Passive immunization with anti-glucosaminidase monoclonal antibodies protects mice from implant-associated osteomyelitis by mediating opsonophagocytosis of Staphylococcus aureus megaclusters

Abstract

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. .

Keywords: electron microscopy; methicillin-resistant Staphylococcus aureus (MRSA); opsonophagocytosis; osteomyelitis; passive immunization.

Figure 1. Passive immunization with 1C11 reduces the severity of S. aureus implant-associated osteomyelitis

(A) Mice were immunized and challenged as described in Methods. All placebo-treated mice displayed a robust BLI signal (top), while half of the 1C11-treated mice had a dramatically reduced BLI signal (bottom). (B) The Day 3 BLI data for each mouse and mean for each group (black line) are presented. The lowest BLI value in the placebo group was used as the threshold value for infection (dashed red line), which revealed a significant protective effect of 1C11 as determined by Fisher's Exact Test (p=0.016).

Figure 2. 1C11 treatment inhibits osteolysis, decreases abscess numbers, and recruits macrophages into SAC

Mice (n= 5) were treated with PBS (A-G) or with 40mg/kg of 1C11 (H-N), and 24h later received a USA300 LAC infected transtibial pin. A third group of mice received an infected transtibial pin with delta-Gmd USA300 LAC (O-U). The mice were euthanized on day 14 post-infection, and the tibiae were harvested for micro-CT and histology. 3D renderings of representative tibiae are shown to illustrate the osteolysis and reactive bone formation (A,H,O). Representative alcian blue hematoxylin/orange G (B-E,I-L,P-S) and Brown & Brenn (F,G,M,N,T,U) stained histology from these tibiae is presented. 12.5× (B,I,P) and 40× (C,J,Q) images are shown to highlight areas that were photographed at 400×, where yellow asterisks mark abscesses (D,E,K,L,R,S); green asterisks mark SAC (F,M,T); and black asterisks mark the colonized necrotic bone fragments in sequestrum (G,N,U). Note that all groups had macrophages in the periphery of the abscess (yellow arrow heads in D,K,R), while macrophages in the center of the abscesses were only present in the 1C11 treated group (yellow arrow heads in L). Conversely, Gram-positive SAC were only present in the marrow and soft tissues of tibiae from mice treated with PBS or delta-Gmd (F&T), as the abscesses in 1C11 treated mice did not contain any bacteria (M). Necrotic bone fragments containing Gram-positive biofilm were detected in all tibiae (G,N,U). (V) The osteolytic area on the medial side was quantified as previously described , and the data for each tibia and mean for the group (bar) are presented (*p<0.05 vs. PBS). (W) Abscess numbers were quantified from the ABH/OG histology as previously described , and the data for each tibia and mean for the group (bar) are presented (*p<0.05 vs. PBS).

Figure 3. 1C11 induces MRSA megacluster formation in vitro by combined inhibition of binary fission and agglutination

(A) Representative micrographs of LAC USA300 cultures grown for 4 hours in the presence of alpha-T2m, 1C11, or PBS, and of LAC delta-Gmd. Of note is that the defective binary fission observed with ΔGmd appears to be phenocopied by 1C11 addition to WT MRSA. (B) O.D. of parallel cultures was measured at the indicated intervals. While no differences were observed among the PBS, alpha-T2m, or delta-Gmd cultures at any time point, the O.D. of the 1C11 treated cultures was significantly lower after 4 hours (**p<0.01 vs. PBS). (C) This decreased O.D. was coincident with a visible bacterial sediment, which revealed large clusters of MRSA as seen by SEM. (D) Quantification of the clusters was performed and presented as the mean +/- SD (*p<0.05 vs. PBS; #p<0.05 vs. delta-Gmd; A: 15,000× (bar=0.5 μm); C: 3,000×).

Figure 4. 1C11 significantly increases internalization of S. aureus and megaclusters

Ultrathin section TEM at 6,000× was performed on RAW cells after 4hr of culture with MRSA opsonized with 1C11 (A) or alpha-T2m (B). Note the megaclusters (arrowheads in A) are connected by a thick matrix (boxed region in A shown at 30,000×), whereas intracellular S. aureus in the control cultures were mostly individual bacterium (boxed region in B). Quantification confirmed that 1C11significantly induced the % of internalized MRSA vs. alpha-T2m (88.5 +/- 7.4 vs. 58.7 +/- 3.3; p<0.006; N=4 randomly chosen fields and the results are representative of duplicate experiments; Nu=nucleus; Cy=cytoplasm). Representative SEM of a macrophage phagocytosing a bacterium (arrow in C), and megaclusters inside of a macrophage (D), on the pins harvested on day 14 from 1C11 treated mice challenged with MRSA, which were not observed on pins harvested from PBS and delta-Gmd treated mice.