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RelA influence on antimicrobial action of t-cinnamaldehyde on Escherichia coli MG1655

ABSTRACT: Antimicrobial resistance poses a global threat. Natural-origin compounds represent a valuable source of antimicrobial agents used in both human and veterinary medicine. However, understanding their mechanisms of action at the molecular level is essential to support their safe and effective application. In this study, we evaluated the antimicrobial potential of trans-cinnamaldehyde (CNMA), a major constituent of cinnamon bark oil, which can account for up to 80% of the oil content in species such as Cinnamomum zeylanicum and cassia. Although CNMA exhibits broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, its precise mode of action remains incompletely understood. To elucidate CNMA's molecular effects, we performed transcriptomic profiling of Escherichia coli MG1655 wild-type (GSE252441) and its ∆relA mutant upon treatment with a sub-inhibitory concentration (0.25×MIC) of CNMA. Total RNA was isolated and assessed using the Agilent Bioanalyzer 2100, and high-throughput sequencing was conducted on the Illumina NovaSeq6000 platform, generating ~30 million paired-end 101 bp reads per sample. The reference genome and annotations of E. coli MG1655 were obtained from GenBank. RNA-seq data were analyzed to identify differentially expressed genes (DEGs) compared to untreated controls at 30 and 60 min post-treatment, using thresholds of p ≤ 0.05 and |log₂FC| ≥ 2. Transcriptomic analysis revealed profound transcriptional remodeling. The most significantly enriched functional categories included genes involved in the tricarboxylic acid (TCA) cycle, flagellar biosynthesis, amino acid transport, and oxidoreductase activity. These findings indicate that CNMA-treated E. coli undergoes a marked metabolic downshift and initiates stress responses. These transcriptomic results were supported by complementary assays showing reduced growth kinetics, cytoplasmic shrinkage, NAD/NADH imbalance, and induction of the stringent response via elevated (p)ppGpp levels. Together, our findings suggest that CNMA disrupts bacterial fitness by impairing core metabolic and regulatory pathways, ultimately leading to loss of viability. Funding: This research was funded by the National Science Center, Poland (grant SONATA UMO-2018/31/D/NZ7/02258 to D.N.)

ORGANISM(S): Escherichia coli

PROVIDER: GSE301628 | GEO | 2025/07/08

REPOSITORIES: GEO

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Project description:Antimicrobial resistance pose a global thread nowadays. Compounds of natural origin are an important source of drugs used in clinical practice. However, it is important to understand both their principles of efficacy and their molecular mechanism of action. In this study we evaluated antimicrobial potential of t-cinnamaldehyde which is an organic compound found in many plant species, especially in the Cinnamomum genus, such as Cinnamomum zeylanicum and cassia. Cinnamon oil extracted from the bark of these plants contains up to 80% trans-cinnamaldehyde. Although CNMA has shown antimicrobial properties against numerous Gram+ and Gram- species, its mode of action against pathogens remains not fuly elucidated. Therefore, this project aims to determine CNMA activity at the level of gene expression. Total RNA was isolated and checked for quality using the Bioanalyzer 2100. The sequencing run was conducted on the Illumina NovaSeq6000 platform. 30 million pair-end reads per samples were assessed with 101 pb read length. Reference E. coli MG1655 genome sequence and annotations were downloaded from GenBank. Differentially expressed analysis of 0.25 x MIC CNMA was performed against untreated control in indicated time with p ≤ 0.001 and log2FC ≥ 1.5. We have discovered many changes bacterial transcriptome. For instance: following the treatment with 0.25×MIC of CNMA, we found 292 and 140 upregulated and 107 and 96 downregulated genes at time points 30 and 60 min, respectively. Among the most enriched genes, were those related to the tricarboxylic acid (TCA) cycle, flagellum synthesis, amino acid transport, and oxidoreductase activity. According to these findings we can conclude that observed transcriprional pattern indicates severe metabolic downshift in treated cells, and consequently activation of stress processes. These was in line with our secondary experiments which revealed drop in growth kinnetic, cytoplasm shrinkage, NAD/NADH level alteration and elevation of stringent response alarmones ((p)ppGpp). Taken together, this suggests that CNMA-treated E. coli bacteria undergo major metabolic changes that finally result in cell death.

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RelA influence on antimicrobial action of t-cinnamaldehyde on Escherichia coli MG1655

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