Role Of Neuraminidase From Trueperella Pyogenes On The Inflammatory Response Of Goat Endometrial Epithelial Cells

Endometritis is a common disease of the reproductive system of domestic animals,occurring mostly after maternal delivery.It causes damage to the ovaries and endometrium and affects estrus and pregnancy in animals,leading to reproductive disorders or even death of animals and seriously compromises the development of health in animal husbandry.Pathogenic microbes,along with their virulence factors,are responsible for causing uterine tissue damage and leading to inflammatory responses in the endometrium.Trueperella pyogenes is one of the main pathogenic bacteria causing endometritis in livestock,and currently,prevention and control of this bacterial infection are difficult due to the lack of an effective vaccine.In depth uncovering of the pathogenic mechanisms of Trueperella pyogenes is key to the development of infection control agents for this bacterium.Bacterial neuraminidase(NA)is one of the important virulence factors of bacteria,which can break down the neuraminic acid residues at the end of the sugar binding complex in the host body and help bacteria colonize,penetrate and spread in the host.Two members of the family,Nan H andNanP,exist outside the cell walls of Trueperella pyogenes.There is little known about this specific virulence,its effect on the virulence of this strain is unclear.Therefore,it is very important to investigate the role of NA in pathogenesis of Trueperella pyogenes in the pathogenesis of endometritis.In this study,by inhibiting the NA activity of Trueperella pyogenes,the role ofNanH andNanP on the growth proliferation,infection pathogenesis and biofilm formation of the bacteria was tested and analyzed.Recombinant protein was purified by constructing recombinant prokaryotic expression vectors of p ET-32a-nan H and p ET-32a-nan P and using E.coli expression system for prokaryotic expression.We then analyzed whetherNanH andNanP induced inflammatory responses in goat endometrial epithelial cells(gEECs)and investigated their association with the TLR4/NF-κB inflammatory signaling pathway.It provides a basis for further investigation of the specific molecular mechanisms of endometritis in goats caused by Trueperella pyogenes.The main test results are as follows:(1)Oseltamivir was used to inhibit neuraminidase activity of Trueperella pyogenes.The growth curve showed that inhibition of NA had no significant effect on the growth and proliferation of the strain(p >0.05).Crystal violet staining showed that the biofilm formation rate of the strain decreased significantly after inhibition of NA(p <0.05).GEECs were respectively infected by NA inhibited and non inhibited Trueperella pyogenes.It was found by cell lysate colony count and immunofluorescence that the ability of bacteria to adhere and invade g EECs was significantly weakened after inhibiting NA(p <0.05),but their survival within g EECs was not affected.QPCR showed that inhibition of NA could significantly down regulate the transcriptional expression of g EECs inflammatory genes IL-6、TNF-α、IL-1β and IL-8 caused by Trueperella pyogenes(p <0.05).(2)The nanH and nanP genes were cloned by PCR,and the recombinant prokaryotic expression vectors of p ET-32a-nan H and p ET-32a-nan P were constructed.The recombinant proteins of rNanH and rNanP with NA activity were induced,expressed and purified.GEECs were treated with 1 μg/m L rNanH and 2.5 μg/m L rNanP respectively.Western Blot and q PCR showed that the treatment of rNanH and rNanP both significantly upregulated the transcriptional expression levels of IL-6,TNF-α,IL-1β and IL-8(p <0.05),significantly upregulated the transcriptional expression level of TLR4,My D88,NF-κB,the protein expression levels of TLR4 and the phosphorylation level of p65 subunit of NF-κB in TLR4/NF-κB pathway(p <0.05),but also significantly upregulated the transcriptional levels of NLRP3 and caspase-1 and the protein expression level of NLRP3(p <0.05).Inhibition of NA activity could significantly down regulate the effects of rNanH and rNanP on the above inflammatory cytokines and signal pathways(p < 0.05).It was illustrated thatNanH andNanP could trigger inflammatory responses through the TLR4/My D88/NF-κB pathway and the formation of NLRP3 inflammasome in g EECs,respectively.When g EECs were treated with rNanH,rNanP alone or in combination at the same final concentration,q PCR showed that the transcript levels of IL-6,TNF-α,IL-1β and IL-8 were not significantly changed in the combined treatment group compared to the rNanH or rNanP alone treatment group(p > 0.05),indicating thatNanH andNanP had no synergistic effect.Using 1 μmol/L TAK-242 specifically inhibits TLR4 activation in g EECs,then treated with rNanH and rNanP respectively.Western Blot,q PCR and immunofluorescence showed that when TLR4 was inhibited,rNanH and rNanP were no longer able to influence g EECs NF-κB or NLRP3-associated inflammatory pathways(p <0.05).These results indicated thatNanH andNanP could recognize by TLR4,and activate NF-κB through TLR4.And the pathway is closely related to the formation of NLRP3 inflammasome.Taken together,this study indicates that NanH and NanP enhance the ability of Trueperella pyogenes to adhere and invade to g EECs,accelerate the formation of the bacterial biofilm.Nan H andNanP can be recognized by TLR4,and induce g EECs inflammatory response through the TLR4/My D88/NF-κB inflammatory signaling pathway and the formation of NLRP3 inflammatory small bodies of this pathway,laying the foundation for uncovering the molecular mechanisms of endometritis caused by Trueperella pyogenes.