RESUMEN
Towards the development of a methicillin-resistant Staphylococcus aureus (MRSA) vaccine we evaluated a neutralizing anti-glucosaminidase (Gmd) monoclonal antibody (1C11) in a murine model of implant-associated osteomyelitis, and compared its effects on LAC USA300 MRSA versus a placebo and a Gmd-deficient isogenic strain (ΔGmd). 1C11 significantly reduced infection severity, as determined by bioluminescent imaging of bacteria, micro-CT assessment of osteolysis, and histomorphometry of abscess numbers (p < 0.05). Histology also revealed infiltrating macrophages, and the complete lack of staphylococcal abscess communities (SAC), in marrow abscesses of 1C11 treated mice. In vitro, 1C11 had no direct effects on proliferation, but electron microscopy demonstrated that 1C11 treatment phenocopies ΔGmd defects in binary fission. Moreover, addition of 1C11 to MRSA cultures induced the formation of large bacterial aggregates (megaclusters) that sedimented out of solution, which was not observed in ΔGmd cultures or 1C11 treated cultures of a protein A-deficient strain (ΔSpa), suggesting that the combined effects of Gmd inhibition and antibody-mediated agglutination are required. Finally, we demonstrated that macrophage opsonophagocytosis of MRSA and megaclusters is significantly increased by 1C11 (p < 0.01). Collectively, these results suggest that the primary mechanism of anti-Gmd humoral immunity against MRSA osteomyelitis is macrophage invasion of Staphylococcal abscess communities (SAC) and opsonophagocytosis of megaclusters. .
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Hexosaminidasas/inmunología , Osteomielitis/prevención & control , Fagocitosis/inmunología , Infecciones Relacionadas con Prótesis/prevención & control , Infecciones Estafilocócicas/prevención & control , Animales , Proliferación Celular/efectos de los fármacos , Femenino , Inmunización Pasiva , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Ratones Endogámicos BALB C , Proteínas Opsoninas/toxicidad , Osteomielitis/microbiología , Infecciones Relacionadas con Prótesis/microbiología , Infecciones Estafilocócicas/microbiologíaRESUMEN
Neutrophils, or polymorphonuclear leukocytes, comprise a crucial component of innate immunity, controlling bacterial and fungal infection through a combination of both oxidative and nonoxidative mechanisms. Indeed, neutrophils are believed to play an important role in controlling infection caused by the major human pathogen Streptococcus pneumoniae. However, the method by which neutrophils kill the pneumococcus as well as other Gram-positive bacteria, is not fully understood. We investigated human neutrophil killing of the pneumococcus in a complement-dependent opsonophagocytic assay. In contrast to other Gram-positive organisms, inhibition of the NADPH oxidase did not affect killing of S. pneumoniae. Supernatant from degranulated neutrophils killed the pneumococcus, suggesting a role for granular products. When neutrophil granule proteases were inhibited with either a protease mixture, or specific serine protease inhibitors 4-(2-Aminoethyl)benzenesulfonylfluoride and diisopropylfluorophosphate, killing by neutrophils was inhibited in a manner that correlated with increased intracellular survival. All three compounds inhibited intracellular activity of the three major neutrophil serine proteases: elastase, cathepsin G, and proteinase 3. Additionally, purified elastase and cathepsin G were sufficient to kill S. pneumoniae in a serine protease dependent-manner in in vitro assays. Inhibition studies using specific inhibitors of these serine proteases suggested that while each serine protease is sufficient to kill the pneumococcus, none is essential. Our findings show that Gram-positive pathogens are killed by human neutrophils via different mechanisms involving serine proteases.