Stauffer. 1993. Regulation of the Salmonella typhimurium metF gene by the MetR protein. J. Bacteriol. 175:5862–5866. 5. Cunnion, K. M., J. C. Lee, and M. M. Frank. 2001. Capsule production and growth phase influence binding of complement to Staphylococcus aureus. Infect. Immun. 69:6796–6803. 6. Herbert, S., S. W. Newell, C. Y. Lee, K. P. Wieland, B. Dassy, J. M. Fournier, C. Wolz, and G. Döring. 2001. Regulation of Staphylococcus aureus type 5 and type 8 capsular polysaccharides by CO2. J. Bacteriol. 183:4609–4613. 7. Higuchi, R. 1989. Using PCR to engineer DNA, p. 61–70. In H. A. Erlich (ed.), PCR technology. Stockton Press, New York, N.Y. 8. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193–197. 9. Kawakawa, W. W., A. Sutton, R. Schneerson, A. Karpas, and W. F. Vann. 1988. Capsular antibodies induce type-specific phagocytosis of capsulated Staphylococcus aureus by human polymorphonuclear leukocytes. Infect. Immun. 56:1090–1095. 10. Kraemer, G. R., and J. J. Iandolo. 1990. High-frequency transformation of Staphylococcus aureus by electroporation. Curr. Microbiol. 21:373–376. 11. Kreiswirth, B. N., S. Lofdahl, M. J. Betley, M. O’Reilly, P. M. Shlievert, M. S. Bergdoll, and R. P. Novick. 1983. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature (London) 305:709–712. 12. Lee, C. Y. 1992. Cloning of genes affecting capsule expression in Staphylococcus aureus strain M. Mol. Microbiol. 6:1515–1522. 13. Lee, C. Y. 2001. Capsule production, p. 35–47. In A. Honeyman, H. Friedman, and M. Bendinelli (ed.), Staphylococcus aureus infection and disease. Klumer Academic/Plenum Publishers, New York, N.Y. 14. Lee, J. C., S. Takeda, P. J. Livolsi, and L. C. Paoletti. 1993. Effects of in vitro and in vivo growth conditions on expression of type 8 capsular polysaccharide by Staphylococcus aureus. Infect. Immun. 61:1853–1858. 15. Lin, W. S., T. Cunneen, and C. Y. Lee. 1994. Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus. J. Bacteriol. 176:7005–7016. 16. Luong, T., S. Sau, M. Gomez, J. C. Lee, and C. Y. Lee. 2002. Regulation of

Editor: E. I. Tuomanen


3395

Staphylococcus aureus capsular polysaccharide expression by agr and sarA. Infect. Immun. 70:444–450. 16a.Luong, T. T., S. Ouyang, K. Bush, and C. Y. Lee. Type 1 capsule genes of Staphylococcus aureus are carried in a staphylococcal cassette chromosome genetic element. J. Bacteriol., in press. 17. Matsubara, K., K. Ohnishi, H. Sadanari, R. Yamada, and S. Fukuda. 2000. A portion of the nucleotide sequence corresponding to the N-terminal coding region of livJ is essential for its transcriptional regulation. Biochim. Biophys. Acta 1494:54–62. 18. Nilsson, I.-M., J. C. Lee, T. Bremell, C. Rydén, and A. Tarkowski. 1997. The role of staphylococcal polysaccharide microcapsule expression in septicemia and septic arthritis. Infect. Immun. 65:4216–4221. 19. Ouyang, S., and C. Y. Lee. 1997. Transcriptional analysis of type 1 capsule genes in Staphylococcus aureus. Mol. Microbiol. 23:473–482. 20. Ouyang, S., S. Sau, and C. Y. Lee. 1999. Promoter analysis of the cap8 operon involved in type 8 capsular polysaccharide production in Staphylococcus aureus. J. Bacteriol. 181:2492–2500. 21. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 22. Sau, S., N. Bhasin, E. R. Wann, J. C. Lee, T. J. Foster, and C. Y. Lee. 1997. The Staphylococcus aureus allelic genetic loci for serotype 5 and 8 capsule expression contain the type-specific genes flanked by common genes. Microbiology 143:2395–2405. 23. Sau, S., and C. Y. Lee. 1996. Cloning of type 8 capsule genes and analysis of gene clusters for the production of different capsular polysaccharides in Staphylococcus aureus. J. Bacteriol. 178:2118–2126. 24. Sau, S., J. Sun, and C. Y. Lee. 1997. Molecular characterization and transcriptional analysis of type 8 genes in Staphylococcus aureus. J. Bacteriol. 179:1614–1621. 25. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503–517. 26. Thakker, M., J. S. Park, V. Carey, and J. C. Lee. 1998. Staphylococcus aureus serotype 5 capsular polysaccharide is antiphagocytic and enhances bacterial virulence in a murine bacteremia model. Infect. Immun. 66:5183–5189. 27. Verbrugh, H. A., P. K. Peterson, B. T. Nguyen, S. P. Sisson, and Y. Kim. 1982. Opsonization of encapsulated Staphylococcus aureus: the role of specific antibody and complement. J. Immunol. 129:1681–1687. 28. Zulowski, M. M., D. G. Gaffiney, D. Speck, M. Kauffmann, A. Findeli, A. Wisecup, and J. P. Lecocq. 1983. Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proc. Natl. Acad. Sci. USA 80:1101–1105.