The Function And Mechanism Study Of LsgB Gene-Mediated Lipooligosaccharide ?2,3-Sialylation In Glaesserella Parasuis Pathogenicity

Glaesserella parasuis(GPS)is a small pleomorphic rod-shaped bacterium in the Haemophilus genus of the family Pasteurellaceae,which is a commensal pathogen colonized the upper respiratory tract of healthy pigs.GPS could invade epithelial cells and enter the bloodstream to causes systemic Gl?sser's disease.When the host was under environmental stresses,weakened immunity or secondary infection.The disease often occurs in weaned piglets,or mixed with other bacteria & viruses to infect other pigs,causing serious economic losses to the breeding industry.At present,the research on this pathogen mainly focused on the virulence factors,and there are few studies on its damage to the respiratory barrier.Studies have shown that the strains causing systemic infections all contain the lsg B gene,which encodes an ?2,3-sialyltransferase protein,but the specific mechanism is still unclear.In this study,we generated a lsg B deletion mutant and we provide evidence that GPS lsg B plays an important role in crossing the monolayer cell barrier,adhesion and invasion,and resistance against the complement alternative pathway-mediated bactericidal effect in the serum.The lsg B-mediated LOS sialylation in GPS induced and bound with Siglec1 on porcine alveolar macrophages as well as triggered the production of TGF-?1 and endotoxin tolerance.TGF-?1 suppressed tracheal epithelial tight junctions as well as extracellular matrix fibronectin(Fn)expression,thus promoting bacterial entry of the respiratory epithelial barrier.The main research contents are as follows:1.lsg B-mediated LOS ?2,3-sialylationUsing the natural transformation,we generated a lsg B deletion mutant in in GPS serotype 5 virulent strain SH0165(the wide type stain),and successfully constructed its complementary strain by supplementing the gene in the deletion strain through a shuttle plasmid.High performance liquid chromatography(HPLC)testing confirmed that compared with wild-type strain and complementary strain,the level of ?2,3-sialylation on the surface of the ?lsg B mutant strains was significantly reduced.Silver staining of LOS in these three strains further confirmed that desialylation reduces the molecular weight of LOS,indicating that the function of the lsg B gene encoded sialyltransferase is to modify the lipo-oligosaccharides of GPS to undergo ?2,3-sialylation.2.lsg B-mediated LOS ?2,3-sialylation contributes to GPS pathogenicityTo further explore the influence of LOS sialylation on the pathogenicity of GPS,we next investigated the in vitro adhesion and invasion abilities of the ?lsg B mutant compared to the wild-type and complementation strains on PK15 and PIEC cells.The invasion of the ?lsg B mutant in both cell types was significantly lower than that of the wild-type and complementation strains,while unexpectedly,adhesion to both cell types by the ?lsg B mutant was significantly higher than that of the wild-type and complementation strains.Since lsg B deletion led to LOS terminal desialylation as well as the galactose residues exposure,when cells were pretreated with galactose,the high level of adhesion by ?lsg B mutant was significantly decreased,showing a similar level as that of the wild-type strain.We therefore questioned whether these attenuated abilities of the ?lsg B mutant are associated with bacterial survival in the blood.Serum resistance assays showed that the ?lsg B mutant was much more sensitive to the sera.WB suggested that the amount of ?lsg B mutant bound f H was much lower than that of the wild-type and complementation strains.We next used flow cytometry to investigate the surface deposition of the deposition of C3 b and formation of the MAC on the bacteria.Compared with the wild-type strain and complementation strain,the deposition of C3 b and MAC on the ?lsg B mutant was significantly increased after incubation with porcine normal serum.The results of animal experiments further confirmed that the deletion of the lsg B gene affects the lethality of GPS in mice.3.?2,3-sialylation promoted the generation of TGF?1In our research,bacterial pull-down demonstrated the r Siglec1 bound more to the surface of wild-type strain than the ?lsg B mutant,Western blot analysis and LOS-ELISA supported the significantly higher binding of r Siglec1 protein to the LOS extracted from the wild-type strain than that from the ?lsg B mutant.Immunoblotting showed significantly higher expression of Siglec1,as well as its downstream molecules Syk and DAP12,in 3D4/21 cells in response to the wild-type strain than those in response to the ?lsg B mutant,and GPS wild-type strain induced higher expression of Siglec1 in the lungs of infected pigs than that induced by the ?lsg B mutant by IHC.Endogenous co-IP supported the protein interactions among Siglec1,DAP12,and Syk in 3D4/21 cells,and these interactions appeared much stronger in response to wild-type strain than the ?lsg B mutant.si RNA interfering with the expression of Siglec1 showed that Siglec1/DAP12/Syk signaling cascades contributed to TGF-?1 generation in 3D4/21.We subsequently explored the downstream molecules that were regulated by Syk activity.p38 was significantly phosphorylated in 3D4/21 cells in response to the wild-type strain,which was much stronger than that being challenged by the ?lsg B mutant,addition of the Syk inhibitor R406 significantly attenuated the wild-type strain-induced activation of p38 and generation of TGF-?1.These data,therefore,indicate that the LOS ?2,3-sialylation-triggered TGF?1 generation is mediated by the Siglec1-DAP12-Syk-p38 signaling cascade in response to GPS.4.TGF?1 promoted GPS invasion and barrier damage of NPTr Cells.We next sought to investigate the potential effects of TGF?1 action on the epithelial barrier.ECIS assay showed a dose-dependent decrease in the resistance of NPTr cells in response to TGF?1 treatment during the incubation,inhibitor SD208 treatment significantly restored the resistance of NPTr cells in response to TGF?1.Immunoblotting analysis further showed TGF-?1 incubation led to significant decrease in the expression of TJ proteins in a dose-dependent manner.Taken together,these findings largely support that TGF?1 promotes GPS entry of the epithelial barrier by reducing TJs expression in respiratory epithelial cells.In addition,TGF-?1 exhibited a dose-dependent increase in invasion of NPTr cells by GPS,and while inhibitor SD208 significantly counteracted this contribution.Luciferase reporter and WB assays supported the negative regulation of Fn transcription by TGF-?1-Smad2/3.Subsequently,we used the CRISPR/Cas9 editing technique to construct Fn knockout(KO)cells,and invasion by GPS of Fn KO cells was significantly higher than that of the normal cells.Taken together,these findings largely support that TGF-?1 promotes GPS invasion by reducing Fn expression in respiratory epithelial cells.5.?2,3-sialylation induced the endotoxin tolerance in macrophageTNF-? is stably down-regulated in all tolerized models and is thus considered to be the most reliable marker of endotoxin tolerance.By constructing endotoxin tolerance models in vitro,we found that desialylation significantly reduced the level of TNF-? in the culture supernatants or mouse serum,and the expression of Siglec1 was also lower expressed.In RAW264.7 cells,WB and co-IP indicated that ?2,3-sialylated LOS up-regulated Siglec1 and SHIP and formed a complex.Confocal experiments further confirmed that ?2,3-sialylated LOS enhanced the interaction between Siglec1 and SHIP.WB confirmed that desialylation reduces p65 phosphorylation and the secretion of TNF-? and NO in endotoxin tolerance cells.si RNA interfering with the expression of Siglec1 showed the up-regulation of SHIP in endotoxin tolerance cells depends on the activation of Siglec1.In vivo,?2,3-sialylation significantly up-regulated the expression of Siglec-1 on spleen cells from endotoxin tolerant mice.In sum,LOS ?2,3-sialylation up-regulated SHIP expression by inducing Siglec1 in endotoxin tolerance.In conclusion,this study confirmed that lsg B gene-mediated ?2,3-sialylation plays an important role in the pathogenicity of GPS.This research deeply explores the important role of ?2,3-sialylation in the process of GPS in evading the host natural immunity,breaking through the barrier,and evading complement killing,which providing a better understanding of the GPS-induced systemic infection.