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Nisin resistance development is one of food safety challenges posed by Listeria monocytogenes, an important foodborne pathogen that causes human listeriosis. The GtcA flippase enzyme is functionally crucial in two separate pathways that glycosylate cell envelope wall teichoic acids (WTA) with N-acetylglucosamine (NAG) and lipoteichoic acids (LTA) with galactose, respectively. This study investigated phenotypic roles and molecular mechanisms underlying GtcA involvement in L. monocytogenes nisin resistance. A GtcA A65V mutation was linked with increased nisin resistance in a food processing environment associated L. monocytogenes strain. Examination of nisin stress survival and growth phenotypes among L. monocytogenes gtcA mutants in different genetic backgrounds showed that GtcA function promoted sensitivity and loss of its function through genetic deletion (ΔgtcA) and a natural GtcA A65V mutation increased nisin resistance. Individual contributions of GtcA WTA NAG and LTA galactose glycosylation functions to nisin resistance modulation were examined through nisin sensitivity analysis of genetic deletion mutants and L. monocytogenes strains complemented using functionally altered GtcA mutants. This revealed WTA NAG glycosylation to be the main functional mechanism that determines GtcA dependent nisin phenotypic sensitization. An examination for mechanisms underlying GtcA involvement in nisin sensitivity revealed that the loss of GtcA function induces changes in the cell envelope carbohydrate composition profiles reducing cell surface hydrophobicity. Overall, our results showed that cell envelope WTA NAG glycosylation promotes nisin susceptibility through facilitation of hydrophobic interactions between nisin and the Listeria cell envelope. There may be practical implications from our observations since nisin resistance could be gained in food associated L. monocytogenes strains that develop phage resistance through acquisition of mutations in genes that cause loss of cell envelope WTA NAG modifications., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)