The cvfC operon of Staphylococcus aureus contributes to virulence via expression of the thyA gene

Microbial Pathogenesis 49 (2010) 1e7

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The cvfC operon of Staphylococcus aureus contributes to virulence via expression of the thyA gene Mariko Ikuo, Chikara Kaito, Kazuhisa Sekimizu* Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3-1, 7-Chome, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 January 2010 Received in revised form 13 March 2010 Accepted 19 March 2010 Available online 27 March 2010

The cvfC operon of Staphylococcus aureus comprises four genes, cvfC1, cvfC2, cvfC3, and cvfC4. The cvfC3disrupted mutant, M1262, produces less hemolysin and has attenuated virulence in silkworms and mice. Although introduction of the cvfC3 gene restores the decreased hemolysin production of M1262, the cvfC operon is more effective for the complementation, suggesting that cvfC operon function is impaired in M1262. In this study, we performed a microarray analysis and identified 21 genes with altered expression in M1262. The expression of virulence genes and metabolic genes, including thyA, was changed in M1262. The decreased expression of thyA in M1262 was restored by introducing a plasmid containing the cvfC operon. Introduction of a thyA gene-containing plasmid in M1262 restored hemolysin production and virulence against silkworms. Both M1262 and a thyA-deletion mutant exhibited slow growth in the presence of the detergent Triton-X 100. The growth defect of M1262 in the presence of Triton-X 100 was restored by the introduction of thyA. These results suggest that the cvfC operon contributes to hemolysin production, detergent resistance, and virulence of S. aureus in host animals via thyA expression. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Staphylococcus aureus Virulence Hemolysin thyA cvfC Detergent

1. Introduction Staphylococcus aureus is a human pathogenic bacterium that causes various diseases, including food poisoning, endocarditis, and toxic shock syndrome. The emergence of methicillin resistant S. aureus and vancomycin-resistant S. aureus is a serious clinical problem. S. aureus possesses many virulence factors such as exotoxins and defensive factors against host immune responses to facilitate their survival and growth in the host microenvironment. Understanding the regulatory mechanisms at the molecular level is important for establishing therapeutic strategies against S. aureus infection. S. aureus has many virulence regulators. The agr locus regulates the expression of virulence factors during growth by a quorum sensing mechanism [1]. Transcription factors, including Sar families and SigB, regulate the expression of virulence genes by sensing various signals [2,3]. SarS regulates the expression of hla that encodes a-hemolysin [4]. The expression of sarS is controlled by sar family genes, rot and sarT [5,6]. Two-component systems such as saeRS, srrAB, and arlRS regulate the expression of many genes by sensing some as-yet unidentified environmental signals [7e9].

* Corresponding author. Tel.: þ81 3 5841 4820; fax: þ81 3 5684 2973. E-mail address: [email protected] (K. Sekimizu). 0882-4010/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2010.03.010

Bacterial metabolic genes contribute to the virulence of S. aureus. The thyA gene, which encodes thymidylate synthase, contributes to the expression of hla and agr in S. aureus [10e12]. Small-colony variants of S. aureus, which require thymine or thymidine for growth, were isolated from cystic fibrosis patients. The hla expression was decreased and the expression of spaencoding protein A was increased in the small-colony variants. Changes in the expression of these genes were restored by introducing thyA or by adding thymidine to the culture medium. In Salmonella typhimurium, the thyA gene contributes to intracellular bacterial growth and virulence in mice [13,14]. In Shigella flexneri, the thyA mutant impairs intracellular growth [14]. Thus, the thyA gene contributes to bacterial virulence, but the regulatory mechanism of thyA expression itself is unclear. We previously identified three novel virulence genes of S. aureus using a silkworm infection model [15e19]. One of them, cvfC, is an operon that comprises four genes, SA1264, SA1263, SA1262, and SA1261, which we named here cvfC1, cvfC2, cvfC3, and cvfC4. Northern blot analysis indicated that the size of the transcript from the cvfC operon is 2.3 kb [15]. The previously isolated M1262 mutant contains a cvfC3 gene disruption (see below). M1262 produces less hemolysin and has attenuated virulence in silkworms and mice [15]. Here, we performed transcriptome analysis of M1262 and found that maintenance of thyA expression by the cvfC operon contributes to hemolysin production and detergent resistance in S. aureus.

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M. Ikuo et al. / Microbial Pathogenesis 49 (2010) 1e7

2. Results 2.1. Structure of the cvfC operon M1262 was constructed by integrational disruption of cvfC3 using a suicide vector [15]. To determine whether the M1262 phenotype that we previously observed is due to the impairment of the cvfC3 gene or to the impairment of the cvfC operon, we performed a complementation analysis. Introduction of the cvfC operon restored the decreased hemolysin production of M1262

(Fig. 1A and B). Introduction of the region containing either cvfC1cvfC2, cvfC3, or cvfC4 partially restored the decreased hemolysin production of M1262. The complementation activities of each region were less effective than that of the entire cvfC operon. These results suggest that the function of the cvfC operon is impaired in M1262, and that cvfC1-cvfC2, cvfC3, and cvfC4 each contribute to the function of the cvfC operon. An amino acid sequence with over 30% identity was found between S. aureus CvfC3, Bacillus subtilis YpgR, and its homologous proteins of Firmicutes (Table 1). The N-terminal amino acid sequence of S. aureus CvfC3 is highly similar to that of S. aureus CvfC2 (Fig. 1C(a)). The conserved amino acid sequences in CvfC3 and CvfC2 show similarity with the N-terminal amino acid sequences of Homo sapiens NFU1 and Brucella melitensis NifU, which are involved in cellular iron-sulfur cluster biosynthesis (Fig. 1A and C (a)). The ion-sulfur cluster is a cofactor of electron carriers, enzymes, and sensor proteins, such as ferredoxin. The C-terminal amino acid sequence of CvfC3 contains a phycobilisome lyase HEAT-like repeat, which shares similarity with phycobilisome lyase of Synechococcus (CpcE) (Fig. 1A and C(b)). There are a number of proteins that show similarity with CvfC4 (Table 1), although no motif has been identified in this protein. 2.2. Transcriptome analysis of M1262 Microarray analysis revealed 95 genes that were differentially expressed between M1262 and the parent strain (Supplemental Table 1). The altered expression of 21 of these genes was confirmed by quantitative RT-PCR analysis (Table 2). M1262 had decreased expression of virulence-associated genes, including RNAIII, which is transcribed from the agr locus and regulates the expression of virulence genes as an RNA molecule [1], capB encoding capsule synthetase, oppB encoding oligopeptide ABC transporter, hla encoding a-hemolysin; and increased expression of sarS, which encodes a transcription factor (Table 2). In addition, there was decreased expression of metabolic genes, including purF encoding phosphoribosylpyrophosphate amidotransferase, asd encoding aspartate semialdehyde dehydrogenase, thyA encoding thymidylate synthase, pflA encoding formate acetyltransferase activating Table 1 Conservation of CvfC1, CvfC2, CvfC3, and CvfC4 among bacteria.

Fig. 1. Structure of the cvfC operon. (A) Schematic representation of the cvfC operon and neighboring genes. Bold lines represent the region inserted into the plasmid. Dotted lines represent the region deleted from pcvfC. (B) The region containing cvfC1 and cvfC2, cvfC3, or cvfC4 partially complements the hemolysin production of M1262. M1262 was transformed with plasmids containing the cvfC1-cvfC2 region (pcvfC12), cvfC3 region (pcvfC3), cvfC4 region (pcvfC4), and the cvfC1-cvfC2-cvfC3-cvfC4 region (pcvfC). Overnight cultures of the mutants were examined for hemolytic activity using sheep red blood cells. *, p ¼ 0.1000; **, p ¼ 0.0356; ***, p ¼ 0.0352. (C) Motifs in CvfC2 and CvfC3. Alignment was performed using ClustalW software. Asterisks under amino acid sequences indicate identical residues and periods indicate similar residues within the aligned sequences. Uniprot ID of proteins were follows: CvfC2, Q7A5M5; CvfC3, Q99U59; Bacillus subtilis YpgR, P54169; Homo sapiens NFU1, Q9UMS0; Brucella melitensis NifU, C0RGJ6.

Phylum

Species

Acidobacteria

Solibacter usitatus Acidobacteria bacterium

Firmicutes

Anoxybacillus flavithermus Bacillus subtilis Geobacillus kaustophilus Oceanobacillus iheyensis Exiguobacterium sibiricum Lysinibacillus sphaericus Staphylococcus aureus Symbiobacterium thermophilum

Bacteroidetes

Flavobacterium johnsoniae Gramella forsetii Cytophaga hutchinsonii Salinibacter ruber Amoebophilus asiaticus

Chloroflexi

Chloroflexus aurantiacus Roseiflexus sp.

CvfC-1

CvfC-2

CvfC-3

CvfC-4 34 31

38 36

41 100

100

43 40 41 37 33 36 100

49 36, 52* 38, 49* 33 33 37, 45* 37, 100* 37 40 41 39 34, 40* 33

16 16

Amino acid identities (%) to S. aureus CvfC1, CvfC2, CvfC3, and CvfC4 indicated in bold letters are shown in each column. Asterisk indicates the existence of two homologous proteins to CvfC4. Proteins homologous with S. aureus CvfC1, CvfC2, CvfC3, and CvfC4 were searched for using the Microbial Genome Database for Comparative Analysis (http://mgbd.genome.ad.jp). The amino acid identities were calculated using ClustalW software.

M. Ikuo et al. / Microbial Pathogenesis 49 (2010) 1e7

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Table 2 Quantitative RT-PCR analysis of the altered gene expression by cvfC3-disruption in S. aureus. ORF No.

SA1007 SAS065 SA0145 SA0404 SA0520 SA1003 SA1935 SA0219 SA0232 SA0922 SA1226 SA1260 SA1272 SA0269 SA0272 SA0420 SA0769 SA0845 SA2133 SA0108 SA1196

Gene

hla RNAIII cap5B lpl8 sdrD hmrA pflA lctE purF asd thyA ald

oppB sarS

Fold

Function

RN4220

NCTC8325-4

Newman

2.2  1.0 5.7  4.9 4.1  1.3 7.3  3.5 2.3  0.6 3.0  1.4 2.2  0.3 5.2  0.9 4.8  2.8 1.7  1.3 4.1  1.9 5.0  1.6 9.7  1.7 3.0  0.3 7.3  1.3 1.7  0.6 2.5  0.7 4.1  1.4 3.4  0.4 þ4.1  1.4 þ15.0  2.4

þ1.4  0.5 þ1.0  1.7 1.1  0.4 1.5  0.4 þ2.1  0.3 þ1.4  0.3 1.1  0.2 þ1.6  0.2 þ1.3  0.4 2.5  0.2 þ1.7  1.5 14.4  0.0 þ1.7  0.3 1.4  0.5 1.2  0.2 þ1.6  1.1 þ1.9  1.0 þ1.2  1.0 þ1.0  1.7 þ3.8  0.2 þ3.3  1.6

þ1.2  0.2 þ1.0  0.1 þ1.0  0.5 þ1.2  0.2 ND þ1.4  0.2 1.1  0.3 1.3  0.1 3.3  0.5 3.6  0.1 þ1.1  0.3 5.2  0.1 þ1.1  0.9 ND 2.8  0.1 1.1  0.2 þ1.3  0.6 1.1  0.2 1.7  0.3 þ1.1  0.4 þ1.4  0.2

Alpha-hemolysin precursor RNAIII, delta-hemolysin Capsular polysaccharide synthesis Hypothetical protein, SaPIn2 Fibrinogen binding protein, bone sialoprotein-binding protein Similar to fibrinogen binding protein Similar to amidase Formate acetyltransferase activating enzyme L-lactate dehydrogenase Phosphoribosylpyrophosphate amidotransferase Aspartate semialdehyde dehydrogenase Thymidylate synthase Alanine dehydrogenase Hypothetical (Cyclin 1) Hypothetical (transmembrane protein) Hypothetical protein, similar to ABC transporter ATP-binding protein ABC transporter ATP-binding protein homologue Oligopeptide transport system permease protein Conserved hypothetical protein Staphylococcal accessory regulator A homologue Hypothetical protein, similar to DNA-damage repair protein

Gene expressions in the cvfC3-disrupted mutants of RN4220, NCTC8325-4, and Newman were measured by quantitative RT-PCR. The data are shown as means  standard deviations from three independent experiments. “Fold þ” indicates increased expression in the cvfC3-disrupted mutants, whereas “Fold -” indicates decreased expression in the cvfC3-disrupted mutants. “ND” indicates not determined.

enzyme, and lctE encoding L-lactate dehydrogenase (Table 2). The expression levels of many hypothetical genes were changed in M1262. Therefore, the cvfC operon affects the expression of various genes encoding virulence factors and metabolic enzymes. 2.3. The cvfC operon contributes to S. aureus hemolysin production and virulence against silkworms in a thyA-dependent manner Expression of thyA affects hemolysin production in S. aureus [10]. Because thyA expression was decreased in M1262, we hypothesized that the cvfC operon contributes to S. aureus virulence via the expression of thyA. The decreased expression of thyA in M1262 was restored by introducing a plasmid containing the cvfC operon (Fig. 2A), confirming that the cvfC operon contributes to thyA expression. We then examined whether the cvfC operon contributes to hemolysin production via thyA expression. Introduction of pNDX1thyA with an anhydrotetracycline-inducible promoter restored the decreased hemolysin production of M1262 (Fig. 2B). The restoration occurred with and without anhydrotetracycline, indicating that the transcription occurred without the inducer, consistent with previous findings [20]. These results suggest that the cvfC operon contributes to hemolysin production via thyA expression. We previously reported that M1262 has decreased virulence in silkworms [15]. To examine whether the cvfC operon contributes to the virulence of S. aureus in animals via thyA expression, we examined the virulence of both the thyA mutant and M1262 harboring a plasmid carrying the thyA gene in silkworms. The LD50 value of the parent strain at 47 h post infection was 1.6  107 CFU, whereas that of thyA mutant was 7.5  107 CFU, an amount 5 times greater than that of the parent strain (Fig. 2C). The introduction of pNDX1thyA into M1262 restored the virulence (Fig. 2D). These results suggest that the cvfC operon contributes to silkworm killing by S. aureus via thyA expression. 2.4. The cvfC operon contributes to resistance against detergents via thyA expression Because an a-hemolysin deletion mutant did not show decreased virulence against silkworms (manuscript in preparation), the

decreased production of hemolysin of M1262 does not explain its decreased virulence in silkworms. Pathogenic bacteria must defend themselves against attack by the host immune response during the infectious process. Detergents and host antimicrobial peptides similarly disrupt bacterial membranes. During the course of our study on the M1262 phenotype, we observed that the mutant had higher sensitivity to detergents. We first examined whether the M1262 and thyA mutants have decreased growth in a medium containing detergent. The growth rates of M1262 and a thyA mutant were decreased in medium with 0.1% Triton X-100 (Fig. 3B), although their growth rates were not distinguishable from that of the parent strain in the absence of detergents (Fig. 3A). The cvfC3 and thyA double mutant also showed a growth defect similar to that of the thyA mutant in the presence of detergent. Moreover, the decreased growth rate of M1262 in the presence of detergent was restored to the parent level by introducing a thyA-expression plasmid (Fig. 3C). Thus, we concluded that the cvfC operon contributes to detergent resistance via thyA expression. 2.5. Presence of alternative pathways to the cvfC operon in NCTC8325-4 and Newman strains The RN4220 strain was constructed to accept a foreign DNA by mutagen treatment and has many artificial mutations [21]. We examined the contribution of the cvfC operon to virulence in other S. aureus strains, NCTC8325-4 and Newman. The cvfC3 disruption did not decrease the virulence of the strains with the NCTC8325-4 and Newman backgrounds in a silkworm model, although the mutation did decrease the virulence of S. aureus with an RN4220 background (Fig. 4). We further examined the contribution of the cvfC operon to gene expressions in NCTC8325-4 and Newman strains. The expressions of thyA and purF were decreased in the cvfC3-disrupted mutants of NCTC8325-4 and Newman (Table 2). The expression of sarS was increased in the cvfC3-disrupted mutant of NCTC8325-4 (Table 2). In contrast, the expressions of hla, RNAIII, cap5B, lpl8, hmrA, pflA, asd, and oppB were not altered in the cvfC3 mutants of NCTC8325-4 and Newman strains (Table 2). These results suggest that the cvfC operon contributes to the expressions of thyA and purF in different genetic backgrounds of S. aureus, although the

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M. Ikuo et al. / Microbial Pathogenesis 49 (2010) 1e7

Fig. 2. Introduction of the thyA gene into M1262 restores hemolysin production and virulence in silkworms. (A) Quantitative RT-PCR analysis was performed to measure thyA expression in M1262 transformed with either a vector or pcvfC. RNA was isolated from the cells grown to A600 ¼ 1. The data are shown as means  standard deviations from three independent experiments. *, p ¼ 0.0477. (B) Hemolysin production of M1262 transformed with thyA-expressing plasmid was measured using sheep erythrocytes. *, p ¼ 0.0053; **, p ¼ 0.0011. (C) Silkworm killing activity of the thyA mutant. The thyA mutant was cultured in tryptic soy broth with 50 mg/ml thymine and serially diluted overnight culture was injected into the hemolymph of silkworms (n ¼ 5). Surviving silkworms at 47 h post infection were counted. (D) Silkworm killing activity of M1262 transformed with thyAexpressing plasmid. Bacterial cells (2.4  106) were injected into silkworms (n ¼ 5) and the survival was monitored.

NCTC8325-4 and Newman strains have alternative functions that compensate for the loss of the function of the cvfC operon. 3. Discussion The present study showed that introduction of the thyA gene into M1262, in which the function of the cvfC operon is impaired, restored hemolysin production, silkworm killing activity, and growth in the presence of a detergent. The thyA gene is known to be involved in the virulence of S. aureus, S. typhimurium, and S. flexneri. This gene is responsible for synthesizing thymidylate under thymine-limited conditions and for upregulating the agr and hemolysin genes [10e14]. Our results demonstrated the role of the thyA gene in detergent resistance, which may protect bacteria against host immunity. Sensitivity of the thyA mutant to detergent was observed in the presence of 50 mg/ml thymine, suggesting that low levels of thymine do not account for the detergent sensitivity of the thyA mutant. We speculate that the thymidilate- and thymine-synthetic pathways have a role in maintaining the homeostasis of the membrane in S. aureus by an unidentified mechanism.

The cvfC operon positively regulates the thyA gene, which increases detergent resistance and hemolysin production, to enhance its defense against the host immune response. In addition to the thyA gene, the expression of several metabolic genes and virulence genes was changed in M1262 (Table 2, Supplemental Table 1). The orchestrated expression of metabolic genes and virulence genes regulated by the cvfC operon would have a significant role in S. aureus infection. Disruption of the cvfC3 gene decreases the expression of purQ, purL, purF, purM, and purN, which encode purine synthetases, and pyrC, pyrAA, pyrF, and pyrE, which encode pyrimidine synthetases. S. aureus purL-disrupted mutants have attenuated virulence in a murine model [22]. In Escherichia coli, Salmonella enterica, and Bacillus anthracis, inactivation of purine and pyrimidine biosynthesis genes prevents bacterial growth in human serum [23], indicating that de novo biosynthesis of nucleosides is required for growth in serum in which nucleosides are limited. We suggest that the cvfC operon positively regulates the genes responsible for nucleoside synthesis, which is needed for growth under nucleoside-limited conditions in the host microenvironment.

M. Ikuo et al. / Microbial Pathogenesis 49 (2010) 1e7

A

0%

10

A600

1

0.1

RN4220 (parent) M1262 (ΔcvfC3) NI8 (ΔthyA) wAC (ΔthyAΔcvfC3)

0.01 0

5

B

10 15 Time (h) Triton X-100

20

A600

C

10 15 Time (h)

20

Triton X-100

0.1%

25

10

A600

1

0.1 RN4220 / pNDX1 (parent) M1262 / pNDX1 (ΔcvfC3) M1262 / pNDX1thyA (ΔcvfC3,thyA+)

0.01 0

5

10 15 Time (h)

20 0 0

10

20

20 30 Time (h)

40

Newman

25

Fig. 3. Growth sensitivity of M1262 and thyA mutants to Triton X-100. (A) Growth curves of RN4220 (parent strain), M1262 (DcvfC3), NI8 (DthyA), and wAC (DthyA, DcvfC) in tryptic soy broth with 50 mg/ml thymine were measured. (B) Growth curves in tryptic soy broth with 50 mg/ml thymine and 0.1% Triton X-100 were measured. (C) Growth curves of M1262 transformed with an empty vector or the thyA-expressing plasmid in medium containing 0.1% Triton X-100 were measured.

80 60 40 20 0 0

C

10

20 30 Time (h)

40

50

RN4220 100

Survival of silkworms (%)

5

40

100

RN4220 (parent) M1262 (ΔcvfC3) NI8 (ΔthyA) wAC (ΔthyAΔcvfC3)

0

60

B 0.1%

1

0.01

80

25

10

0.1

NCTC8325-4 100

Survival of silkworms (% )

Triton X-100

Survival of silkworms (%)

A

5

80 60 40 20 0 0

10 wild type

20 30 Time (h) ΔcvfC3

40

50

no bacteria

Fig. 4. Effects of cvfC3-disruption of NCTC8325-4 and Newman in a silkworm model. The cvfC3 mutants were injected into the hemolymph of silkworms and survival was monitored. (A), NCTC8325-4 strain; 2.2  106 CFU was injected. (B), Newman strain; 1.9  107 CFU was injected. (C), RN4220 strain; 4.4  107 CFU was injected.

4. Materials and methods 4.1. Bacterial strains and growth conditions

The Nfu_N domain, which is found in CvfC2 and CvfC3, exists in various proteins in many organisms, although its function is unknown. CvfC3 has another motif, PBS lyase HEAT-like repeat, which has adhesive activity against RNA and DNA [24]. Analysis of these motifs of CvfC2 and CvfC3 will further elucidate how the cvfC operon affects gene expression in S. aureus.

The JM109 strain of E. coli was used as a host for pHY300C, pNDX1, and their derivatives. E. coli strains transformed with the plasmids were cultured at 37  C in Luria-Bertani broth containing 25 mg/ml chloramphenicol. S. aureus strains were aerobically cultured in tryptic soy broth at 37  C, and chloramphenicol (12.5 mg/ ml), erythromycin (10 mg/ml), or kanamycin (30 mg/ml) was added

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M. Ikuo et al. / Microbial Pathogenesis 49 (2010) 1e7

to the medium to maintain the plasmids. Details of the bacterial strains and plasmids used in this study are shown in Table 3.

[15]. Disruption of the cvfC3 gene was confirmed by Southern blot analysis (Fig. S1).

4.2. DNA manipulation

4.4. Microarray analysis

Transformation of E. coli, extraction of plasmid DNA from E. coli, PCR, and Southern blot analyses were performed as previously reported [25]. To construct plasmids harboring the cvfC operon or the thyA gene, the cvfC operon and the thyA gene were amplified by PCR using oligonucleotide primers cvfC-F, cvfC-R, thyA-F, and thyAR (Table S2). The amplified DNA fragments were inserted into the pHY300C and pNDX1 plasmids, resulting in pcvfC and pNDX1thyA (Table 3). To construct pcvfC12 and pcvfC4, DNA fragments were amplified by PCR using pcvfC as a template and the primers listed in Table S2, and were self ligated. To construct pcvfC3, the DNA fragment containing cvfC1, cvfC2, and cvfC3 was amplified by PCR using primers FccvfC and cvfC-F and the RN4220 genome DNA as a template. The DNA fragment was inserted into pHY300C, resulting in pHY300C-cvfC-B. DNA fragments were amplified by PCR using pHY300C-cvfC-B and primers cvfC3-F and cvfC3-R. The amplified DNA fragments were self ligated, resulting in pcvfC3.

Overnight cultures of RN4220 and M1262 were inoculated into tryptic soy broth, and further cultured under aerobic conditions. Cells were collected at A600 ¼ 1.0 and total RNA was extracted using an RNeasy Mini Kit (Qiagen, Gaithersburg, MD). cDNA was prepared according to the protocol recommended by Affymetrix. RNA was reverse-transcribed to cDNA using Super Script II reverse transcriptase with random primers. cDNA was digested with DNase I and labeled with biotin using Gene Chip DNA labeling reagents (Affymetrix, Santa Clara, CA) and deoxynucleotidyl transferase. Biotin-labeled cDNA was hybridized to a S. aureus array (Affymetrix, Santa Clara, CA). The array was stained with streptavidin-phycoerythrin and the fluorescent signal was scanned. The signal intensities were analyzed using GeneChip Operating Software and the presence of each signal was determined. Obtained signal intensities were normalized using GeneSpring 4.0.

4.3. Construction of double disruption mutant for the cvfC3 and thyA genes A thyA and cvfC3 double-disruption mutant was constructed by transformation of a thyA-disrupted mutant NI8 [20] with pT1262

Table 3 Bacterial strains and plasmids used. Strain or plasmid

Genotypes or characteristicsa

Strains S. aureus NCTC8325-4

NCTC8325 cured of phage11, phage12, and phage13 RN4220 8325e4, restriction mutant, partially agr suppressedb Newman Laboratory strain, high level of clumping factor M1262 RN4220 cvfC::pT1262 M1262NC NCTC8325-4 cvfC::pT1262 M1262N Newman cvfC::pT1262 NI8 RN4220 thyA19:: pSF151kan wAC RN4220 thyA19:: pSF151kan, cvfC::pT1262 RN4220pHY300C RN4220 harboring with pHY300C M1262pHY300C M1262 harboring with pHY300C M1262pHY300CcvfC M1262 harboring with pHY300CcvfC RN4220pNDX1 M1262 harboring with pNDX1 M1262pNDX1 M1262 harboring with pNDX1 M1262pNDX1thyA M1262 harboring with pNDX1thyA E. coli JM109 Plasmids pT1262 pHY300C pcvfC pcvfC12 pcvfC3 pcvfC4 pNDX1

pNDX1thyA a b

Source

[27]

[29] [15] Present study Present study [30] Present study

Serially diluted supernatants of overnight cultures were incubated with sheep red blood cells at 37  C for 1 h, and the reaction mixture was centrifuged and the OD405 of the supernatant was measured. The activity was expressed by hemolytic units corresponding to the reciprocal of the dilution of supernatant that yielded 50% erythrocyte lysis [26]. 4.7. Measurement of detergent resistance of bacteria

study study study study

General purpose host strain for cloning

Takara Bio

pMutinT3 with partial cvfC from RN4220; Ermr E. coli-S. aureus shuttle vector; Cmr pHY300C with intact cvfC operon from RN4220 pHY300C with intact cvfC1 and cvfC2 from RN4220 pHY300C with intact cvfC3 from RN4220 pHY300C with intact cvfC4 from RN4220 pND50-based S. aureus-E. coli shuttle vector carrying TetR and xyl/tet from pWH353; Cmr pNDX1 with intact thyA from RN4220

[15]

Erm, erythromycin; Cm, chloramphenicol; Kan, kanamycin. The expression of RNAII of the agr locus is low.

RNA was reverse-transcribed to cDNA using Multiscribe Reverse Transcriptase (Roche, Basel, Switzerland). Quantitative real-time PCR was performed using cDNA as template and SYBR Premix ExTaq (Takara Bio, Tokyo, Japan) and primers (Table S1). The signals were detected by ABI PRISM 7700 Sequence Detector (Applied Biosystems, Tokyo, Japan). The reaction mixture was incubated at 95  C for 10 s and at 40 cycles (95  C, 5 s; 60  C, 31 s). The data were normalized to 16S rRNA. 4.6. Measurement of hemolytic activity

[28]

[17] Present Present [20] Present Present

4.5. Confirmation of gene expression by quantitative real-time PCR analysis

Overnight culture of bacterial strains was inoculated into tryptic soy broth containing 50 mg/ml thymine and 0.1% Triton X-100 and cultured under aerobic conditions. For bacterial strains harboring pNDX1 and pNDX1thyA, the medium was supplemented with 0.1 mg/ml anhydrotetracycline 1 h before the addition of Triton X-100. 4.8. Infection experiment using silkworms

[17] Present study Present study Present study Present study [20]

Present study

Bacterial infection experiments using silkworms were performed according to the previously established methods [15]. Briefly, serially diluted bacterial cultures were injected into the hemolymph. The injected larvae were maintained without food in a safety cabinet (BHC-1303IIA; Airtech Japan) at 27  C with 50% humidity and survival was monitored. For measurement of the bacterial numbers injected, serially diluted bacterial culture was spread onto tryptic soy agar and incubated overnight and the colonies were counted. LD50 values were determined as the number of bacteria that killed 50% of silkworms (n ¼ 5).

M. Ikuo et al. / Microbial Pathogenesis 49 (2010) 1e7

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