Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles

Abstract

All Gram-negative bacteria are believed to produce outer membrane vesicles (OMVs), proteoliposomes shed from the outermost membrane. We previously separately engineered E. coli to produce and package two organophosphate (OP) hydrolyzing enzymes, phosphotriesterase (PTE) and diisopropylfluorophosphatase (DFPase), into secreted OMVs. From this work, we realized a need to thoroughly compare multiple packaging strategies to elicit design rules for this process, focused on (1) membrane anchors or periplasm-directing proteins (herein "anchors/directors") and (2) the linkers connecting these to the cargo enzyme; both may affect enzyme cargo activity. Herein, we assessed six anchors/directors to load PTE and DFPase into OMVs: four membrane anchors, namely, lipopeptide Lpp', SlyB, SLP, and OmpA, and two periplasm-directing proteins, namely, maltose-binding protein (MBP) and BtuF. To test the effect of linker length and rigidity, four different linkers were compared using the anchor Lpp'. Our results showed that PTE and DFPase were packaged with most anchors/directors to different degrees. For the Lpp' anchor, increased packaging and activity corresponded to increased linker length. Our findings demonstrate that the selection of anchors/directors and linkers can greatly influence the packaging and bioactivity of enzymes loaded into OMVs, and these findings have the potential to be utilized for packaging other enzymes into OMVs.

Keywords: diisopropyl fluorophosphatase (DFPase); outer membrane vesicles (OMVs); phosphotriesterase (PTE).

Figure 1

Schematic representation of the OMVs in this study. PTE or DFPase was expressed as a fusion protein with various anchors/directors, including to the anchor Lpp’ with increasing linker length (Lpp’, L3, L4, and L34), a truncated form of transmembrane porin protein (OmpA) as an anchor fused to SpyTag (ST) that binds PTE/DFPase fused to SpyCatcher (SC), PTE/DFPase-SC as a non-directed control, to periplasmic proteins as directors including maltose-binding protein (MBP) and vitamin B12-binding protein (BtuF), and to two additional lipoprotein anchors SLP and SlyB.

Figure 2

Western blot of purified OMVs from various constructs demonstrating the abundance of DFPase fused to Lpp’-based linkers. (A) Representation of the different DFPase fusion constructs. The orange box represents Lpp’ and the blue circle represents DFPase (B) Immunoblot lane 1: Lpp’-DFPase, lane 2: L3-DFPase, lane 3: L4-DFPase, lane 4: L34-DFPase, lane 5: BL21(DE3), lane 6: molecular weight marker. Upper panel shows the PonceauS-stained membrane, and the lower panel shows immunoblot with anti-6× His antibody.

Figure 3

OMV size and count distributions. (Left) NanoSight count in particles/mL measured from each of the OMVs averaged over three 60 s sample reads of a 1:1000 diluted sample (in CHES pH 8.5). (Right) NanoSight size distribution from each of the OMVs.

Figure 4

Enzyme activity of OMVs categorized by fusion and enzyme. Boxplot representations of the initial kinetic rates for the degradation of paraoxon by PTE (top) and DFPase (bottom) are shown (n ≥ 8), ordered by sample. Dots represent outliers. Initial rate was calculated from the first 20 min of the curve. Buffer did not contain OMVs, while BL21 was the BL21(DE3) OMV sample that did not contain PTE or DFPase.