Engineered Living Materials Based on Adhesin-Mediated Trapping of Programmable Cells

Abstract

Engineered living materials have the potential for wide-ranging applications such as biosensing and treatment of diseases. Programmable cells provide the functional basis for living materials; however, their release into the environment raises numerous biosafety concerns. Current designs that limit the release of genetically engineered cells typically involve the fabrication of multilayer hybrid materials with submicrometer porous matrices. Nevertheless the stringent physical barriers limit the diffusion of macromolecules and therefore the repertoire of molecules available for actuation in response to communication signals between cells and their environment. Here, we engineer a novel living material entitled "Platform for Adhesin-mediated Trapping of Cells in Hydrogels" (PATCH). This technology is based on engineered E. coli that displays an adhesion protein derived from an Antarctic bacterium with a high affinity for glucose. The adhesin stably anchors E. coli in dextran-based hydrogels with large pore diameters (10-100 μm) and reduces the leakage of bacteria into the environment by up to 100-fold. As an application of PATCH, we engineered E. coli to secrete the bacteriocin lysostaphin which specifically kills Staphyloccocus aureus with low probability of raising antibiotic resistance. We demonstrated that living materials containing this lysostaphin-secreting E. coli inhibit the growth of S. aureus, including the strain resistant to methicillin (MRSA). Our tunable platform allows stable integration of programmable cells in dextran-based hydrogels without compromising free diffusion of macromolecules and could have potential applications in biotechnology and biomedicine.

Figure 1

Overview of the design elements for PATCH. (a) Escherichia coli (E. coli, orange ovals with black outlines) expressing a cell-surface adhesin (orange-black sticks) with a high affinity for glucose are retained inside dextran-based hydrogels. (b) Cell surface display of the MpA adhesin is regulated by cotransforming E. coli with two expression vectors (MpA⁺; yellow). MpA was cloned into a pET24a vector (pET24a-mpa) under the control of a T7 promoter (pT7-Lac) inducible by IPTG. The engineered adhesin contains the membrane anchor (orange), extender (orange), and sugar-binding domains (SBD, pink circle) derived from MpIBP of the Antarctic bacterium, Marinomonas primoryensis. The 23-kDa C-terminal fragment of hemolysin protein A (HlyAc, dark blue circle) functions as a Type I Secretion Pathway (T1SS) sequence specific to E. coli, which was grafted to the end of MpA to promote its cell-surface-display. The other two E. coli T 1SS components HlyB (dark green) and HlyD (light green) were expressed from a pSTV28 vector (pSTV28-hlyb/d) under the control of a lac promoter (pLac) inducible by IPTG. The T1SS outer-membrane component (TolC) constitutive to E. coli BL21 cells is colored brown. (c) X-ray crystal structure of the MpA sugar-binding domain (SBD), which is responsible for binding to the dextran component of the hydrogel matrix. Ca²⁺ are illustrated as large blue-green spheres, waters are indicated by small red spheres. The sugar-binding site is occupied by a glucose molecule (green backbone; stick representation). The figure was rendered using UCSF Chimera. (d) E. coli (green; MpA⁺/lyso⁺) is re-engineered to surface-display the MpA adhesin and secrete the bacteriocin lysostaphin via the T1SS. HlyAc (small dark-blue circles) was added to the C-terminus of lysostaphin (small red circles) via a cleavable linker. The lysostaphin-HlyAc fusion construct was under the control of a pBAD promoter (inducible by arabinose) on the pSTV28 vector that also contains the pLac-HlyB/D. The modified vector is named pSTV28-hlyb/d_lyso.e) Schematics of the designed living material with anti-S. aureus activity. Left: Surface-expression of MpA allows E. coli to bind to the dextran matrix, thereby retaining the bacteria in the hydrogel (boundary marked by blue dashed lines). The engineered E. coli inside the hydrogel can secrete lysostaphin (small red circles), which diffuses freely to the exterior environment to inhibit the growth of S. aureus (gold spheres), including a methicillin-resistant strain (MRSA). Gold spheres with dashed boundaries indicate dead S. aureus cells killed by lysostaphin.

Figure 2

Construct design of the engineered MpA adhesins and its localization to E. coli cell surface. (a) Linear domain map of the native 1.5-MDa MpIBP with four of its functional regions illustrated as membrane anchor (orange), central extenders (orange repeats), sugar-binding domain (pink), and M. primoryensis T1SS sequence (brown). Dotted lines indicate the ∼100 central repeats omitted in the figure. (b) Linear domain maps of engineered constructs of MpA expressed and surface-displayed by E. coli. (c) Immuno-detection of MpA by flow cytometry. Top schematic shows immunodetection of MpA through an antibody raised against its central extender domains. Histograms (bottom panel) illustrating fluorescence distribution of the immuno-detection experiments done with MpA^–/lyso⁺ (gray) and MpA⁺/lyso⁺ (red) cells. (d) Fluorescence localization after immunostaining (green) of MpA-mRuby2 (red) obtained using confocal microscopy. Top schematic shows the immunodetection of MpA through an antibody raised against its central extender domains. Bottom panels show images obtained from confocal microscopy. The MpA-mRuby2⁺ cells are displayed in the top two panels, while the detection of MpA on E. coli outer surface by immuno-staining with fluorescently labeled secondary antibodies (Alexa 488, green patches on the cells) are shown in the bottom two panels. Scale bar indicates 2 μm, and is representative for all confocal images in this panel.

Figure 3

Characterization of the MpA adhesin mediated retention of E. coli inside a dextran-based hydrogel matrix. (a) Experimental design to quantify bacterial migration from the hydrogel matrix into liquid medium. Leakage of MpA⁺ (orange) from a hydrogel cube was assessed by counting the colony forming units (CFUs) present in the LB medium surrounding the hydrogel, and compared with the leakage obtained from MpA^– cells (blue, negative control). (b) Bacterial migration was quantified immediately after bacterial loading (initial leakage) and over the following day. Right panel: representative images of plates at the same dilution are shown to illustrate the difference in migration between MpA⁺ and MpA^– samples after 24 h (Day 1). Dots on the bar graph represent the results of individual experiments. (c) Scanning electron microscopy images obtained from the dextran-based hydrogel inoculated with the MpA⁺E. coli inside of the bacterial loading chamber (cross-section). (d) Zoomed-in view of an area of panel c, showing a bacterial microcolony of MpA⁺ formed on the lamina of the hydrogel matrix. Scale bars (green) in panels c and d indicate 10 μm. (e) Representative images showing the migration of E. coli through the matrix of a disk-shaped hydrogel, which was assessed by estimating the surface area colonized by the bacteria (MpA⁺/lyso⁺ or MpA^–/lyso⁺) on the outside of the hydrogel disk over time. Arrowheads indicate the presence of nascent colonies of E. coli (MpA^–/lyso in blue) and (MpA⁺/lyso⁺ in orange). Scale bar represents 2 mm. (f,g,h) Quantification of the time-dependent migration of engineered E. coli (MpA⁺/lyso⁺ in blue and MpA^–/lyso⁺ in orange) through the hydrogel matrix. Average area and standard deviation of the bacteria-colonized regions outside of the hydrogel were calculated from replicates of that in panel e over 72 h (n = 4). (f) Experiments performed in the presence of both inducing agents, IPTG for adhesin surface display, and arabinose for lysostaphin expression (Ara). (g) Experiments performed in the presence of IPTG, but in the absence of Ara (n = 3). (h) Experiments performed in the absence of IPTG or Ara (n = 3).

Figure 4

PATCH with Anti-S. aureus activity. (a) Schematic of the adapted Kirby-Bauer test for assessing the anti-S. aureus activity of lysostaphin-secreting E. coli as an application of PATCH. (b) Evaluation of bactericidal activity of the cell-free media collected from MpA^–/lyso⁺, MpA^–, MpA⁺/lyso⁺ and MpA⁺/lyso^– cells against Staphylococcus aureus and Streptococcus agalactiae. White arrows indicate where the samples were applied. Black arrows indicate the target bacteria streaked on the plates. Growth inhibition zones are marked by a red arrow. (c) Evaluation of bactericidal activity of MpA^–/lyso⁺, MpA^–, MpA⁺/lyso⁺ and MpA⁺/lyso^– (no lysostaphin-producing capability) E. coli cells against Methicillin-resistant Staphylococcus aureus (MRSA) and S. agalactiae. Images were captured after an incubation time of approximately 48 h. d) Evaluation of bactericidal activity of the living hydrogels with engineered MpA⁺/lyso⁺E. coli against MRSA. MpA⁺/lyso⁺E. coli cells were spotted to the centers of the hydrogel disks, which were placed onto Mueller-Hinton agar plates streaked with MRSA. The images were captured after an incubation period of 96 h. The adhesin- and lysostaphin-functionalities were controlled by induction by IPTG and/or Arabinose (Ara), respectively. White arrowheads point to the edge of the hydrogel disks as outlined by thin white circles.