Structural and biophysical characterisation of the repetitive regions from biofilm-mediating cell-wall anchored proteins

Staphylococci and streptococci form microbial accumulations, defined as biofilms, on in-dwelling medical devices or damaged tissue. Biofilms are implicated in serious infections, such as infective endocarditis, with a mortality rate of 30%. Cell wall-anchored (CWA) proteins, containing a repetitive B region that putatively projects a functional A region from the bacterial surface, mediate biofilms, independent of other mechanisms. This highlights the need to better understand the mechanism of biofilm formation/accumulation by CWA proteins. Staphylococcus aureus surface protein C (SasC) mediates biofilm accumulation, but the structure and function of the B region, containing domains of unknown function (DUF1542), remains undetermined. SGO0707 from Streptococcus gordonii mediates surface adhesion putatively via its A domains; the function of the B region remains unknown. Here, the B regions of SasC and SGO0707 are biophysically characterised and their ability to form elongated stalks is assessed. Work presented in this thesis enables the redefinition of the domain boundaries for the repeats comprising the B region of SasC and renaming these as DUF1542 rigid extracellular surface structural (DRESS) domains. Tandem DRESS domains have tightly connected inter-domain interfaces that are essential for tandem domain stability and which mediate long-range stability. Importantly, DRESS domains form an extended, rigid rod and have remarkable mechanical stability, compared to other helical proteins. The B region of SGO0707 comprises SGO0707 high identity repeat tandem (SHIRT) domains with an extended tandem domain architecture, determined prior to this work. Here, SHIRT domains are shown to lack flexible loops and notably, the linker between SHIRT domains has limited flexibility, implying that tandem SHIRT domains have an extended, rod-like conformation in solution. Importantly, both domain architectures form a rigid rod, suggesting their ability to project the functional A region from the cell surface. Thus, staphylococci and streptococci have both evolved structurally distinct stalks-like CWA proteins to mediate biofilms.