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. 2017 Jun 20;85(7):e00177-17.
doi: 10.1128/IAI.00177-17. Print 2017 Jul.

Galectin-3 Is a Target for Proteases Involved in the Virulence of Staphylococcus aureus

Jonas Elmwall  1 , Jakub Kwiecinski  1   2 , Manli Na  1 , Abukar Ahmed Ali  1 , Veronica Osla  1 , Lindsey N Shaw  3 , Wanzhong Wang  4 , Karin Sävman  5 , Elisabet Josefsson  1   6 , Johan Bylund  7 , Tao Jin  1 , Amanda Welin  1 , Anna Karlsson  8
Affiliations

Galectin-3 Is a Target for Proteases Involved in the Virulence of Staphylococcus aureus

Jonas Elmwall et al. Infect Immun. .

Abstract

Staphylococcus aureus is a major cause of skin and soft tissue infection. The bacterium expresses four major proteases that are emerging as virulence factors: aureolysin (Aur), V8 protease (SspA), staphopain A (ScpA), and staphopain B (SspB). We hypothesized that human galectin-3, a β-galactoside-binding lectin involved in immune regulation and antimicrobial defense, is a target for these proteases and that proteolysis of galectin-3 is a novel immune evasion mechanism. Indeed, supernatants from laboratory strains and clinical isolates of S. aureus caused galectin-3 degradation. Similar proteolytic capacities were found in Staphylococcus epidermidis isolates but not in Staphylococcus saprophyticus Galectin-3-induced activation of the neutrophil NADPH oxidase was abrogated by bacterium-derived proteolysis of galectin-3, and SspB was identified as the major protease responsible. The impact of galectin-3 and protease expression on S. aureus virulence was studied in a murine skin infection model. In galectin-3+/+ mice, SspB-expressing S. aureus caused larger lesions and resulted in higher bacterial loads than protease-lacking bacteria. No such difference in bacterial load or lesion size was detected in galectin-3-/- mice, which overall showed smaller lesion sizes than the galectin-3+/+ animals. In conclusion, the staphylococcal protease SspB inactivates galectin-3, abrogating its stimulation of oxygen radical production in human neutrophils and increasing tissue damage during skin infection.

Keywords: Staphylococcus aureus; galectin-3; neutrophils; protease; skin infection; staphopain; virulence; virulence factors; virulence regulation.

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Figures

FIG 1
FIG 1
Galectin-3-induced ROS production in neutrophils is prevented by secreted factors from S. aureus strain 8325-4. (A) Galectin-3 (400 μg/ml) was incubated in the presence or absence of 2.5% S. aureus 8325-4 culture supernatant for 48 h. The preincubated galectin-3 was added to TNF-α-primed neutrophils at a 1:10 dilution. Production of ROS (y axis; AU, arbitrary units) was followed over time by isoluminol-amplified chemiluminescence. The results of a representative experiment out of six experiments are shown. The inset shows a comparison of the levels of ROS production upon stimulation with galectin-3 (40 μg/ml) and with PMA (50 nM), a well-defined and potent NADPH oxidase activator. (B) Galectin-3 incubated with or without culture supernatant (see above) was subjected to immunoblotting with an antibody directed toward the CRD. For reference, recombinant galectin-3 (32 kDa) and CRD (16 kDa) are shown. (C) Primed neutrophils were exposed to galectin-3 (40 μg/ml) in the presence or absence of culture supernatant without prior incubation or to culture supernatant only, and ROS production was measured as described above.
FIG 2
FIG 2
Specific S. aureus proteases are responsible for cleavage of galectin-3 into lower-mass fragments. (A) Culture supernatants from S. aureus strain 8325-4 were incubated with galectin-3 (100 μg/ml) in the presence or absence of protease inhibitor ScpB, SspC, EDTA, or Pefabloc at the given concentrations for 1 h at 37°C. The samples were then analyzed for content of galectin-3 and fragments thereof by immunoblotting using the anti-CRD antibody. (B) Culture supernatants (diluted 1:40) from strain 8325-4 were incubated with galectin-3 (400 μg/ml) in the presence or absence of SspC (8 μM). The mixture was added to TNF-α-primed neutrophils at a 1:10 dilution, and ROS production (y axis; AU, arbitrary units) was followed over time by isoluminol-amplified CL. (C) Galectin-3 (2 μM; 640 μg/ml) was incubated at 37°C with or without 0.2 μM isolated S. aureus protease ScpA, SspB, Aur, or SspA in KRG with 1% BSA for the indicated times. Samples were analyzed as described above. All samples were analyzed on the same blot that was then cut and reassembled with the lanes in another order, to increase legibility. (D) Galectin-3 (400 μg/ml) was preincubated with SspB (4 μM) for 24 h at 37°C. The proteolytic activity of SspB was then inactivated by the addition of SspC (8 μM; necessary due to cytotoxic effects of SspB at the given concentrations) before the mixture was added to neutrophils at a 1:10 dilution. ROS production was measured as described above.
FIG 3
FIG 3
Galectin-3 is cleaved by S. aureus strains expressing SspB. Culture supernatants from strain 8325-4 and ΔscpA, ΔsspB, ΔsspAB, and Δaur mutants were incubated with galectin-3 (100 μg/ml) for 1 or 2 h, and the content of galectin-3 and fragments thereof in each was analyzed by immunoblotting with anti-CRD antibody.
FIG 4
FIG 4
Clinical isolates of S. aureus and S. epidermidis but not S. saprophyticus cleave galectin-3. Culture supernatants of 10 strains of S. aureus isolated from invasive infections and 10 strains from superficial skin infections (A) or of isolates of two strains each of S. saprophyticus (S. sapro A and S. sapro B) and S. epidermidis (S. epid A and S. epid B) (B) were incubated with galectin-3 (100 μg/ml) for 16 h (A) or 1, 2, and 5.5 h (B). Galectin-3 fragmentation was analyzed by immunoblotting using the anti-CRD antibody.
FIG 5
FIG 5
SspB enhances S. aureus virulence in a skin infection model in the presence of galectin-3. Gal-3+/+ or Gal-3−/− mice were injected with 1 × 107 CFU of S. aureus 8325-4 or the ΔsspB mutant on either flank and observed for 3 days, after which they were sacrificed. (A and B) Skin biopsy specimens of healthy skin (A) and S. aureus 8325-4-infected lesions (B). Histological biopsy specimens were immunohistochemically analyzed for galectin-3 using an anti-galectin-3 antibody. (A) 1, muscle tissue; 2, fat tissue; 3, squamous epithelial cells; 4, adnexa epithelial cells; 5, dermal macrophages or dendritic cells. (B) 6, normal tissue; 7, infiltrating macrophages; 8, S. aureus cells; 9, necrotic tissue. (C) Bacterial loads in skin biopsy specimens collected at day 3 were determined by viable count. (D) Lesion sizes caused by strain 8325-4 and the ΔsspB mutant, respectively, were recorded, and the sizes at day 3 are shown. Horizontal bars designate median values. Statistical analysis was performed using the Wilcoxon matched-pairs signed-rank test to compare the two strains inoculated on the same mouse and the Mann-Whitney test to compare the strains between the two groups of mice. Data from the two experiments are pooled. *, P < 0.05; **, P < 0.01; ns, no significant difference.
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