Fructose 1,6-Bisphosphate Aldolase, a Novel Immunogenic Surface Protein on Listeria Species

Abstract

Listeria monocytogenes is a ubiquitous food-borne pathogen, and its presence in food or production facilities highlights the importance of surveillance. Increased understanding of the surface exposed antigens on Listeria would provide potential diagnostic and therapeutic targets. In the present work, using mass spectrometry and genetic cloning, we show that fructose-1,6-bisphosphate aldolase (FBA) class II in Listeria species is the antigen target of the previously described mAb-3F8. Western and dot blot assays confirmed that the mAb-3F8 could distinguish all tested Listeria species from close-related bacteria. Localization studies indicated that FBA is present in every fraction of Listeria cells, including supernatant and the cell wall, setting Listeria spp. as one of the few bacteria described to have this protein on their cell surface. Epitope mapping using ORFeome display and a peptide membrane revealed a 14-amino acid peptide as the potential mAb-3F8 epitope. The target epitope in FBA allowed distinguishing Listeria spp. from closely-related bacteria, and was identified as part of the active site in the dimeric enzyme. However, its function in cell surface seems not to be host cell adhesion-related. Western and dot blot assays further demonstrated that mAb-3F8 together with anti-InlA mAb-2D12 could differentiate pathogenic from non-pathogenic Listeria isolated from artificially contaminated cheese. In summary, we report FBA as a novel immunogenic surface target useful for the detection of Listeria genus.

Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies

“…www.nature.com/scientificreports/ the target, and made it possible to determine the MSR of GSM134-C1, which showed no result with ORFeome display. Even though ORFeome also has been reported to allow epitope mapping in the case of a hybridomaderived monoclonal antibody with considerably high resolution 31 , the results from the present study indicate that single-gene display can significantly improve the resolution of the detected epitope region. This corroborates with the fact that most of the antibodies mapped against diphtheria toxin using a similar procedure had relatively short MSR (14-38 amino acids) 59 .…”

“…Similarly, the PDC in Gram-negatives, as well as in Gram-positives, is mostly found in the cytosol. Nevertheless, some other metabolic proteins, such as 1,6-fructose bisphosphate aldolase (FBA), that were described to be mainly cytoplasmic or attached to the membrane, were also found in the cell wall and, thus, accessible on the cell surface 31,62 . PDC-E2 in Listeria may behave similarly to FBA, being a protein that is mainly present in the cytoplasm but makes its way through the membrane reaching the cell wall and surface.…”

“…For this, the cell wall, cytoplasm, and membrane fractions were diluted in phosphate-buffered saline (PBS) 1:100, 1:100, and 1:400, respectively, and were coated on ELISA Costar plates (Corning). Plates were blocked with 2% (w/v) milk powder diluted in PBS supplemented with 0.05% (v/v) Tween-20 (2% MPBS-T), and incubated with monoclonal antibodies 2D12 (mouse IgG anti-InlA, 1 µg/mL) 22 and 3F8 (mouse IgM anti-FBA, 1 µg/mL) 31 . Finally, the plates were incubated either with anti-mouse IgG Fc specific (Sigma) for 2D12, or anti-mouse IgA, G, M (Antibodies Online) for 3F8, both HRP conjugated.…”

“…A phage library built from random genome fragments of L. monocytogenes ATCC 7644 was used to perform panning on obtained antibodies (GSM130-H1, GSM133-A4, GSM133-E2, and GSM134-C1) in order to identify the antibodies' targets. The used library was described in a previous study 31 . Briefly, 1 µg of the antibodies was coated on an ELISA plate.…”

Immunoinformatics Prediction of Epitope Based Peptide Vaccine Against Listeria Monocytogenes Fructose Bisphosphate Aldolase Protein

“…Mendonça et al, (2016) mentioned that the surface exposed antigens of fructose-1,6-bisphosphate aldolase (FBA) class II in Listeria species is the antigen target of the previously described a hybridoma-derived antibody (mAb-3F8). They reported FBA as a novel immunogenic surface target useful for the detection of the genus Listeria [7]. 30-kDa protein is a fructose-1,6-bisphosphate aldolase (FBA), an enzyme of the glycolytic pathway that catalyzes the cleavage of its substrate fructose-1,6-bisphosphate (FBP) into glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP).…”