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

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:

• P. aeruginosa
• reverse transcriptase
• prokaryotes
• antibiosis
• CRISPRi
• bioinformatics

View correction statement:

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

Data Sharing Statement

The raw sequence data of RNA-seq were deposited in NCBI with the accession number PRJNA824013.

Consent for Publication

We confirm that the manuscript and any images, videos, audio recordings, etc. are not currently under public review and publication. All co-authors have read the manuscript and agreed to submit it to Infection and Drug Resistance.

Acknowledgments

We gratefully acknowledge the gift of the shuttle vector (pHERD20T) from Dr. Schweizer (University of Florida). In the whole transcriptome sequencing and transcriptome data analysis, we are especially grateful for the help provided by Novogene Bioinformatics Technology Co., Ltd(Beijing, China).

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

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).