Impacts of Penicillin Binding Protein 2 Inactivation on β-Lactamase Expression and Muropeptide Profile in <i>Stenotrophomonas maltophilia</i>

Abstract

Penicillin binding proteins (PBPs) are involved in peptidoglycan synthesis, and their inactivation is linked to β-lactamase expression in <i>ampR</i>-β-lactamase module-harboring Gram-negative bacteria. There are seven annotated PBP genes, namely, <i>mrcA</i>, <i>mrcB</i>, <i>pbpC</i>, <i>mrdA</i>, <i>ftsI</i>, <i>dacB</i>, and <i>dacC</i>, in the <i>Stenotrophomonas maltophilia</i> genome, and these genes encode PBP1a, PBP1b, PBP1c, PBP2, PBP3, PBP4, and PBP6, respectively. In addition, <i>S. maltophilia</i> harbors two β-lactamase genes, L1 and L2, whose expression is induced via β-lactam challenge. The impact of PBP inactivation on L1/L2 expression was assessed in this study. Inactivation of <i>mrdA</i> resulted in increased L1/L2 expression in the absence of β-lactam challenge, and the underlying mechanism was further elucidated. The roles of <i>ampNG</i>, <i>ampD</i>_<i>I</i> (the homologue of <i>Escherichia coli ampD</i>), <i>nagZ</i>, <i>ampR</i>, and <i>creBC</i> in L1/L2 expression mediated by a <i>ΔmrdA</i> mutant strain were assessed via mutant construction and β-lactamase activity determinations. Furthermore, the strain <i>ΔmrdA</i>-mediated change in the muropeptide profile was assessed using liquid chromatography mass spectrometry (LC-MS). The mutant Δ<i>mrdA</i>-mediated L1/L2 expression relied on functional AmpNG, AmpR, and NagZ, was restricted by AmpD_I, and was less related to the CreBC two-component system. Inactivation of <i>mrdA</i> significantly increased the levels of total and periplasmic <i>N</i>-acetylglucosaminyl-1,6-anhydro-<i>N</i>-acetylmuramyl-l-alanyl-d-glutamyl-<i>meso</i>-diamnopimelic acid-d-alanine (GlcNAc-anhMurNAc tetrapeptide, or M4N), supporting that the critical activator ligands for mutant strain Δ<i>mrdA</i>-mediated L1/L2 expression are anhMurNAc tetrapeptides. <b>IMPORTANCE</b> Inducible expression of chromosomally encoded β-lactamase(s) is a key mechanism for β-lactam resistance in <i>Enterobacter cloacae</i>, <i>Citrobacter freundii</i>, <i>Pseudomonas aeruginosa</i>, and <i>Stenotrophomonas maltophilia</i>. The muropeptides produced during the peptidoglycan recycling pathway act as activator ligands for β-lactamase(s) induction. The muropeptides 1,6-anhydromuramyl pentapeptide and 1,6-anhydromuramyl tripeptide are the known activator ligands for <i>ampC</i> β-lactamase expression in <i>E. cloacae</i>. Here, we dissected the type of muropepetides for L1/L2 β-lactamase expression in an <i>mrdA</i> deletion mutant of <i>S. maltophilia</i>. Distinct from the findings with the <i>ampC</i> system, 1,6-anhydromuramyl tetrapeptide is the candidate for <i>ΔmrdA</i>-mediated β-lactamase expression in <i>S. maltophilia</i>. Our work extends the understanding of β-lactamase induction and provides valuable information for combating the occurrence of β-lactam resistance.