Autotransporters (ATs) are virulence factors found in Gram-negative bacteria that consist of three domains: an N-terminal signal peptide, a central passenger domain, and a C-terminal β-barrel domain. Here, we used RpeA from rabbit enteropathogenic Escherichia coli as a model AT. RpeA belongs to the serine protease ATs of Enterobacteriaceae (SPATEs) subfamily and contains a characteristic serine protease motif (GDSGSP) in the passenger domain. RpeA also contains a proline-rich region (PRR) in the form of tandem repeats [PPE(S/T)EKPV] towards the C-terminus of the passenger domain. RpeA, unlike other SPATEs, lacks a conserved cleavage site (NN) within the linker between the passenger and β-barrel domains, which suggested that RpeA is a non-secreted SPATE.
This study showed that the RpeA passenger domain is not cleaved from its β-barrel domain and is exposed on the bacterial cell surface. Through sequence analysis followed by point mutations, we showed that the N-terminal region of the RpeA passenger domain contains a highly conserved catalytic triad at positions His124Asp159Ser265 that contributes to RpeA protease activity. Through qualitative and quantitative functional assays, we showed that RpeA does not mediate bacterial aggregation yet does mediate biofilm formation, although the PRR has no role in this process. In contrast, the PRR contributes to the adhesion of bacterial cells expressing RpeA to epithelial cells. Through pulse-chase expression in bacteria and biophysical analyses on in vitro refolded proteins, we showed that the PRR has no role in the assembly and folding of the RpeA passenger domain.
Sequence analysis of ~1435 putative ATs revealed that 25% of ATs have PRRs where 71% of these are, like in RpeA, located towards the C-terminus of the passenger domain. Moreover, among the PRR-containing ATs, the majority (63%) are adhesins like RpeA, which suggests that our findings can be applied more broadly.