Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan

<jats:title>ABSTRACT</jats:title><jats:p>The C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. The<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Listeria monocytogenes</jats:named-content>Ply118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure of<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Listeria</jats:named-content>. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding using<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">L. monocytogenes</jats:named-content>serovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundant<jats:italic>tagO</jats:italic>homologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of either<jats:italic>tagO1</jats:italic>(EGDe<jats:italic>lmo0959</jats:italic>) or<jats:italic>tagO2</jats:italic>(EGDe<jats:italic>lmo2519</jats:italic>) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficient<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Listeria</jats:named-content>cells. Substitution of<jats:italic>tagO</jats:italic>from an isopropyl-β-<jats:sc>d</jats:sc>-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from other<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Listeria</jats:named-content>phage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains.</jats:p>