Design of a novel affinity probe using the cell wall-binding domain of a <i>Listeria monocytogenes</i> autolysin for pathogen detection
Bibliographic record
Abstract
ABSTRACT Rapid identification of foods and food-processing environments contaminated with Listeria monocytogenes is crucial for controlling human listeriosis, a leading cause of fatality related to foodborne illnesses. The L. monocytogenes autolysin IspC anchors to the bacterial surface non-covalently via its C-terminal cell wall-binding domain (CWBD), which contains conserved Gly-Trp (GW) dipeptides. This study explored the binding property of CWBD IspC to design an affinity probe for detecting L. monocytogenes . Immunogold electron microscopy analysis of a ∆ ispC mutant and its wild type (WT) revealed that exogenously added CWBD IspC bound to surface ligands that were evenly distributed and remained mostly unoccupied in the wild type. CWBD IspC detected, via recognizing the cell wall component lipoteichoic acid, L. monocytogenes strains primarily belonging to the serotype 4 group, as well as Listeria innocua , and Listeria welshimeri but did not detect three other Listeria spp. ( Listeria seeligeri , Listeria ivanovii, and Listeria grayi ) nor any non- Listeria spp. tested. CWBD IspC , when tagged with the enhanced green fluorescent protein, was capable of detecting L. monocytogenes by fluorescence microscopy, a colony lift filter assay, and a microtube binding assay. CWBD IspC , when covalently attached to magnetic beads, showed the ability to separate L. monocytogenes cells with a maximum capture rate of 90 to nearly 100% from 1 × 10 3 to 1 × 10 5 target cells. This study demonstrated for the first time that the CWBD derived from a GW surface protein IspC is a novel antibody alternative for the rapid detection and isolation of L. monocytogenes despite binding to two other Listeria species. IMPORTANCE Human listeriosis is caused by consuming foods contaminated with the bacterial pathogen Listeria monocytogenes , leading to the development of a severe and life-threatening foodborne illness. Detection of L. monocytogenes present in food and food processing environments is crucial for preventing Listeria infection. The L. monocytogenes peptidoglycan hydrolase IspC anchors non-covalently to the bacterial surface through its C-terminal cell wall-binding domain (CWBD), CWBD IspC . This study explored the surface binding property of CWBD IspC to design, construct, characterize, and assess an affinity molecular probe for detecting L. monocytogenes . CWBD IspC recognized a cell wall ligand lipoteichoic acid that remains evenly displayed and mostly unoccupied on the bacterial surface for interaction with the exogenously added CWBD IspC . CWBD IspC , when fused to the enhanced green fluorescent protein reporter or covalently conjugated onto magnetic beads, exhibited the functionality as an antibody alternative for rapid detection and efficient separation of the pathogen.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".