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

Penicillin binding proteins (PBPs) are involved in peptidoglycan synthesis, and their inactivation is linked to beta-lactamase expression in ampR-13-lactamase module-harboring Gram-negative bacteria. There are seven annotated PBP genes, namely, mrcA, mrcB, pbpC, mrdA, ftsl, dacB, and dacC, in the Stenotrophomonas maltophilia genome, and these genes encode PBP1a, PBP1b, PBP1c, PBP2, PBP3, PBP4, and PBP6, respectively. In addition, S. maltophilia harbors two beta-lactamase genes, L1 and L2, whose expression is induced via beta-lactam challenge. The impact of PBP inactivation on L1/L2 expression was assessed in this study. Inactivation of mrdA resulted in increased L1/L2 expression in the absence of beta-lactam challenge, and the underlying mechanism was further elucidated. The roles of ampNG, ampD, (the homologue of Escherichia coli ampD), nagZ, ampft, and creBC in L1/L2 expression mediated by a mrdA mutant strain were assessed via mutant construction and beta-lactamase activity determinations. Furthermore, the strain AmrdA-mediated change in the muropeptide profile was assessed using liquid chromatography mass spectrometry (LC-MS). The mutant Delta mrdA-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 mrdA significantly increased the levels of total and periplasmic N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyk-alanylo-glutamyl-meso-diamnopimelic acid-o-alanine (GlcNAc-anhMurNAc tetrapeptide, or M4N), supporting that the critical activator ligands for mutant strain Delta mrdA-mediated L1/L2 expression are anhMurNAc tetrapeptides. IMPORTANCE Inducible expression of chromosomally encoded beta-lactamase(s) is a key mechanism for beta-lactam resistance in Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The muropeptides produced during the peptidoglycan recycling pathway act as activator ligands for beta-lactamase(s) induction. The muropeptides 1,6-anhydromuramyl pentapeptide and 1,6-anhydromuramyl tripeptide are the known activator ligands for ampC beta-lactamase expression in E. cloacae. Here, we dissected the type of muropepetides for L1/L2 beta-lactamase expression in an mrdA deletion mutant of S. maltophilia. Distinct from the findings with the ampC system, 1,6-anhydromuramyl tetrapeptide is the candidate for Delta mrdA-mediated beta-lactamase expression in S. maltophilia. Our work extends the understanding of beta-lactamase induction and provides valuable information for combating the occurrence of beta-lactam resistance.