Proteomic Investigation of the Antibacterial Mechanism of trans-Cinnamaldehyde against Escherichia coli.

Trans-Cinnamaldehyde (TC) is a widely used food additive, known for its sterilization, disinfection, and antiseptic properties. However, its antibacterial mechanism is not completely understood. In this study, quantitative proteomics was performed to investigate differentially expressed proteins (DEPs) in Escherichia coli in response to TC treatment. Bioinformatics analysis suggested aldehyde toxicity, acid stress, oxidative stress, interference of carbohydrate metabolism, energy metabolism, and protein translation as the bactericidal mechanism. E. coli BW25113ΔyqhD, ΔgldA, ΔbetB, ΔtktB, ΔgadA, ΔgadB, ΔgadC, and Δrmf were used to investigate the functions of DEPs through biochemical methods. The present study revealed that TC exerts its antibacterial effects by inducing the toxicity of its aldehyde group producing acid stress. These findings will contribute to the application of TC in the antibacterial field.

Novel Mechanistic Insights into Bacterial Fluoroquinolone Resistance.

Advancements in studies on the evolutionary mechanisms underlying bacterial antibiotic resistance are unclear. This study aimed to investigate the evolutionary mechanism underlying bacterial antibiotic resistance using isobaric tags for relative and absolute quantitation-based quantitative proteomics along with functional validation. Quantitative analysis revealed 101, 325, and 428 differentially expressed proteins (DEPs) at three drug resistance levels (low-R, 0.2 µg/mL; medium-R, 5 µg/mL; high-R, 15 µg/mL). Continuous adjustment of metabolic patterns to enhance nucleotide synthesis and energy generation may underlie evolution. Indeed, nucleotide levels were elevated and strengthened ciprofloxacin resistance. Quorum sensing (QS) genes were upregulated in the early growth phase, thus potentially improving survival. Further, a thicker cell wall potentially serves as a stronger barrier and reduces drug permeation. The aforementioned three drug resistance levels displayed continuity and differences; the low-resistant level displayed no prominent mechanism; medium, a more focused change in nucleotide metabolism; and high, a thorough evolution to a complete systematic mechanism with higher adenosine 5'-triphosphate levels, serving as a defense mechanism for reducing drug-induced stress. Thus, gradual increments in nucleotide synthesis, energy synthesis, cell wall synthesis, QS, and biofilm formation may direct the evolution of bacterial resistance.

YjgM is a crotonyltransferase critical for polymyxin resistance of Escherichia coli.

Lysine crotonylation has attracted widespread attention in recent years. However, little is known about bacterial crotonylation, particularly crotonyltransferase and decrotonylase, and its effects on antibiotic resistance. Our study demonstrates the ubiquitous presence of crotonylation in E. coli, which promotes bacterial resistance to polymyxin. We identify the crotonyltransferase YjgM and its regulatory pathways in E. coli with a focus on crotonylation. Further studies show that YjgM upregulates the crotonylation of the substrate protein PmrA, thereby boosting PmrA's affinity for binding to the promoter of eptA, which, in turn, promotes EptA expression and confers polymyxin resistance in E. coli. Additionally, we discover that PmrA's crucial crotonylation site and functional site is Lys 164. These significant discoveries highlight the role of crotonylation in bacterial drug resistance and offer a fresh perspective on creating antibacterial compounds.

Proteomic Investigation of the Antibacterial Mechanism of Cefiderocol against Escherichia coli.

This study aimed to investigate the antibacterial mechanism of cefiderocol (CFDC) using data-independent acquisition quantitative proteomics combined with cellular and molecular biological assays. Numerous differentially expressed proteins related to the production of NADH, reduced cofactor flavin adenine dinucleotide (FADH2), NADPH and reactive oxygen species (ROS), iron-sulfur cluster binding, and iron ion homeostasis were found to be upregulated by CFDC. Furthermore, parallel reaction monitoring analysis validated these results. Meanwhile, we confirmed that the levels of NADH, ROS, H2O2, and iron ions were induced by CFDC, and the sensitivity of Escherichia coli to CFDC was inhibited by the antioxidant vitamin C, N-acetyl-l-cysteine, and deferoxamine. Moreover, deferoxamine also suppressed the H2O2 stress induced by CFDC. In addition, knockout of the NADH-quinone oxidoreductase genes (nuoA, nuoC, nuoE, nuoF, nuoG, nuoJ, nuoL, nuoM) in the respiratory chain attenuated the sensitivity of E. coli to CFDC far beyond the effects of cefepime and ceftazidime; in particular, the E. coli BW25113 ΔnuoJ strain produced 60-fold increases in MIC to CFDC compared to that of the wild-type E. coli BW25113 strain. The present study revealed that CFDC exerts its antibacterial effects by inducing ROS stress by elevating the levels of NADH and iron ions in E. coli. IMPORTANCE CFDC was the first FDA-approved siderophore cephalosporin antibiotic in 2019 and is known for its Trojan horse tactics and broad antimicrobial activity against Gram-negative bacteria. However, its antibacterial mechanism is not fully understood, and whether it has an impact on in vivo iron ion homeostasis remains unknown. To comprehensively reveal the antibacterial mechanisms of CFDC, data-independent acquisition quantitative proteomics combined with cellular and molecular biological assays were performed in this study. The findings will further facilitate our understanding of the antibacterial mechanism of CFDC and may provide a theoretical foundation for controlling CFDC resistance in the future.

Proteomic investigation into the action mechanism of berberine against Streptococcus pyogenes.

Berberine is an isoquinoline alkaloid found in many plants. Although berberine is known to possess the antibacterial activity against Streptococcus pyogenes, the mechanism underlying it is not fully understood. In the current study, to investigate the molecular mechanism how berberine exerts its antibacterial effects, quantitative proteomics was conducted to investigate differential expressed proteins in S. pyogenes in response to berberine treatment. KEGG pathways analysis revealed that berberine regulated proteins were mainly involved in carbohydrate metabolism, fatty acid biosynthesis, pyrimidine metabolism, RNA degradation, ribosome, purine metabolism, DNA replication and repair and oxidative phosphorylation pathways. Moreover, we found that berberine induced the accumulation of reactive oxygen species (ROS), whereas inhibition of ROS generation with antioxidant N-acetyl L-cysteine could block the berberine induced antibacterial effects. Collectively, we demonstrated that berberine exerts its antibacterial effects by perturbing carbohydrate metabolism, which therefore generate ROS to damage the DNA, protein and lipids biosynthesis, ultimately trigger cell lethality. These findings provide novel insights into the mechanism of berberine as an antimicrobial drug to control diseases caused by S. pyogenes. SIGNIFICANCE: Streptococcus pyogenes is the major cause of invasive bacterial disease in human, which leads to hundreds of million cases annually and over 500,000 deaths due to severe infections. Berberine is an isoquinoline alkaloid from medicinal plants, which possesses a variety of pharmacological effects including antibacterial. In this work, proteomic analysis revealed that berberine affected carbohydrate metabolism, DNA, protein and fatty acid biosynthesis and oxidative phosphorylation pathways in S. pyogenes. And further experimental results showed that berberine exerts its antibacterial effects against Streptococcus pyogenes by stimulated the generation of reactive oxygen species (ROS). These data provide novel insights into the effect of berberine on oxidative stress as an antimicrobial drug.

SPD_1495 Contributes to Capsular Polysaccharide Synthesis and Virulence in Streptococcus pneumoniae.

Streptococcus pneumoniae, a Gram-positive human pathogen, causes a series of serious diseases in humans. SPD_1495 from S. pneumoniae is annotated as a hypothetical ABC sugar-binding protein in the NCBI database, but there are few reports on detailed biological functions of SPD_1495. To fully study the influence of SPD_1495 on bacterial virulence in S. pneumoniae, we constructed a deletion mutant (D39Δspd1495) and an overexpressing strain (D39spd1495+). Comparative analysis of iTRAQ-based quantitative proteomic data of the wild-type D39 strain (D39-WT) and D39Δspd1495 showed that several differentially expressed proteins that participate in capsular polysaccharide synthesis, such as Cps2M, Cps2C, Cps2L, Cps2T, Cps2E, and Cps2D, were markedly upregulated in D39Δspd1495 Subsequent transmission electron microscopy and uronic acid detection assay confirmed that capsular polysaccharide synthesis was enhanced in D39Δspd1495 compared to that in D39-WT. Moreover, knockout of spd1495 resulted in increased capsular polysaccharide synthesis, as well as increased bacterial virulence, as confirmed by the animal study. Through a coimmunoprecipitation assay, surface plasmon resonance, and electrophoretic mobility shift assay, we found that SPD_1495 negatively regulated cps promoter expression by interacting with phosphorylated ComE, a negative transcriptional regulator for capsular polysaccharide formation. Overall, this study suggested that SPD_1495 negatively regulates capsular polysaccharide formation and thereby enhances bacterial virulence in the host. These findings also provide valuable insights into understanding the biology of this clinically important bacterium.IMPORTANCE Capsular polysaccharide is a key factor underlying the virulence of Streptococcus pneumoniae in human diseases. Thus, a deep understanding of capsular polysaccharide synthesis is essential for uncovering the pathogenesis of S. pneumoniae infection. In this study, we show that protein SPD_1495 interacts with phosphorylated ComE to negatively regulate the formation of capsular polysaccharide. Deletion of spd1495 increased capsular polysaccharide synthesis and thereby enhanced bacterial virulence. These findings further reveal the synthesis mechanism of capsular polysaccharide and provide new insight into the biology of this clinically important bacterium.