A bacteriomimetic inhibitor based on the adhesin MAM7 decreases bacterial burden on burn wounds

The adhesin MAM7 is an outer membrane protein that is widely expressed by Gram-negative pathogens. It consists of seven protein domain repeats that are responsible for attachment to host cells during an infection. A recombinant fragment of MAM7 containing these repeats is expressed as a Glutathione-S-Transferase (GST) fusion protein, GST-MAM7. Purified, recombinant GST-MAM7 is chemically coupled to a micron-sized, spherical polystyrene scaffold and this composition provides a mimic for its endogenous presentation on the surface of a bacterium (Fig. 1). Multivalent surface display significantly enhances binding avidity and thus, the material’s competitive effect against pathogens (Hawley et al.17). GST-MAM7 functionalized beads and control microbeads functionalized with only GST were synthesized on a milligram scale and tested in a quality control inhibition assay using an in vitro infection model as previously described17,18,19,20.

Figure 1 Schematic of inhibitor synthesis. (A) Carboxy-functionalized polystyrene micro-beads of 1 μm diameter are activated using EDC/NHS, and covalently coupled to GST (control beads) or GST-MAM7 (inhibitor beads) using Sulfo-SMPB. This results in directional coupling of recombinant proteins to the bead surface via the cysteine-containing GST domain. (B) Schematic of GST-MAM7 bead mimicry of bacterial MAM7 presentation. Full size image

To assess whether GST-MAM7 beads could decrease the bacterial load in burns, we used a burn and excision infection rat model. For severe burns, early excision, followed by application of wound dressings and/or grafting is currently the standard of care1. Early surgical excision to remove necrotic tissue within the first few days following injury is routinely used in treatment of extensive burns, and has significantly contributed to decreased occurrence of burn wound sepsis and improved survival21,22. For this study, dorsal burn wounds covering 40% of the total body surface area were induced onto the back of adult rats and two days after injury, eschar tissue was surgically excised (Fig. 2A)23. Excision prior to treatment with the MAM7 inhibitor increased the treatment efficacy, compared to unexcised, necrotic burns (Fig. 3). Coincidently, on day two, a suspension of 5·106 CFU of P. aeruginosa Xen5, a bioluminescent derivative of a multidrug-resistant septicemia blood isolate24, were introduced into the excision. Suspensions containing 3·108 GST beads or GST-MAM7 beads in saline were applied topically to the wound bed at the same time and, then, applied every 24 hours after excision. The bacterial loads of the infection were monitored by biophotonic imaging over 6 days (Fig. 2A).

Figure 2 Treatment of excised burn wounds with a GST-MAM beads decreases bacterial burden and spatially constrains the spread of infection. (A) Schematic of infection and dosing schedule. (B) Representative bioluminescence images of control bead treated (upper row) or MAM7 inhibitor treated (bottom row) P. aeruginosa infected excisions over 6 days after infection (d.p.i.). (C) Quantification of bacterial loads in control bead-treated (shades of blue) and inhibitor treated (shades of red) animals using IVIS biophotonic imaging. Different shading indicates three sets of independent trials. Data for individual animals, means ± s.e.m. for each treatment group are depicted. Analysis of variance (ANOVA), followed by Tukey’s post hoc test, was used to test for significance. (*) indicates p ≤ 0.05, ns, not significant. (D) Quantitative analysis of the spread of infection to adjacent tissues. Full size image

Figure 3 Inhibition of P. aeruginosa in unexcised burns. Schematic of infection and dosage regimen (A). Representative bioluminescence images of MAM7 inhibitor treated (bottom row) and control bead treated (upper row) rats infected with P. aeruginosa (B). Quantification of bacterial burden by IVIS photon flux analysis (C). Full size image

In the infection that was treated with control GST beads, the bacteria were initially constrained to the edge of the surgical excision, but gradually spread across the entire burn area over the course of 6 days (Fig. 2B). By contrast, in wounds treated with GST-MAM7 beads, the bacteria remained on the edge of the wound (Fig. 2B). Over the first 24 hours, GST-MAM7 bead treatment reduced the bacterial burden below the initial inoculum and, then, remained lower than the control treatment for an additional three days. In control treated animals, the bacterial burden increased approximately 10-fold over the course of the 6 day experiment (Fig. 2C). Treatment with the MAM7 inhibitor beads significantly decreased bacterial colonization of the burn for four days following infection when compared to mock treatment, although leveled off towards the end of the experiment. These studies demonstrate that fresh wounds can be treated with GST-MAM7 functionalized beads as an early intervention and the bacterial load will be reduced in the excision and surrounding burnt area (Fig. 2).

Treatment of excised burn wounds with GST-MAM beads decreases bacterial spread

We considered the possibility that the anti-adhesion activity of MAM7 may simply displace burn-colonizing bacteria to adjacent tissue sites. To address this, we used biophotonic imaging to follow the spread of the infection over the wounded area (Fig. 2D; the peaks represent bacterial burden across the wound). In the control animals treated with GST-beads, we observed a gradual spread of bacteria (light blue to dark blue) over the wound during the course of the 6 day experiment. By contrast, in the GST-MAM7 bead treated animals, the spread and amount of bacterial colonization (pink to red) is substantially lower during the experimental time course. This demonstrates that GST-MAM7 beads not only lower the total bacterial burden in the wound, but spatially confine bacterial colonization and inhibit the spread of infection.

Beads functionalized with MAM7 adhesin do not perturb angiogenesis or wound closure

Antimicrobial treatments for wounds must not impair the natural wound healing process. Thus, to assess the impact of GST-MAM7 beads on vascularization and wound closure, we analyzed H&E sections of burned tissue on day 6 of the infection. In Fig. 4A, a section of skin containing a burn is shown in three parts: 1. unburned, normal tissue; 2. burned tissue with eschar intact; 3. burned tissue with the eschar excised. Analysis of an uninfected tissue sample from the burned tissue with the eschar excised shows normal granulation, vascularization and initiation of wound healing (Fig. 4A). When the GST-beads or GST-MAM7 beads are applied to the burned tissue with the eschar excised, similar granulation, vascularization and initiation of wound healing is observed (Fig. 4C and D). Therefore, the beads alone did not disrupt normal healing. Burned tissue with the eschar excised that was treated with P. aeruginosa and the GST-beads or GST-MAM7 beads also appeared to heal in a normal manner (Fig. 4E and F). Further quantitative analysis of wound closure revealed no significant differences in healing between GST-beads or GST-MAM7 beads treated groups, with excision areas decreasing by approximately 22 and 30%, respectively, over 6 days (Fig. 4G). Thus, MAM7 bead treatment does not impair normal burn wound healing.

Figure 4 Angiogenesis and wound closure are unaffected by treatment with GST-MAM7 beads. (A) H&E-stained skin tissue sampled post-mortem (6 d.p.i.) depicting normal skin (left section), burned skin with eschar (middle section), and burned skin with excised eschar (right section). Boxed area highlights region that was used to analyze vascularization in the following samples: (B) burned skin only, (C) burned skin with GST-beads, (D) burned skin with GST-MAM7-beads, (E) burned skin with GST-beads + P. aeruginosa, and (F) burned skin with GST-MAM7-beads + P. aeruginosa. Arrows point to blood vessels. Scale bar equals to 1000 μm in (A) and 100 μm in (B–F). (G) Analysis of excision closure from excised, uninfected animals. Full size image

Normal neutrophil infiltration of burned skin is observed in GST-MAM7 bead treatment

Severe burns generally elicit a strong inflammatory response, both locally and in the tissues adjacent to the burn25. A moderate pro-inflammatory response encourages wound healing, but an excessive inflammatory response leads to organ damage and dysfunction26. We therefore tested if application of control beads or adhesin beads to burns without and with P. aeruginosa would have an effect on local inflammation. Adjacent sections of tissues shown in Fig. 3 were used to assess neutrophil infiltration. In untreated animals, a normal migration of neutrophils moving toward the wounded surface area is observed (Fig. 5).

Figure 5 Neutrophil infiltration on burned skin is unimpaired by treatment with GST-MAM7 beads. Confocal micrographs of burned skin area as in Fig. 1A was used to analyze neutrophil infiltration for the following samples: (A) burned skin only, (B) burned skin with GST-beads, (C) burned skin with GST-MAM7-beads, (D) burned skin with GST-beads + P. aeruginosa, and (E) burned skin with GST-MAM7-beads + P. aeruginosa. Hoechst DNA (blue) and mpo staining for neutrophils (green). Scale bar equals to 150 μm. Samples were taken post-mortem (6 d.p.i.). Full size image

In addition, as observed with the green channel background, vascularization is occurring in the healing tissue (Fig. 6A). The application of GST beads or GST-MAM7 beads to uninfected or P. aeruginosa infected burned and excised wounds did not lead to a change near newly formed blood vessels (Fig. 6A–E) or neutrophil migration towards the wound (Figs 5A–E and 6A–E). Overall, the observation that GST-MAM7 does not hinder wound healing are consistent with our in vitro infection studies showing that, despite MAM7 binding to host surface receptors including fibronectin27, MAM7 inhibitor treatment does not interfere with processes intimately associated with wound healing, including proliferation and extracellular matrix formation17.

Figure 6 Neutrophil infiltration and vascularization of burned skin is unimpaired by GST-MAM7 beads. Confocal micrographs of burned skin (as in boxed area depicted in Fig. 1A) were used to analyze neutrophil infiltration (bright green fluorescent clumps) and vascularization (observed through green channel background) for the following samples: (A) burned skin only, (B) burned skin with GST-beads, (C) burned skin with GST-MAM7-beads, (D) burned skin with GST-beads + P. aeruginosa, and (E) burned skin with GST-MAM7-beads + P. aeruginosa. Arrows point to blood vessels. Scale bar equals to 150 μm. (F) Serum cytokine levels of burned only (grey), burned inhibitor (red) or burned control (blue) treated uninfected animals. (G) Serum cytokine levels of inhibitor (red) or control (blue) treated animals with P. aeruginosa infected burns. Data presented are means ± stdev (n = 6 for inhibitor and bead treated groups, n = 5 for burn only group, each sample measured in triplicate against cytokine standards). All samples were taken post-mortem (6 d.p.i.). Full size image

Application of adhesin functionalized beads does not alter systemic inflammatory reponsens

Although wound healing was visually normal in adhesin-bead treated wounds, we considered the possibility that the application of these beads may provoke a systemic inflammatory response that could be deleterious to the host. We therefore tested levels of circulating serum cytokines in the test animals. However, in all cases animals that were untreated or treated with GST beads or GST-MAM7 beads expressed similar levels of circulating serum cytokines (Fig. 5F). In animals infected with P. aeruginosa, the induction of circulating serum cytokines was unaltered by application of MAM7 adhesin beads, compared to control beads (Fig. 5G). Therefore, the application of MAM7 inhibitor to P. aeruginosa infected wounds did not alter systemic inflammatory responses.