Reversible assembly and amyloidogenesis of the staphylococcal biofilm protein, Aap

The human skin commensal, Staphylococcus epidermidis, is the bacterium most commonly responsible for hospital-acquired infections. This microbe has a very strong capacity for forming bacterial communities known as biofilms. These communities are well-structured and often involve a slime-like matrix of extracellular polysaccharide which assists in bacterial accumulation. A well-known protein factor, the accumulation-associated protein (Aap), can also mediate intercellular adhesion, contributing the biofilm formation. Interestingly, Aap has been shown to be critical for infection in a rat catheter model, whereas the extracellular polysaccharide was irrelevant in infection.

Aap is a large, multi-functional, cell wall-anchored protein expressed by S. epidermidis. The N-terminus of the protein contains a region of short repeats called the A-repeats, followed by a globular lectin domain. The lectin domain can mediate attachment of bacteria to a surface. A proteolytic cleavage site downstream of the lectin domain leads to the release of the A-repeats and lectin domain, allowing Aap to function in accumulation. There are 5 - 17 B-repeats downstream of the cleavage site, which can assembly with Aap on adjacent cells in the presence of Zn²⁺. At the C-terminus of Aap lies a region of low complexity, which is rich in proline and glycine residues. After this region is a cell wall-anchoring motif that results in the covalent attachment of Aap to the bacterial cell wall.

Much of the progress made toward understanding the structure and biological function of the B-repeats has utilized a minimal construct containing one and a half B-repeats (Brpt1.5). Previous members of the Herr Lab have determined that Brpt1.5 can assemble into an anti-parallel dimer in the presence of Zn²⁺. Interestingly, while the Brpt1.5 dimer would disassociate in the presence of Zn²⁺-chelator, mature biofilms were unaffected by addition of the chelator. This led members of the Herr Lab to express and characterize longer B-repeat constructs, which more closely resemble the number of B-repeats observed in Aap and show Zn²⁺-dependent assembly beyond dimer, eventually forming amyloid-like fibers. Amyloid fibers are often associated with toxicity, however, the fibers formed by Aap's B-repeats are utilized by the bacterium in a functionally beneficial way. In several other bacteria, functional amyloids have been shown to provide added strength to the biofilm structure. In the work presented here, we show Aap forms fibers in S. epidermidis biofilms and are responsible for the biofilm's resistance toward Zn²⁺-chelator. We also characterize a recently discovered protein, small basic protein (Sbp), which is able to reduce the amount of Zn²⁺ required for B-repeat aggregation. We propose that Sbp is a nucleating or accessory protein for Aap-amyloidogenesis.

Finally, a secondary interest of this dissertation work is to characterize the structure of the proline/glycine-rich stalk-like region of Aap and other cell wall-anchored proteins. Interestingly, several of these regions have a very high propensity to remain extended in solution, primarily due to the high polyproline type II helix propensity. Overall, this dissertation work has led to an increased understanding of the mechanism of Aap-dependent accumulation in biofilm formation.