NagX, a periplasmic β- <i>N</i> -acetylglucosaminidase, contributes to NagZ-independent β-lactamase expression in <i>Stenotrophomonas maltophilia</i>

ABSTRACT Beta-lactam resistance in Stenotrophomonas maltophilia is primarily mediated by the expression of L1 and L2 β-lactamases. L1/L2 expression is closely linked to peptidoglycan recycling through the AmpNG-NagZ-AmpR pathway, in which NagZ plays a dominant role for L1/L2 expression. Although a NagZ-independent β-lactamase expression has been suggested, its molecular basis remains unclear. Here, we identify NagX, a periplasmic β- N -acetylglucosaminidase, as a key contributor to the NagZ-independent β-lactamase pathway. Using S. maltophilia KJ and its derived nagZ in-frame deletion mutant, KJΔNagZ, we confirm the presence of NagZ-independent β-lactamase expression and an additional β- N -acetylglucosaminidase in addition to NagZ. Determination of β-lactamase and β- N -acetylglucosaminidase activities in various engineered strains of S. maltophilia KJ demonstrated that NagX is a periplasmic β- N -acetylglucosaminidase and drives NagZ-independent β-lactamase expression in an AmpNG- and AmpR-dependent manner. To reveal how NagX processes the released peptidoglycan sacculus, the periplasmic muropeptides profile of wild-type KJ, KJΔAmpNG (an ampNG mutant), and KJΔAmpNGΔNagX (an ampNG and nagX double mutant) were determined by LC-MS/MS. The results demonstrated that NagX can remove the N -acetylglucosamine (GlcNAc) moiety from the GlcNAc-anhMurNAc-peptide and GlcNAc-anhMurNAc, yielding anhMurNAc-peptide and anhMurNAc, respectively. AmpNG permease system shuttled GlcNAc-anhMurNAc-peptide, GlcNAc-anhMurNAc, and anhMurNAc-peptide from periplasm to cytosol. Then, the anhMurNAc-peptide served as activator ligands that cooperate with AmpR to induce β-lactamase expression. Our findings uncover a novel NagX-AmpNG-AmpR circuit underlying NagZ-independent β-lactamase expression in S. maltophilia .