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

Date Published: January 10, 2013

Publisher: Public Library of Science

Author(s): Rhys A. Dunstan, Eva Heinz, Lakshmi C. Wijeyewickrema, Robert N. Pike, Anthony W. Purcell, Timothy J. Evans, Judyta Praszkier, Roy M. Robins-Browne, Richard A. Strugnell, Konstantin V. Korotkov, Trevor Lithgow, Tomoko Kubori.


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.

Partial Text

Bacterial outer membranes incorporate proteins of at least three well-characterized architectures: β-barrel proteins, lipoproteins and secretins. The integral membrane proteins having a β-barrel architecture are targeted to the outer membrane and assembled by the β-barrel assembly machinery, the BAM complex [1]–[3]. Lipoproteins, anchored in the outer membrane by covalently attached lipid modifications, are inserted into the outer membrane by the receptor LolB after being ferried across the periplasm by factors of the Lol machinery [4], [5]. Secretins are integral proteins which assemble to form multimeric secretion channels in the outer membrane, with examples including outer membrane proteins of the Type II Secretion Systems (T2SS) and Type III Secretion Systems (T3SS), Type IV fimbrae and the filamentous phage extrusion machinery [6]–[9]. In the case of the T2SS, the secretin multimer in the outer membrane docks onto a platform of inner membrane proteins that energize its function in the selection and/or secretion of one or a few substrate proteins across the outer membrane into the external milieu[10]–[12].

A “piggyback” model for the targeting of PulD to the outer membrane of K. oxytoca has found general credence in the targeting of secretins for T2SS [19]. This system relies on (i) the outer membrane targeting characteristics of a small lipoprotein, the pilotin, which will be recognized and ferried to the outer membrane by the general lipoprotein targeting “Lol pathway” and (ii) a selective and tight binding of the S-domain of the secretin by the pilotin prior to leaving the inner membrane surface. It has been generally assumed that in the case of T2SS secretins only members of the PulS-OutS family of proteins function in the role of pilotins, and in organisms like V. cholerae where no obvious PulS-OutS proteins could be found, targeting of secretins had been thought to be pilotin-independent and mediated by other factors, such as GspA and GspB using functionally-distinct mechanisms [39]. We have clarified this apparent discrepancy by showing that there are at least two classes of T2SS secretins, each having distinguishing targeting sequences and each being targeted by distinct families of pilotin proteins: either the PulS-OutS family or the AspS family.




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