Glycosylation Of T9SS Substrate Proteins Of Cytophaga Hutchinsonii And The Study Of Related Genes Chu＿3842 And Chu＿0012

Protein secretion systems are crucial for bacteria to grow and survive.The TypeⅨ secretion system（T9SS）is exclusively widespread in the Bacteroidetes phylum.It involves the secretion of abundant important proteins.It is a common feature that most cargo proteins of the T9SS have an N-terminal signal peptide（SP）and a conserved Cterminal domain（CTD）.The N-terminal signal peptide assists the T9SS substrate protein to enter the periplasmic space through the Sec system.The CTD,the signal recognized by the T9SS,assists cargo proteins across the outer membrane.As the recognition transport signal for T9SS,CTD plays a role in the secretion and outer membrane anchoring of substrate proteins.However,the mechanism by which T9SS recognizes the substrate protein CTD remains unclear.Cytophaga hutchinsonii is an aerobic Gram-negative cellulolytic bacterium and widely distributed in the soil.The cellulose degradation mechanism of C.hutchinsonii is unique and unknown.It has all homologous genes encoding T9SS component proteins.Bioinformatics analysis revealed that at least 147 proteins in C.hutchinsonii were processed and secreted by T9SS.CHU₃220 has been shown to be secreted by T9SS and participate in the degradation of crystalline cellulose.According to the characteristics of T9SS substrate protein,the N-terminal signal peptide of T9SS substrate protein and CTD fusion reporter protein GFP were expressed in C.hutchinsonii to explore the mechanism of T9SS recognition of substrate protein CTD.CHU 2708 is the only reported T9SS cargo protein with a clear CTD cleavage site in C.hutchinsonii,CTD of CHU₂708 was linked to the C terminal of GFP to study its function in the secretion of T9SS cargo proteins.Western blotting results showed that a GFP-related protein of about 40 kDa occurred in the periplasm of the WTGFP-CTD strain,which is about 5 kDa bigger than the GFP-CTDCHU₂708 fusion protein.Mass spectrometry identification,periodate-Schiff staining experiment and lectin affinity experiment showed that the GFP-CTDCHU₂708 fusion protein in the periplasm of WTGFPCTD strain was glycosylated to 40 kDa.The glycosylated GFP is susceptible to PNGase F enzymatic deglycosylation suggested that it may be N-glycosylated.In addition,the asparagine site in the CTD sequence of CHU 2708 that conforms to the traditional Nglycosylation modification motif N-X-S/T（X≠P）were mutated.Mutation at the N296 site of CTDCHU₂708 significantly reduced glycosylated GFP in the periplasmic space.and the secretion and localization of GFP-related proteins were defective,suggesting that the N-glycosylation of CTDCHU₂708 played a role in the recognition and transport of substrate proteins by T9SS.Cellulase CHU₁336 as T9SS substrate protein has type A CTD.When the GFPCTDCHU₁336 recombinant protein was expressed in C.hutchinsonii,a protein band with molecular weight close to 40 kDa was also detected in the periplasm of the expression strain.This protein is responsive to ConA lectin detection and sensitive to PNGase F enzymatic deglycosylation.Site-directed mutagenesis showed that the glycosylated GFP recombinant protein in the periplasm disappeared completely when the N900 site of CTDCHU₁336 was mutated,indicating that N900 is the N-glycosylation site of CTDCHU₁336.In addition,when the amino acid D898 or S902 near the N-glycosylation site of CTDCHU₁336 was mutated,it also affected the glycosylated GFP recombinant protein in the periplasm,indicating that the N-glycosylation motif of CTDCHU₁336 in C.hutchinsonii was D-X-N-X-S,consistent with N-glycosylation motif D/E-X-N-X-S/T（X≠P）in Campylobacter jejuni.There is a 28%amino acid sequence identity between Campylobacter jejuni PglA and CHU₃842.The content of N-glycosylated proteins in the periplasm of the Δ3842 mutant strain was significantly reduced.In addition,the N-glycosylation of CTDCHU₂708 and CTDCHU₁336 were both affected in the Δ3842 mutant strain,indicating that chu₃842 is involved in the N-glycosylation of proteins in C.hutchinsonii.Deletion of chu₃842 affected cellulose degradation,cell motility,and cell resistance to some chemicals.The glycosyltransferase-related protein CHU₀012 has a conserved domain that binds to UDP-N-acetylglucosamine.The content of glycosylated proteins in the periplasm of the Δ0012 mutant was decreased.Deletion of chu₀012 affected not only the N-glycosylation of CTD of T9SS cargo protein,but also the secretion of the T9SS cargo.In addition,chu₀012 was also involved in cellulose degradation,affecting the abundance of outer membrane proteins.The above experimental results show that C.hutchinsonii,a member of Bacteroidetes,has an N-glycosylation system involved in important physiological functions such as cellulose degradation,and also indicates that the bacterial N-glycosylation system is not limited to Proteobacteria and Campylobacter.There is no N-glycosylation motif that conforms to N-X-S/T in the CTD sequence of the T9SS substrate protein cellulase CHU₁107.The GFP-CTDCHU₁107 recombinant protein was expressed in C.hutchinsonii,a protein band with molecular weight close to 40 kDa was also detected in the periplasm of the expression strain.This protein was identified as glycosylated protein and could be digested by glycosidase that removes O-linked glycans,indicating that CTDCHU₁107 was O-glycosylated.Combining the Nglycosylated CTDCHU₂708 and CTDCHU₁336,it shows the diversity of CTD modifications of T9SS substrate proteins.The homologous protein of B.fragilis BF4305 in C.hutchinsonii is CHU₀890,and the sequence similarity between them is 29%.CHU₀895 is a protein near CHU₀890,and annotated as glycosyltransferaserelated protein.Deletion of chu₀890 and chu₀895 affected the accumulation of Oglycosylated GFP fusion proteins in the periplasm of C.hutchinsonii and the ability to degrade cellulose.In addition,the deletion of chu₀895 also affected the cell motility of C.hutchinsonii.These experimental results indicated that the O-glycosylation system played an important role in C.hutchinsonii.When the type A CTD of cellulase CHU₁655 or CHU₁075 was fused to GFP,no GFP-related protein with increased molecular weight was found in the periplasmic fraction of the expression strain,indicating that CTDCHU₁655 and CTDCHU₁075 were not glycosylated.We constructed a cell surface display system with the reporter protein LacZ using CTDCHU₁655 and CTDCHU₁075 as the T9SS recognition elements,and detected LacZ enzyme activity,suggesting the potential of C.hutchinsonii T9SS as a cell surface protein display system.We found that the CTD of T9SS substrate protein was modified by Nglycosylation or O-glycosylation in the periplasm in C hutchinsonii,showing the diversity of T9SS substrate protein modifications.In C.h utchinsonii,N-glycosylation of the T9SS substrate protein CTD affects its secretion and localization.In addition,the N-glycosylation system is also closely related to various phenotypes of C.utchinsonii,such as cellulose degradation ability,cell motility ability and stress resistance.These findings provide a new perspective for revealing the recognition mechanism of substrate proteins by C.hutchinsonii T9SS and its unique cellulose degradation mechanism.This finding not only extends the bacterial N-glycosylation system to Bacteroidetes,but also enriches our understanding of the physiological functions of the bacterial N-glycosylation system.