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

• Published in: PLoS pathogens Vol. 18; no. 9; p. e1010713
• Main Authors: Gelfat, Ilia, Aqeel, Yousuf, Tremblay, Jacqueline M., Jaskiewicz, Justyna J., Shrestha, Anishma, Lee, James N., Hu, Shenglan, Qian, Xi, Magoun, Loranne, Sheoran, Abhineet, Bedenice, Daniela, Giem, Colter, Manjula-Basavanna, Avinash, Pulsifer, Amanda R., Tu, Hann X., Li, Xiaoli, Minus, Marilyn L., Osburne, Marcia S., Tzipori, Saul, Shoemaker, Charles B., Leong, John M., Joshi, Neel S.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.09.2022; Public Library of Science (PLoS)
• Subjects: Antibodies; Antigens; Antigens, Surface; Antiinfectives and antibacterials; Bacteria; Bacterial Toxins; Binding; Biofilms; Biology and Life Sciences; Colonization; Congo Red; Cryptosporidiosis; Cryptosporidium; Disease; Dosage and administration; E coli; Electron micrographs; Enterocytes; Enteropathogenic Escherichia coli; Escherichia coli; Escherichia coli infections; Fibers; Flagellin; Glycoproteins; Health aspects; Humans; Infant mortality; Infectious diseases; Intimin; Low income areas; Medicine and Health Sciences; Microorganisms; Mortality; Motility; Nanobodies; Pathogens; Patient outcomes; Prevention; Probiotics; Protein A; Proteins; Public health; Research and Analysis Methods; Risk factors; Shiga toxin; Shigella; Single-Domain Antibodies; Toxin A; Toxins; Translocated intimin receptor; Type III Secretion Systems; Viral antibodies; Virulence; Virulence (Microbiology); Virulence factors; Virulence Factors - genetics; United States
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1010713

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.