Split fluorescent protein-mediated multimerization of cell wall binding domain for highly sensitive and selective bacterial detection

Shirley Xu; Inseon Lee; Seok-Joon Kwon; Eunsol Kim; Liv Nevo; Lorelli Straight; Hironobu Murata; Krzysztof Matyjaszewski; Jonathan S Dordick

doi:10.1016/j.nbt.2024.05.004

Split fluorescent protein-mediated multimerization of cell wall binding domain for highly sensitive and selective bacterial detection

N Biotechnol. 2024 May 15:82:54-64. doi: 10.1016/j.nbt.2024.05.004. Online ahead of print.

Authors

Shirley Xu¹, Inseon Lee¹, Seok-Joon Kwon¹, Eunsol Kim¹, Liv Nevo¹, Lorelli Straight², Hironobu Murata³, Krzysztof Matyjaszewski³, Jonathan S Dordick⁴

Affiliations

¹ Department of Chemical and Biological Engineering, and Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY, USA.
² Department of Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY, USA.
³ Department of Chemistry, Carnegie Mellon University, 5000 Forbes Ave, Pittsburgh, PA, USA.
⁴ Department of Chemical and Biological Engineering, and Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY, USA; Department of Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY, USA. Electronic address: dordick@rpi.edu.

PMID: 38750815
DOI: 10.1016/j.nbt.2024.05.004

Abstract

Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial binding, and thus, have potential as biorecognition molecules for nondestructive bacterial detection. Here, a novel design for a self-complementing split fluorescent protein (FP) complex is proposed, where a multimeric FP chain fused with specific CBDs ((FP-CBD)_n) is assembled inside the cell, to improve sensitivity by enhancing the signal generated upon Staphylococcus aureus or Bacillus anthracis binding. Flow cytometry shows enhanced fluorescence on the cell surface with increasing FP stoichiometry and surface plasmon resonance reveals nanomolar binding affinity to isolated peptidoglycan. The breadth of function of these complexes is demonstrated through the use of CBD modularity and the ability to attach enzymatic detection modalities. Horseradish peroxidase-coupled (FP-CBD)_n complexes generate a catalytic amplification, with the degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 10³ cells/droplet (approximately 0.1 ng S. aureus or B. anthracis) within 15 min on a polystyrene surface. These fusion proteins can be multiplexed for simultaneous detection. Multimeric split FP-CBD fusions enable use as a biorecognition molecule with enhanced signal for use in bacterial biosensing platforms.

Keywords: Bacteria detection; Cell wall binding domain; Enzymatic biosensors; Split fluorescent protein.