Carly A. Dillen

Polymeric nanofiber coating with tunable combinatorial antibiotic delivery prevents biofilm-associated infection in vivo

Significance Biofilm infections are a major complication associated with implantable medical devices and prostheses, which are exceedingly difficult to treat. To date, there has been no effective clinical solution that combines antibacterial efficiency with excellent osseointegration. Here, a nanofiber-based conformal coating capable of controlled and independent local delivery of two or more combinatorial antibiotics was developed to provide optimal antimicrobial activity for the prevention of biofilm-associated infections. In a preclinical animal model of orthopedic-implant infection, this technology demonstrated complete bacterial clearance from the implant and surrounding bone/joint tissue while promoting osseointegration. This tunable nanofiber composite coating could be highly effective in preventing medical device infections in patients. Bacterial biofilm formation is a major complication of implantable medical devices that results in therapeutically challenging chronic infections, especially in cases involving antibiotic-resistant bacteria. As an approach to prevent these infections, an electrospun composite coating comprised of poly(lactic-coglycolic acid) (PLGA) nanofibers embedded in a poly(ε-caprolactone) (PCL) film was developed to locally codeliver combinatorial antibiotics from the implant surface. The release of each antibiotic could be adjusted by loading each drug into the different polymers or by varying PLGA:PCL polymer ratios. In a mouse model of biofilm-associated orthopedic-implant infection, three different combinations of antibiotic-loaded coatings were highly effective in preventing infection of the bone/joint tissue and implant biofilm formation and were biocompatible with enhanced osseointegration. This nanofiber composite-coating technology could be used to tailor the delivery of combinatorial antimicrobial agents from various metallic implantable devices or prostheses to effectively decrease biofilm-associated infections in patients.

Mouse model of hematogenous implant-related Staphylococcus aureus biofilm infection reveals therapeutic targets

Significance Hematogenous implant-related infections are an important clinical problem because bacteria spread from the bloodstream to a previously well-functioning implant and result in infectious complications and failure of a medical device or prosthesis. To study these infections, we developed a preclinical animal model of a Staphylococcus aureus hematogenous implant infection with the capability to monitor noninvasively and longitudinally the dissemination of the bacteria from the blood to a surgically placed orthopedic implant. Using this model, α-toxin and clumping factor A were identified as key factors that contributed to the pathogenesis of these infections by promoting biofilm formation. Finally, neutralizing antibodies against these factors provided a targeted, nonantibiotic alternative approach to help prevent these difficult-to-treat and costly infections. Infection is a major complication of implantable medical devices, which provide a scaffold for biofilm formation, thereby reducing susceptibility to antibiotics and complicating treatment. Hematogenous implant-related infections following bacteremia are particularly problematic because they can occur at any time in a previously stable implant. Herein, we developed a model of hematogenous infection in which an orthopedic titanium implant was surgically placed in the legs of mice followed 3 wk later by an i.v. exposure to Staphylococcus aureus. This procedure resulted in a marked propensity for a hematogenous implant-related infection comprised of septic arthritis, osteomyelitis, and biofilm formation on the implants in the surgical legs compared with sham-operated surgical legs without implant placement and with contralateral nonoperated normal legs. Neutralizing human monoclonal antibodies against α-toxin (AT) and clumping factor A (ClfA), especially in combination, inhibited biofilm formation in vitro and the hematogenous implant-related infection in vivo. Our findings suggest that AT and ClfA are pathogenic factors that could be therapeutically targeted against S. aureus hematogenous implant-related infections.

Neutralizing α-toxin accelerates healing of Staphylococcus aureus-infected wounds in normal and diabetic mice

ABSTRACT Staphylococcus aureus wound infections delay healing and result in invasive complications such as osteomyelitis, especially in the setting of diabetic foot ulcers. In preclinical animal models of S. aureus skin infection, antibody neutralization of alpha-toxin (AT), an S. aureus-secreted pore-forming cytolytic toxin, reduces disease severity by inhibiting skin necrosis and restoring effective host immune responses. However, whether therapeutic neutralization of alpha-toxin is effective against S. aureus-infected wounds is unclear. Herein, the efficacy of prophylactic treatment with a human neutralizing anti-AT monoclonal antibody (MAb) was evaluated in an S. aureus skin wound infection model in nondiabetic and diabetic mice. In both nondiabetic and diabetic mice, anti-AT MAb treatment decreased wound size and bacterial burden and enhanced reepithelialization and wound resolution compared to control MAb treatment. Anti-AT MAb had distinctive effects on the host immune response, including decreased neutrophil and increased monocyte and macrophage infiltrates in nondiabetic mice and decreased neutrophil extracellular traps (NETs) in diabetic mice. Similar therapeutic efficacy was achieved with an active vaccine targeting AT. Taken together, neutralization of AT had a therapeutic effect against S. aureus-infected wounds in both nondiabetic and diabetic mice that was associated with differential effects on the host immune response.

NMR structure-based optimization of Staphylococcus aureus sortase A pyridazinone inhibitors

Staphylococcus aureus is a leading cause of hospital‐acquired infections in the USA and is a major health concern as methicillin‐resistant S. aureus and other antibiotic‐resistant strains are common. Compounds that inhibit the S. aureus sortase (SrtA) cysteine transpeptidase may function as potent anti‐infective agents as this enzyme attaches virulence factors to the bacterial cell wall. While a variety of SrtA inhibitors have been discovered, the vast majority of these small molecules have not been optimized using structure‐based approaches. Here we have used NMR spectroscopy to determine the molecular basis through which pyridazinone‐based small molecules inhibit SrtA. These inhibitors covalently modify the active cysteine thiol and partially mimic the natural substrate of SrtA by inducing the closure of an active site loop. Computational and synthetic chemistry methods led to second‐generation analogues that are ~70‐fold more potent than the lead molecule. These optimized molecules exhibit broad‐spectrum activity against other types of class A sortases, have reduced cytotoxicity, and impair SrtA‐mediated protein display on S. aureus cell surface. Our work shows that pyridazinone analogues are attractive candidates for further development into anti‐infective agents, and highlights the utility of employing NMR spectroscopy and solubility‐optimized small molecules in structure‐based drug discovery.

α-Toxin Regulates Local Granulocyte Expansion from Hematopoietic Stem and Progenitor Cells in Staphylococcus aureus–Infected Wounds

The immune response to Staphylococcus aureus infection in skin involves the recruitment of polymorphonuclear neutrophils (PMNs) from the bone marrow via the circulation and local granulopoiesis from hematopoietic stem and progenitor cells (HSPCs) that also traffic to infected skin wounds. We focus on regulation of PMN number and function and the role of pore-forming α-toxin (AT), a virulence factor that causes host cell lysis and elicits inflammasome-mediated IL-1β secretion in wounds. Infection with wild-type S. aureus enriched in AT reduced PMN recruitment and resulted in sustained bacterial burden and delayed wound healing. In contrast, PMN recruitment to wounds infected with an isogenic AT-deficient S. aureus strain was unimpeded, exhibiting efficient bacterial clearance and hastened wound resolution. HSPCs recruited to infected wounds were unaffected by AT production and were activated to expand PMN numbers in proportion to S. aureus abundance in a manner regulated by TLR2 and IL-1R signaling. Immunodeficient MyD88-knockout mice infected with S. aureus experienced lethal sepsis that was reversed by PMN expansion mediated by injection of wild-type HSPCs directly into wounds. We conclude that AT-induced IL-1β promotes local granulopoiesis and effective resolution of S. aureus–infected wounds, revealing a potential antibiotic-free strategy for tuning the innate immune response to treat methicillin-resistant S. aureus infection in immunodeficient patients.

The fungal ligand chitin directly binds and signals inflammation dependent on oligomer size and TLR2

Chitin is a highly abundant polysaccharide and linked to fungal infection and asthma. Unfortunately, its polymeric structure has hampered the identification of immune receptors directly binding chitin and signaling immune activation and inflammation, because purity, molecular structure and molarity are not well definable for a polymer typically extracted from biomass. Therefore, by using defined chitin (N-acetyl-glucosamine) oligomers, we identified six subunit long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR) 2 as the primary fungal chitin receptor on human and murine immune cells. Chitin oligomers directly bound TLR2 with nanomolar affinity and showed both overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Conversely, chitin oligomers shorter than 6 subunits were inactive or showed antagonistic effects on chitin/TLR2-mediated signaling, hinting to a size-dependent sensing/activation system unexpectedly conserved in plants and humans. Since blocking the chitin-TLR2 interaction effectively prevented chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease.

Injury, dysbiosis, and filaggrin deficiency drive skin inflammation through keratinocyte IL-1α release

Background Atopic dermatitis (AD) is associated with epidermal barrier defects, dysbiosis, and skin injury caused by scratching. In particular, the barrier‐defective epidermis in patients with AD with loss‐of‐function filaggrin mutations has increased IL‐1&agr; and IL‐1&bgr; levels, but the mechanisms by which IL‐1&agr;, IL‐1&bgr;, or both are induced and whether they contribute to the aberrant skin inflammation in patients with AD is unknown. Objective We sought to determine the mechanisms through which skin injury, dysbiosis, and increased epidermal IL‐1&agr; and IL‐1&bgr; levels contribute to development of skin inflammation in a mouse model of injury‐induced skin inflammation in filaggrin‐deficient mice without the matted mutation (ft/ft mice). Methods Skin injury of wild‐type, ft/ft, and myeloid differentiation primary response gene–88–deficient ft/ft mice was performed, and ensuing skin inflammation was evaluated by using digital photography, histologic analysis, and flow cytometry. IL‐1&agr; and IL‐1&bgr; protein expression was measured by means of ELISA and visualized by using immunofluorescence and immunoelectron microscopy. Composition of the skin microbiome was determined by using 16S rDNA sequencing. Results Skin injury of ft/ft mice induced chronic skin inflammation involving dysbiosis‐driven intracellular IL‐1&agr; release from keratinocytes. IL‐1&agr; was necessary and sufficient for skin inflammation in vivo and secreted from keratinocytes by various stimuli in vitro. Topical antibiotics or cohousing of ft/ft mice with unaffected wild‐type mice to alter or intermix skin microbiota, respectively, resolved the skin inflammation and restored keratinocyte intracellular IL‐1&agr; localization. Conclusions Taken together, skin injury, dysbiosis, and filaggrin deficiency triggered keratinocyte intracellular IL‐1&agr; release that was sufficient to drive chronic skin inflammation, which has implications for AD pathogenesis and potential therapeutic targets. Graphical abstract Figure. No Caption available.

Mouse model of Gram-negative prosthetic joint infection reveals therapeutic targets

Bacterial biofilm infections of implantable medical devices decrease the effectiveness of antibiotics, creating difficult-to-treat chronic infections. Prosthetic joint infections (PJI) are particularly problematic because they require prolonged antibiotic courses and reoperations to remove and replace the infected prostheses. Current models to study PJI focus on Gram-positive bacteria, but Gram-negative PJI (GN-PJI) are increasingly common and are often more difficult to treat, with worse clinical outcomes. Herein, we sought to develop a mouse model of GN-PJI to investigate the pathogenesis of these infections and identify potential therapeutic targets. An orthopedic-grade titanium implant was surgically placed in the femurs of mice, followed by infection of the knee joint with Pseudomonas aeruginosa or Escherichia coli. We found that in vitro biofilm-producing activity was associated with the development of an in vivo orthopedic implant infection characterized by bacterial infection of the bone/joint tissue, biofilm formation on the implants, reactive bone changes, and inflammatory immune cell infiltrates. In addition, a bispecific antibody targeting P. aeruginosa virulence factors (PcrV and Psl exopolysaccharide) reduced the bacterial burden in vivo. Taken together, our findings provide a preclinical model of GN-PJI and suggest the therapeutic potential of targeting biofilm-associated antigens.

The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size

Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N‐acetyl‐glucosamine) oligomers, we here identify six‐subunit‐long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll‐like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size‐dependent system of immuno‐modulation that appears conserved in plants and humans. Since blocking of the chitin‐TLR2 interaction effectively prevents chitin‐mediated inflammation in vitro and in vivo, our study highlights the chitin‐TLR2 interaction as a potential target for developing novel therapies in chitin‐related pathologies and fungal disease.