School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK
Correspondence
Keith T. Holland
K.T.Holland{at}leeds.ac.uk
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ABSTRACT |
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INTRODUCTION |
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HL, a long-neglected potential virulence determinant of S. aureus, is produced by the organism as an extracellular enzyme capable of degrading the acidic mucopolysaccharide hyaluronic acid (HA), a major component of the intercellular ground substance of human and animal connective tissue (Arvidson, 1983). Duran-Reynals (1933)
observed that culture extracts of S. aureus increased permeability of rabbit tissue to vaccinia virus, toxins and dyes. Subsequently, the spreading factor was identified as a HL (Linker et al., 1955
). A study (Pritchard & Lin, 1993
) with group B streptococci (GBS), indicated that the enzyme, previously reported to be a neuraminidase, was in fact a HL. Clearly, previous reports describing an association of elevated levels of this enzyme with virulent strains of GBS add support to the hypothesis that HL is also a virulence factor of staphylococci. In addition, the observation that 91·2 % of S. aureus strains produce HL (Choudhuri & Chakrabarty, 1969
) strengthens the case for a role in virulence. The production of HL from other staphylococcal strains is either negative or variable, though Staphylococcus hyicus is positive for HL production and is also an opportunistic pathogen in animals (Devriese, 1990
).
Studies on HL regulation were previously initiated by Taylor & Holland (1991). In addition the HL gene, hysA, has been previously cloned from a genomic library of S. aureus (Farrell et al., 1995
). The present investigation was undertaken in order to assess the importance of HL in the virulence of S. aureus in vivo. A mutant deficient in HL activity was compared with wild-type S. aureus, using an animal model. The roles of the S. aureus global regulators, agr and sar, on the in vitro production of HL were also investigated. In the longer term such studies are relevant to the potential future of research directed at designing agents which interfere with virulence determinant expression in this important pathogen.
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METHODS |
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Construction of a temperature-sensitive (TS) shuttle plasmid.
Plasmid pUL5032 carries a 2·6 kb EcoRI fragment which contains most of the HL gene's ORF, hysA, with the start site and an 80 bp segment downstream from it missing (Farrell et al., 1995). A mutation was constructed in this incomplete hysA gene by insertion of the 1·44 kb erythromycin resistance (Emr) determinant (ermC) from plasmid pE194 (Villafane et al., 1987
). Forward (5'-GATCTACGTATCGAATTGCATCTG-3'; nucleotides 3386 to 3373) and reverse (5'-GCAGTACGTAATCGATTCACAAA-3'; nucleotides 1937 to 1951) primers were designed according to the published sequence of pE194 (Horinouchi & Weisblum, 1982
), with the additional restriction site NsiI introduced at the 5' end of each primer (underlined). The tails added to the 5' ends of the primers are shown in bold. The amplified ermC fragment was ligated with the PCR vector pCR2.1 (Invitrogen) and the DNA was transformed into E. coli selecting Apr. The erythromycin-resistance determinant was excised from positive recombinants with NsiI and was ligated with NsiI-digested and dephosphorylated pUL5032. The ligation products were transformed into E. coli selecting Apr. The desired recombinant plasmid, designated pGM02, was digested with BamHI, dephosphorylated, and ligated with the BamHI-cleaved TS plasmid pTS2tet (Fitzgerald & Foster, 2000
). The DNA was transformed into E. coli selecting Apr. A 13·5 kb plasmid, designated pGM03, was isolated from putative transformants and was mapped by restriction enzyme analysis.
Inactivation of the chromosomal hysA gene of S. aureus 8325-4.
The TS shuttle plasmid pGM03 was electroporated into S. aureus RN4220, selecting Emr at 30 °C for 48 h. Positive recombinants were confirmed by demonstrating Cmr and Tcr. Plasmid pGM03 was prepared from RN4220 and established into S. aureus 8325-4 in a similar manner. Recombinants in which the hysA : : ermC mutation had replaced the wild-type (w.t.) chromosomal allele were selected as follows. A single colony of 8325-4 (pGM03) was inoculated into 5 ml TYEg containing Em and was grown overnight at 30 °C. The culture was diluted 1 : 100 into 25 ml fresh TYEg in the absence of antibiotic selection and was grown at 45 °C for 17 generations in order to facilitate the reduction of pGM03 copy number per cell. During this time the culture was subcultured four times into fresh TYEg medium lacking antibiotic. At the end of the extended growth cycle, and in order to select for the first recombination event, dilutions of the culture were plated on agar containing Em and were incubated at 45 °C. Plasmid excision was induced by subculturing several colonies on agar containing Em and incubating them at 30 °C. Any resulting single colonies were then screened on Cm and Tc agar plates. Isolates in which allele replacement had occurred and which were cured of the TS shuttle plasmid were phenotypically Emr Cms Tcs. The replacement of the w.t. gene by the disrupted allele was verified by Southern blot hybridization using the 1·4 kb TaqI fragment from pE194 (ermC) and a 0·7 kb NsiINcoI fragment from pUL5032, and by PCR analysis using forward (5'-CTTCTCATATGACTCGTACCTATCG-3'; nucleotides 458 to 482) and reverse (5'-TAGGTGTTCCAATTTCATAATCCCA-3'; nucleotides 676 to 700) primers designed based on the published sequence of hysA (Farrell et al., 1995).
Construction of a vector carrying the active hysA gene.
A 7·2 kb HindIII fragment containing the full hysA ORF, including upstream regulatory regions, was excised from pUL5030 (Farrell et al., 1995) and following dephosphorylation it was ligated with HindIII-cleaved pCU1 shuttle vector (Augustin et al., 1992
). The ligation mix was transformed into E. coli selecting Apr. A 12·2 kb plasmid, designated pGM04, was isolated from putative transformants and was mapped by restriction enzyme analysis. Plasmid pGM04 was passaged through S. aureus RN4220 and was then electroporated into electrocompetent HL-deficient cells selecting Emr and Cmr. Several colonies were screened for HL production on Cm plates containing HA.
Phage transduction.
This was performed as described by Foster (1998) using
11 (Novick, 1991
) as the transducing phage. Briefly, 500 µl of an overnight w.t. S. aureus 8325-4 culture was transferred to a plastic universal containing 500 µl phage lysate (1091010 p.f.u. ml1) and 1 ml LK broth. The lysate/cell mixture was incubated at 37 °C without shaking for 25 min, followed by 15 min with shaking at 250 r.p.m. Then 1 ml ice-cold sodium citrate (0·02 M) was added and the cells were pelleted at 5000 r.p.m. for 10 min, resuspended in 1 ml ice-cold sodium citrate (0·02 M) and incubated on ice for 2 h. The lysate/cell mixture was spread on LK bottom agar plates containing 0·05 % (w/v) sodium citrate and 0·15 µg Em ml1, and was incubated at 37 °C for 90 min. The plates were overlaid with 5 ml LK top agar containing 25 µg Em ml1 and 25 µg Lm ml1, and following setting, they were incubated at 37 °C until the appearance of putative phage transductants (1872 h).
HL assays.
All chemicals were purchased from Sigma unless otherwise stated. HL-deficient isolates were screened from strains with the w.t. phenotype by the plate assay of Grenier & Michaud (1993). Briefly, BHI agar plates supplemented with HA (0·4 mg ml1) were spot inoculated with several candidate colonies and incubated at 37 °C. Following growth, the plate was flooded with 10 % (w/v) cetylpyridinium chloride (BDH) and was left to stand at room temperature for 2030 min. The cetylpyridinium chloride was then rinsed off the plate with distilled water. Colonies producing HL were surrounded by a zone of clearing.
HL in culture supernatants was assayed by measuring the production of N-acetylamino end-group sugars by the method of Reissig et al. (1955). A standard curve was constructed for the subsequent determination of HL activity units. Briefly, 0·1 ml potassium tetraborate solution (0·8 M, pH 9·1) was mixed with 0·5 ml N-acetyl-D-glucosamine (NAG) standard sample (0·050·5 µmol ml1) and with 0·5 ml distilled water. The samples were boiled for exactly 3 min and cooled immediately under cold water. Colour production was initiated by the addition of 3 ml 0·1x DMAB reagent [10 % (w/v) p-dimethylaminobenzaldehyde, 12·5 % (v/v) 10 M HCl (BDH), 87·5 % (v/v) glacial acetic acid (BDH)] and was completed by incubating the resulting solutions at 37 °C for exactly 20 min. The A544 of each sample was measured using an SP6-450 UV/VIS spectrophotometer (Pye Unicam). A standard curve of A544 against NAG concentration (µmol ml1) was plotted using a best-fit straight line. The procedure was repeated with 0·5 ml of test sample or blank (50 mM sodium acetate, pH 5·2). Each sample was mixed with 1 ml HA solution [200 mM NaCl, 1 % (w/v) sodium azide, 0·6 % (w/v) HA] and allowed to react at 37 °C. Reactions were stopped immediately (t0) or exactly 15 minutes (t15) following the addition of the substrate by mixing 0·5 ml of the reaction mixture with 0·1 ml potassium tetraborate solution (0·8 M, pH 9·1). Each sample was assayed in duplicate and the enzyme specific activity was expressed as 103xµmol NAG released ml1 min1 per OD600 unit and was calculated according to the following formula:
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Pathogenicity study.
The virulence of S. aureus strains was tested in an established murine abscess model of infection (Chan et al., 1998). S. aureus strains were grown in BHI to stationary phase (20 h) as described above. The bacteria were harvested and washed twice in an equal volume of ice-cold sterile PBS (pH 7·4). The concentration of the bacterial cells was adjusted to approximately 1x108 c.f.u. ml1 using a counting chamber (Thoma). Female, 5- to 8-week hairless mice (Harlan) were injected subcutaneously (n=12) with 0·2 ml of bacterial suspension. Serial dilutions of the samples were plated on BHI agar. The precise sizes of the inocula were confirmed by counting the resulting growth of colonies. After 7 days, the mice were killed and skin lesions were aseptically removed. Lesions destined for histological examination were measured and stored in sterile PBS supplemented with 10 % (v/v) formalin. The remaining lesions were measured, chopped and homogenized in 3 ml sterile ice-cold PBS in a mini-blender for approximately 5 min. The total number of bacteria recovered from the lesions was determined by viable cell count on BHI agar. Statistical significance of the data was evaluated by ANOVA. All values are reported as means ±95 % confidence limits.
Histology.
Skin lesions were fixed in 10 % (w/v) formalin saline solution for 48 h and were then embedded in paraffin wax (Paraplast). Each wax-embedded lesion was sectioned (6 µm) using stainless steel microtome blades (RaLamb). A total of 10 sections per lesion were prepared, stained with haematoxylin and eosin using routine histological procedures (Bancroft & Stevens, 1996) and analysed by microscopy (Olympus BX40).
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RESULTS AND DISCUSSION |
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Transduction of the hysA : : ermC allele
The hysA : : ermC mutation present on the chromosome of HL-6 was transduced to the w.t. 8325-4 strain via 11. The expression of the ermC gene was initially induced with low levels of Em. However, in order to minimize the selection of point mutations for Em, the cells were screened on Lm as well as Em. Two transductants, HL-13T and HL-16T, were isolated and their identity was verified using the Pastorex Staph-Plus (PSP) latex agglutination test (Bio-Rad). Culture supernatants from both isolates were HL negative when tested with the plate and the colorimetric assays (data not shown).
Growth characteristics of staphylococcal strains
The growth characteristics, i.e. maximum specific growth rate (µmax) and growth yield, of HL-6, HL-13T and HL-16T were compared to those of the w.t. strain during growth in BHI medium. All four strains reached a maximum specific growth rate of 1·4 h1. In addition, they reached similar growth yields, which ranged between 10 and 12 OD600 units.
Virulence studies with HL-deficient mutants
In order to determine whether HL is a virulence factor of S. aureus, mice were challenged subcutaneously with strains 8325-4 (w.t.) and HL-6. Following the 7 day infection period the majority of the animals challenged with the w.t. strain developed a red, open and crusty lesion. In contrast, the HL-negative strain caused a less severe white, raised pustule (Fig. 3). These results suggested a role for HL in subcutaneous infections.
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These results strongly suggest that the disruption of the hysA gene of S. aureus has a negative effect on the degree of pathology caused by the organism.
Role of global regulators agr and sar on HL production
Although, to date, there are no reports on the role of the agr and sar loci in the regulation of HL, the results of previous studies by Taylor & Holland (1991) and Rogers (1954)
suggest that its regulation is distinct from that of other S. aureus exoproteins, such as toxic shock syndrome toxin-1 (TSST-1), which are expressed during late exponential phase.
The w.t. S. aureus 8325-4 strain and its derivatives, WA250 (agr) and PC1839 (sar), were grown in CDM in the absence of selection. HL activity and OD600 were measured as described in Methods. Although the three strains had similar growth kinetics (Fig. 5), strain WA250 demonstrated approximately a 16-fold reduction in HL activity compared to the w.t. (0·9 vs 14·0 specific activity units per OD600 unit). In contrast, strain PC1839 exhibited a 35-fold (487 specific activity units per OD600 unit) increased activity compared to the w.t. It is of interest that the activity from all three strains decreased to undetectable levels during the post-exponential phase of growth. It is possible that proteases in the supernatant fluid may cleave HL post-translationally, thus reducing its activity. This effect was exaggerated in the sar strain, in which although HL specific activity was the highest of the strains tested, it diminished at the highest rate. Previous investigations have reported that proteases, which are overexpressed in the sarA mutant strain PC1839, are responsible for the inactivation of
-haemolysin in the same strain (Lindsay & Foster, 1999
). However, additional factors may be responsible for this observed decrease in HL specific activity since it occurred even in the agr mutant strain, which is expected to have a reduced protease production.
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The results presented in this work were obtained using the standard laboratory S. aureus strain 8325-4, which is defective for rsbU, the gene required for the activation of B. The results were therefore obtained in the absence of
B function and hence may not be representative of the behaviour of a true w.t. clinical strain. However, since strain 8325-4 has been used for much work on the regulation of virulence of S. aureus, it is important that the regulation of HL be analysed in this background for comparative purposes. Furthermore, the results obtained from in vivo experiments using the isogenic hysA mutant are valid because the positive control w.t. 8325-4 strain was of an identical genomic background apart from the hysA gene. Future work should investigate the regulation of HL in an rsbU+ derivative of S. aureus 8325-4. Such a derivative has recently been described by Horsburgh et al. (2002)
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 25 November 2003;
revised 17 February 2004;
accepted 2 March 2004.
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