Elucidation On Physiological Mechanisms Of Heavy Metal Cadmium Tolerance Of Lactobacillus Plantarum

Cadmium（Cd）contamination and intoxicity incidents have occurred frequently these years,which has caused much public concern.In previous study,our group has screened one Lactobacillus plantarum（L.plantarum）CCFM8610 with high Cd tolerance and binding ability,proposing the dietary strategy to apply lactic acid bacteria（LAB）on bioremoval of Cd toxicity.The results of animal experiments indicated that L.plantarum CCFM8610 could significantly alleviate Cd toxicity in vivo,and this protection was largely dependent on its Cd tolerance ability.Thus,it is of great interest and necessity to elucidate the mechanism of high Cd resistance ability of CCFM8610,thus providing theoretical guidelines for the application of this strain on the Cd toxicity alleviation.Till now,very limited study has focused on heavy metal tolerance mechanisms of LAB.In addition,proteomics and metabolomics methods have achieved marked success on elucidating the global stress response of microorganisms,which inspires us to combine these two omics methods to construct the global stress response network of L.plantarum,finally elucidating the related Cd tolerance mechanisms.The results of this study have been listed as follows:Firstly,the screening of L.plantarum species with different Cd tolerance abilities was carried out by the liquid medium method.The results indicated that there was significant Cd tolerance diversity among all 20 tested L.plantarum strains.The 20 strains could be categorized into four general groups according their respective minimum inhibitory concentrations（MICs）of Cd which were MIC>50 mg/L,MIC=50 mg/L,MIC=20 mg/L and MIC=10 mg/L.L.plantarum CCFM8610 belonged to one of the eight strains with the highest MIC values,which was in agreement with our previous study on the tolerance of LAB strains tested on Cd-containing agar plates.CCFM191 belonged to one of the three strains with the lowest MIC value（10 mg/L）,and it could be regarded as a Cd-sensitive strain.Therefore,the Cd-tolerant strain CCFM8610 and Cd-sensitive strain CCFM191 were finally chosen to conduct comparative omics analysis.Through isobaric tags for relative and absolute quantitation（iTRAQ）based comparative proteomics analysis,totally 1415 proteins identified in both Cd-tolerant strain CCFM8610 and Cd-sensitive strain CCFM191.Among these proteins,206 were differentially expressed for the comparison of natural proteomic profiles of CCFM8610 and CCFM191,27 were differently regulated in CCFM8610 after Cd exposure,and 111 were changed in CCFM191 in response to Cd stress.These differentially expressed proteins involved in several biological processes,including energy metabolism,purine and pyrimidine metabolism,global stress responses,lipid and amino acid metabolism,metal binding properties,cell wall biosynthesis and transporters.Through liquid chromatography-mass spectrometry（LC-MS）based comparative metabolomics analysis,49 metabolites were identified to be differentially expressed between CCFM8610 and CCFM191 in natural conditions,and 48 and 36 metabolites were changed significantly in CCFM8610 and CCFM191 after Cd exposure,respectively.The pathway enrichment analysis indicated that these changed metabolites involved in several metabolic processes,including amino acid metabolism,carbon metabolism,lipid metabolism,nucleic acid metabolism and transportation.The underlying mechanisms of the intra-species distinction between CCFM8610 and CCFM191 on Cd tolerance can be attributed to the following aspects: CCFM8610 possesses an inherent energy-conservation survival mode in natural condition;CCFM8610 can cope with environmental stress with mild induction of the cellular defense and repair system;CCFM8610 has inherent superior Cd binding ability,and effective cell wall structures in response to Cd stress;Several key proteins,including prophage P2 b protein 18,CadA,mntA and lp₃327,also play a potential role in Cd tolerance in CCFM8610;CCFM8610 has more rigid cell membrane structure with lower fluidity.In general,CCFM8610 shows inherited Cd tolerance properties in its natural condition and represents specific response mode upon Cd exposure,which determines its high Cd tolerance ability.Finally,the reverse transcription quantitative polymerase chain reaction（RT-qPCR）method was adopted to quantify several differentially expressed proteins in proteomics data,and the results indicated that the differences in the mRNA expressions were in accordance with the corresponding differences in protein level,thus verifying the results of the proteomics study.Some experiments on bacterial physiological properties have been conducted,including intracellular metal accumulation,Cd binding,bacterial hydrophobicity,autoaggregation and morphological characterization,intracellular reactive oxygen species（ROS）production,cellular components involved in Cd binding,glucose consumption and hydrophobic amino acid production,which further validated the conclusions from the omics analysis.