A Natural Antimicrobial Agent: Analysis of Antibacterial Effect and Mechanism of Compound Phenolic Acid on Escherichia coli Based on Tandem Mass Tag Proteomics

The objective of this study was to evaluate the antibacterial mechanisms of phenolic acids as natural approaches against multi-drug resistant Escherichia coli (E. coli). For that purpose, five phenolic acids were combined with each other and 31 combinations were obtained in total. To select the most potent and effective combination, all of the obtained combinations were examined for minimum inhibitory concentration (MIC) and it was found that the compound phenolic acid (CPA) 19 (protocatechuic acid, hydrocinnamic acid, and chlorogenic acid at concentrations of 0.833, 0.208, and 1.677 mg/mL, respectively) showed better efficacy against E. coli compared to other combinations. Furthermore, based on tandem mass tag (TMT) proteomics, the treatment of CPA 19 significantly downregulated the proteins associated with resistance (Tsr, Tar, CheA, and CheW), OmpF, and FliC of multidrug-resistant E. coli. At the same time, we proved that CPA 19 improves the sensitivity of E. coli to antibiotics (ceftriaxone sodium, amoxicillin, fosfomycin, sulfamonomethoxine, gatifloxacin, lincomycin, florfenicol, cefotaxime sodium, and rifampicin), causes the flagellum to fall off, breaks the structure of the cell wall and cell membrane, and leads to macromolecules leaks from the cell. This evidence elaborated the potential therapeutic efficacy of CPA 19 and provided a significant contribution to the discovery of antibacterial agents.

Tandem Mass Tag labelling quantitative acetylome analysis of differentially modified proteins during mycoparasitism of Clonostachys chloroleuca 67–1

Lysine acetylation (Kac) is an important post-translational modification (PTM) of proteins in all organisms, but its functions have not been extensively explored in filamentous fungi. In this study, a Tandem Mass Tag (TMT) labelling lysine acetylome was constructed, and differentially modified Kac proteins were quantified during mycoparasitism and vegetative growth in the biocontrol fungus Clonostachys chloroleuca 67–1, using liquid chromatography-tandem mass spectrometry (LC–MS/MS). A total of 1448 Kac sites were detected on 740 Kac proteins, among which 126 sites on 103 proteins were differentially regulated. Systematic bioinformatics analyses indicate that the modified Kac proteins were from multiple subcellular localizations and involved in diverse functions including chromatin assembly, glycometabolism and redox activities. All Kac sites were characterized by 10 motifs, including the novel CxxKac motif. The results suggest that Kac proteins may have effects of broadly regulating protein interaction networks during C. chloroleuca parasitism to Sclerotinia sclerotiorum sclerotia. This is the first report of a correlation between Kac events and the biocontrol activity of C. chloroleuca. Our findings provide insight into the molecular mechanisms underlying C. chloroleuca control of plant fungal pathogens regulated by Kac proteins.

Tandem Mass Tag-Based Quantitative Proteomics and Virulence Phenotype of Hemolymph-Treated Bacillus thuringiensis kurstaki Cells Reveal New Insights on Bacterial Pathogenesis in Insects

After ingestion by a susceptible insect and damaging its midgut epithelium, the bacterium Bacillus thuringiensis (Bt) reaches the insect blood (hemolymph), where it propagates despite the host’s antimicrobial defenses and induces insect death by acute septicemia. Although the hemolymph stage of the Bt toxic pathway is determinant for the infested insects’ fate, the response of Bt to hemolymph and the latter’s role in bacterial pathogenesis has been poorly explored.

Tandem Mass Tag-Based Quantitative Proteomic Analysis of ISG15 Knockout PK15 Cells in Pseudorabies Virus Infection

Pseudorabies virus (PRV) is recognized as one of the most important pathogens of swine and poses a serious threat to the swine industry worldwide. Available commercial vaccines fail to protect against the emergence of new PRV strains. Therefore, the new protein targets against PRV highlight the urgent need for uncovering the molecular determinants of host cellular proteins following PRV infection. Interferon-stimulated gene 15 (ISG15) demonstrates an outstanding antiviral response. However, the molecular mechanism of ISG15 that affects PRV replication is incompletely known. Here, we performed a tandem mass tag (TMT)-based approach to quantitatively identify protein expression changes in PRV-infected ISG15 knockout PK15 (ISG15−/−-PK15) cells. In total, 4958 proteins were identified by using TMT coupled with LC-MS/MS in this study. In the PRV- and mock-infected groups, 241 differentially expressed proteins (DEPs) were identified, 162 upregulated and 79 downregulated proteins at 24 h post-infection (hpi), among which AFP, Vtn, Hsp40, Herc5, and Mccc1 may play important roles in PRV propagation. To ensure the validity and reliability of the proteomics data, the randomly selected DEPs were verified by RT-qPCR and Western blot analysis, and the results were consistent with the TMT results. Bioinformatics analyses further demonstrated that the DEPs are mainly involved in various biological processes and signaling pathways, such as signal transduction, the digestive system, and the PI3K-AKT pathway. These findings may provide new insight into molecular mechanisms for PRV infection, which is helpful for identifying potential protein targets for antiviral agents.