Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS

Abstract

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS.

Multiple Structures Disclose the Secretins' Secrets

“…The N-domain was less well resolved than the upper secretin part, suggesting that there is a relatively high flexibility/movement among the N-domains. Based on sequence analysis, the authors previously proposed that the EPEC secretin belongs to the Vibrio-type subfamily (14). In agreement with sequence similarities, a similar structural architecture can be seen between the two Vibrio-type secretins, i.e., the EPEC structure from the work of Hay et al (6) and the Vibrio structure from the work of Yan et al (15), with loops extending from the extracellular side to form the typical cap gate (Fig.…”

“…Overall, with their structure-based BLAST experiment, Hay et al (6) revealed that the previous T2SS secretin classification, i.e., Vibrio and Klebsiella types (14), is definitely structurally relevant. Moreover, in providing an additional high-resolution 3D structure of a secretin, these authors are giving us a reminder of the structural diversity existing among T2SS secretins and are lending support to the existence of subtle functional differences.…”

The Type II Secretion Pathway in Vibrio cholerae Is Characterized by Growth Phase-Dependent Expression of Exoprotein Genes and Is Positively Regulated by σ E

“…Similarly, transcription of T2S genes was induced in Yersinia enterocolitica cultured at 26°C in comparison to those cultured at 37°C (38), whereas in enterotoxigenic E. coli, expression of the T2S gene cluster was repressed at lower temperature, which was mediated by the global regulatory proteins H-NS and StpA (39). Considering the recently revealed phylogenetic differences between the T2S secretins, it is possible that there is a broader bifurcation of T2S gene expression control across bacterial species (40). It is also tempting to speculate that the temperature-dependent T2S transcription patterns and the stimulatory effect of HapR in vibrios could contribute to their environmental fitness.…”

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

“…After pulse-chase protein expression, cells were harvested and prepared for sucrose density gradient centrifugation as described previously 50 . In heat modifiability experiments, 40 mg of total membranes were mixed with SDS-PAGE loading buffer and then heated at temperatures between 25°C and 90°C.…”