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

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

Dapeng Zhou #  1   2 , Guangtao Huang #  1   2   3   4 , Guangchao Xu  1   2 , Lijuan Xiang  5 , Siyi Huang  1   2 , Xinchong Chen  1   2 , Yixin Zhang  4 , Dali Wang  1   2
Affiliations

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

Dapeng Zhou et al. Infect Drug Resist. .

Erratum in

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.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
An arabinose-inducible CRISPRi system for tunable repression of PA0715 expression in P. aeruginosa.
Figure 2
Figure 2
PA0715 and its putative expression product.
Figure 3
Figure 3
Effect of PA0715 suppression on the proliferation and activity of PAO1 strains.
Figure 4
Figure 4
Changes in pyocyanin production and biofilm formation due to PA0715 inhibition.
Figure 5
Figure 5
SEMimages of PA0715 being or not being inhibited by PAO1 cells.
Figure 6
Figure 6
Survival of Galleria mellonella inoculated with PA0715-suppressed or control strains.
Figure 7
Figure 7
Volcano map of genes regulated by PA0715, and functional classification based on the Gene Ontology (GO) database (http://www.geneontology.org).
Figure 8
Figure 8
KEGG functional annotation and RT-qPCR validation of differential genes in PAO1 strains with or without PA0715 being repressed.
See this image and copyright information in PMC

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