Involvement of a creBC−anmK−L1/L2 regulatory circuit in β-lactamase expression and β-lactam resistance of Stenotrophomonas maltophilia

Beta-lactams are among the most successful antibiotics for treating bacterial infections, but its use is impeded by increasing resistance. The contribution of BlrAB and CreBC two-component regulatory systems (TCSs) to β-lactam resistance is reported in Aeromonas spp. and Pseudomonas aeruginosa, respectively. Stenotrophomonas maltophilia, an opportunistic pathogen, is resistant to most β-lactams due to the chromosomally encoded β-lactamases L1 and L2. The role of CreBC TCS in the β-lactam resistance of S. maltophilia is still unclear. In this article, we aimed to address this issue. The susceptibility of KJΔBC, a creBC mutant of S. maltophilia KJ, to ceftazidime (CAZ) and ticarcillin-clavulanate increased compared to that of the wild-type KJ. Consistently, the CAZ-induced β-lactamase activity of KJΔBC was lower than that of the wild-type KJ. To find out the candidate responsible for the ΔcreBC-mediated β-lactam susceptibility increase and β-lactamase activity decrease, transcriptome results of wild-type KJ and KJΔBC were analyzed, focusing on the genes whose encoding proteins are involved in peptidoglycan (PG) homeostasis. Among the genes surveyed, anmK was the highest upregulated in KJΔBC. anmK encodes an anhydro-N-acetylmuramic acid kinase, which converts anhMurNAc to MurNAc-6P in the PG recycling pathway. The anmK gene and its upstream gene Smlt0416 (annotated as anmH) formed a two-member operon. Promoter activity of anmHK operon was upregulated in KJΔBC. However, the CAZ challenge hardly altered creBC and anmHK operons expression. anmK, but not anmH, deletion from the KJΔBC chromosome reverted CAZ resistance and CAZ-induced β-lactamase activity to the wild-type level. In unstressed S. maltophilia, CreBC TCS is activated and partially represses anmHK operon, favoring PG recycling toward the NagK-dependent pathway. In the CAZ-treated creBC mutant, anmHK operon is derepressed and the elevated AnmK activity may enhance the AnmK–MupP–AmgK–MurU–MurCDEF pathway, thereby increasing UDP-MurNAc-pentapeptide levels and decreasing β-lactam-induced β-lactamase activity. A creBC–anmK–L1/L2 regulatory circuit responsible for CAZ resistance has been established in S. maltophilia.