Effect Of Glutamate Dehydrogenase Deletion On Biofilm Formation,Virulence And Extracellular Proteins Expression Of Listeria Monocytogenes

Listeria monocytogenes is an opportunistic foodborne pathogen causing listeriosis in both humans and animals.The extracellular proteins secreted by bacteria play an important role in the survival and infection of pathogenic bacteria.Glutamate dehydrogenase is widely exist in all organisms such as plants,animals,and microorganisms and plays an important role in carbon and nitrogen metabolism and energy metabolism.GDH has been shown to be associated with virulence in Streptococcus suis serotype 2.In this paper,the effects of GDH on the physiological characteristics,autolysis ability,virulence,biofilm formation of L.monocytogenes were studied,and the proteomic analysis of the extracellular proteins from L.monocytogenes wild strain EGDe and its glutamate dehydrogenase deletion mutant EGDe?gdhA were carried out using iTRAQ technique.Bacterial morphology and growth were compared among the wild-type EGDe,GDH-deficient strain EGDe?gdhA and revertant strain strain EGDe?gdhA+pERL3-gdhA.The data showed that the deletion of GDH had no significant effect on bacterial morphology.In the brain heart infusion medium,the deletion strain grew slower than the wild strain at the end of logarithmic phase,and the revertant strains grew slower than the deletion strain throughout the logarithmic phase.However,in the minimal essential medium,EGDe?gdhA grew slowly at the end of logarithmic phase and the early stationary phase,and the biomass decreased significantly,but the biomass of EGDe?gdhA+pERL3-gdhA was significantly higher than that of the deletion strain at the same phase.The effect of GDH deletion on the formation of L.monocytogenes biofilms was examined using a microtiter plate method.The result showed that compared with the wild strain,the biofilm formation ability of EGDc?gdhA was significantly reduced(P?0.01)when keeping the growth of the two strains at 24 and 48 hours without significant difference.The autolysis ability of the strain was detected by Triton X-100.The autolysis degree of EGDe?gdhA was significantly increased(P<0.01),which was 1.25 times of the wild strain and 1.45 times of EGDe?gdhA+pERL3-gdhA.These results suggest that the loss of GDH will increase bacterial autolysis and decrease the biofilm formation of L.monocytogenes.The hemolytic activity,the semi-lethal dose of Helicoverpa armigera(Hubner),the transcriptional levels of the major virulence factor LLO(coding gene hly)and the virulence gene regulator PrfA(coding gene prfA)in EGDe,EGDeAgdhA and EGDe?gdhA+pERL3-gdhA were compared.The results showed that compared with the wild strain,the transcription level of the prfA gene in EGDe?gdh4 was reduced,and the half-lethal dose to the Helicoverpa armigera(Hubner)was also increased.It is interesting that although the transcription level of the hly gene in the GDH-deleted strain was increased,the hemolytic activity was significantly decreased(P?0.01).These data suggest that deletion of GDH leads to a reduction in the virulence of the bacteria.Using iTRAQ technology to isolate and identify the extracellular proteins of wild strain and EGDe?gdhA and to perform bioinformatics analysis on the differentially expressed proteins.The functions of all sixty-two differential expressed extracellular proteins identified were cataloged into 16 functional groups,including carbohydrate transport and metabolism,energy synthesis and transport,transcription,cell membrane protein related to cell wall and so on.Among them,the most dominant groups including 24 proteins,accounting for 38.71%of total differential expressed proteins,are related to carbon and nitrogen metabolism as well as energy transport,suggesting that glutamate dehydrogenase is a key enzyme in carbon and nitrogen metabolism as well as energy synthesis and transport for L.monocytogenes.The slow growth and reduced biomass of EGDe?gdhA compared to EGDe in minimal essential medium were in agreement well with these proteomic results.At the same time,we found that the expression of three proteins associated with bacterial adhesion and biofilm formation were reduced.This result also explains the decrease in biofilm formation after GDH deletion.The glutamate dehydrogenase protein was purified by constructing a GDH expression strain pET28a-gdhA(BL21),which facilitates to further study the function of GDH in L.monocytogenes.In conclusions,GDH plays a crucial role in biofilm formation,virulence and extracellular proteins expression in L.monocytogenes.