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LysGH15 Kills Staphylococcus Aureus Without Being Affected by the Humoral Immune Response or Inducing Inflammation

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LysGH15 Kills Staphylococcus Aureus Without Being Affected by the Humoral Immune Response or Inducing Inflammation

Lei Zhang et al. Sci Rep.

Abstract

The lysin LysGH15, derived from the staphylococcal phage GH15, exhibits a wide lytic spectrum and highly efficient lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we found that LysGH15 did not induce resistance in MRSA or methicillin-sensitive S. aureus (MSSA) strains after repeated treatment. Although LysGH15 triggered the generation of LysGH15-specific antibodies in mice, these antibodies did not block lytic activity in vitro (nor the binding capacity of LysGH15). More importantly, when the antibody titre was highest in mice immunized with LysGH15, a single intravenous injection of LysGH15 was sufficient to protect mice against lethal infection with MRSA. These results indicated that LysGH15-specific antibodies did not affect the killing efficiency of LysGH15 against MRSA in vitro or in vivo. LysGH15 also reduced pro-inflammatory cytokines in mice with lethal infections. Furthermore, a high-dose LysGH15 injection did not cause significant adverse effects or pathological changes in the main organs of treated animals. These results provide further evidence for the administration of LysGH15 as an alternative strategy for the treatment of infections caused by MRSA.

Figures

Figure 1
Figure 1. The sensitivity of different generation cells to LysGH15.
The CFU/mL descent was used to evaluate the antibacterial activity of LysGH15 (50 μg/mL) on different generation cells (1010 CFU/mL). The values represent the mean ± SD (n = 3).
Figure 2
Figure 2. LysGH15 induced specific IgG antibodies.
Serum samples from immunized mice and control mice were collected every week for 10 weeks, and the titres (A) and concentrations of IgG, IgM and IgE isotypes (B) were measured using ELISA. (C) Western blotting assay of LysGH15. Mouse serum at 3 weeks post-immunization was used as the primary antibody (dilution 1:500), and HRP-labeled anti-mouse IgG antibody was used as the secondary antibody. The left lane shows the results for the LysGH15-injected mouse. The right lane shows the LysGH15 buffer-injected mouse. (n = 6 mice per group per experiment). *P < 0.05 compared with unimmunized mice. The data are representative of three experiments.
Figure 3
Figure 3. Anti-LysGH15 serum did not neutralize the activity of LysGH15 in vitro.
(A) The influence of anti-LysGH15 serum on the lytic activity of LysGH15. LysGH15 was pre-incubated with serum (dilution 1:500) from immunized mice for 10 min or 1 h, and this mixture or LysGH15 alone or control buffer was added to cultures of the MRSA strain (YB57). CFU numbers were counted at different time points as indicated. (n = 3 per group per experiment). *P < 0.05 compared with the buffer control. The data are representative of 3 experiments. (B) The influence of anti-LysGH15 serum on the binding activity of C54S-LysGH15. YB57 cells were dyed with Hoechst No. 33342 fluorescent dye, and then C54S-LysGH15 or buffer was pre-incubated with serum (dilution 1:500) prior to the addition to YB57. TRITC-conjugated goat anti-mouse IgG was used. The images were acquired using laser scanning confocal microscopy (LSCM) as described in the Methods section. 1. Localization at 405 nm (blue fluorescence, emitted from Hoechst No. 33342 fluorescent dye). 2. Localization at 543 nm (red fluorescence, emitted from TRITC-conjugated Goat-anti-mouse IgG). 3. Image of an ordinary ray (normal light). 4. Overlay of the pictures shown in 1, 2, and 3.
Figure 4
Figure 4. LysGH15 protected the mice from lethal MRSA infection.
(A) The rescue of mice from lethal MRSA (YB57) infection using LysGH15. BALB/c mice immunized with lysin or control buffer were challenged intravenously with YB57 MRSA (1 × 1010 CFU/mouse). One hour later, 50 μg of LysGH15 or control buffer was injected into the mice. (B) Colony counts in peripheral blood samples. At the indicated times, the bacterial counts (CFU/mL) in the mice were determined from peripheral blood samples (10 μL) obtained from the caudal vein (n = 6 mice per group per experiment). *P < 0.05 compared with the buffer-treated control. The data are representative of 3 experiments.
Figure 5
Figure 5. LysGH15 reduced pro-inflammatory cytokines.
The BALB/c mice immunized with LysGH15 were injected intravenously with MRSA strain YB57 (1 × 1010 CFU/mouse). One hour later, these mice were treated with LysGH15 or the control buffer. At the indicated time points, the levels of TNF-α, IFN-γ, IL-1β, and IL-5 in the serum were determined (n = 6 mice per group per experiment). *P < 0.05 compared with the buffer-treated control; #P < 0.05 compared with the uninfected control. The data are representative of 3 experiments.
Figure 6
Figure 6. LysGH15 did not cause significant adverse effects.
The mice were immunized with LysGH15 at 3 weeks prior to the intravenous administration of large amounts of LysGH15 or buffer. The mice were monitored for 10 days and euthanized on day 11. (A) The health score was assessed as described in the Methods section. (B) Pathological changes and histopathology of the organs. The hearts, livers, spleens, lungs, kidneys, and colons were stained with haematoxylin and eosin. n = 6 mice per group per experiment. *P < 0.05 compared to the buffer-treated control; N.S. not significant. The data are representative of three experiments.

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References

    1. Blodkamp S. et al. . In vitro activity of human and animal cathelicidins against livestock-associated methicillin-resistant Staphylococcus aureus. Veterinary microbiology, 10.1016/j.vetmic.2015.09.018 (2015). - DOI - PubMed
    1. Knox J., Uhlemann A. C. & Lowy F. D. Staphylococcus aureus infections: transmission within households and the community. Trends Microbiol 23, 437–444, 10.1016/j.tim.2015.03.007 (2015). - DOI - PMC - PubMed
    1. Miller L. G. et al. . Necrotizing fasciitis caused by community-associated methicillin-resistant Staphylococcus aureus in Los Angeles. The New England journal of medicine 352, 1445–1453, 10.1056/NEJMoa042683 (2005). - DOI - PubMed
    1. Tenover F. C. et al. . Vancomycin-resistant Staphylococcus aureus isolate from a patient in Pennsylvania. Antimicrobial agents and chemotherapy 48, 275–280, 10.1128/AAC.48.1.275-280.2004 (2004). - DOI - PMC - PubMed
    1. Weigel L. M. et al. . Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus. Science 302, 1569–1571, 10.1126/science.1090956 (2003). - DOI - PubMed

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