Autonomous bacterial localization and gene expression based on nearby cell receptor density

Abstract

Escherichia coli were genetically modified to enable programmed motility, sensing, and actuation based on the density of features on nearby surfaces. Then, based on calculated feature density, these cells expressed marker proteins to indicate phenotypic response. Specifically, site-specific synthesis of bacterial quorum sensing autoinducer-2 (AI-2) is used to initiate and recruit motile cells. In our model system, we rewired E. coli's AI-2 signaling pathway to direct bacteria to a squamous cancer cell line of head and neck (SCCHN), where they initiate synthesis of a reporter (drug surrogate) based on a threshold density of epidermal growth factor receptor (EGFR). This represents a new type of controller for targeted drug delivery as actuation (synthesis and delivery) depends on a receptor density marking the diseased cell. The ability to survey local surfaces and initiate gene expression based on feature density represents a new area-based switch in synthetic biology that will find use beyond the proposed cancer model here.

Figure 1

A bacterial ‘dirigible’ concept. Biological nanofactories (NFs) that synthesize bacterial AI-2 are targeted to epidermal growth factor receptor (EGFR) on the surface of squamous cell carcinoma of head and neck (SCCHN) line, PCI-15B. AI-2 molecules are emitted from the cell surface and recognized by reprogrammed bacteria, which swim to the site of signal generation and decide, based on AI-2 level (which is proportional to the EGFR surface density), whether or not to initiate gene expression.

Figure 2

In situ generation of AI-2 though surface assembled NF and QS-activated gene expression from ‘docked’ bacterial cells. (A) Scheme for NF targeting on various densities of avidin chips. NFs are fusion proteins comprising Pfs, LuxS (for AI-2 synthesis) and protein G (IgG-binding motif, for antibody conjugation). Anti-avidin (FITC) coupled NFs target to avidin-coated chips and catalyze SAH to AI-2. (B) NF targeting and subsequent in vitro AI-2 synthesis on avidin plate wells. NF loading was determined by FITC fluorescence and AI-2 concentrations were measured by Ellman’s assay (Fernandes et al, 2010). (C) E. coli W3110 (ΔlsrFG ΔluxS) response to in vitro AI-2 synthesized by NF assembled onto avidin plate wells. Bacterial suspensions were added directly to wells and DsRed intensities were measured via fluorescence plate reader. The negative control is without SAH addition. (D) Conceptual bacterial docking via surface-expressed protein G and outer membrane assembled anti-avidin IgG facilitating binding to avidin on chip surface. Subsequent application of AI-2 triggers QS switch (lsr gene expression) as indicated by DsRed fluorescence. (E) E. coli W3110 cells with surface-expressed protein G decorated with anti-avidin IgG were applied to wells with various avidin densities. Unbound bacteria were washed followed by 40 μM AI-2 addition (this concentration is above QS threshold). After 12 h, fluorescence images depict immobilized cells. (F) Image analysis (ImageJ) revealed number of bacteria on avidin surfaces. Both cell number and DsRed fluorescence were linear with avidin loading. Source data for this figure is available on the online supplementary information page.

Figure 3

E. coli targeted to SCCHN cell line PCI-15B via EGFR and QS activation of gene expression. (A) Scheme for targeting and docking bacteria to receptors on mammalian cell surfaces and subsequent NF conjugation to targeted bacteria for local AI-2 synthesis and delivery bacteria for triggering QS switch. The protocol follows: (i) anti-EGFR IgG decorated bacteria facilitate cell binding to EGFR on PCl-15B cell surfaces; (ii) secondary Ab-NFs are directed to immobilized bacteria; and (iii) NFs convert SAH into AI-2 for initiating gene expression (lsr operon). (B) Indicated densities of PCI-15B or HEK293 cells were seeded to wells followed by mouse anti-EGFR (AF488-labeled) bacteria, anti-mouse-NF targeting and subsequent SAH addition. DsRed expression was measured after 12 h. (C) Analyses of fluorescent images yielded linear increase in both cell number and DsRed fluorescing cells with PCI-15B loading (reflective of EGFR density). The HEK293 control sample exhibited 3.4 × 10⁶ AF488 fluorescence, which indicates bacterial docking ∼1/3 that of PCI-15B at seeding density of 20 000 cells/well. Source data for this figure is available on the online supplementary information page.

Figure 4

Bacterial motility directed to AI-2. (A) Simulation (COMSOL) of AI-2 concentration profile over time within the transwell apparatus. AI-2 was loaded in the upper chamber where it could diffuse through the membrane to the lower chamber (initially buffer). The top four plots represent the concentration gradients in time. The bottom chart depicts the dynamics of the local concentration gradient at specific sites (A, B, C, and D). Chemotaxis experiments were performed as follows: 40 μM AI-2 or SAH (control) was added to the upper chamber of the 3 μm 6-well inserts. W3110 cells (OD ∼1–1.5) rinsed with DPBS three times were then resuspended into DPBS (OD ∼0.14) and applied into bottom wells. The inserts were incubated under static conditions at 37 °C for 2 h. Samples were obtained from each chamber via pipette. (B) Determination of bacterial migration under bright field microscopy. (C) Systematic optical density measurements (OD₆₀₀) were conducted to assess migration from the source (bottom) upward toward the chemoattractant (top). Source data for this figure is available on the online supplementary information page.

Figure 5

Autonomously functioning sense and respond system. (A) Schematic depicts nanofactories assembled onto EGFRs on surface of PCI-15B cells. These cells, in turn, are cultivated on an inverted transwell insert exposing cell surfaces and nanofactories to the bacterial cells initially seeded on the bottom of the transwell apparatus. The bacteria then migrate vertically toward mammalian cells and subsequently initiate gene expression by sensing the in vitro AI-2 gradient, which is locally synthesized by the NF located on the mammalian cell surfaces. (B) After 9 h, bacteria and AI-2 levels were assayed in the upper chamber. (C) PCI-15B cells were rinsed and both QS-activated bacteria (DsRed) and NF (AF488) were imaged and assayed (ImageJ, D). Source data for this figure is available on the online supplementary information page.