A LytA-Derived Biosensor for Identifying Choline-Containing Streptococcal Pathogens

Abstract Rapid detection of choline-containing streptococcal pathogens is important for timely diagnosis and antimicrobial treatment. Here, we developed gCBMLytA, a GFP-fused choline-binding module derived from the pneumococcal autolysin LytA, as a fluorescence-based biosensor. gCBMLytA specifically labeled all 14 tested Streptococcus pneumoniae strains and also recognized Streptococcus oralis, while showing no detectable binding to nontarget ESKAPE pathogens or Haemophilus influenzae. These results indicate that gCBMLytA recognizes choline within the structural context of streptococcal teichoic acids rather than choline-containing structures in unrelated bacteria. In mouse pneumonia models, intravenously administered gCBMLytA selectively accumulated in the lungs of S. pneumoniae-infected mice, but not in healthy controls or Staphylococcus aureus-infected mice. Together, these findings demonstrate that gCBMLytA is a selective fluorescence-based probe for choline-containing streptococcal pathogens and may serve as a versatile recognition module for diagnostic platforms, including fluorescence imaging, lateral flow assays, ELISA, and flow-cytometry-based detection systems.