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

Xianggui Yang,Xuejing Yu,Qin Zhou,Yingzi Fan,Fang Nie,Ying Xu,Hongfei Du,Xueli Pang,Fuying Wang,Dan Wang,Tingting Bai,Yuanxiu Zhong,Jun Zeng

doi:10.3389/fmicb.2020.586729

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 (ΔnagZ EC) was constructed by homologous recombination. Conversely, ampC mRNA and protein levels were down-regulated, and resistance to β-lactam antibiotics was attenuated in ΔnagZ EC, while specific complementation of nagZ was able to rescue ampC expression and resistance in ΔnagZ 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.

Highlights

Enterobacter cloacae complex (ECC), including E. cloacae (EC), Enterobacter asburiae, Enterobacter hormaechei, Enterobacter kobei, Enterobacter ludwigii, and Enterobacter nimipressuralis (Guerin et al, 2015), are widely distributed in nature
To determine whether NagZ was involved in developing resistance in EC, nagZ mRNA expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-quantitative PCR (qPCR)), as indicated in Figure 1A. nagZ mRNA expression was significantly enhanced in the resistant isolates compared with susceptible ones
To further detect the different protein expressions of nagZ between susceptible and resistant strains, we prepared anti-NagZ antibody for the first time, and its specificity was verified by nagZ-knockout EC model (Supplementary Figure 1). nagZ protein expressions were detected by western blot in six resistant and six susceptible strains, as indicated in Figures 1B,C, protein expressions of nagZ were dramatically up-regulated in six resistant EC isolates compared to susceptible ones

Summary

Introduction

Enterobacter cloacae complex (ECC), including E. cloacae (EC), Enterobacter asburiae, Enterobacter hormaechei, Enterobacter kobei, Enterobacter ludwigii, and Enterobacter nimipressuralis (Guerin et al, 2015), are widely distributed in nature. They are parts of commensal microbiota in human gastrointestinal tract as well. The chromosomal AmpC β-lactamase is highly inducible in presence of some β-lactams, such as imipenem, cefoxitin, and clavulanate (Jacoby, 2009; Gomez-Simmonds et al, 2018), but it is still not clear about underlying genetic regulation in AmpC β-lactamase associated with peptidoglycan recycling in E. cloacae clinical isolates

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