Removing an S-layer enables endolysin-derived probes to detect Paenibacillus thiaminolyticus in mixed bacterial populations

Abstract

The emerging neonatal bacterial pathogen Paenibacillus thiaminolyticus causes sepsis and postinfectious hydrocephalus, yet few tools are available to study or specifically detect it. We developed fluorescent probes based on cell wall-binding domains (CBDs) from bacteriophage-derived cell-wall hydrolases. CBDs can target structurally conserved cell-wall glycans, making them attractive affinity reagents for bacterial detection. None of the 11 probes we designed labeled intact cells under standard conditions, and we identified a surface layer (S-layer) that prevented access to their cell-wall targets. Brief acidic treatment (pH 2.0) released abundant high-molecular-mass proteins from the cell surface and enabled labeling by five distinct CBD probes, each containing a single C-terminal S-layer homology domain. Mass spectrometry identified the main released protein as an ortholog of the P. alvei SpaA S-layer protein, which we designate SlsA. Across 26 surveyed P. thiaminolyticus genomes, we identified four distinct SlsA variants with 58–79% pairwise amino acid identity, encoded within a conserved cluster homologous to the P. alvei S-layer-associated locus. After either the acidic treatment or a quick flame fixation, the lead probe mGL-BDPt9 labeled six P. thiaminolyticus strains, including three Ugandan clinical isolates, as well as a P. dendritiformis clinical isolate from a US infant, and three additional Paenibacillus species. mGL-BDPt9 also distinguished P. thiaminolyticus from five gram-positive sepsis pathogens in pairwise mixtures and detected it in a five-species suspension. Together, these findings establish the presence of an S-layer in P. thiaminolyticus and provide a rapid fluorescent labeling strategy for multiple Paenibacillus pathogens associated with neonatal paenibacilliosis. IMPORTANCE Paenibacillus species are increasingly recognized as causes of devastating neonatal infections, but little is known about the surface structures of these pathogens or how those structures affect direct detection. Here, we identify an S-layer in P. thiaminolyticus, revealing a major cell-surface component that may contribute to pathogenesis, since S-layers mediate host interactions and contribute to immune evasion and virulence in other bacterial pathogens. We also show that the S-layer can hide conserved cell-wall targets and can be rapidly removed with a simple one-minute treatment that is readily incorporated into labeling protocols. Probes built from the binding domains of prophage-encoded enzymes labeled clinical isolates of two Paenibacillus species associated with infant disease and distinguished P. thiaminolyticus within mixed bacterial suspensions. S-layers are common among bacterial pathogens, and similar removal steps may expose additional cell-wall targets for affinity-probe development in those organisms.