Effect Of GlnR And GlnA Deletion On Growth,Biofilm Formation And Virulence In Listeria Monocytogenes

Listeria monocytogenes is a Gram-positive bacterium capable of causing listeriosis,the disease known for the highest mortality rate in human and animals among food-borne pathogens.Nitrogen metabolism is of utmost importance for bacterial survival and is therefore strictly regulated.Nitrogen-dependent regulation in Gram-positive bacteria is governed mainly by GlnR(for Gln synthesis regulator).The expression of the GInR regulon plays a central role in both nitrogen metabolism and cellular activities such as carbohydrate metabolism,virulence,and stress responses.The gene glnA encodes glutamine synthetase GlnA,which forms glutamine out of glutamate and ammonium while hydrolyzing ATP.In Streptococcus suis Serotype 2 and Salmonella enterica serovar Typhimurium,GlnA not only takes part in glutamine metabolism,but also is essential for bacterial virulence.GlnR and GlnA in L.monocytogenes are highly conserved like in other bacteria,but until now no detailed report about their functions in L.monocytogenes has been published.In this study,the following works were carried out to elucidate the roles of GlnR and GlnA in L.monocytogenes.In first part,the glnR complement strain EGDe?glnR+pLSV16-glnR was constructed based on the glnR deletion strain EGDe?glnR.Then,EGDe,EGDe?glnR and EGDe?glnR+pLSV16-glnR were compared with each other in growth,biofilm formation and virulence.The results were shown as follows:1)There were no obvious difference among the three strains in bacterial morphology under the microscope.However,no matter in rich medium of brain heart infusion broth(BHI),in Luria Bertani(LB)or in poor minimal essential medium(MEM),EGDe always grew faster than EGD?AglnR,which indicated that deletion of GlnR in L.monocytogenes inhibited bacterial growth.2)The biomass of biofilm formed by EGDe?glnR were significantly lower than that of EGDe,indicating that GlnR is important for biofilm formation in L.monocytogenes.3)The hemolytic activity of EGDc?glnR was significantly lower than that of EGDe.The median lethal doses LD50 of EGDe and EGDe?glnR against Helicoverpa armigera(Hiibner)larvae were 2.69×105 cfu and 12.6x 105 cfu,repectively.The transcriptional levels of two major virulent genes hly and prfA in EGDc?glnR measured by RT-qPCR were significantly lower than that in EGDe.Above all,those results indicated that the loss of GlnR reduced virulence in L.monocytogenes.In the second part,the glnA-inserted mutant EGDe::glnA was compared with EGDe to investigate the role of GlnA in growth,biofilm formation,virulence and resistance to the acidity,alkalinity and salinity stress in L.monocytogenes.The genome-wide transcription sequencing of EGDe::glnA and EGDe were also analysed in this study.The results were shown as follows:1)EGDe grew faster than EGDe::glnA in MEM.2)The biomass of biofilm formed by EGDe::glnA was significantly lower than that of EGDe.3)The hemolytic activity of EGDe::glnA was significantly lower than that of EGDe,and the median lethal dose LD50 of EGDe::glnA against Helicoverpa armigera(Hubner)larvae was 3.6 times lower than that of EGDe,indicating that the deficiency of GlnA reduced significantly virulence of L.monocytogenes.4)EGDe grew better than EGDe::glnA under the acidity,alkalinity and salinity environment.5)Compared with EGDe,the genome-wide transcription sequencing of EGDe::glnA shew that there were 98 genes expressed differently,including 57 genes down expressed and 41 genes up expressed.Those genes belonged to 14 functional categories.The most of genes related to carbon and nitrogen metabolism were down expressed in EGDe::glnA,indicating that glutamine synthetase in L.monocytogenes is the key enzyme for carbon and nitrogen metabolism and energy transfermation.Those would also be reason that EGDe::glnA strain grew slowly and produced less biomass.Meanwhile,the transcriptional levels of virulent genes such as sigB,lmo2672,lmo0051 and inlB in EGDe::glnA were significantly lower,which was consistent well with the bacterial reduced hemolysis activity and toxicity against the worm.The genes related to stress resistance in EGDe::glnA were down-expressed,which was consistent with the deceased abilities of biofilm formation and resistance to acidity,alkalinity and salinity stress.All of those above shew that GlnA played an important role in biofilm formation and stress resistance in L.monocytogenes.In summary,both GlnR and GlnA were essencial to L.monocytogenes in growth,biofilm formation and virulence.