| Salmonella enterica serovar Enteritidis（S.Enteritidis）is a food-borne bacterial pathogen that can cause human salmonellosis predominately by contaminating eggs and egg products.Egg white is an adverse environment for bacterial survival due to its unfavourable alkalinity,high viscosity,antimicrobial proteins and peptides.Most of Salmonella serovars hardly survive in egg white,but only S.Enteritidis can resist the antibacterial components of egg white to survive and multiply in eggs,posing a great threat to food safety.Hence,it is of great significance to elucidate the resistance mechanisms of S.Enteritidis to egg white in order to provide new ideas and strategies for controlling this pathogen in eggs.Survival mechanisms of S.Enteritidis in egg white have been partially explored via some molecular biological approaches such as transponson insertional mutagenesis,in vivo expression,and DNA array.However,these investigations were mainly focused on elucidating the global response of S.Enteritidis to egg white at the m RNA level.To our knowledge,little information is available regarding characterization of molecular mechanisms on S.Enteritidis survival in egg white at the proteomics level.In fact,proteins are the ultimate executor of gene function in pathogens.Changes at the protein level are important strategies for pathogens to resist external stresses.Therefore,the aim of this study was to reveal the survival mechanisms of S.Enteritidis SJTUF 10978 in egg white by comparative proteomics analysis,functional identification of stress tolerance candidate proteins,and functioinal analysis of key stress tolerance proteins.The major findings regarding the above-mentioned aspects are as follows.1.Proteomics analysis of S.Enteritidis cells in response to egg white.The proteomic profile of S.Enteritidis in response to different concentrations of egg white was explored by i TRAQ techology to acquire the differential proteins.Results of proteomics analysis were further verified by RT-q PCR.Furthermore,GO functional annotation,KEGG pathway enrichment,STRING protein interaction and expression pattern clustering analysis were carried out on differential proteins,and a regulatory network regarding the resistance of S.Enteritidis to egg white was then constructed.The main results are as follows.（i）A total of 273（114 up-regulated and 159down-regulated）,284（145 up-regulated and 139 down-regulated）,and 303（166up-regulated and 137 down-regulated）differential proteins were acquired from S.Enteritidis grown in 50%,80%,and 100%egg white compared with that in Luria-Bertani（LB）broth,respectively.（ii）Alteration trends in the 30 differential proteins at the m RNA level were in accordance with those in the corresponding protein levels,demonstrating the reliability of the data derived from proteomics analysis.（iii）Differential proteins were involved in 14 biological processes（e.g.metabolic process and cellular process）,10 cellular components（e.g.cell membrane and macromolecular complex）,and 8 molecular functions（e.g.binding activity and molecular transducer activity）.Significant enrichment analysis of KEGG pathway showed that differential proteins were primarily involved in 24 metabolic pathways such as amino acids biosynthesis,ABC transporters,tricarboxylic acid cycle,bacterial chemotaxis,and two-component system.（iv）Proteomic characterization demonstrated that multiple strategies employed by S.Enteritidis to survive in egg white included stress response,iron acquisition system,amino acid biosynthesis,cofactor and vitamins biosynthesis,transporters,regulation,energy metabolism,bacterial motility,and chemotaxis as well as virulence.2.Exploration of stress tolerance protein YbgC in S.Enteritidis cells in response to egg white.Proteins that are up-regulated or play a potential important role in the above-mentioned pathways were selected for functional identification to explore key stress tolerance proteins.To this aim,we tested four proteins,namely,stress response-related thioesterase YbgC,nitrogen regulating protein Gln K,and iron acquisition system-related esterases Fes as well as Iro D.The main results are as follows.（i）S.Enteritidis mutants（i.e.⊿ybg C,⊿gln K,⊿fes,and⊿iro D）were successfully constructed according to homologous recombination knockout procedures using the cloning vector p MD19-T and the sucrose suicide plasmid p RE112.（ii）Deletion of ybg C,gln K,fes,and iro D had no significant effect on S.Enteritidis growth in LB broth.（iii）Deletion of ybg C significantly decreased the survival ability of S.Enteritidis in egg white,but no significant difference in survival ability between the wild-type strain and the three mutants⊿gln K,⊿fes,and⊿iro D in egg white was found.（iv）There was no significant difference in survival ability between the wild-type and⊿ybg C-C complemented strains in egg white.These results indicated the crucial role of YbgC for S.Enteritidis survival in egg white.3.Mechanism analysis on YbgC-mediated resistance to egg white in S.Enteritidis.Antibacterial proteins and peptides have been commonly suggested to account for the ability of egg white to prevent bacterial growth,and the cell membrane is the main target of these antibacterial components.In order to reveal the role of YbgC in the resistance of S.Enteritidis to egg white,S.Enteritidis wild-type,⊿ybg C mutant,and⊿ybg C-C complemented strains were utilized to analyze their survival ability and cell membrane property after treatment with egg white antibacterial components.The main results are as follows.（i）Cells lacking ybg C showed significantly reduced survival ability in lysozyme,whereas no significant difference in cell number was observed between the wild-type and△ybg C-C complemented strains.Additionally,deletion of ybg C had no significant effect on the survival of S.Enteritidis in ovalbumin,ovotransferrin,avidin,or ovomucoid.These results demonstrated that ybg C played an important role in the response of S.Enteritidis to lysozyme.（ii）An apparent alteration in gross morphology from a rod to a spherical shape with cell lysis was observed in the⊿ybg C mutant after exposure to lysozyme.Nevertheless,treatment with lysozyme failed to induce morphological changes in the wild-type and⊿ybg C-C complemented strains.（iii）The fluorescence in the⊿ybg C mutant was significantly higher than that in the wild-type and⊿ybg C-C complemented strains after exposure to lysozyme,indicating that deletion of ybg C contributed to an increase in outer membrane permeability of S.Enteritidis in the presence of lysozyme.（iv）The amounts of C14:0 and C17:0 cyclo fatty acids were significantly decreased,while those of iso-C15:0 3-OH,C18:1ω7c,and C19:0cycloω8c fatty acids were significantly increased in response to lysozyme by the deletion of ybg C.Moreover,it was found that the ratio of total unsaturated fatty acids to total saturated fatty acids in the⊿ybg C mutant was significantly higher than that in the wild-type strain in response to lysozyme.（v）The genes responsible for fatty acid（acp P,cfa,acc ACD,fad R,fab BDG,and tes AB）,phospholipid（ass,pls B,and pls C）,and lipid A（lpx A）synthesis were up-regulated in the⊿ybg C mutant after exposure to lysozyme.In summary,S.Enteritidis employed multiple pathways to cope with antibacterial egg white,including stress response,iron acquisition system,amino acid biosynthesis,cofactor and vitamins biosynthesis,transporters,regulation,energy metabolism,bacterial motility,and chemotaxis as well as virulence.Moreover,stress response-related protein YbgC was indeed an essential enzyme contributing to the survival of S.Enteritidis in the presence of egg white lysozyme via regulating cell membrane fatty acids composition and cell membrane integrity.Collectively,these results provide new ideas for a systematical characterization of molecular mechanisms on S.Enteritidis resistance to egg white at the proteomics level. |
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