Staphylococcus aureus osteomyelitis: Characterization of bacterial antigens and utilization as vaccine candidates and imaging targets

Abstract

Osteomyelitis, or infection of the bone, is a devastating illness that is most often caused by the Gram-positive bacterium Staphylococcus aureus. Staphylococcus aureus causes these persistent, recurrent infections by forming biofilms. Though biofilms exhibit increased resistance to the host immune system, little is known about the antibody-mediated response to these infections. To survey this, the tibias of rabbits were infected with methicillin-resistant S. aureus to produce chronic osteomyelitis. Serum was collected prior to infection and 14, 28, and 42 days post-infection. The serum was used to perform immunoblots on total protein from biofilm grown in vitro and separated by two-dimensional gel electrophoresis. Those proteins recognized by host antibodies in the harvested sera were identified via MALDI-ToF analysis. Using protein from total and fractionated biofilm protein samples, we identified 26 and 22 immunogens, respectively, of which approximately 75% of the cell wall immunogens were also up-regulated transcriptomically during biofilm growth. Five proteins were recombinantly expressed, including four cell wall-associated, biofilm-specific proteins and one extracellular protein, and were shown to cause a robust polyclonal IgG response when used to immunize rabbits. Antibodies against these recombinant proteins bound to the native forms of each protein as harvested from MRSA in vitro-grown biofilms, both via Western blot and in immunofluorescence confocal microscopy. These IgGs could be utilized as imaging tools that localize to areas of specific protein production within a biofilm. Finally, these same proteins were administered prior to bacterial challenge in a tibial osteomyelitis model to evaluate protective efficacy. In vaccinated animals, clinical and radiological signs of osteomyelitis were significantly lessened compared to controls, while viable bacterial counts were lowered 85%. This is the first work to identify immunogens during chronic S. aureus biofilm infections and to simultaneously show the global relationship between the antigens expressed during an in vivo infection and the corresponding in vitro transcriptomic and proteomic gene expression levels. This work also illustrates that immunogenic, cell wall-associated, biofilm-up-regulated proteins are promising for in vitro visualization of biofilm growth, architecture, and spatial-functional relationships, as well as for the development of an anti-S. aureus biofilm vaccine.