A molecular toolkit for heterologous protein secretion across Bacteroides species

Abstract

Bacteroides species are abundant and prevalent stably colonizing members of the human gut microbiota, making them a promising chassis for developing long-term interventions for chronic diseases. Engineering these bacteria as on-site production and delivery vehicles for biologic drugs or diagnostics, however, requires efficient heterologous protein secretion tools, which are currently lacking. To address this limitation, we systematically investigated methods to enable heterologous protein secretion in Bacteroides using both endogenous and exogenous secretion systems. Here, we report a collection of secretion carriers that can export functional proteins across multiple Bacteroides species at high titers. To understand the mechanistic drivers of Bacteroides secretion, we characterized signal peptide sequence features as well as post-secretion extracellular fate and cargo size limit of protein cargo. To increase titers and enable flexible control of protein secretion, we developed a strong, self-contained, inducible expression circuit. Finally, we validated the functionality of our secretion carriers in vivo in a mouse model. This toolkit should enable expanded development of long-term living therapeutic interventions for chronic gastrointestinal disease.

Keywords: Bacteroides; antibody fragments; bacterial therapeutics; commensal bacteria; drug delivery; live biotherapeutic products; outer membrane vesicles; protein secretion; synthetic biology; therapeutic proteins.

Figure 1.. Engineering B. theta to secrete sdAb-TcdA

(A) Design of genetic constructs for protein expression and secretion. (B) Protein secretion (top row) and growth (bottom row) of B. theta expressing BT_2472, BT_3382, and BT_3769 with their native secretion signals, measured from supernatant samples taken every 4 hrs for 48 hrs. Protein levels were measured by dot blot and bacterial growth was measured by optical density at 600 nm (OD₆₀₀). For strains using the P1T_DP-GH023 promoter, the inducer aTc (100 or 200 ng/ml) was included in the medium at the time of inoculation. Error bars represent standard deviation of three biological replicates. a.u., arbitrary units. (C) Schematic representation of secretion strategies explored in this study. (D) Design of genetic constructs for secretion carrier screening in B. theta. (E) Relative levels of secretion of sdAb-TcdA in culture supernatants of B. theta harboring sixty different expression/secretion constructs, measured by dot blot. Inset shows representative dot blot with effective secretion carriers (above detection limit) labeled. Detection limit (dotted line) was set at the signal intensity of the faintest dot visible by unaided eye on the membrane, which is around 7 arbitrary units (a.u.). Error bars represent one standard deviation of triplicate biological samples. Significance was determined using unpaired two-tailed Welch’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. WT, wild-type B. theta; NC, negative control B. theta expressing sdAb-TcdA with no secretion carrier fusion.

Figure 2.. Rational engineering enables ineffective lipoprotein SPs to secrete sdAb-TcdA

(A) Comparison of the length of five ineffective (black) and nineteen effective (green) lipoprotein SP sequences. (B) Comparison of amino acid sequences of the five ineffective lipoprotein SPs with a prototypical effective sequence. Positively charged residues are colored blue, negatively charged residues are colored red, and cysteine residue cleavage sites are colored green. (C) Secretion of sdAb-TcdA driven by native (SP) and domain-swapped (SP-N, SP-H, and SP-NH) ineffective lipoprotein SPs, measured by ELISA. BT_3630 SP-sdAb-TcdA is included as positive control. Residue coloring is the same as for (B), and hydrophobic regions are boxed in gray. Error bars represent one standard deviation of triplicate biological samples. Significance was determined using unpaired two-tailed Welch’s t test. **p < 0.01, ***p < 0.001, ****p < 0.0001

Figure 3.. B. theta-derived secretion carriers function across multiple heterologous proteins

(A) Relative levels of antibody fragments and reporter proteins secreted into culture supernatant by B. theta secretion carriers. Bubble size corresponds to average blot intensity of triplicate experiments with p < 0.05 indicated by the blue color scale and p > 0.05 shown in gray. Significance was determined using unpaired two-tailed Welch’s t test. (B) Functional assays of antibody fragments and reporter proteins secreted into culture supernatant by B. theta secretion carriers. Binding of antibody fragments (sdAbs and scFv) to their respective targets was determined by ELISA. Enzymatic activity of reporter proteins Nluc and BLac was determined by bioluminescence assay and colorimetric assay, respectively. Following log transformation of luminescence data, all functional assay readouts were converted to values between zero and one by cargo-wise min-max normalization.

Figure 4.. B. theta-derived secretion carriers mediate export of diverse, functional cargoes from multiple Bacteroides species

(A) Relative levels of six cargo proteins detected in the culture supernatants of three Bacteroides species, driven by each of the ten highest performing native B. theta secretion carriers. Bubble size corresponds to average blot intensity of triplicate experiments with p < 0.05 indicated by the blue color scale and p > 0.05 shown in gray. Significance was determined using unpaired two-tailed Welch’s t test. (B) Functional assays of antibody fragments and reporter proteins secreted into culture supernatant by secretion carriers. Binding of antibody fragments (sdAb-TcdA, sdAb-TNFα, sdAb-EGFR, and scFv-HER2) to their respective targets was determined by ELISA. Enzymatic activity of reporter proteins (Nluc and BLac) was determined by bioluminescence assay and colorimetric assay, respectively. Following log transformation of luminescence data, all functional assay readouts were converted to values between zero and one by cargo-wise min-max normalization. All functional assays were performed in triplicate. (C) Quantification of protein secretion titers mediated by the two secretion carriers that yielded the highest functional protein levels of each cargo in each species. Error bars represent the standard deviation of triplicate experiments.

Figure 5.. Development of a strong, aTc-inducible expression cassette for enhanced control of protein secretion across multiple Bacteroides species

(A) Low-activity promoter and RBS sequences in the original P2-A21-tetR-P1T_DP-GH023 inducible expression cassette (left) were replaced with high-activity variants to generate the modified P_BT1311-tetR-P1T_DP-A21 expression cassette (right). (B) Modified inducible expression cassette drives expression of Nluc reporter at levels similar to high-level constitutive promoter P_BfP1E6-RBS8 in cultures diluted at 1:100 (top) or 1:10 (bottom). (C) Correlation between bacterial growth and Nluc secretion levels shown in Fig. 5B, top. (D) Modified inducible system mediates tightly controlled protein expression across multiple Bacteroides species. For all experiments, luminescence measurements were obtained from clarified culture supernatant and thus represent the secreted fraction of total expressed Nluc. Nluc secretion in this study was mediated by the highly active secretion carrier BT_3630 SP. Error bars represent one standard deviation of triplicate biological samples. Significance was determined using unpaired two-tailed Welch’s t test. **p < 0.01, ***p < 0.001, ****p < 0.0001

Figure 6.. Characterization of the post-secretion extracellular fate and size limit of cargo proteins for secretion carriers

(A) Western blot analysis of Nluc abundance in different fractions of B. theta liquid cultures expressing four carrier-Nluc constructs. P, cell pellet; T, total supernatant; S, soluble fraction of total supernatant; and O, OMV fraction of total supernatant. P and O are 2.5-fold and 20-fold more concentrated than supernatant of equivalent volume, respectively. (B) Enzymatic activity of secreted Nluc in soluble and OMV fractions, measured by luminescence assay. To normalize the efficiency difference between the four secretion carriers, the luminescence in soluble and OMV fractions was divided by the luminescence in total supernatants to calculate the relative abundance of secreted Nluc in soluble and OMV fractions. Normalized luminescence of concentrated OMV fractions was divided by 20 to correct for concentration during sample prep. Error bars represent one standard deviation of triplicate biological samples. Significance was determined using unpaired two-tailed Welch’s t test. *p < 0.05, **p < 0.01 (C) Western blot analysis of proteinase K assay of OMV fractions from B. theta cultures expressing BT_0569-Nluc (Sec/SPI SP; predicted localization to OMV lumen) and BT_3630-Nluc (lipoprotein SP; predicted localization to OMV surface). PK, proteinase K (D) Schematic representation of post-secretion extracellular fate of Nluc mediated by BT_0169, BT_0569, and BT_3630 SP. BT_0922-Nluc is predicted to have a similar pattern as BT_3630 SP-Nluc as both have a lipoprotein SP and demonstrated similar abundance and localization in 7A and 7B. (E) Set of seven expression constructs generated to test the ability of BT_3630 SP to mediate secretion of different sized protein cargoes to the outer surface of OMVs in B. theta. The molecular weight of each protein is shown on the right. (F) Western blot analysis of liquid culture supernatants and cell pellets from B. theta expressing seven proteins of varying size fused to BT_3630 SP.

Figure 7.. Direct intestinal delivery of heterologous protein cargo by B. theta in mice

(A) Design of in vivo experiments. Mice were monitored, and fecal samples were collected and analyzed for two months following inoculation. (B) The weight of mice in all groups increased similarly over time, indicating no adverse health effects. (C) Engineered B. theta strains persisted at high levels in the mouse intestine, as determined by fecal CFU counts. (D) The functionality of intestinally delivered protein cargo (Nluc) persisted over time, as determined by luminescence measurements of fecal homogenates. Error bars represent one standard deviation of the results of four mice for each group.