John C. March

luxS-Dependent Gene Regulation in Escherichia coli K-12 Revealed by Genomic Expression Profiling

The bacterial quorum-sensing autoinducer 2 (AI-2) has received intense interest because the gene for its synthase, luxS, is common among a large number of bacterial species. We have identified luxS-controlled genes in Escherichia coli under two different growth conditions using DNA microarrays. Twenty-three genes were affected by luxS deletion in the presence of glucose, and 63 genes were influenced by luxS deletion in the absence of glucose. Minimal overlap among these gene sets suggests the role of luxS is condition dependent. Under the latter condition, the metE gene, the lsrACDBFG operon, and the flanking genes of the lsr operon (lsrR, lsrK, tam, and yneE) were among the most significantly induced genes by luxS. The E. coli lsr operon includes an additional gene, tam, encoding an S-adenosyl-l-methionine-dependent methyltransferase. Also, lsrR and lsrK belong to the same operon, lsrRK, which is positively regulated by the cyclic AMP receptor protein and negatively regulated by LsrR. lsrK is additionally transcribed by a promoter between lsrR and lsrK. Deletion of luxS was also shown to affect genes involved in methionine biosynthesis, methyl transfer reactions, iron uptake, and utilization of carbon. It was surprising, however, that so few genes were affected by luxS deletion in this E. coli K-12 strain under these conditions. Most of the highly induced genes are related to AI-2 production and transport. These data are consistent with the function of LuxS as an important metabolic enzyme but appear not to support the role of AI-2 as a true signal molecule for E. coli W3110 under the investigated conditions.

In vitro 3D human small intestinal villous model for drug permeability determination

We present a novel method for testing drug permeability that features human cells cultured on hydrogel scaffolds made to accurately replicate the shape and size of human small intestinal villi. We compared villous scaffolds to more conventional 2D cultures in paracellular drug absorption and cell growth experiments. Our results suggest that 3D villous platforms facilitate cellular differentiation and absorption more similar to mammalian intestines than can be achieved using conventional culture. To the best of our knowledge, this is the first accurate 3D villus model offering a well-controlled microenvironment that has strong physiological relevance to the in vivo system.

Secretion of Insulinotropic Proteins by Commensal Bacteria: Rewiring the Gut To Treat Diabetes

ABSTRACT Here, we show that commensal bacteria can stimulate intestinal epithelial cells to secrete insulin in response to glucose. Commensal strains were engineered to secrete the insulinotropic proteins GLP-1 and PDX-1. Epithelia stimulated by engineered strains and glucose secreted up to 1 ng ml−1 of insulin with no significant background secretion.

Engineered Commensal Bacteria Reprogram Intestinal Cells Into Glucose-Responsive Insulin-Secreting Cells for the Treatment of Diabetes

The inactive full-length form of GLP-1(1-37) stimulates conversion of both rat and human intestinal epithelial cells into insulin-secreting cells. We investigated whether oral administration of human commensal bacteria engineered to secrete GLP-1(1-37) could ameliorate hyperglycemia in a rat model of diabetes by reprogramming intestinal cells into glucose-responsive insulin-secreting cells. Diabetic rats were fed daily with human lactobacilli engineered to secrete GLP-1(1-37). Diabetic rats fed GLP-1–secreting bacteria showed significant increases in insulin levels and, additionally, were significantly more glucose tolerant than those fed the parent bacterial strain. These rats developed insulin-producing cells within the upper intestine in numbers sufficient to replace ∼25–33% of the insulin capacity of nondiabetic healthy rats. Intestinal tissues in rats with reprogrammed cells expressed MafA, PDX-1, and FoxA2. HNF-6 expression was observed only in crypt epithelia expressing insulin and not in epithelia located higher on the villous axis. Staining for other cell markers in rats treated with GLP-1(1-37)–secreting bacteria suggested that normal function was not inhibited by the close physical proximity of reprogrammed cells. These results provide evidence of the potential for a safe and effective nonabsorbed oral treatment for diabetes and support the concept of engineered commensal bacterial signaling to mediate enteric cell function in vivo.

Expression of an Anaplerotic Enzyme, Pyruvate Carboxylase, Improves Recombinant Protein Production in Escherichia coli

ABSTRACT Anaplerotic enzyme reactions are those which replenish tricarboxylic acid intermediates that are withdrawn for the synthesis of biomass. In this study, we examined recombinant protein production in Escherichia coli containing activity in an additional anaplerotic enzyme, pyruvate carboxylase. In batch fermentations, the presence of pyruvate carboxylase resulted in 68% greater production of the model protein, β-galactosidase, 41% greater cell yield, and 57% lower acetate concentration. We discuss why these results indicate that acetate concentration does not limit cell growth and protein synthesis, as predicted by other researchers, and suggest instead that the rate of acetate formation represents an inefficient consumption of glucose carbon, which is reduced by the presence of pyruvate carboxylase.

Synthetic small intestinal scaffolds for improved studies of intestinal differentiation

In vitro intestinal models can provide new insights into small intestinal function, including cellular growth and proliferation mechanisms, drug absorption capabilities, and host‐microbial interactions. These models are typically formed with cells cultured on 2D scaffolds or transwell inserts, but it is widely understood that epithelial cells cultured in 3D environments exhibit different phenotypes that are more reflective of native tissue. Our focus was to develop a porous, synthetic 3D tissue scaffold with villous features that could support the culture of epithelial cell types to mimic the natural microenvironment of the small intestine. We demonstrated that our scaffold could support the co‐culture of Caco‐2 cells with a mucus‐producing cell line, HT29‐MTX, as well as small intestinal crypts from mice for extended periods. By recreating the surface topography with accurately sized intestinal villi, we enable cellular differentiation along the villous axis in a similar manner to native intestines. In addition, we show that the biochemical microenvironments of the intestine can be further simulated via a combination of apical and basolateral feeding of intestinal cell types cultured on the 3D models. Biotechnol. Bioeng. 2014;111: 1222–1232.

Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models

Bacteria can be genetically engineered to kill specific pathogens or inhibit their virulence. We previously developed a synthetic genetic system that allows a laboratory strain of Escherichia coli to sense and kill Pseudomonas aeruginosa in vitro. Here, we generate a modified version of the system, including a gene encoding an anti-biofilm enzyme, and use the probiotic strain Escherichia coli Nissle 1917 as host. The engineered probiotic shows in vivo prophylactic and therapeutic activity against P. aeruginosa during gut infection in two animal models (Caenorhabditis elegans and mice). These findings support the further development of engineered microorganisms with potential prophylactic and therapeutic activities against gut infections.

Interrupting Vibrio cholerae infection of human epithelial cells with engineered commensal bacterial signaling

Vibrio cholerae El Tor serotypes are largely responsible for outbreaks of cholera in the developing world. The infection cycle for some strains of V. cholerae is coordinated, at least in part, through quorum sensing. That is, the expression of virulence genes depends on the concentration of V. cholerae autoinducers cholera autoinducer 1 (CAI‐1) and autoinducer 2 (AI‐2). High concentrations of CAI‐1 and AI‐2 have been shown previously to inhibit virulence gene expression. We have demonstrated here that a commensal bacterium, E. coli Nissle 1917 (Nissle), can be engineered to express CAI‐1 (Nissle expresses AI‐2 natively) and effectively interrupt V. cholerae virulence. We engineered Nissle to express CAI‐1 under control of the lac promoter, and demonstrated inhibition of V. cholerae expression of cholera toxin (CT, as indicated by presence of the CT subunit B (CTB)) and of the toxin co‐regulated pilus (TCP, as indicated by the relative transcript of TCP subunit A (TCPA)) in both monocultures of V. cholerae and co‐cultures with epithelial cells, Nissle, and V. cholerae. In the model system of Caco‐2 epithelia incubated with V. cholerae, we demonstrated that co‐cultures with Nissle expressing CAI‐1 activity reduced CTB binding to Caco‐2 cells by 63% over co‐cultures with wild‐type Nissle. Further, cultures with Nissle expressing CAI‐1 had significantly lower TCPA transcription than controls with wild‐type Nissle. These results represent a significant step towards a prophylactic method for combating enteric disease through engineered quorum signaling within a commensal bacterial strain. Biotechnol. Biotechnol. Bioeng. 2008;101: 128–134.

Intestinal stem cell growth and differentiation on a tubular scaffold with evaluation in small and large animals

AIMS To investigate the growth and differentiation of intestinal stem cells on a novel tubular scaffold in vitro and in vivo. MATERIALS & METHODS Intestinal progenitor cells from mice or humans were cultured with myofibroblasts, macrophages and/or bacteria, and evaluated in mice via omental implantation. Mucosal regeneration was evaluated in dogs after rectal mucosectomy followed by scaffold implantation. RESULTS Intestinal progenitor cells differentiated into crypt-villi structures on the scaffold. Differentiation and scaffold coverage was enhanced by coculture with myofibroblasts, macrophages and probiotic bacteria, while the implanted scaffolds enhanced mucosal regeneration in the dog rectum. CONCLUSION Intestinal stem cell growth and differentiation on a novel tubular scaffold is enhanced through addition of cellular and microbial components, as validated in mice and dogs.

3-D intestinal scaffolds for evaluating the therapeutic potential of probiotics.

Biomimetic in vitro intestinal models are becoming useful tools for studying host–microbial interactions. In the past, these models have typically been limited to simple cultures on 2-D scaffolds or Transwell inserts, but it is widely understood that epithelial cells cultured in 3-D environments exhibit different phenotypes that are more reflective of native tissue, and that different microbial species will preferentially adhere to select locations along the intestinal villi. We used a synthetic 3-D tissue scaffold with villous features that could support the coculture of epithelial cell types with select bacterial populations. Our end goal was to establish microbial niches along the crypt–villus axis in order to mimic the natural microenvironment of the small intestine, which could potentially provide new insights into microbe-induced intestinal disorders, as well as enabling targeted probiotic therapies. We recreated the surface topography of the small intestine by fabricating a biodegradable and biocompatible villous scaffold using poly lactic-glycolic acid to enable the culture of Caco-2 with differentiation along the crypt–villus axis in a similar manner to native intestines. This was then used as a platform to mimic the adhesion and invasion profiles of both Salmonella and Pseudomonas, and assess the therapeutic potential of Lactobacillus and commensal Escherichia coli in a 3-D setting. We found that, in a 3-D environment, Lactobacillus is more successful at displacing pathogens, whereas Nissle is more effective at inhibiting pathogen adhesion.