Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB

Abstract

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.

New Insights into the Assembly of Bacterial Secretins

“…Prior to our study, the working paradigm for secretin structure has been that secretin monomers assemble under C 12 symmetry to construct the dodecameric rings as visualized by electron microscopy (11,15,37). In contrast, our study supports a hexameric assembly of dimers, a model that was also recently suggested by other studies (17,18).…”

“…Furthermore, negative stain EM images of pseudocrystals of XcpQ from Pseudomonas alcaligenes (11) clearly show hexagonal particles, consistent with our proposed model. In addition, a recent study by EM of the structure of BfpB, the secretin for type IV pili, proposed a dodecameric secretin that can be assembled as a set of six dimers (17). Finally, we note that the crystal structure of the periplasmic domain of EscC, the T3SS secretin from enteropathogenic E. coli, also forms dimers in the crystal lattice (16), but the possible relevance of dimers in the oligomeric assembly of T3SS with an even number of subunits has not been addressed.…”

“…An important consensus from these studies has been the proposal that monomeric GspD secretins assemble under C 12 symmetry to construct the functional GspD dodecamer. However, several recent studies (17,18) suggested that the assembly of secretin dodecamers might proceed via oligomerization of dimers of secretin subunits.…”

PilMNOPQ from the Pseudomonas aeruginosa Type IV Pilus System Form a Transenvelope Protein Interaction Network That Interacts with PilA

“…7). Alternatively, data from the T2SS suggests that the secretin is assembled as a hexamer of dimers (40)(41)(42); in this case, the arrangement may be one PilMNOP complex for every two PilQ monomers (1:1:1:1:2 stoichiometry). Differences in orientation of the N0 domain of PilQ in a dimer versus monomer configuration might explain why we saw differences in PilPQ interactions when fragments versus full periplasmic domains of PilQ were used for pulldowns.…”

A Single Amino Acid Substitution Changes the Self-Assembly Status of a Type IV Piliation Secretin

“…The BfpB secretin of an Escherichia coli T4PS is also a lipoprotein, but lipid modification is not required for targeting to the OM (31). The secretin PilQ from the T4PS of Neisseria meningitidis requires a lipoprotein chaperone, PilW, for multimer stability (3) and the Bam complex for correct assembly in the OM (45) and cannot assemble in vitro (20).…”