Staphylococcus epidermidis is one of the most frequent nosocomial pathogens which cause implant-associated infections through biofilm formation. It's biofilm assembly relies on the production of an extracellular matrix. In effort to understand specific molecular interactions within this matrixome, a so far uncharacterized, 18 kDa protein was identified named small basic protein (Sbp).

The aim is to characterize and to elucidate the temporal-spatial distribution of Sbp in S. epidermidis biofilms.

To investigate the dynamic Sbp matrix three Sbp-isoforms were constructed in which Sbp is fused to an ALFA-tag located at different positions within the protein. The constructs were cloned in two plasmids (constitutive promotor and xylose-inducible promotor) and were tested (growth curves, biofilm formation, WB, CLSM).

Bioinformatic analysis revealed a structured protein head with ß-loops and α-helices, along with an unstructured N-terminal tail. Analysis of the three cloned constructs by WB revealed no degradation products and no effect on growth rate. CLSM revealed a unique spatial distribution pattern of Sbp within matrices, accumulating at early timepoints at the biofilm-substratum interface (forming a carpet-like structure). Similar patterns were observed for sbp-ALFA at positions 508 and 466. In mature biofilms, Sbp, regardless of isoform, is enriched in both the biofilm matrix and the substrate. In higher parts of the biofilm Sbp partially co-localizes with ALFA independent of tag position whereas these carpet like structure of ALFA could only be detected in sbp-ALFA 508.

All Sbp-ALFA fusion constructs are stable and reconstitute the wildtype biofilm phenotype in Δsbp strains. Sbp-ALFA nt508 recapitulates wildtype Sbp distribution in biofilms the best, so this construct can be used for high resolution microscopy and to decipher specific Sbp- matrix interactions and their functional importance. At present, it is unclear whether and how Sbp can be exchanged between S. epidermidis sbp-producing and non-producing cells. Co-culture experiments will help understand Sbp production, repositioning, and exchange dynamics between different subpopulations and strains.