Reciprocal Packaging of the Main Structural Proteins of Type 1 Fimbriae and Flagella in the Outer Membrane Vesicles of "Wild Type" Escherichia coli Strains

Abstract

Fundamental aspects of outer membrane vesicle (OMV) biogenesis and the engineering of producer strains have been major research foci for many in recent years. The focus of this study was OMV production in a variety of Escherichia coli strains including wild type (WT) (K12 and BW25113), mutants (from the Keio collection) and proprietary [BL21 and BL21 (DE3)] strains. The present study investigated the proteome and prospective mechanism that underpinned the key finding that the dominant protein present in E. coli K-12 WT OMVs was fimbrial protein monomer (FimA) (a polymerizable protein which is the key structural monomer from which Type 1 fimbriae are made). However, mutations in genes involved in fimbriae biosynthesis (ΔfimA, B, C, and F) resulted in the packaging of flagella protein monomer (FliC) (the major structural protein of flagella) into OMVs instead of FimA. Other mutations (ΔfimE, G, H, I, and ΔlrhA-a transcriptional regulator of fimbriation and flagella biosynthesis) lead to the packaging of both FimA and Flagellin into the OMVs. In the majority of instances shown within this research, the production of OMVs is considered in K-12 WT strains where structural appendages including fimbriae or flagella are temporally co-expressed throughout the growth curve as shown previously in the literature. The hypothesis, proposed and supported within the present paper, is that the vesicular packaging of the major FimA is reciprocally regulated with the major FliC in E. coli K-12 OMVs but this is abrogated in a range of mutated, non-WT E. coli strains. We also demonstrate, that a protein of interest (GFP) can be targeted to OMVs in an E. coli K-12 strain by protein fusion with FimA and that this causes normal packaging to be disrupted. The findings and underlying implications for host interactions and use in biotechnology are discussed.

Keywords: Escherichia coli; FimA; Flagellin; FliC; OMV.

FIGURE 1

Comparison of the OMV yield (A) and proteome (B) from E. coli K-12 wild type MG1655 strain, FimB-LacZ fusion strain (where fimbriae production is locked off) vs. B [BL21 and BL21 (DE3)] strains. OMVs were purified concurrently for a direct comparison. Samples were from the same harvest points and with equivalent gel loading concentrations. It is noted that OMVs from E. coli K-12 strains were co-purified with either flagella (Δ) or fimbriae (□).

FIGURE 2

Enrichment and location of preferentially packaged proteins in the OMVs of E. coli. The co-purification of either fimbriae (□) or flagella (Δ) but not both is highlighted next to TEM images of OMV preparations derived from K12 strains (A). FimA and Flagellin are enriched in E. coli K-12 OMVs compared to levels in the periplasm and whole cell (B,C).

FIGURE 3

The effect of mutations in various genes of the fim operon using WT (E. coli BW25113) and mutants from the Keio collection. (A) OMV co-purification with either fimbriae (□) or flagella (Δ); (B) Proteome of the OMVs labeled with proteins identified by mass spectrometry (C) Western blot using anti-FimA (monomer and polymerized) and anti-Flagellin antibodies.

FIGURE 4

The effect of regulatory mutants on the composition and co-purified appendages in E. coli WT (BW25113). (A) OMV co- purification with either fimbriae (□) or flagella (Δ). (B) Proteome of the OMVs and proteins identified by mass spectrometry (C) Western blot using anti-FimA (monomer and polymerized) and anti-Flagellin antibodies.

FIGURE 5

The ubiquity of mutual exclusivity using E. coli WT, deletion mutants (ΔfimA and ΔfliC) and a range of E. coli clinical isolates (described in section “Materials and Methods”). (A) OMV co-purification with either fimbriae (□) or flagella (Δ). (B) Proteome of the OMVs and (C) Western blot using anti FimA (monomer and polymerized) and anti-Flagellin antibodies. Proteins identified in panel (B): 1, Flagellin; 2, FimA; 3, Antigen 43α chain; 4, OmpA; 5, FimH; 6, KS71A fimbrillin; 7, F7-2 fimbrial protein precursor.

FIGURE 6

The transport and packaging of a GFP-FimA fusion protein in E. coli MG1655. (A) OMV co-purification with either fimbriae or flagella; (B) Proteome of the OMVs and (C) Western blot using anti FimA (monomer and polymerized) and anti-Flagellin antibodies. (D,E) TEM analysis of thin-sectioned OMVs (D) and E. coli FimA-GFP strain cells (E) embedded in resin. The sections were immunogold labeled and probed with: anti-GFP antibody, anti-FimA monomer antibody, anti-FimA monomer/anti-GFP antibodies mixed and anti-Flagellin antibody. As a negative control, the embedded OMVs were incubated in TBST only (no primary antibody). The samples were then incubated with the following secondary antibodies: 15 nm gold label or 10 nm gold label.

FIGURE 7

The transport and packaging of a mNeonGreen-FimA fusion protein in E. coli BW25113 and a ΔfimA mutant. (A) TEM images show purified OMVs from the E. coli BW25113 and ΔfimA mutant strains containing the pSB001 plasmid (encoding the mNeonGreen-FimA fusion protein) in presence or absence of IPTG. (B) Presence of mNeon green in either the cells or OMVs of the WT and ΔfimA mutant both in the presence or absence of IPTG induction. (C) Western blot using anti-Neon green on whole cells and subcellular [periplasm, outer membrane (OM) and OMV] fractions in the presence and absence of IPTG induction. (D) As C using anti-FimA monomer and anti-Flagellin antibodies.