CRISPRi-Mediated Gene Suppression Reveals Putative Reverse Transcriptase Gene PA0715 to Be a Global Regulator of Pseudomonas aeruginosa

Dapeng Zhou; Guangtao Huang; Guangchao Xu; Lijuan Xiang; Siyi Huang; Xinchong Chen; Yixin Zhang; Dali Wang

doi:10.2147/IDR.S384980

CRISPRi-Mediated Gene Suppression Reveals Putative Reverse Transcriptase Gene PA0715 to Be a Global Regulator of Pseudomonas aeruginosa

Infect Drug Resist. 2022 Dec 22:15:7577-7599. doi: 10.2147/IDR.S384980. eCollection 2022.

Authors

Dapeng Zhou^#^{1

2}, Guangtao Huang^#^{1

2

3

4}, Guangchao Xu^{1

2}, Lijuan Xiang⁵, Siyi Huang^{1

2}, Xinchong Chen^{1

2}, Yixin Zhang⁴, Dali Wang^{1

2}

Affiliations

¹ Department of Burns and Plastic Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi, People's Republic of China.
² The Collaborative Innovation Center of Tissue Damage Repair and Regeneration Medicine of Zunyi Medical University, Zunyi, People's Republic of China.
³ Department of Burn and Plastic Surgery, Department of Wound Repair, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, People's Republic of China.
⁴ Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
⁵ Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, People's Republic of China.

^# Contributed equally.

Abstract

Purpose: Pseudomonas aeruginosa is a common pathogen of infection in burn and trauma patients, and multi-drug resistant P. aeruginosa has become an increasingly important pathogen. Essential genes are key to the development of novel antibiotics. The PA0715 gene is a novel unidentified essential gene that has attracted our interest as a potential antibiotic target. Our study aims to determine the exact role of PA0715 in cell physiology and bacterial pathogenicity, providing important clues for antibiotic development.

Patients and methods: The shuttle vector pHERD20T containing an arabinose inducible promoter was used to construct the CRISPRi system. Alterations in cellular physiology and bacterial pathogenicity of P. aeruginosa PAO1 after PA0715 inhibition were characterized. High-throughput RNA-seq was performed to gain more insight into the mechanisms by which PA0715 regulates the vital activity of P. aeruginosa.

Results: We found that down-regulation of PA0715 significantly reduced PAO1 growth rate, motility and chemotaxis, antibiotic resistance, pyocyanin and biofilm production. In addition, PA0715 inhibition reduced the pathogenicity of PAO1 to the greater galleria mellonella larvae. Transcriptional profiling identified 1757 genes including those related to amino acid, carbohydrate, ketone body and organic salt metabolism, whose expression was directly or indirectly controlled by PA0715. Unexpectedly, genes involved in oxidative phosphorylation also varied with PA0715 levels, and these findings support a hitherto unrecognized critical role for PA0715 in oxidative respiration in P. aeruginosa.

Conclusion: We identified PA0715 as a global regulator of the metabolic network that is indispensable for the survival and reproduction of P. aeruginosa. Our results provide a basis for future studies of potential antibiotic targets for P. aeruginosa and offer new ideas for P. aeruginosa infection control.

Keywords: CRISPRi; P. aeruginosa; antibiosis; bioinformatics; prokaryotes; reverse transcriptase.

Grants and funding

This research was funded by the National Natural Science Foundation of China (Grant No. 8196080379 and 8217081828, respectively), Collaborative Innovation Center of Chinese Ministry of Education(2020-39), Science and Technology Support Project of Guizhou Province(Grant No. 2020-1Y332), China Postdoctoral Science Foundation(GrantF No. 2020M670112ZX), Guizhou Province Graduate Education Innovation Project (Grant No. 2020-173) and Guizhou Province Graduate Education Innovation Project(fzc120220679).