Induction of Neutral Lipid-Containing Granules by Staphylococcal Lipase from Clinical Isolates

Shigeki Kamitani1,2*, Masami Miyake2,3, Michiko Hatano2, Takashi Yutsudo4, Wakio Minamide4, Iwao Kato2, Masatoshi Noda2 1Department of Clinical Nutrition, Faculty of Comprehensive Rehabilitations, Osaka Prefecture University, Habikino, Osaka, Japan 2Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, Japan 3Department of Veterinary Environmental Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, Japan 4Shionogi and Co., Ltd., Toyonaka, Osaka, Japan SOJ Microbiology & Infectious Diseases Open Access Research Article


Introduction
Staphylococcus aureus is an important pathogen causing a wide range of infections in humans and other animals.Possible virulence factors, including various proteinaceous toxins and enzymes, and their roles in the bacterial infection have been the main foci of investigations [1,2].Among them, alpha-toxin, which is one of the major virulence determinants of microorganisms, and enterotoxins have been extensively examined and characterized biochemically and biologically [2].However, a precise understanding of the enzymes secreted by S. aureus upon bacterial infection has yet to be obtained.

Staphylococcal lipases have been purified from several
Staphylococcus spp.and their enzymatic properties have been well documented [3][4][5][6].Molecular biological studies and threedimensional structure analysis revealed that amino acid residues

Characterization of the strain
The strain MRSA 10-123 was characterized as follows.Many kinds of drug sensitivity including for oxacillin were tested by MIC determination, disk diffusion and agar screen methods, respectively.Hemolytic activity by α-toxin was detected on rabbit blood plate [23].Producibility and typing of staphylococcal enterotoxin, TSST-1 and coagulase were investigated by using, respectively, SET-RPLA, TST-RPLA and Coagulase Antisera (Seiken, Tokyo, Japan).Specific primers for the amplification of toxA and lukS genes were designed: 5'-CAT TGC TGG TCA ATA TAG AG-3' and 5'-GTT GGG CTC TCT AAA ATT GT-3' (for toxA), and 5'-CTA CAA CTT TAT CTG TGA GC-3' and 5'-TTA TAT TGG AAT GGC CAT CG-3' (for lukS).The mecA gene was identified according to Murakami et al. [24].The nucA gene was detected by the method of Brakstad et al. [25].Detection of genes for enterotoxins and toxic shock syndrome toxin 1 was performed on the basis of the method of Johnson et al. [26].

Cytopathicity assay
UV r -1 human embryonic clonal cells [27], Chinese hamster ovary (CHO) cells, were maintained with Eagle's minimum essential medium (MEM) supplemented with 10% newborn calf serum throughout the experiments.The cells were inoculated at 1.5 x 10 3 cells/well (150 ml/well) in a 96-well plate.A total of 50 µl of the supernatant or chromatography effluents was added into the well and the mixture was incubated at 37°C for 24 h.Morphological changes of UV r -1 cells were examined under phase-contrast light microscopy.

Lipase assay
Lipase activities in the supernatant were determined with Lipase Kit S (Dainippon Pharmaceutical, Co. Ltd., Osaka, Japan) with a colorimetric substrate.The supernatant was serially diluted with a dilution buffer supplied in the kit and subjected to the lipase assay in according with the manufacturer's instructions.The reaction was terminated with termination buffer, and optical density at 412 nm (OD 412 ) of the reaction mixtures was determined.One unit was defined as the activity that shows an increase of OD 412 of 1.0 for 30 min at 30°C.The experiments were performed at least three times and the data are representative of three experiments and each point represents the mean of duplicate measurements.

Purification of cytopathic substance
MRSA10-123 culture supernatant (10 L) was precipitated by adding 75 mM ZnCl 2 and centrifuged at 8,000 x g for 15 min at 4°C.The precipitate was dissolved in 2 L of 0.4 M Na 2 HPO 4 and centrifuged at 8,000 x g for 15 min.The supernatant was dialyzed against 20 L of TEN buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM NaN 3 ) overnight.The dialysate was applied to a DEAE-TOYOPEARL column (3 x 90 cm).After washing away unbound materials with TEN buffer, cytopathic fractions were eluted with TEN buffer containing 0.5 M NaCl.The effluent was collected and dialyzed against TEN buffer, and re-applied to DEAE-TOYOPEARL.Elution was performed with TEN buffer containing a 0-0.3 M NaCl gradient.The cytopathic fractions were concentrated with ultrafiltration membrane (PM-10 membrane, Amicon) to a final volume of 1.0 ml.Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified preparation revealed two major protein bands with molecular masses of 43 and 45 kDa.In order to identify the proteins, ZnCl 2 -imidazole staining was performed [28] and then recovery was carried out by electroelution (The Centrilutor Micro-Electroelutor, Amicon).

Preparation of the recombinant lipase and mutant lipase
MRSA10-123 was grown with shaking overnight at 37°C in 5 ml of tryptic soy broth and recovered by centrifugation.After washing, the chromosomal DNA was prepared as described previously [31].For cloning of the mature part of the lipase gene geh [32], PCR primers with BamHI or EcoRI sites were used: 5'-CT GGA TCC AAA GCG AAT CAA GTA CAA CCA CTT A-3' (LIP-S1) and 5'-GC GAA TCC TTA ACT TGC TTT CAA TTG TGT TCC TT-3' (LIP-A1).A 1,188-bp DNA fragment was amplified by PCR and digested with BamHI and EcoRI.The digested fragment was cloned into pGEX-2T, a glutathione-S-transferase (GST) fusion vector plasmid (Pharmacia, Sweden).BL21 (DE3) cells transformed with pGH 26 (a plasmid with an insert encoding the GST-lipase fusion protein) were incubated until the early log phase and the recombinant lipase was induced with 0.1 mM IPTG.After 3-5 h, the cells were collected by centrifugation and lysed by sonication in PBS-1% Triton X-100.After centrifugation and filtration, the recombinant GST-lipase fusion protein was absorbed to a Glutathione Sepharose 4B column, and cleaved by using thrombin at 4°C overnight.Recombinant lipase was eluted with 0.5 M NaCl-50 mM Tris HCl, pH 8.0.The plasmid expressing the recombinant mutant lipase was constructed by site-directed mutagenesis.Mutated primers containing a SphI site are: 5'-CCA CCC ATA GCA TGC CCT AC-3' (LIP-MA1) and 5'-GTA GGG CAT GCT ATG GGT GG-3' (LIP-MS1).A 362-bp DNA fragment for LIP-S1/ LIP-MA1 and an 850-bp DNA fragment for LIP-MS1/ LIP-A1 were amplified and each PCR product was digested with SphI.These two fragments were ligated into pGEX-2T.The recombinant mutant lipase was purified by the same method.

Induction of Neutral Lipid-Containing Granules by Staphylococcal Lipase from Clinical Isolates
Copyright: © 2014 Kamitani et al.

Preparation of anti-lipase antibody
BALB/C female mice were injected with either 5 or 25 µg of the purified cytopathic fraction.Several injections were performed over 2 months.The serum was recovered after 3 months and the anti-recombinant lipase titer was checked.The anti-recombinant lipase antibody raised in rabbit (Japanese White) was purified by protein A-coupled Sepharose column chromatography 3 months after immunization.A total of 5 mg of protein A (EY Laboratories, Inc., San Mateo, CA) was coupled with a pre-packed HiTrap NHS-activated (Pharmacia Biotech) column according to the manufacturer's instructions.The column was equilibrated with PBS before use and 1 ml of rabbit serum (anti-recombinant lipase serum or pre-immune serum) was applied to it.After the column was washed with 5 ml of PBS, the bound antibody was eluted with 3 ml of 0.2 M glycine -HCl, pH 2.8-0.5 M NaCl.The effluent was neutralized with 1 M Tris-HCl, pH 8.0, immediately, concentrated by ultrafiltration and dialyzed against PBS.

Western blot analysis
The specificity of the antibodies purified by protein A-coupled affinity chromatography was examined by Western blot analysis.The effluent from DEAE-TOYOPEARL chromatography or the recombinant lipase was run in an SDS-PAGE gel (12%) and the separated proteins were transferred onto a PVDF membrane [29].After blocking with 3% bovine serum albumin and 2% skim milk in washing buffer (PBS-0.1% Tween 20), the membrane was incubated for 2 h with 5 µg of either affinity-purified antirecombinant lipase antibody or pre-immune antibody per ml of washing buffer.The membrane was probed with peroxidaseconjugated anti-rabbit IgG antibody (Organon Teknika Corp., West Chester, PA) for 2 h at room temperature, followed by enzyme detection with ECL Western blotting detection reagents (Amersham International plc, Buckinghamshire, England).

Neutralization study
A total of 100 µl of the supernatant from MRSA 10-123 was incubated for 30 min at room temperature with 100 µl of affinity purified anti-recombinant lipase antibody or antibody from preimmune serum.The mixture was subjected to the cytopathicity assay.Briefly, 5 x 10 3 cells were treated with the mixture and incubated.The experiments were performed three times and the data are representative of three experiments and each point represents the mean of duplicate measurements.

Determination of protein concentration
Protein concentration was determined by the Bradford method [33] (Protein Assay, BioRad Laboratories, Hercules, CA).Bovine serum albumin served as a standard.

General properties of S. aureus MRSA (methicillinresistant S. aureus) 10-123 strain
MRSA 10-123 showed TSST-1, enterotoxin C and type II coagulase activities, but not α-toxin and β-lactamase activities, by bacteriological examinations.Genes of nucA, mecA, sec, luk S and toxA were detected in MRSA 10-123 by PCR (data not shown).The filtered supernatant from overnight culture of MRSA 10-123 was examined for cytotoxicity to UV r -1 [27] and CHO cells.Both cells were severely damaged by the supernatant at high concentration (4-to 16-fold dilution), and then lysed by 24 h after incubation.However, at low concentration (more than 32-fold dilution), intracellular granules accumulated inside of cells.Oily vesicles accumulated in UV r -1 cells after 12-h incubation with the supernatant (Figure 1B) in contrast to the case for PBS (Figure 1A).After 24-h incubation, the cells had become round and detached from the plate.Histochemical examination by Sudan III staining [34] showed that neutral lipid was present in the vesicles (Figure 1C).The morphological changes of the cells treated with the supernatant were caused by the formation of neutral lipid vesicles.The cytopathic substrate of the supernatant was heat-stable because the supernatant did not lose cytopathicity after heating at 80°C for 30 min (data not shown).Hereafter, this vesicle formation was regarded as the source of cytopathicity in this study.

The cytopathic substance is a staphylococcal lipase
Next, we attempted to purify and identify the cytopathic substance in the bacterial culture supernatant.Cytopathicity was recognized in the fractions eluted with 0.30-0.35M NaCl by DEAE-TOYOPEARL chromatography (Figure 2A).SDS-PAGE revealed that the fractions included two major proteins with molecular masses of 43 and 45 kDa (Figure 2B).The protein bands visualized by ZnCl 2 -imidazole staining were then recovered by electroelution and tested for cytopathicity.Both 43-and 45-kDa proteins showed cytopathicity to UV r -1 cells.The 45 kDa protein was digested with lysyl-endopeptidase.Two peptides separated by high-performance liquid chromatography (HPLC) were then sequenced by using a protein sequencer.The amino acid sequence of one peptide was identical to the N-terminal sequence of Staphylococcus aureus lipase-2 (SAL-2), and the sequence from the other peptide corresponded to the internal sequence of the same lipase (Figure 3), indicating that

Induction of Neutral Lipid-Containing Granules by Staphylococcal Lipase from Clinical Isolates
Copyright: © 2014 Kamitani et al.
the 45-kDa cytopathic protein is a lipase.The 43-kDa protein was also subjected to the digestion by a proteinase and peptide sequencing.The N-terminal and internal amino acid sequences of the 43-kDa polypeptide were identical to those of the 45-kDa protein.As previously reported the staphylococcal lipase is secreted into the culture supernatant as a 76-kDa proenzyme and activated extracellularly to produce a 45-kDa mature protein [8].These factors indicates that the 45-kDa protein identified as a cytopathic substance is staphylococcal lipase and that the 43-kDa cytopathic protein is presumably generated by degradation of the 45-kDa lipase by limited proteolysis at its C-terminal portion because the N-terminal and internal sequences of the two proteins were shown to be identical.

Recombinant staphylococcal lipase is cytopathic
To obtain a further confirmation that the cytopathic substance in the supernatant was staphylococcal lipase, the gene encoding the lipase, geh, from MRSA 10-123 was cloned and its gene product was expressed in Escherichia coli as a glutathione-Stransferase (GST) fusion protein.The fusion protein was purified with a glutathione-Sepharose 4B column, and its GST portion was cleaved by thrombin digestion.The recombinant lipase induced morphological changes of UV r -1 cells as well as 45-to 43-kDa cytopathic protein from MRSA 10-123 (Table 1; Figure 1D).Next, we examined whether the cytopathicity in the supernatant was neutralized by the rabbit polyclonal antibody against the recombinant lipase.A total of 40 µg of the antibody completely neutralized the cytopathicity of the supernatant, whereas the same amount of affinity-purified antibody from pre-immune serum failed to do so (data not shown).
These results suggested that the cytopathicity in the supernatant mostly involved the lipase.The specificity of this antibody against staphylococcal lipase was confirmed by western blotting (Figure 4).In addition, mouse polyclonal anti-lipase antibodies obtained upon immunization with purified native lipase completely inhibited the cytopathicity (Figure 5), whereas pre-immune serum did not.

Lipase activity was essential for the cytopathicity
In general, the catalytic site of lipase consists of Ser-Asp-His amino acid residues.To examine whether the lipase activity was essential for the cytopathicity, the serine residue in the highly conserved pentapeptide sequence GXSXG was replaced with alanine by site-directed mutagenesis of the wild-type recombinant lipase.As shown in (Table 1), the mutant lipase lost the lipase activity.In contrast, the wild-type recombinant lipase and purified staphylococcal lipase were more active than human pancreatic lipase or Rhizopus lipase.The mutant lipase showed no cytopathicity to the UV r -1 cells (Table 1), whereas Rhizopus lipase and human pancreatic lipase were slightly cytopathic.These results indicate that the cytopathicity is identical to the lipase activity.

Lipase activity in the supernatant from S. aureus clinical isolates
To investigate the lipase productivity of various types of S. aureus strain, ten clinical isolates including four strains of methicillin-sensitive S. aureus (MSSA) and six strains of MRSA were examined for the lipase activity of each supernatant (Table 2).Three strains of the four MSSA and one strain of the seven MRSA showed lower lipase activities (<3 units); in contrast, one strain of the four MSSA and six strains including MRSA 10-123 of the seven MRSA had higher lipase activities (>3 units).These results indicate that S. aureus strains were classified into two different groups possessing high or low lipase activities.

Discussion
We identified granule-inducing activity of the staphylococcal culture supernatant on human and animal cell lines.Our observation clearly demonstrated that cytopathicity in the supernatant from MRSA 10-123 was caused by the staphylococcal lipase.Staphylococcal lipase has both chemotactic and chemokinetic properties for human granulocytes [35] and causes monophasic aggregation accompanied by the release of lactoferrin [17].Moreover, lipase interferes with human granulocyte phagocytic killing of Staphylococcus aureus but not Staphylococcus pneumoniae or Streptococcus agalactioae [16,36,37].Staphylococcal strains from deep infections produce more lipase than strains from superficial locations [38].Such strains produce lipases in excess of the amounts necessary to elicit an aggregation response in granulocytes.In this study, the staphylococcal lipase was purified without using a detergent such as Triton X-100.Therefore, the biological effect of staphylococcal lipase was precisely confirmed.Others reported that patients with bacterial endocarditis showed positive reactivity with antibody

Induction of Neutral Lipid-Containing Granules by Staphylococcal Lipase from Clinical Isolates
Copyright: © 2014 Kamitani et al.
to staphylococcal lipase or a significant change in antibody titer [39].These reports suggest that lipase serves as a virulence factor in severe infection of S. aureus including septicemia.Our data also showed that there are two groups of S. aureus with high and low lipase activities.More recently, in an intraperitoneal challenge mouse model of S. aureus, peritoneal abscess formation was reduced by the lipase-deleted mutant [40].Taking these findings together, staphylococcal lipase may have a role in determining the survival of S. aureus in lesions and the cytopathicity may be involved in the pathogenesis of staphylococcal disease.

Figure 1 :
Figure 1: Cytopathicity by the supernatant of MRSA 10-123 and by recombinant lipase to the human embryonic UV r -1 cells.Cells were passaged at 1.5 X10 3 cells/well.A total of 50 µl of either PBS (A) or the supernatant (B) was added to the well.After 24-h incubation, the cell viability was examined by light microscopy.Furthermore, the cells treated with the supernatant were stained for neutral fat with Sudan III (C).UV r -1 cells were treated with 50 µl of recombinant lipase (D).

Figure 2 :"Figure 3 :Table 1 :
Figure 2: The elution profile of DEAE-TOYOPEARL chromatography and the migration profile on SDS-PAGE.(A) DEAE-TOYOPEARL chromatography was performed.The cytopathicity was recognized in the fractions eluted with 0.3 to 0.35 M NaCl.The cytopathicity is described in units; the number of units in fractions is numerically equal to the dilution at CD 50 .Protein concentration was revealed by OD 280 (open circle) and the cytopathicity was scored (closed circle).(B) The fractions with cytopathicity were analyzed by 10% SDS-PAGE.

Figure 5 :
Figure 5: Neutralization of the cytopathicity with anti-lipase antibody: The cytopathicity assay was performed as described in Material and Methods.The recombinant lipase was previously mixed with either normal serum or anti-lipase antibody for neutralization assay.Neutralizing effects of normal serum (closed square) and anti-lipase antibody (open square) are shown.The experiments were performed at least three times and the data are representative of three experiments and each point represents the mean of duplicate measurements.

Table 2 :
Lipase activity in the culture supernatant from S. aureus clinical isolates.