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

ABSTRACT

β-N-acetylglucosaminidase (NagZ), a cytosolic glucosaminidase, plays a pivotal role in peptidoglycan recycling. Previous research demonstrated that NagZ knockout significantly eradicated AmpC-dependent β-lactam resistance in Enterobacter cloacae. However, NagZ’s role in the virulence of E. cloacae remains unclear. Our study, incorporating data on mouse and Galleria mellonella larval mortality rates, inflammation markers, and histopathological examinations, revealed a substantial reduction in the virulence of E. cloacae following NagZ knockout. Transcriptome sequencing uncovered differential gene expression between NagZ knockout and wild-type strains, particularly in nucleotide metabolism pathways. Further investigation demonstrated that NagZ deletion led to a significant increase in cyclic diguanosine monophosphate (c-di-GMP) levels. Additionally, transcriptome sequencing and RT-qPCR confirmed significant differences in the expression of ECL_03795, a gene with an unknown function but speculated to be involved in c-di-GMP metabolism due to its EAL domain known for phosphodiesterase activity. Interestingly, in ECL_03795 knockout strains, a notable reduction in the virulence was observed, and virulence was rescued upon complementation with ECL_03795. Consequently, our study suggests that NagZ’s function on virulence is partially mediated through the ECL_03795→c-di-GMP pathway, providing insight into the development of novel therapies and strongly supporting the interest in creating highly efficient NagZ inhibitors.

KEYWORDS:

• E. cloacae
• NagZ
• virulence
• ECL_03795
• c-di-GMP

Acknowledgements

We are very grateful for technical guidance provided by ChinaPeptides Co., Ltd (Shanghai, China) in antibody preparation and technical guidance provided by Knogen Biotech Co., Ltd (Guangzhou, China) in genetic modification.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Xianggui Yang: Funding acquisition, Data curation, Investigation, Writing – original draft. Jun Zeng: Data curation, Supervision. Dan Wang and Qin Zhou: Investigation, Methodology, Data curation. Xuejing Yu and Zhenguo Wang: Data curation, Formal analysis. Tingting Bai: Conceptualization. Guangxin Luan and Ying Xu: Conceptualization, Supervision, Writing – review & editing.

Data availability statement

The data that support the findings of this study are openly available in the figshare repository at https://doi.org/10.6084/m9.figshare.25898071.v1. The raw data of Transcriptome Sequencing data have been deposited in the GenBank repository under BioProject ID PRJNA1113565 (http://www.ncbi.nlm.nih.gov/bioproject/1113565)

Ethics statement

All animal study was reviewed and approved by the Animal Ethics Committee of Clinical Medical College and the First Affiliated Hospital of Chengdu Medical College prior to the study (IACUC-23-063) and the procedures adhere to the ARRIVE guidelines (https://arriveguidelines.org). All methods were carried out in accordance with the Helsinki Declaration of 1975.

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2024.2367652

Additional information

Funding

This work was financially supported by projects from the Natural Science Foundation of Sichuan, China [2023NSFSC0623] and the Chengdu Municipal Health Commission, China [2023392].