High-Throughput Transcriptomic Profiling Reveals the Inhibitory Effect of Hydroquinine on Virulence Factors in Pseudomonas aeruginosa

Abstract

Hydroquinine is an organic alkaloid compound that exhibits antimicrobial activity against several bacterial strains including strains of both drug-sensitive and multidrug-resistant P. aeruginosa. Despite this, the effects of hydroquinine on virulence factors in P. aeruginosa have not yet been characterized. We therefore aimed to uncover the mechanism of P. aeruginosa hydroquinine-sensitivity using high-throughput transcriptomic analysis. We further confirmed whether hydroquinine inhibits specific virulence factors using RT-qPCR and phenotypic analysis. At half the minimum inhibitory concentration (MIC) of hydroquinine (1.250 mg/mL), 254 genes were differentially expressed (97 downregulated and 157 upregulated). We found that flagellar-related genes were downregulated by between −2.93 and −2.18 Log2-fold change. These genes were consistent with the analysis of gene ontology and KEGG pathway. Further validation by RT-qPCR showed that hydroquinine significantly suppressed expression of the flagellar-related genes. By analyzing cellular phenotypes, P. aeruginosa treated with ½MIC of hydroquinine exhibited inhibition of motility (30–54% reduction) and pyocyanin production (~25–27% reduction) and impaired biofilm formation (~57–87% reduction). These findings suggest that hydroquinine possesses anti-virulence factors, through diminishing flagellar, pyocyanin and biofilm formation.

Hydroquinine Inhibits the Growth of Multidrug-Resistant Pseudomonas aeruginosa via the Suppression of the Arginine Deiminase Pathway Genes

“…In general, L-arginine in P. aeruginosa is an essential molecule in the regulation of biofilm formation [ 39 ]. This supports the previous findings by Rattanachak et al [ 17 ] that hydroquinine could suppress QS-related gene expression, reduce virulence factor production, and impair biofilm formation in P. aeruginosa [ 17 ]. For another possible reason, hydroquinine may affect the production of carbamoyl phosphate, which is required for pyrimidine production [ 30 ].…”

“…According to the work of Rattanachak et al (2022), there were several changes in the transcriptomes of P. aeruginosa ATCC 27853 when treated with a hydroquinine concentration of 1.25 mg/mL for 1 h. This study revealed 157 upregulated genes and 97 downregulated genes in response to the treatment [ 17 ]. Several differentially regulated genes have already been investigated relating to virulence factors, such as quorum sensing and flagella assembly.…”

“…For example, the arginine/ornithine antiporter (AOA) encoded by the arcD gene was the most downregulated in the transcriptomic analysis by a −4.24 Log 2 -fold change in response to hydroquinine. Consistently, the other arginine deiminase (ADI)-pathway-related genes, including arcA (arginine deiminase; ADI) , arcB (ornithine transcarbamylase; OTC) , and arcC (carbamate kinase; CK) were also significantly downregulated in response to hydroquinine, by −3.85, −3.32, and −3.41 Log 2 -fold changes, respectively [ 17 ].…”

“…Rattanachak et al (2022) mechanistically investigated the inhibitory effects of ½ MIC hydroquinine through the evaluation of the global transcripts of P. aeruginosa using RNA sequencing with high-throughput transcriptomic analysis [ 17 ]. They found that hydroquinine inhibited several virulence factors, such as downregulating flagellar-related genes, which govern bacterial motility, and reducing QS-related genes, affecting the reduction of pyocyanin production and biofilm formation [ 17 ]. Moreover, several additional genes were identified as having up- or downregulated expression with high confidences ( Figure S1 ).…”

“…Moreover, several additional genes were identified as having up- or downregulated expression with high confidences ( Figure S1 ). The observation of significant downregulation of four genes from the arginine deiminase (ADI) pathway (namely, arcA, arcB, arcC, and arcD ) in response to hydroquinine treatment is intriguing ( Table S1 ) [ 17 ]. For example, the arginine/ornithine antiporter (AOA) encoded by the arcD gene was the most downregulated in the transcriptomic analysis by a −4.24 Log 2 -fold change in response to hydroquinine.…”