Elevating NagZ Improves Resistance to β-Lactam Antibiotics via Promoting AmpC β-Lactamase in Enterobacter cloacae

Abstract

Enterobacter cloacae complex (ECC), one of the most common opportunistic pathogens causing multiple infections in human, is resistant to β-lactam antibiotics mainly due to its highly expressed chromosomal AmpC β-lactamase. It seems that regulation of chromosomal AmpC β-lactamase is associated with peptidoglycan recycling. However, underlying mechanisms are still poorly understood. In this study, we confirmed that NagZ, a glycoside hydrolase participating in peptidoglycan recycling in Gram-negative bacteria, plays a crucial role in developing resistance of E. cloacae (EC) to β-lactam antibiotics by promoting expression of chromosomal AmpC β-lactamase. Our data shows that NagZ was significantly up-regulated in resistant EC (resistant to at least one type of the third or fourth generation cephalosporins) compared to susceptible EC (susceptible to all types of the third and fourth generation cephalosporins). Similarly, the expression and β-lactamase activity of ampC were markedly enhanced in resistant EC. Moreover, ectopic expression of nagZ enhanced ampC expression and resistance to β-lactam antibiotics in susceptible EC. To further understand functions of NagZ in β-lactam resistance, nagZ -knockout EC model (Δ nagZ EC) was constructed by homologous recombination. Conversely, ampC mRNA and protein levels were down-regulated, and resistance to β-lactam antibiotics was attenuated in Δ nagZ EC, while specific complementation of nagZ was able to rescue ampC expression and resistance in Δ nagZ EC. More interestingly, NagZ and its hydrolyzates 1,6-anhydromuropeptides (anhMurNAc) could induce the expression of other target genes of AmpR (a global transcriptional factor), which suggested that the promotion of AmpC by NagZ is mediated AmpR activated by anhMurNAc in EC. In conclusion, these findings provide new elements for a better understanding of resistance in EC, which is crucial for the identification of novel potential drug targets.

NagZ modulates the virulence of E. cloacae by acting through the gene of unknown function, ECL_03795

“…In the case of Pseudomonas aeruginosa , previous studies have reported a significant reduction in β-lactam resistance when peptidoglycan recycling is impeded [ 13 , 36 ]. Our earlier research findings also pointed to a noticeable decrease in β-lactam resistance in E. cloacae following NagZ deletion [ 20 ]. Nevertheless, the impact of NagZ on the virulence of E. cloacae remains unclear.…”

“…For instance, inhibiting NagZ has been demonstrated to decrease bacterial resistance to β-lactam antibiotics in various bacteria, including Pseudomonas aeruginosa , Stenotrophomonas maltophilia , and Yersinia enterocolitica [ 16–19 ]. Our previous research has substantiated NagZ’s critical role in regulating the expression of chromosomal AmpC β-lactamase and subsequent resistance in E. cloacae [ 20 ]. Additionally, limited studies have also reported NagZ’s involvement in bacterial biofilm synthesis and virulence regulation, such as NagZ triggers Neisseria gonorrhoeae biofilm disassembly [ 21 ] and knockout of NagZ disrupts the peptidoglycan recycling pathway, leading to reduced virulence in Pseudomonas aeruginosa [ 14 , 22 ].…”

Cefazolin and Imipenem Enhance AmpC Expression and Resistance in NagZ-Dependent Manner in Enterobacter Cloacae

“…Some studies indicate that metabolism and recycling of peptidoglycan take part in the expression of AmpC [39,40], one of the key enzymes in peptidoglycan recycling is NagZ and its main function is to form anhMurNAc from GlcNAc-1,6-anhydroMurNAc-peptides by cleaving non reducing GlcNAc residues. 43 In the process of peptidoglycan metabolism, anhMurNAc promote the expression of AmpC by binding to the transcription factor AmpR. According to genetic research, NagZ is required for the induction of AmpC β-lactamases in Pseudomonas aeruginosa [7].…”

“…For CFZ and IMP treatment assays, the reagents were employed at the subculture stage. Western blot analysis was carried out using the standard methodology as elaborated earlier [43]. The detailed attributes of antibodies employed in western blot assay are as follows: rabbit anti-NagZ (preparation by ourselves), mouse anti-DnaK (Abcam, Cambridge, MA, USA), rabbit anti-AmpC (Abnova Taipei, Taiwan, China), goat antimouse IgG-HRP and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).…”

Cefazolin and imipenem enhance AmpC expression and resistance in NagZ-dependent manner in Enterobacter cloacae complex

“…For CFZ and IMP treatment assays, the reagents were employed at the sub-culture stage. Western blot analysis was carried out using the standard methodology as elaborated earlier [ 36 ]. The detailed attributes of antibodies employed in western blot assay are as follows: rabbit anti-NagZ (preparation by ourselves), mouse anti-DnaK (Abcam, Cambridge, MA, USA), rabbit anti-AmpC (Abnova Taipei, Taiwan, China), goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).…”