Targeting surface-layer proteins with single-domain antibodies: a potential therapeutic approach against Clostridium difficile-associated disease.
Clostridium difficile is a leading cause of death from gastrointestinal infections in North America. Antibiotic therapy is effective, but the high incidence of relapse and the rise in hypervirulent strains warrant the search for novel treatments. Surface layer proteins (SLPs) cover the entire C. difficile bacterial surface, are composed of high-molecular-weight (HMW) and low-molecular-weight (LMW) subunits, and mediate adherence to host cells. Passive and active immunization against SLPs has enhanced hamster survival, suggesting that antibody-mediated neutralization may be an effective therapeutic strategy. Here, we isolated a panel of SLP-specific single-domain antibodies (VHHs) using an immune llama phage display library and SLPs isolated from C. difficile hypervirulent strain QCD-32g58 (027 ribotype) as a target antigen. Binding studies revealed a number of VHHs that bound QCD-32g58 SLPs with high affinity (K D = 3-6 nM) and targeted epitopes located on the LMW subunit of the SLP. The VHHs demonstrated melting temperatures as high as 75 °C, and a few were resistant to the gastrointestinal protease pepsin at physiologically relevant concentrations. In addition, we demonstrated the binding specificity of the VHHs to the major C. difficile ribotypes by whole cell ELISA, where all VHHs were found to bind 001 and 027 ribotypes, and a subset of antibodies were found to be broadly cross-reactive in binding cells representative of 012, 017, 023, and 078 ribotypes. Finally, we showed that several of the VHHs inhibited C. difficile QCD-32g58 motility in vitro. Targeting SLPs with VHHs may be a viable therapeutic approach against C. difficile-associated disease.
Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, K1A 0R6, Canada.
Hiba
H
Kandalaft
Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, K1A 0R6, Canada.
Greg
G
Hussack
Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, K1A 0R6, Canada.
Annie
A
Aubry
Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, K1A 0R6, Canada.
Henk
H
van Faassen
Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, K1A 0R6, Canada.
Yonghong
Y
Guan
Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, K1A 0R6, Canada.|Department of Biology, Carleton University, Ottawa, ON, K1S 5B6, Canada.|School of Environmental Sciences, University of Guelph, Guelph, ON, N1G 2W1, Canada.
Mehdi
M
Arbabi-Ghahroudi
Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, K1A 0R6, Canada.|School of Environmental Sciences, University of Guelph, Guelph, ON, N1G 2W1, Canada.
Roger
R
MacKenzie
Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, K1A 0R6, Canada.|Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, K1N 6N5, Canada.
Susan M
SM
Logan
Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, K1A 0R6, Canada. jamshid.tanha@nrc-cnrc.gc.ca.|School of Environmental Sciences, University of Guelph, Guelph, ON, N1G 2W1, Canada. jamshid.tanha@nrc-cnrc.gc.ca.|Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, K1N 6N5, Canada. jamshid.tanha@nrc-cnrc.gc.ca.
Jamshid
J
Tanha
D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism*; D000907:Antibodies, Bacterial / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D016360:Clostridioides difficile / Q000187:drug effects* / Q000502:physiology; D000939:Epitopes / Q000378:metabolism; D008124:Locomotion / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors*; D019151:Peptide Library; D011485:Protein Binding; D061905:Single-Domain Antibodies / Q000302:isolation & purification / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D019151:Peptide Library; C055764:S-layer proteins; D061905:Single-Domain Antibodies
Clostridium difficile; Nanobody; Single-domain antibody; Surface layer protein; VHH