Constraints on lateral gene transfer in promoting fimbrial usher protein diversity and function

Abstract

Fimbriae are long, adhesive structures widespread throughout members of the family Enterobacteriaceae. They are multimeric extrusions, which are moved out of the bacterial cell through an integral outer membrane protein called usher. The complex folding mechanics of the usher protein were recently revealed to be catalysed by the membrane-embedded translocation and assembly module (TAM). Here, we examine the diversity of usher proteins across a wide range of extraintestinal (ExPEC) and enteropathogenic (EPEC) Escherichia coli, and further focus on a so far undescribed chaperone-usher system, with this usher referred to as UshC. The fimbrial system containing UshC is distributed across a discrete set of EPEC types, including model strains like E2348/67, as well as ExPEC ST131, currently the most prominent multi-drug-resistant uropathogenic E. coli strain worldwide. Deletion of the TAM from a naive strain of E. coli results in a drastic time delay in folding of UshC, which can be observed for a protein from EPEC as well as for two introduced proteins from related organisms, Yersinia and Enterobacter We suggest that this models why the TAM machinery is essential for efficient folding of proteins acquired via lateral gene transfer.

Keywords: fimbriae; outer membrane; translocation and assembly module.

Figure 1.

Phylogeny of usher proteins in a large E. coli collection. The E. coli genomes (electronic supplementary material, table S3) were searched using HMMER and the Pfam profile for usher proteins and subjected to tree calculation using RAxML. Following manual assessment visually, monophyletic groups are coloured according to their described members (electronic supplementary material, table S4); four groups without described members as in Wurpel et al. [23] are based on the annotation of similar sequences in UniProt (LPF-like 2, AggC (AAF/I), FedC (F18), MrkH (Type 3)).

Figure 2.

The distribution of ushers across the E. coli pangenome. Given the recent increase in publicly available EPEC/UPEC genomes [–29], we investigated the distribution of ushers across E. coli. The tree is based on a core gene alignment of E. coli genomes with a focus on EPEC and ExPEC strains, but also including a variety of reference strains for other pathovars. The inner rings show the respective usher families, the other rings show, from inside to outside, the main sequence types according to multi-locus sequence typing (MLST), and the pathotypes. The presence of UshC and YraJ are again highlighted in the outermost ring. This highlights the uneven distribution of the two closely related usher proteins UshC and YraJ, both across the E. coli diversity but also within the respective pathovars; branches are coloured according to the pathovars scheme as indicated in the legend. The tree representation was performed using iTOL [30]. Pathotypes: EPEC, enteropathogenic E. coli; ATEC, atypical EPEC; EHEC, enterohaemorrhagic E. coli; ETEC, enterotoxigenic E. coli; UPEC, uropathogenic E. coli; ExPEC, extraintestinal pathogenic E. coli; EAEC, enteroaggregative E. coli; other, see details in electronic supplementary material, table S3.

Figure 3.

The phylogenetic relationships of usher proteins in Enterobacteriaceae reference strains. The tree was generated using RAxML and shows the diversity of usher proteins across several genera as given in the electronic supplementary material, table S1 and highlights that these proteins are very widely distributed across various species within the Enterobacteriaceae. The colours indicate the different genera (inset). Chaperone–usher systems are assigned as in figure 1, but only for the representative E. coli proteins as shown in the middle and outer ring fragments.

Figure 4.

The Ush/Yra clade ushers. (a) Phylogenetic tree of the respective usher sequences calculated with MrBayes shows the various adhesins associated with the respective usher sequence. The nonlinear evolution of the chaperone–usher systems is apparent from the different monophyletic groups displaying a mixed distribution of associated adhesin sequences in the operons. (b) Similarity network of the sequences as in the electronic supplementary material, table S5, highlights two different types of adhesins associated with the different operons; one group comprises stalk-like adhesins, which can also form the tip, whereas the other group includes a second adhesion protein different to the stalk-like sequences.

Figure 5.

Usher biogenesis in E. coli. Escherichia coli cells harbouring (a) pCJS39, (b) pCJS75 or (c) pCJS77 were assessed by pulse chase analysis. Aliquots were taken at 10 s, 2, 4, 8, 16 and 32 min, treated with (+Try) or without (−Try, last timepoint only) 20 μg ml⁻¹ trypsin. Analysis was by SDS–PAGE, storage phosphor-imaging and immunoblotting. Representative autoradiograms and immunoblots are shown, from three independent experiments (n = 3). The time increment is indicated as a graded triangle above the autoradiogram. SurA is a periplasmic protein used to assess the integrity of the outer membrane. (d) The usher densities at each timepoint (a–c) were used to calculate the observed rate constants (k_obs). Calculations were as per Stubenrauch et al. [14]. Error bars represent s.e.m. (n = 3), and all folding rates of mutants were significantly slower than the respective wild-type folding rate, as assessed by one-way ANOVA (p < 0.05).

Figure 6.

Schematic of fimbriae biogenesis. Nascent protein is translocated across the inner membrane (IM) via the SecYEG apparatus. The TAM is thought to promote protein insertion through destabilization of the lipid bilayer [50,51]. TamA (pdb: 4C00) acts as a lever, pushing onto TamB, to distort the outer membrane (OM). Once assembled, the fimbrial usher acts as an anchor and pore for fimbrial subunits to thread through. Initially, the dedicated chaperone transfers the tip adhesion subunit to initiate fimbrial biogenesis. The chaperone subsequently transfers hundreds to thousands of the major fimbrial subunits, allowing the growing pilus to extend from the cell surface [12].