GC-TOF/MS-based metabolomics analysis to investigate the changes driven by N-Acetylcysteine in the plant-pathogen Xanthomonas citri subsp. citri

Abstract

N-Acetylcysteine (NAC) is an antioxidant, anti-adhesive, and antimicrobial compound. Even though there is much information regarding the role of NAC as an antioxidant and anti-adhesive agent, little is known about its antimicrobial activity. In order to assess its mode of action in bacterial cells, we investigated the metabolic responses triggered by NAC at neutral pH. As a model organism, we chose the Gram-negative plant pathogen Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker disease, due to the potential use of NAC as a sustainable molecule against phytopathogens dissemination in citrus cultivated areas. In presence of NAC, cell proliferation was affected after 4 h, but damages to the cell membrane were observed only after 24 h. Targeted metabolite profiling analysis using GC–MS/TOF unravelled that NAC seems to be metabolized by the cells affecting cysteine metabolism. Intriguingly, glutamine, a marker for nitrogen status, was not detected among the cells treated with NAC. The absence of glutamine was followed by a decrease in the levels of the majority of the proteinogenic amino acids, suggesting that the reduced availability of amino acids affect protein synthesis and consequently cell proliferation.

Potential and Metabolic Pathways of Eugenol in the Management of Xanthomonas perforans, a Pathogen of Bacterial Spot of Tomato

“…Interestingly, comparing the pathways affected by eugenol from this study to other studies utilizing the small molecule N-Acetylcysteine (NAC) against Xanthomonas citri subsp. citri showed similar results in the affected pathways [ 46 ]. This study and two previous studies utilizing carvacrol and NAC against Xanthomonas species showed that the pathways of glutathione metabolism; cyanoamino acid metabolism; sulfur metabolism; aminoacyl t-RNA metabolism; glyoxylate and decarboxylate metabolism; glycine, serine, and threonine metabolism; and alanine, aspartate, and glutamate metabolism were regulated by carvacrol, eugenol, and NAC [ 45 , 46 ].…”

“…citri showed similar results in the affected pathways [ 46 ]. This study and two previous studies utilizing carvacrol and NAC against Xanthomonas species showed that the pathways of glutathione metabolism; cyanoamino acid metabolism; sulfur metabolism; aminoacyl t-RNA metabolism; glyoxylate and decarboxylate metabolism; glycine, serine, and threonine metabolism; and alanine, aspartate, and glutamate metabolism were regulated by carvacrol, eugenol, and NAC [ 45 , 46 ]. It therefore appears that small molecules frequently target these pathways in Xanthomonas species.…”

“…The pathway analyses suggested that eugenol targets several pathways in X. perforans . The pathways that were significantly regulated by the putatively annotated metabolites in both untargeted search and when targeted against the Pseudomonas putida KT2440 are widely present in Gram-negative bacteria, including X. perforans [ 45 , 46 ]. At least 10 pathways and 12 pathways were significantly affected by the putatively annotated metabolites in the positive and negative ion phases, respectively.…”

“…For example, while metabolites in the glutathione metabolism pathway were listed as upregulated in the effects of NAC against X. citri subsp. citri, most metabolites in this pathway were downregulated for the effects of carvacrol and eugenol on X. perforans [ 45 , 46 ]. Interestingly, isoleucine, a branched-chain amino acid (BCAA, which includes leucine and valine) which is an essential amino acid that regulates protein synthesis [ 62 ], showed upregulation in the treated strains in this study, suggesting that the pathway of protein synthesis may not be the primary target of eugenol activity.…”

“…A recent study further showed the pathways altered by NAC activities in Xanthomonas citri subsp. citri , suggesting the role of NAC in the downregulation of proteinogenic amino acids and its possible influence on protein synthesis [ 46 ]. In our study, the small molecule, eugenol, was evaluated for its potential in managing bacterial spot of tomato.…”