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

Abstract

Inducible expression of chromosomally encoded β-lactamase(s) is a key mechanism for β-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 β-lactamase(s) induction. The muropeptides 1,6-anhydromuramyl pentapeptide and 1,6-anhydromuramyl tripeptide are the known activator ligands for ampC β-lactamase expression in E. cloacae . Here, we dissected the type of muropepetides for L1/L2 β-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 ΔmrdA -mediated β-lactamase expression in S. maltophilia . Our work extends the understanding of β-lactamase induction and provides valuable information for combating the occurrence of β-lactam resistance. Penicillin binding proteins (PBPs) are involved in peptidoglycan synthesis, and their inactivation is linked to β-lactamase expression in ampR –β-lactamase module–harboring Gram-negative bacteria. There are seven annotated PBP genes, namely, mrcA , mrcB , pbpC , mrdA , ftsI , 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 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 mrdA resulted in increased L1/L2 expression in the absence of β-lactam challenge, and the underlying mechanism was further elucidated. The roles of ampNG , ampD I (the homologue of Escherichia coli ampD ), nagZ , ampR , and creBC in L1/L2 expression mediated by a ΔmrdA mutant strain were assessed via mutant construction and β-lactamase activity determinations. Furthermore, the strain ΔmrdA -mediated change in the muropeptide profile was assessed using liquid chromatography mass spectrometry (LC-MS). The mutant Δ 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 -acetylmuramyl- l -alanyl- d -glutamyl- meso -diamnopimelic acid- d -alanine (GlcNAc-anhMurNAc tetrapeptide, or M4N), supporting that the critical activator ligands for mutant strain Δ mrdA -mediated L1/L2 expression are anhMurNAc tetrapeptides. IMPORTANCE Inducible expression of chromosomally encoded β-lactamase(s) is a key mechanism for β-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 β-lactamase(s) induction. The muropeptides 1,6-anhydromuramyl pentapeptide and 1,6-anhydromuramyl tripeptide are the known activator ligands for ampC β-lactamase expression in E. cloacae . Here, we dissected the type of muropepetides for L1/L2 β-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 ΔmrdA -mediated β-lactamase expression in S. maltophilia . Our work extends the understanding of β-lactamase induction and provides valuable information for combating the occurrence of β-lactam resistance.