Staphylococcus aureus Protein A, a Newly Identified Lectin, Promotes Aerobic Biofilm Formation

Abstract

Staphylococcus aureus forms biofilms in a variety of human infections. These infections span a wide range of environments in the body. The environment of the infection determines the physiological state of the bacteria. There exists a gap between studying biofilms in a laboratory setting and the physiology of cells in a clinical biofilm. To bridge this gap, we designed a high-throughput biofilm assay to mimic the environment of a clinical infection. Using this assay, we measured the formation of two different classes of biofilms in five clinical isolates. These two classes of biofilms differed in their location: the ring class formed at the air-water interface on the sides of the wells and the bottom class formed at the bottom of the wells. Each class exhibited different phenotypes in response to environmental demands. Biofilms were grown aerobically and anaerobically on plasma-coated surfaces to evaluate the role of environmental oxygen. Protein A (SpA) and polysaccharide amounts were analyzed to elucidate the relationship between proteins, polysaccharides, and biomass accumulation in these two classes of biofilms. The bottom biofilms were disrupted by degradation of polysaccharides, enhanced under hypoxic environments, and their SpA content increased inversely with biomass. In contrast, the ring biofilms were not disrupted by polysaccharide degradation, were enhanced under aerobic environments and their SpA content increased proportionally with biomass. Furthermore, SpA promotion of biofilm formation in ring biofilms was found to be limited to biofilms grown aerobically. Additionally, some results depended on whether the strains were nasal or pathogenic isolates. The results of this study suggest that the influence of environmental and genetic factors on biofilms changes with the physiological state of the biofilm. Because the location of biofilms in S. aureus diseases in patients vary in their environment, our ability to observe these two different classes provides useful insight. This study also evaluated the mechanism by which SpA promotes aerobic ring biofilms. We measured SpA binding to several biofilm matrix components. Isothermal titration calorimetry (ITC) analysis revealed that SpA binds preferentially to β-linked polysaccharides, in particular the major biofilm exopolysaccharide poly-β-1,6-N-acetylglucosamine (PNAG). Biolayer interferometry (BLI) revealed that the multivalency of SpA increases the apparent affinity for β-linked glucans. BLI results also indicated preferential binding of SpA to β-1,6 linked glucans, as shown by ITC, but suggests that SpA can facilitate weaker interactions with the other β-linked glucans. Therefore, we propose that SpA-PNAG binding facilitates cellular aggregation and promotes biofilm formation. Additionally, we proposed that SpA binds other β-linked glucans secreted by other organisms facilitating cellular adhesion in multispecies biofilm infections. Understanding the role of SpA in both single- and multispecies biofilms is important to targeting and combating these types of infections.