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Targeting of Alpha-Hemolysin by Active or Passive Immunization Decreases Severity of USA300 Skin Infection in a Mouse Model

  1. Adam D. Kennedy1,
  2. Juliane Bubeck Wardenburg2,3,
  3. Donald J. Gardner4,
  4. Daniel Long4,
  5. Adeline R. Whitney1,
  6. Kevin R. Braughton1,
  7. Olaf Schneewind2 and
  8. Frank R. DeLeo1
  1. 1Laboratory of Human Bacterial Pathogenesis, National Institutes of Health, Hamilton, Montana
  2. 2Department of Microbiology, University of Chicago, Chicago, Illinois
  3. 3Department of Pediatrics, University of Chicago, Chicago, Illinois
  4. 4Veterinary Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana
  1. Reprints or correspondence: Dr Frank R. DeLeo, Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, NIAID, NIH, 903 South 4th St, Hamilton, MT 59840 (fdeleo{at}niaid.nih.gov).
 
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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 alphahemolysin (Hla) contributes to the severity of USA300 skin infections in mice and determined whether vaccination against Hla reduces disease severity. Isogenic hla-negative (Δhla) 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 Δhla 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.

Staphylococcus aureus is a leading cause of infection in hospitals, as well as in the community. Communityassociated methicillin-resistant S. aureus (CA-MRSA) strains typically cause infection in otherwise healthy individuals and, therefore, may be considered to be highly virulent. This notion is supported by data from animal infection models, in which prominent CAMRSA strains, such as USA300, are more virulent than are traditional hospital-associated strains [1, 2]. The enhanced virulence phenotype of USA300 is likely to be attributable in part to the relatively high expression of virulence factors, such as phenol soluble modulins and alpha-hemolysin (α-hemolysin, α-toxin, and Hla) [1]. Hla is a secreted pore-forming toxin that has cytolytic activity toward a variety of host cell types, including human keratinocytes, epithelial cells, and lymphocytes [3–8]. Expression of Hla is regulated, at least in part, by the 2-component agr and saeR/S signal-transduction systems [9].

Hla is lethal to animals, especially rodents and rabbits [10], and S. aureus strains that are deficient in hla have significantly reduced virulence in animal infection models [6, 11–18]. Despite this previous work, the role of Hla in the pathogenesis of CA-MRSA infections was unknown until recently. With use of USA300 and USA400 wild-type and isogenic hla-negative mutant (Δhla) strains, Bubeck Wardenburg and colleagues [6, 13] demonstrated that Hla is essential for pathogenesis in a mouse model of CA-MRSA pneumonia. Subsequent studies showed that vaccination against Hla protects mice from lethal USA300 or USA400 pneumonia [6, 13]. More recent studies by Bartlett et al [19] have demonstrated that Hla elicits production of CXC chemokines by host cells during experimental S. aureus pneumonia, thereby promoting severe lung inflammation.

Inasmuch as CA-MRSA infections are primarily those affecting the skin and soft tissues, we determined the role played by Hla in a mouse model of USA300 skin infection and, in turn, tested whether a vaccine approach directed at Hla moderated disease severity.

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Methods

S. aureus strains and culture conditions. S. aureus strains used in this study were characterized previously [13]. To prepare inocula, S. aureus was cultured to mid-exponential phase of growth, washed twice in Dulbecco's phosphate-buffered saline (DPBS), and diluted in DPBS to the appropriate concentration. Inocula were stored on ice until used.

Mouse skin infection model. We used a previously described mouse skin infection model [2, 20, 21]. Animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee at Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Shaved female Balb/c mice were anesthetized with isoflurane and inoculated by subcutaneous injection in the right flank with 1×107 S. aureus in 50 µL of DPBS. Mice were weighed before inoculation, and mass and abscess formation were monitored at 24-h intervals for 14 days. The size of the abscesses was calculated using a standard formula for area (A = [π/2] × l × w), as described elsewhere [2, 20, 21]. Dermonecrosis was scored as present or absent. Fifteen mice were used for each treatment (bacterial strain) or control group, unless otherwise indicated.

Histological examination of mouse tissues. Mouse skin was harvested on days 1, 2, and 3 after inoculation and fixed in 10% neutral-buffered formalin for 48 h. Fixed tissues were dehydrated in a graded series of ethanol, cleared in xylene, infiltrated, and embedded in Paraplast Extra (Thermo Fisher Scientific) after a routine 12-h schedule in a Tissue Tek processor (Sakura). Tissue blocks were sectioned at 5 µm, and slides were stained with hematoxylin-eosin. Tissue sampleswere evaluated by an experienced veterinary pathologist (D.J.G.).

Passive immunization. Rabbit polyclonal Hla-specific antisera was generated using purified HlaH35L as an immunogen, as described elsewhere [6]. Female Balb/c mice (∼7 weeks of age) received 100 µL of rabbit pre-immune serum or polyclonal Hla-specific rabbit antisera via intraperitoneal injection 4 h before S. aureus challenge and 2 days after S. aureus challenge. Animal weight and abscess formation were monitored once per day for 14 days, as noted above. Passive immunization with rabbit anti-Hla sera typically results in circulating rabbit antibody titers of ∼1:500, as detected by enzyme-linked immunosorbent assay (ELISA), which was shown previously to protect against the effects of staphylococcal Hla [6].

Active immunization. Female Balb/c mice (4 weeks of age) were administered 20 µg of endotoxin free glutathione-S-transferase (GST)-HlaH35L in complete Freund's adjuvant (CFA; Difco Laboratories) via intramuscular injection, followed by a boost with 20 µg of endotoxin free GST-HlaH35L in incomplete Freund's adjuvant (IFA; Difco Laboratories) 10 days later. Animals were challenged with S. aureus 21 days after the initial vaccination and monitored once per day for 14 days. Serum samples were collected on day 20 (24 h prior to infection) to assess Hla-specific antibody titer by ELISA.

Anti-Hla ELISA. Blood was obtained from 5 control mice and 10 infected mice via submandibular bleeding, as described by Golde et al [22]. Mouse serum antibody titers were determined by ELISA using MaxiSorp microtiter plates (Thermo- Fisher Scientific) coated with 1 µg/mL HlaH35L, as described elsewhere [6]. Antibody reactivity to Hla was detected with horseradish peroxidase-conjugated secondary antibodies and Opti-EIA (BD Biosciences) using a microplate spectrophotometer (GENios; Tecan).

Statistical analyses. Statistics for abscess or lesion area were performed using a 1-way analysis of variance (ANOVA) with a Dunnett's post-test (Figure 1) or 2-way ANOVA and Bonferroni's post-test, as indicated. Student's t test or 1-way ANOVA and Dunnett's post-test were used to compare the number of mice with dermonecrosis over the course of 14 days (aggregate analysis). Results are expressed as mean ± standard error of the mean, unless otherwise indicated. Analyses were done using GraphPad Prism 5 (GraphPad Software) or SigmaPlot2001 for Windows, version 7.0 (SPSS).

Figure 1
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Figure 1

hla contributes to the pathogenesis of USA300 skin infections. Results are presented as mean values ± standard error of the mean for all groups. The number in parenthesis is the number of mice in each group. A and B, Mouse abscess size monitored once per day after subcutaneous infection with 1 × 107 of the indicated bacteria. *P < .05 versus wild-type USA300 (LAC) or Newman strains using a 2- way analysis of variance (ANOVA) and Bonferroni's post-test. C, Size of abscesses at maximum. *P < .05 using an ANOVA and Dunnett's posttest. PBS, phosphate buffered saline.

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Results

Expression of hla enhances virulence in skin and soft-tissue infections. As a first step toward understanding the role played by hla in USA300 skin infections, we compared skin abscess formation in mice after infection with LAC (USA300) or Newman (non-USA300) wild-type, isogenic hla-negative mutant (Δhla), and Δhla-complemented mutant strains (Δhla:: phla) (Figure 1). Infection of mice with LAC or Newman caused skin abscess or lesions that reached maximum size by day 5 or 6 (Figure 1A and 1B). Mice infected with LACΔhla had significantly smaller lesions than did animals infected with the wild-type strain (Figure 1A and 1C). Unexpectedly, there was no difference in lesion size between Newman wild-type- infected or Δhla—infected mice (Figure 1B and 1C). Animals infected with the complemented mutant strain (LACΔhla::phla) had abscesses that were similar in appearance to those infected with the wild-type strain, but lesions were significantly larger and took much longer to resolve (Figure 1A–C). LACΔhla::phla and NewmanΔhla::phla expressed more Hla than did the wild-type strains [6], thereby providing an explanation for the larger abscess size observed in the mice that were infected with complemented mutant strains (Figure 1). Consistent with differences in abscess size between wild-type and Δhla-infected mice, animals infected with wild-type or complemented mutant strains in general lost more body mass over the course of the experiment than did those infected with Δhla strains (data not shown).

S. aureus skin infections in this animal model manifested as either raised abscesses and lesions or relatively flat dermonecrotic lesions (Figure 2A and 2B). One of the more notable differences in lesion phenotype between wild-type-infected and Δhla-infected mice was in the formation of dermonecrotic skin lesions (Figure 2A and 2B). Mice infected with wild-type strains had dermonecrotic lesions, whereas those infected with LACΔhla and NewmanΔhla had lesions with little or no dermonecrosis (Figure 2B and 2C). Dermonecrosis was defined by necrosis of the epidermis and dermis, and significant numbers of polymorphonuclear leukocytes and cell debris adjacent to this region (Figure 2A). This finding was especially striking in mice that were infected with complemented mutant strains (Figure 2B and 2C). Taken together, these data demonstrate that Hla contributes to the severity of USA300 skin infections.

Figure 2
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Figure 2

hla promotes dermonecrosis. A, Representative histological sections showing normal mouse skin tissue (phosphate-buffered saline; PBS), mouse USA300 abscess with dermonecrosis (LAC), and an hla-deficient USA300 abscess without dermonecrosis (LACΔhla). Mice were inoculated subcutaneously 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 < .001 for per mice infected with either LAC or Newman wild-type strains or complemented mutant strains versus Δhla strains over the 14-day time course. Data were analyzed using a 1-way analysis of variance and Dunnett's post-test.

Passive immunization with Hla-specific antisera reduces skin lesion size and severity. Inasmuch as Hla contributed to disease severity in the mouse skin infection model, we next determined whether passive immunization with rabbit antisera directed against Hla would moderate the severity of skin disease in infected mice. Previous studies have demonstrated that intraperitoneal injection of Hla-specific rabbit antisera protects mice from lethal S. aureus pneumonia [6]. Therefore, we used this vaccination model to test whether passive immunization against Hla prevents or moderates severe skin infection. Skin lesions of mice infected with LAC or Newman strains were significantly smaller after passive immunization with Hla-specific rabbit antisera, compared with lesions of mice that received pre-immune serum samples (Figure 3A and 3B). Moreover, the clinical course of disease in immunized mice is altered, because the peak lesion size is reached by day 2, followed by gradual resolution of the abscess by days 12–14 (Figure 3A and 3B). In contrast, mice treated with pre-immune serum samples prior to infection with LAC experience progressive disease until day 5, after which resolution occurs by days 12–14 (Figure 3A). Similar results were seen for mice infected with strain Newman (Figure 3B). Immunized mice infected with LAC also lost less weight than did those that were given pre-immune serum samples (data not shown).

Figure 3
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Figure 3

Passive immunization with alpha-hemolysin (Hla)-specific rabbit anti-sera (anti-Hla) reduces size of lesions caused by USA300 or Newman strains of Staphylococcus aureus. A and B, Mice received 100 µL of pre-immune rabbit serum samples (pre-immune) or Hla-specific rabbit anti-serum samples (anti-Hla) 4 h before subcutaneous infection with 1 × 107 with LAC or Newman strains and on day 2 after infection. Results are the mean value ± standard error of the mean for all groups; n = 15 mice per group. *P < .05 versus wild-type LAC or Newman strains using a 2-way analysis of variance and Bonferroni's post-test.

Mice that received Hla-specific rabbit antisera either failed to develop dermonecrotic lesions after infection with either LAC or Newman strains or the area of dermonecrosis was limited (Figure 4). Abscesses or lesions of wild-type infected mice that were given Hla-specific antisera developed skin lesions that were comparable in appearance and size to those of mice infected with Δhla strains in the absence of passive vaccination (compare Figure 2 with Figure 4). We note that rabbit pre-immune serum contains naturally occurring antibodies that are specific for S. aureus, and therefore, skin disease caused by wild-type strains in the presence of pre-immune serum was less severe than was skin disease in untreated animals (compare Figure 2B and Figure 4B).

Figure 4
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Figure 4

Passive immunization with alpha-hemolysin (Hla)-specific rabbit anti-sera prevents dermonecrosis. A, Percentage of mice per group that had dermonecrosis on each day. P < .001 for mice administered pre-immune versus anti-Hla serum after infection with either LAC or Newman strains 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. anti-Hla, Hla-specific rabbit anti-sera; Pre-immune, Pre-immune rabbit serum samples. Black arrows indicate dermonecrosis, and white arrows indicate abscess formation without dermonecrosis

Immunization with HlaH35L moderates severity of USA300 skin infections. Previous studies have shown that a non-cytolytic mutant form of Hla (HlaH35L) can be used as protective immunogen against S. aureus infections in animal infection models [6, 17]. To determine whether active immunization with HlaH35L protects mice from severe S. aureus skin infections, mice were vaccinated intramuscularly with HlaH35L 21 days before infection with LAC or Newman strains. Mice vaccinated with HlaH35L produced antibodies specific for the toxin, as determined by ELISA (half-maximal anti-Hla antibody titers were 1:491 ± 49 and 1:596 ± 87 for each of 2 groups of 5 vaccinated mice tested, whereas anti-Hla was undetectable in 5 unvaccinated mice). Correspondingly, S. aureus abscess size was reduced significantly in mice vaccinated with HlaH35L (Figure 5A and 5B). The modest or limited protective effect observed in mice infected with strain Newman may reflect a limited contribution of Hla to the severity of skin disease caused by this strain (Figure 1B). In addition, there was little or no dermonecrosis in infected mice that had been vaccinated (Figure 6), which demonstrates that active immunization with HlaH35L moderates severity of S. aureus skin infections.

Figure 5
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Figure 5

Active immunization with alpha-hemolysin (Hla) H35L decreases the size of abscesses caused by USA300 or Newman strains of Staphylococcus aureus. Mice were injected intramuscularly with complete Freund's adjuvant (CFA) plus Dulbecco's phosphate-buffered saline (DPBS) at 4 weeks of age followed by incomplete Freund's adjuvant (IFA) plus DPBS 10 days later (sham) or CFA plus HlaH35L at 4 weeks of age and IFA plus HlaH35L 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 value ± standard error of the mean; n = 15 mice per group. *P < .05 versus wild-type LAC or Newman strains using a 2-way analysis of variance and Bonferroni's post-test

Figure 6
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Figure 6

Active immunization with alpha-hemolysin (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 < .001 for sham versus immunized mice after infection with either LAC or Newman strains over the 14-day time course. B, Representative mouse skin lesions on day 5. Black arrows indicate dermonecrosis, and white arrows indicate abscess formation without dermonecrosis.

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Discussion

USA300 is the leading cause of community-associated bacterial infections in the United States [23]. In addition, USA300, which is epidemic in the United States, appears to have enhanced virulence, compared with traditional hospital-associated MRSA strains [1, 24]. Although the pathogen can cause severe or fatal invasive disease [25, 26], the vast majority of USA300 infections are those of skin and soft tissue [27, 28]. There has been an intense effort to better understand the mechanisms of USA300 virulence and transmission [29–32]. Recent work indicates that Hla is produced by USA300 at relatively high levels in vitro [33], and studies in animal models have revealed a prominent role for this secreted toxin in the pathogenesis of USA300 pneumonia [6, 13]. However, the contribution of Hla to the severity of USA300 skin infections had not, to our knowledge, been evaluated until now.

Herein, we demonstrate that Hla contributes significantly to the severity of USA300 skin infection in a mouse model. Compared with strain Newman, the contribution of Hla to disease severity was more pronounced in mice that were infected with USA300 (LAC) (Figures 1 and 3). LAC is a widely used clinical isolate representative of the USA300 epidemic clone [12, 24], whereas Newman is a methicillin-susceptible S. aureus strain originally isolated from an individual with secondarily infected tubercular osteomyelitis in (or before) 1952 [34]. Newman expresses less Hla than does LAC [6], and consistent with this observation, deletion of Hla from the Newman strain minimally changed the size of skin lesions (Figure 1B). These data are in accordance with the recent studies of Li et al [1], which indicates that differential expression of virulence molecules, rather than presence or absence in the core genome, dictates (at least in part) differences in virulence among S. aureus strains. Thus, the findings here suggest that factors other than Hla in strain Newman play a more prominent role in the pathogenesis of skin infections, whereas the toxin is a major determinant of severity in USA300 skin infections. It is also evident that multiple S. aureus molecules contribute to skin infections in general, because deletion or neutralization of Hla did not completely ablate formation of abscesses.

Previous studies have not attempted to correlate expression of Hla with severity of skin infections. However, production of Hla was recently shown to correlate with refractory S. aureus skin colonization in patients with atopic dermatitis [35]. Inasmuch as hla is present in the genome of many S. aureus strains, it is likely the toxin contributes to severity of human S. aureus infection, which is a notion that has been confirmed in animal infection models [6, 11, 13, 14, 17, 36]. Although the mouse skin infection model cannot mimic all of the features of human skin infection, such as the size of inoculum, the primary readouts of our mouse skin infection model (skin necrosis and abscess size) are 2 parameters that are used to classify severe human skin and soft-tissue infections [37].Moreover, USA300 is known to cause necrotizing skin and soft-tissue infections in humans [25]. Therefore, it is reasonable to conclude that the mouse skin infection model described herein can be used as a general or rudimentary approximation of S. aureus skin infection in humans.

There is also little information on anti-Hla antibody titers in human skin infections. In 1962, Lack and Towers [38] reported that only 48% of patients with proven S. aureus infection had relatively high anti-Hla antibody titers. More notably, anti- Hla antibody titers did not consistently increase in patients with S. aureus infections, and when they did increase after acute infection, antibody levels then decreased rapidly over time [38]. These early observations suggest that humans may lack or have limited protection against Hla, albeit the anamnestic antibody response to Hla should be robust in individuals reinfected with Hla-producing S. aureus. The passive and active immunization data presented here suggest that Hla is a potential target for vaccines or therapeutics designed to moderate the severity of S. aureus skin infections. Inasmuch as Hla is a core genomeencoded toxin present in virtually all CA-MRSA strains, a therapeutic approach directed at Hla would be relatively broad in scope.

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Acknowledgements

We thank Anita Mora, Austin Athman, and Gary Hettrick (RML), for help with photography, and Ralph Larson and Laura Talley, (RML) for assistance with mouse vaccinations

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), and Region V “Great Lakes” Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Consortium (NIH Award 1-U54-AI-057153 to J.B.W. and O.S.).

  • Received December 30, 2009.
  • Accepted April 9, 2010.
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  1. J Infect Dis. (2010) 202 (7): 1050-1058. doi: 10.1086/656043
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