Targeting of alpha-hemolysin by active or passive immunization decreases severity of USA300 skin infection in a mouse model

Abstract

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are predominantly those affecting skin and soft tissues. Although progress has been made, our knowledge of the molecules that contribute to the pathogenesis of CA-MRSA skin infections is incomplete. We tested the hypothesis that alpha-hemolysin (Hla) contributes to the severity of USA300 skin infections in mice and determined whether vaccination against Hla reduces disease severity. Isogenic hla-negative (Deltahla) strains caused skin lesions in a mouse infection model that were significantly smaller than those caused by wild-type USA300 and Newman strains. Moreover, infection due to wild-type strains produced dermonecrotic skin lesions, whereas there was little or no dermonecrosis in mice infected with Deltahla strains. Passive immunization with Hla-specific antisera or active immunization with a nontoxigenic form of Hla significantly reduced the size of skin lesions caused by USA300 and prevented dermonecrosis. We conclude that Hla is a potential target for therapeutics or vaccines designed to moderate severe S. aureus skin infections.

Figure 1

hla contributes to the pathogenesis of USA300 skin infections. Results are the mean +/- SEM for all groups; (n) = number of mice per group. A and B, Mouse abscess size was monitored once per day after subcutaneous infection with 1 × 10⁷ of the indicated bacteria. *, p < 0.05 versus wild-type LAC or Newman strains using a two-way ANOVA and Bonferroni's posttest. C, Size of abscesses at maximum. *, p < 0.05 using an ANOVA and Dunnett's posttest.

Figure 2

hla promotes dermonecrosis. A, Representative histological sections showing normal mouse skin tissue (PBS), mouse USA300 abscess with dermonecrosis (LAC), and an hla-deficient USA300 abscess without dermonecrosis (LACΔhla). Mice were inoculated s.c. with either PBS, LAC, or LACΔhla as described in Methods and skin was harvested on day 3. Original magnification of images labeled as PBS, LAC and LACΔhla is ×200; that for LAC (lower magnification) is ×100. B, Representative mouse skin lesions (day 5). Black arrows indicate dermonecrosis and white arrows indicate abscess formation without dermonecrosis. C, Percentage of mice per group that had dermonecrosis on each day. p < 0.001 for mice infected with either LAC or Newman wild-type or complemented mutant strains versus Δhla strains over the 14-day time course. Data were analyzed using a one-way ANOVA and Dunnett's posttest.

Figure 3

Passive immunization with Hla-specific rabbit anti-sera (anti-Hla) reduces size of lesions caused by USA300 or Newman. A and B, Mice received 100 μl of pre-immune rabbit sera (Pre-immune) or Hla-specific rabbit anti-sera (anti-Hla) 4 h before subcutaneous infection with 1 × 10⁷ with LAC or Newman and on day 2 post-infection. Results are the mean +/- SEM for all groups; n = 15 mice per group. *, p < 0.05 versus wild-type LAC or Newman strains using a two-way ANOVA and Bonferroni's posttest.

Figure 4

Passive immunization with Hla-specific rabbit anti-sera prevents dermonecrosis. A, Percentage of mice per group that had dermonecrosis on each day. p < 0.0001 for mice administered pre-immune versus anti-Hla serum following infection with either LAC or Newman over the 14-day time course. B, Representative skin lesions of mice on day 3 for each of the treatment conditions. Passive immunization was performed as described in Methods and the legend of figure 3. Pre-immune rabbit sera (Pre-immune); Hla-specific rabbit anti-sera (anti-Hla). Black arrows indicate dermonecrosis and white arrows indicate abscess formation without dermonecrosis.

Figure 5

Active immunization with Hla_H35L decreases size of abscesses caused by USA300 or Newman. Mice were injected i.m. with CFA + DPBS at 4 wks of age followed by IFA + DPBS 10 days later (sham) or CFA + Hla_H35L at 4 wks of age and IFA + Hla_H35L 10 days later (immunized). A and B, Abscess formation was monitored once per day after subcutaneous infection with 1 × 10⁷ of the indicated bacteria 21 days after primary immunization. Results are the mean +/- SEM and n = 15 mice per group. *, p < 0.05 versus wild-type LAC or Newman strains using a two-way ANOVA and Bonferroni's posttest.

Figure 6

Active immunization with Hla_H35L prevents dermonecrosis caused by USA300 or Newman skin infections. A, Percentage of mice per group that had dermonecrosis on each day. Immunization is described in the legend of figure 5. p < 0.0001 for sham versus immunized mice following infection with either LAC or Newman over the 14-day time course. B, Representative mouse skin lesions, day 5. Black arrows indicate dermonecrosis and white arrows indicate abscess formation without dermonecrosis.