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

Abstract

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.

Enteric pharmacokinetics of monomeric and multimeric camelid nanobody single-domain antibodies

“…expressed in patients by probiotics or, possibly by engineering their own microbiome. Such products have already been reported to show some therapeutic benefits in numerous animal models of enteric diseases [2][3][4][5][6][7][8][9][10][11]. Because the GI tract is a hostile environment for protein therapeutics due to its role in proteolytic food digestion and its rapid luminal flow leading to elimination, optimal exploitation of VHH-based agents as enteric therapeutics will require a good understanding of the factors that influence the functional stability of these agents while resident within this environment.…”

“…Thus, in enteric situations when VHH monomers are not sufficient, it may prove necessary to employ alternative approaches in which the VHH fusion proteins are continuously expressed and thus replenished within the GI tract such as by living probiotic bacteria. This strategy has been successfully demonstrated recently in two systems; one expressing VHH/CsgA-fusions within probiotic E. coli Nissle which produced VHHs as stacked multimers within curli fibers [11], and another displaying VHHs fused to a surface protein of Lactobacillus paracasei within the GI tract [25].…”

“…A number of reports have emerged in which VHH agents have been successfully employed as oral prophylactic agents to prevent rotavirus infections [2][3][4], ETEC [5], inflammatory bowel disease [6,7] and pathology related to Clostridium difficile infection [8]. To bypass potential damage to VHHs within the stomach, in some cases oral VHH delivery employed encapsulation in nanoparticles [7], delivery as fusions to IgA Fc domains within plant seeds [9] or expression by genetically modified blue-green algae (spirulina) [10] and probiotic bacteria such as Lactobacillus and Lactococcus [4,6,8,11].…”

Engineering a novel probiotic toolkit inEscherichia coli Nissle1917for sensing and mitigating gut inflammatory diseases

“…Thus, EcN is a promising chassis for targeted gut therapies 25 . Over the last decade, this strain has been extensively engineered to produce biomolecules at disease sites, particularly for treating intestinal diseases [26][27][28][29][30][31][32][33] . However, despite recent developments in expanding the tools and biological parts for engineering EcN, there remains a shortage of self-regulating genetic circuits that can recognize specific biomarkers and respond by producing therapeutic molecules 34 .…”

Enteric pharmacokinetics of monomeric and multimeric camelid nanobody single-domain antibodies

“…A number of reports have emerged in which VHH agents have been successfully employed as oral prophylactic agents to prevent rotavirus infections (2)(3)(4), ETEC (5), inflammatory bowel disease (6,7) and pathology related to Clostridium difficile infection (8). To bypass potential damage to VHHs within the stomach, in some cases oral VHH delivery employed encapsulation in nanoparticles (7), delivery as fusions to IgA Fc domains within plant seeds (9) or expression by genetically modified blue-green algae (spirulina) (10) and probiotic bacteria such as Lactobacillus and Lactococcus (4,6,8,11).…”