Cold Plasma-mediated Oxidative Stress Triggers The Inactivation Of Listeria Monocytogenes And Associated Molecular Response Mechanisms

Food safety caused by food-borne pathogenic microorganisms is a public health issue,which has attracted worldwide attention.Among them,the fatality rate of food-borne diseases caused by Listeria monocytogenes（LM）is at the forefront,which has been listed by WHO as one of the top five food-borne pathogens.The novel cold plasma,due to its resulting strong oxidative environment mediated by high-energy reactive oxygen and nitrogen species（RONS）,has been demonstrated to be highly effective in inactivating microorganisms.However,the current research on microbial inactivation and response mechanisms mostly focuses on biological characteristics and some certain functional genes,while the systematic and global research on the molecular response mechanisms is still deficient.Adopting high-throughput multi-omics technologies to elucidate the molecular regulation mechanisms of LM inactivation in plasma-mediated oxidative stress is an urgent scientific issue to ensure food safety and public health.Therefore,based on the intense and mild oxidative stress systems mediated by the dielectric barrier discharge（DBD）and radio frequency（RF）,with the help of the physiology,transcriptomics and metabolomics and joint multi-omics techniques,and integrating with plasma chemistry,food microbiology,molecular biology and other disciplines,we explored the inactivation mechanisms of LM triggered by the novel cold plasma mediated oxidative stress as well as the molecular functions,biological processes,and omics responses in its sublethal condition,aiming to reveal the molecular response mechanisms of LM in plasma-mediated inactivation.The main conclusions are listed as follows:（1）Construction of cold plasam mediated oxidative stress system and analysis of LM inactivation kinetics.The optical diagnostics using optical emission spectroscopy（OES）confirmed that RF and DBD plasma-mediated mild and intense plasma stress systems were obvious different in gas-phase emission spectra.The bulk of the observed emission of RF and DBD were in the near infrared and UV regions,respectively,thus mediating different oxidative stress and inactivation kinetics.Namely,after DBD plasma treatment for 4 min,the intracellular reactive oxygen species（ROS）level of LM was 2.18-fold that of RF treatment,and the inactivation was 2.76-fold that of RF treatment.The Weibull model（RMSE=0.074）and S-like model（RMSE=0.033）with three phases（shoulder region,log-linear phase and trailing phase）described the most optimal inactivation dynamics mediated by RF and DBD plasma,respectively,showing similar initial resistance and different terminal heterogeneous population resistance.（2）Inactivation biology and sublethal injury of LM mediated by cold plasma.Results shown that exogenous cold plasma induced a burst of intracellular endogenous ROS and the accumulation of Fe²⁺/Cu.Following biological characteristics and partial functional gene analysis indicated that both the germicidal effect and associated oxidative lesions mediated by RF and DBD plasma exhibited a time-dependent behavior.The membrane integrity was uncompromised but with obviously profound shrinkage and deformation,and accompanied by the reduction of respiration in oxygen consumption rates（OCRs）and extracellular acidification rates（ECARs）,cytoplasmic enzymatic activity and severe DNA fragmentation.Finally,binding RT-q PCR technique identified a significant upregulation of rec A（encoding DNA repair gene）.Therefore,the inactivation of LM was mainly attributed to intracellular ROS and Fe²⁺/Cu accumulation toxicity-mediated DNA lesions and esterase inactivation.Ultrasonic-assisted pretreatment time for 15 min followed by 2 min plasma treatment effectively weakened the cell membrane and mediated a higher intracellular ROS and associated collapse and partial rupture,which finally contributed to a 2.14-fold elevation of inactivation efficiency.However,bacterial inactivation was accompanied by the formation of sublethal injury,which was further highlighted to 49.23%after post-storage for 24 h.And there was a replication recovery in milk and an enhancement after respiratory depression at the physiological levels,as well as an urgent need for replication（19.7-fold）and up-regulated expression of stress genes（e.g.,sig B,per R,lmo2344,lmo2770）,indicating residual metabolic viability,resistance,and resuscitation potential of sublethally injured cells.（4）Transcriptomics-based study to reveal DBD plasma triggered pathogenicity attenuation mechanisms of LM.Results indicated that the pathogenicity attenuation of sublethal LM subjected to DBD plasma-mediated oxidative stress involved the extensive regulation of multiple virulence gene groups,including the general downregulation of genes encoding ribosomal protein（e.g.,L15（rplO）,L20（rplT）,L11（rplK））,the intracellular infection life cycle（e.g.,hly,iap,plcB）,biofilm formation（e.g.,agrA,dltABC）,quorum sensing（e.g.,luxS、plcB、luxS、lacD、oppB）,flagellum biosynthesis（e.g.,fliC）,as well as the significant up-regulation of genes related to galactose metabolism（e.g.,gatC-3,gatC-1）and Fe²⁺/Cu efflux pump（e.g.,isd G,sal A,fieF-1,fieF-2,copA）.Meanwhile,a consistent significant inhibition of extracellular virulence protein secretion and hemolytic activity（3.70-fold）were noticed at the physiological level.（4）Multi-omics-based analysis to reveal the molecular response mechanisms of inactivated LM triggered by DBD plasma.Results indicated that the response of sublethal LM was more positive after DBD plasma treated post-storage for 24 h,in which 706 genes and 50 metabolites were significantly different(|log2（fold change）|>1,p_adj<0.05).Subsequent bioinformatics analysis revealed that those significantly expressed genes and metabolites were mainly involved in depressed ribosomal proteins,enhanced transmembrane transport,disturbance of the phosphotransferase system and two-component regulatory system,limitation of amino acid metabolism,depletion of nucleotide metabolism,re-configuration of central carbon metabolism flux,and homeostasis of cytoplasmic pH,etc.However,typical antioxidant systems such as the TrxR-Trx system and common antioxidant genes（e.g.,sodA,katA,ahpC,trxA,spxA）were inhibited.Prominent antioxidant pathways included methionine metabolism,pentose phosphate pathway and glutathione metabolism as well as the DNA repair system（e.g.,recombinational repair,nucleotide excision repair,SOS response）,which systematically revealed the plasma-mediated intracellular damage and metabolic deprivation of LM.This study provides new ideas and methods for the construction of multi-dimensional mechanisms of bacterial inactivation and pathogenicity attenuation,and also provides scientific basis and theoretical support for the precise control and inactivation of microorganisms in plasma non-thermal processing.