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-1ß 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-1ß 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.

Engineering immunomodulatory nanoplatforms from commensal bacteria-derived polysaccharide A.

Capsular zwitterionic polysaccharides (CZPs), typically found on the surfaces of commensal gut bacteria, are important immunomodulatory molecules due to their ability to produce a T cell dependent immune response upon processing by antigen presenting cells (APCs). Their immunological activity makes them potentially useful for generating material constructs that are applicable for the treatment of diseases, or as vaccines. Herein, we explored synthetic strategies to generate immunologically active polymer-carbohydrate conjugates and nanomaterials of the CZP, Polysaccharide A (PSA) derived from Bacteroides fragilis. Initially, we addressed the purification of PSA, which is critical for the realization of materials applicable for biomedical purposes. Anion exchange high performance liquid chromatography in the presence of a surfactant (CHAPS) enabled the isolation of pure PSA. Through modification of purified PSA with azide groups, we demonstrated that polymers or antigens could be incorporated with PSA via click chemistry reactions to generate conjugates that can be fabricated into nanoparticles. By conjugation of PSA with a DBCO end functionalized polyphosphoester polymer with hydrophobic pendant terminal alkyne groups, an amphiphilic conjugate was obtained which formed nanoparticles of about 100 nm in aqueous solution. Moreover, terminal alkyne groups could be modified with charged thiol molecules (amine/carboxylate) via thiol-yne radical chemistry to generate conjugates, which could be incorporated into nanoparticles via electrostatic interactions building onto a charged nanoparticle template. The conjugates and nanoparticles exhibited immunological activity as assessed by the toll-like receptor 2 (TLR2) activation assay and positive cytokine production (IL-10) following their co-incubation with APCs and T cells. Summarily, this work plainly demonstrates chemical biology strategies for fabricating immunomodulatory nanomaterials from commensal microorganisms that can potentially be novel vaccines or immunotherapeutics.