Function And Mechanism Of Gb3 In Bacillus Cereus Infection

Bacillus cereus(B.cereus)is an opportunistic pathogen that can cause emetic or diarrheal foodborne illness.In addition,B.cereus also leads to local or systemic nongastrointestinal infections,such as bacteremia,pneumonia,endophthalmitis and central nervous system infections,especially in immunocompromised persons.Moreover,B.cereus infection can sometimes cause death,especially in patients with bacteremia.Globotriaosylceramide(Gb3),also known as CD77,is an important surface receptor of human cells and characterized by binding to several components of different pathogens.Some pathogens were confirmed keep capacity of binding with Gb3,such as Shiga toxins of Shigella dysenteriae,P fimbriae of Escherichia coli,lectin I(Lec A)of Pseudomonas aeruginosa and Fhb of Streptococcus suis.Although Gb3 functions diversely in different pathogens,we speculated that the Gb3 may be a common recognition receptor/site for several bacteria.Whether this hypothesis also applies to B.cereus remains to be determined.Herein,we evaluated the relevance of the B.cereus and Gb3 in the context of acute infection caused by B.cereus.We used Gb3-deficient mice to establish a B.cereus infection model,and found that Gb3 contributes to the B.cereus acute infection.Then we screened and identified that the flagellin of B.cereus can bind to Gb3.Adhesion assay proved that the interaction between flagellin and Gb3 contributes to the adherence of B.cereus.Subsequently,we found that flagellin is involved in the process of B.cereus infection with the presence of Gb3,and anti-flagellin antibody can protect mice against B.cereus infection.Finally,we conducted a preliminary study on the signaling pathways that may be affected by the interaction between flagellin and Gb3.The experimental contents and results divided into the following three parts.1.Gb3 contributes to B.cereus acute infectionIn order to explore the role of Gb3 in B.cereus infection,we infected WT and Gb3-deficient mice with B.cereus HN001,and found that the Gb3-deficient mice displayed lower mortality,lower bacterial loads in the spleens and livers,and reduced IL-1β,IL-6,and TNFα levels in serum compared with WT mice.These results indicated that Gb3 contributes to B.cereus acute infection.To study the effect of virulence factors secreted by B.cereus on B.cereus infection,we constructed a B.cereus strain HN001-Δplc R with suppressed virulence factors expression.We infected mice with HN001,HN001-Δplc R or the culture supernatants of them,and found that the secreted virulence factors are responsible for the rapid death of the infected mice.Then,we injected wild-type and Gb3-deficient mice with HN001 culture supernatant,both mice suffered acute death,however,considering the mortality of HN001 infected Gb3-deficient mice was lower than the mortality of HN001 infected WT mice,these results indicated that the secreted virulence factors do not directly cause the death of mice through Gb3.Therefore,we speculated that a certain surface component of B.cereus can interact with the host cell Gb3.Then,we evaluated the adhesion of B.cereus to WT and Gb3-deficient h CMEC/D3 cells,and found that B.cereus may adhere to cells by Gb3,which verified our speculation.2.The interaction between flagellin and Gb3Gb3 as a cell surface receptor,its hydrophilic oligosaccharide chain can be exposed outside the cell membrane.The oligosaccharide chain of Gb3 contains the disaccharide structure of Galα1-4Gal(galabiose),which is the binding site of various pathogen components.We labeled the pigeon ovomucoid(POVO)containing Galα1-4Gal on the Dynabeads to capture the surface protein of B.cereus bound to it,and then identified the B.cereus flagellin as a candidate protein by mass spectrometry.This result was also verified by bio-layer interferometry technology.Then we used flow cytometry to prove that B.cereus flagellin can be combined with Gb3 on the cell surface,and observed the adhesion of B.cereus to cell surface by scanning electron microscopy and laser confocal scanning microscope.In order to study the interaction of B.cereus flagellin with Gb3,we constructed the B.cereus flagellin knockout strain HN001-Δfla.Then we conducted a bacterial adhesion assay and found that knockout of flagellin or treatment with PDMP,Lec A,flagellin or disaccharide Galα-4Gal will significantly reduce the adhesion ability of B.cereus,these results indicated that the binding of flagellin to cell surface Gb3 contributes to the adherence of B.cereus.To investigate the effect of flagellin-Gb3 interaction on B.cereus infected mice,WT mice and Gb3-deficient mice were infected with HN001 and HN001-Δfla respectively.For WT mice,HN001-Δfla infected mice displayed lower mortality,lower bacterial loads in the spleens and livers,and reduced IL-1β,IL-6,and TNFα levels in serum compared with HN001 infected mice.However,for Gb3-deficient mice,there is no significant difference between the results of HN001-Δfla infected mice and HN001 infected mice.These results suggested that flagellin is involved in the B.cereus infection in mice with the presence of Gb3.The passive antibody therapy,which targets to the virulence factors or bacterial surface molecules,has been used to prevent some bacterial infectious diseases.One of the ways that antibacterial antibodies work is to inhibit the bacterial adhesion.In this study,we collected anti-flagellin serum from flagellin immunized rabbits and then purified the anti-flagellin antibody.Mice were treated with anti-flagellin or non-specific antibody before the B.cereus infection,anti-flagellin antibody treated mice displayed lower mortality,lower bacterial loads,and reduced IL-1β,IL-6,and TNFα levels.These results indicated that anti-flagellin antibody can protect mice against B.cereus infection.3.Preliminary study on the mechanism of Bc-SMaseIn view of the fact that the flagellin of B.cereus can bind to Gb3,we conducted a preliminary study on the signaling pathways that may be affected by the interaction between flagellin and Gb3.It has been reported that the interaction between P fimbriae and Gb3 can significantly increase the release of ceramide and result in the activation of the TLR4.Recombinant B.cereus sphingomyelinase(Bc-SMase)can hydrolyze sphingomyelin on the surface of the host cell and increase the level of ceramide.Therefore,we speculated that after flagellin binds to Gb3,the sphingomyelin on the cell surface may be hydrolyzed to release ceramide by B.cereus secreted Bc-SMase.We constructed the Bc-SMase knockout strain HN001-Δsmase,and found that BcSMase produced by B.cereus can significantly increase the release of ceramide during B.cereus infection,and this process is dependent on the presence of Gb3.These results indicated that the interaction between flagellin and Gb3 may increase the release of ceramide by Bc-SMase and result in the activation of the related signaling pathway,however,further research is needed to verify this conclusion.In conclusion,we found that Gb3 of host cell contributes to B.cereus acute infection,and identified the interaction between flagellin and Gb3 which promoted the adhesion of B.cereus to host cells.Besides,the interaction between flagellin and Gb3 may be involved in the B.cereus infection process of mice,and anti-flagellin antibody can protect mice against B.cereus infection.Finally,we conducted a preliminary study on the signaling pathways that may be affected by the interaction between flagellin and Gb3,and found that the interaction may increase the release of ceramide by Bc-SMase and result in the activation of the related signaling pathway.Collectively,these results uncovered the interaction between flagellin and Gb3 and provided useful information for the development of therapies to treat infection of B.cereus.