Outer Membrane Vesicle Production by Escherichia coli Enhances Its Defense against Phage Infection

Abstract

Several studies have investigated the multifunctional characteristics of outer membrane vesicles (OMVs), but research on their role in mediating phage-bacteria interactions is limited. Employing Escherichia coli as a model, we engineered a mutant strain overproducing OMVs for protective experiments against phage infections. The addition of exogenous OMVs proved highly effective in safeguarding the bacterial host against various phages, mitigating predatory threats. Screening for phage-resistant strains and adsorption experiments revealed that inhibiting phage adsorption is a crucial pathway through which OMVs protect against phage predation. Although OMVs conferred tolerance to the phage-sensitive strains (those easily infected by phages), they could not restore the phage-resistant strains (those that effectively resist phage infection) to a sensitive phenotype. This study provides valuable insights for the future development of novel biotechnological approaches aimed at utilizing OMVs to protect fermentative strains and reduce the risk of phage contamination.

Keywords: E. coli; adsorption; outer membrane vesicles; phage; resistance.

Figure 1

The E. coli MG1655∆nlpI∆tolA strain overproduces OMVs. (A) Comparison of the yield of OMVs between MG1655∆nlpI∆tolA and wild-type MG1655. Mean values ± standard deviation were calculated from three independent experiments. A t-test was performed (***, 0.0001 < p < 0.001). (B) TEM images of OMVs from MG1655∆nlpI∆tolA compared to wild-type MG1655 (left image: MG1655; right image: MG1655∆nlpI∆tolA). The red arrow indicates the formed OMVs.

Figure 2

Preparation and characterization of OMVs. (A) Preparation process of OMVs from E. coli. (B) Size distribution of E. coli OMVs. (C) TEM images of E. coli OMVs. The red arrow indicates the formed OMVs.

Figure 3

Host survival under phage predation pressure was markedly improved by the external introduction of prepared OMVs. (A) Assessing the influence of exogenous OMV addition on phage plaque formation on E. coli. Series dilutions of 4 μL of phage IME339 were spotted on MG1655 for plaque-forming ability determination. (B) Relative EOP of phage IME339 on E. coli MG1655 before and after addition of OMVs. Mean values ± standard deviation were calculated from three independent experiments. A t-test was performed (****, p < 0.0001). (C) Growth curves were determined at different time points after infecting E. coli MG1655 strains with phage IME339 under the conditions of exogenously added OMVs. The optical density (OD) at 600 nm was measured using a SynergyH1 microplate reader in a 96-well plate.

Figure 4

Examining the role of exogenously added OMVs in the defense of host E. coli MG1655 against infections by different phages. Growth curves were determined at different time points after infecting E. coli MG1655 strains with (A) phage IME347, (B) phage IME281, (C) phage PZJ0206, and (D) phage IME390 under the conditions of exogenously added OMVs.

Figure 5

OMVs can provide multiple binding targets for phages, thereby disrupting phage invasion of the host. (A) Plaque assay characterizing the phage-resistant strain MG1655∆nlpI∆tolA−R. (B) Co-culture growth curves testing the phage-resistant strain MG1655∆nlpI∆tolA−R. (C) Analysis of the OMV production capacity in strains MG1655∆nlpI∆tolA and MG1655∆nlpI∆tolA−R. (D) Adsorption experiment of phage IME339 on MG1655∆nlpI∆tolA and MG1655∆nlpI∆tolA−R. (E) Adsorption experiment of phage IME339 on the OMVs secreted by strains MG1655∆nlpI∆tolA and MG1655∆nlpI∆tolA−R. (F) Co-culture growth curve analysis determining the impact of OMVs from MG1655∆nlpI∆tolA and MG1655∆nlpI∆tolA−R on E. coli’s resistance to phages. Mean values ± standard deviation were calculated from three independent experiments. A t-test was performed (“ns”, no significance; *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, 0.0001 < p < 0.001).

Figure 6

Phage adsorption assays. (A) TEM observation of phage IME339 adsorption on OMVs. (B) OMVs were introduced into the culture of E. coli MG1655, and changes in the adsorption efficiency of phage IME339 were assessed. Mean values ± standard deviation were calculated from three independent experiments. A t-test was performed (**, 0.001 < p < 0.01).

Figure 7

OMVs do not facilitate the effective restoration of phage-resistant strains to a sensitive phenotype. Co-culture growth curve experiments were conducted to analyze the impact of varying OMV concentrations on the conversion between phage-resistant and phage-sensitive phenotypes. The OD_600nm was measured using a SynergyH1 microplate reader in a 96-well plate.