Single domain antibodies against enteric pathogen virulence factors are active as curli fiber fusions on probiotic E. coli Nissle 1917.

Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic E. coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eukaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.

Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium, commonly employed to treat certain gastrointestinal disorders. It is fast emerging as an important target for the development of therapeutic engineered bacteria, benefiting from the wealth of knowledge of E. coli biology and ease of manipulation. Bacterial synthetic biology projects commonly utilize engineered plasmid vectors, which are simple to engineer and can reliably achieve high levels of protein expression. However, plasmids typically require antibiotics for maintenance, and the administration of an antibiotic is often incompatible with in vivo experimentation or treatment. EcN natively contains plasmids pMUT1 and pMUT2, which have no known function but are stable within the bacteria. Here, we describe the development of the pMUT plasmids into a robust platform for engineering EcN for in vivo experimentation, alongside a CRISPR-Cas9 system to remove the native plasmids. We systematically engineered both pMUT plasmids to contain selection markers, fluorescent markers, temperature sensitive expression, and curli secretion systems to export a customizable functional material into the extracellular space. We then demonstrate that the engineered plasmids were maintained in bacteria as the engineered bacteria pass through the mouse GI tract without selection, and that the secretion system remains functional, exporting functionalized curli proteins into the gut. Our plasmid system presents a platform for the rapid development of therapeutic EcN bacteria.

Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut.

Mucosal healing plays a critical role in combatting the effects of inflammatory bowel disease, fistulae and ulcers. While most treatments for such diseases focus on systemically delivered anti-inflammatory drugs, often leading to detrimental side effects, mucosal healing agents that target the gut epithelium are underexplored. We genetically engineer Escherichia coli Nissle 1917 (EcN) to create fibrous matrices that promote gut epithelial integrity in situ. These matrices consist of curli nanofibers displaying trefoil factors (TFFs), known to promote intestinal barrier function and epithelial restitution. We confirm that engineered EcN can secrete the curli-fused TFFs in vitro and in vivo, and is non-pathogenic. We observe enhanced protective effects of engineered EcN against dextran sodium sulfate-induced colitis in mice, associated with mucosal healing and immunomodulation. This work lays a foundation for the development of a platform in which the in situ production of therapeutic protein matrices from beneficial bacteria can be exploited.

Fructose-Enhanced Antimicrobial Activity of Silver Nanoparticle-Embedded Polymersome Nanocarriers.

In recent years, an increasing body of research has indicated that the antimicrobial activity of certain antibiotic drugs can be enhanced by the addition of specific metabolites. This study aimed to incorporate these findings into polymersomes (novel polymer-based nanoscale drug delivery vehicles) which can be loaded with various therapeutic molecules and nanoparticles. Polymersome technology has shown promising results in treating antibiotic-resistant infections by co-encapsulating the antibiotic methicillin with silver nanoparticles. Here, silver nanoparticle-embedded polymersomes (AgPs) were synthesized in a similar fashion with gentamicin replacing methicillin as the antibiotic agent and supplemented with fructose to promote efficacy. Two clinically-isolated strains of methicillin-resistant Staphylococcus aureus (MRSA) (ATCC #43300 and ATCC #25923) were cultured and treated with the new AgP formulations, with the former strain being susceptible to gentamicin and the latter strain being resistant to gentamicin. The treatment of the non-resistant strain yielded promising results with the polymersomes without fructose supplementation inducing a maximal growth rate reduction of up to 40% and an increase in lag time of up to 141% relative to the untreated control. Impressively, the fructose-loaded polymersomes completely eliminated the bacterial growth over the observed time period at the higher doses and outperformed the no-fructose treatment at all concentrations. However, despite significantly reducing bacterial growth, the treatment of the gentamicin-resistant strain did not seem to be enhanced by the addition of fructose. Lastly, the present study demonstrated that the presence of fructose in the polymersomes seemed to slightly ameliorate the cytotoxic effect of the treatment on human dermal fibroblasts (a model mammalian cell). In addition to developing and testing a new polymersome formulation with fructose resulting in increased efficacy, the results of this study also demonstrated the variability inherent to developing novel antimicrobial treatments for different bacterial strains.

Superparamagnetic iron oxide-encapsulating polymersome nanocarriers for biofilm eradication.

The rising prevalence and severity of antibiotic-resistant biofilm infections poses an alarming threat to public health worldwide. Here, biocompatible multi-compartment nanocarriers were synthesized to contain both hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) and the hydrophilic antibiotic methicillin for the treatment of medical device-associated infections. SPION co-encapsulation was found to confer unique properties, enhancing both nanocarrier relaxivity and magneticity compared to individual SPIONs. These iron oxide-encapsulating polymersomes (IOPs) penetrated 20 µm thick Staphylococcus epidermidis biofilms with high efficiency following the application of an external magnetic field. Three-dimensional laser scanning confocal microscopy revealed differential bacteria death as a function of drug and SPION loading. Complete eradication of all bacteria throughout the biofilm thickness was achieved using an optimized IOP formulation containing 40 µg/mL SPION and 20 µg/mL of methicillin. Importantly, this formulation was selectively toxic towards methicillin-resistant biofilm cells but not towards mammalian cells. These novel iron oxide-encapsulating polymersomes demonstrate that it is possible to overcome antibiotic-resistant biofilms by controlling the positioning of nanocarriers containing two or more therapeutics.