Structural And Functional Analysis Of A Novel BSAPs Toxin From Gut Bacteroides

Bacteroides,the intestinal symbiotic bacterium,plays an important role in human health.It can exist stably for a long time and occupy a dominant position in the intestinal ecosystem.The competition antagonism mechanism plays an important role in the process of bacteria competing for ecological niche.Competitive antagonisms in bacteroides are generally divided into two categories:contact-dependent antagonism mediated by T6SS,which targets a wide range but can only affect neighboring cells,and contactless antagonism secreted by vesicles or unknown means,which targets a narrow range but can affect distant cells.BSAPs are a class of toxins identified and named by Comstock lab.BSAPs toxin produced by bacteroidetes for intraspecific antagonism is a MACPF bacterial toxin and it is screated by outer membrane vesicles.MACPF protein is widely found in natural organisms and plays an important role in the immune system,cell development,bacterial disease,and other processes.In most cases,it functions through oligomerization.BSAPs are the first to be discovered as the bactericidal MACPF proteins secreted by bacteria.Studies have shown that BSAPs are important factor affecting the composition level of human intestinal flora.Because the producing and sensitive strains of BSAPs toxin cannot coexist in the same host intestinal flora.So it has important biological significance to study the molecular mechanism between bacterias caused by BSAPs.At present,the sequence similarity of the four known BSAPs toxins is only about 20%.BSAP-1 and BSAP-4 come from B.fragilis,and their receptors are different outer membrane proteins(OMPs);BSAP-2 and BSAP-3 are derived from B.uniformis and B.vulgatus,respectively,and their receptors are lipopolysaccharide(LPS)from different strains.Due to the lack of three-dimensional structural information of BSAPs toxin,the specificity of receptor recognition and the molecular mechanism of bactericidal activity remain unclear.In our study,we found a new BSAP toxin in B.fragilis,which is the fifth kind of BSAPs toxin found so far.We named it BSAP-X and explored its bactericidal function through agar spot experinrent.We predicted that the receptor of BSAP-X was LPS through bioinformatic analysis.Gel filtration experiments showed that LPS of the sensitive strain could change the aggregation state of BSAP-X.After solving the BSAP-X structure,we concluded through structural analysis that the N-terminal may be responsible for the recognition between monomers,and the C-terminal may have a membrane insertion function.The membrane insertion function of the C-terminal domain of this protein was verified through liposome binding experiments.In order to understand the molecular mechanisms underlying the recognition specificity of different BSAPs receptors,we further solved the structure of BSAP-2 targeting LPS in B.uniformis and the structure of BSAP-4 targeting OMP in B.fragilis 638R.Through structure comparison,we found that there are significant differences in the C-terminal structure of BSAPs toxin proteins targeting different types of receptors(LPS or OMP).So we speculated that the C-terminal domain of BSAPs toxin proteins may be involved in the recognition of specific receptors.Through the C-terminal domain of BSAP-X and BSAP-2 toxin proteins using LPS as receptors are highly similar,BSAP-X from B.fragilis and BSAP-2 from B.uniformis can only play intraspecific antagonism.This may be related to the difference in potential and hydrophobicity at the bottom of the C-terminal of BSAP-X and BSAP-2.This study identified a new BSAP-X toxin targeting LPS receptor that is present in a variety of B.fragilis.This was the fifth known BSAPs toxin.In addition,we solved the BSAPs toxin with LPS and OMP as receptors for the first time.Through structural comparison and analysis,we proposed a possible molecular mechanism for BSAPs to recognize different receptors and provided a structural basis for further research on the bactericidal mechanism of BSAPs toxin proteins.