Autotransporters (ATs) are virulence factors found in Gram-negative bacteria that consist of three domains: a N-terminal signal peptide, a central passenger domain, and a C-terminal β-barrel domain. Here, we used RpeA as a model AT. RpeA was shown to mediate bacterial colonization to the rabbit intestine. RpeA belongs to the serine protease ATs of Enterobacteriaceae (SPATEs) subfamily and contains a characteristic serine protease motif (GDSGSP) in the passenger domain. Moreover, the RpeA passenger also contains a proline-rich region (PRR) in the form of tandem repeats [PPE(S/T)EKPV]. RpeA, unlike other SPATEs, lacks a conserved autocatalytic cleavage site (NN) within the linker between the passenger and β-barrel domains, which suggested that RpeA is a non-secreted SPATE.
Here, we show that unlike other SPATEs, the RpeA passenger domain is not cleaved from its β-barrel domain and is exposed on the bacterial cell surface. Furthermore, protease activity assays using purified RpeA passenger revealed that RpeA has serine protease activity that could be inhibited by serine protease inhibitors and through mutation of the catalytic serine in the serine protease motif. Through qualitative and quantitative functional assays comparing E. coli TOP10 cells expressing wild-type RpeA, RpeAΔPRR or empty vector, we show that RpeA does not mediate bacterial aggregation, yet does mediate biofilm formation although the PRR has no role in biofilm production. However, the PRR was necessary for the adhesion of RpeA to epithelial cells.
Through in vivo pulse-chase assays and biophysical analysis on refolded wild-type RpeA and RpeAΔPRR, we show that the PRR has no role in the folding of the RpeA passenger domain. Furthermore, sequence analysis of ~1435 putative ATs revealed that 36% of ATs have PRRs where 66% of these are, like in RpeA, located towards the C-terminus of the passenger domain. Therefore, our findings have the potential to be applied more broadly.
These findings increase our knowledge about the biological role of RpeA and the contribution of its PRR to RpeA structure and function. Furthermore, our work suggests that PRRs in ATs could be exploited as a therapeutic target to reduce the virulence levels of bacteria expressing AT adhesins.