Function Research On O-antigen Of Burkholderia Pseudomallei In The Infection And Pathogenesis

Burkholderia pseudomallei（B.pseudomallei）,is a Gram-negative rod bacterium that can cause the zoonotic disease commonly known as melioidosis.The clinical treatment of this disease presents significant challenges.Because of the high mortality rate（with a case fatality rate up to 40%）,the resistance to a large number of antibiotics,and the lack of an effective vaccine,B.pseudomallei has been classifitied as a category B bioterrorism agent by WHO,and a Class Ⅰ pathogen organism by the USA Centres for Disease Control and Prevention.Melioidosis is a tropical medical disease.With the increasing international communications,especially the implementation of national strategic projects such as“The Belt and Road”initiative,China（Hainan）Pilot Free Trade Zone development,and International Tourism Island construction,it is an imperative for strengthening the prevention and control measures against melioidosis.As a facultative intracellular pathogen,the ability to escape efficiently to host immune clearance from the host cells of B.pseudomallei is the key for its intracellular survival,infection and pathogenesis.B.pseudomallei has a large bacterial genome（7.2 Mb）,encoding numerous virulence factors,including capsule polysaccharides,lipopolysaccharides（LPS）,flagellin proteins,outer membrane proteins,adhesion molecules,quorum-sensing systems and protein secretion systems.LPS is the primary component of the outer membrane and a crucial virulence factor and surface antigen of B.pseudomallei,playing an important role in the protection from environmental stress factors,including infection,drug resistance,pathogenesis,and symbiosis.Based on the diversity of O-antigen structure,B.pseudomallei is grouped by serotype into types A,B,and B₂,which serotype A is the most common type of all screened isolates.Studies have shown that LPS of B.pseudomallei exhibits negative regulation of host innate immune responses by interfering with macrophage activation,and facilitates the pathogen’s evading.LPS of B.pseudomallei consists of lipid A,core oligosaccharide（OS）,and O-specific polysaccharide（O-antigen）.The lipid A portion is responsible for the endotoxin activity of LPS and its structure is highly conserved among species.It interacts with Toll-like receptors and other molecules in the host’s immune system,which triggers strong inflammatory responses that can be lethal.Additionally,the O-antigen,as a highly specific structural component within LPS,can be recognized by the host’s innate immune system.It is thought to play an important role in complement-mediated immune responses and may have a significant impact on LPS-mediated innate immune escape,however its exact function is still unclear.This research focuses on the O-antigen biosynthesis and relationship with the biological characteristics and virulence of B.pseudomallei.This study aims to provide new targets and strategies for effective prevention and treatment of melioidosis.【Objective】To explore the biological function of O-antigen of B.pseudomallei in the infection and pathogenesis.【Methods】（1）The serotype of BPC006 was determined with a combination of methods,including sequence homology alignment,PCR amplification,and characterization of the lipopolysaccharide structure.Furthermore,we conducted a systematic analysis of the distribution of serotypes in strains from major epidemic areas in China.（2）The functions of the key genes involved in O-antigen synthesis in B.pseudomallei were predicted by bioinformatics analysis method.Subsequently,knockout strains were constructed to explore their specific roles in type A O-antigen biosynthesis,using silver staining and GC-MS.（3）The morphology of bacteria and the ultrastructure of their cell walls were observed using Gram staining and transmission electron microscopy.Growth curves of strains were compared between wild and mutant in vitro.The permeability of the outer membrane and surface hydrophobicity of the knockout strains were analyzed using fluorescent probes and microbial adhesion to hydrocarbons experiments.The ability of the bacterial biofilms forming was evaluated through crystal violet staining and scanning electron microscopy.The antimicrobial susceptibility test that target cell wall were detected by broth microdilution method.（4）A macrophage model infected with B.pseudomallei was used to observe the adhesion,invasion,and intracellular survival of knockout and wild strains.The bacterial quantification was accomplished by counting single colonies.Additionally,we detected the cytotoxicity of the knockout and wild strains using a lactate dehydrogenase release assay.（5）A mouse model infected with the bacteria was used to evaluate the impact of the O-antigen on the virulence of B.pseudomallei.We employed various methods to analyze the virulence comprehensively,including plotting mouse survival curves,counting bacterial loads in infected organs,measuring endotoxin levels in serum,verifying chip analysis of serum inflammatory factor expression,and observing organ lesions and inflammatory infiltration by HE staining of pathological sections.【Results】（1）The O-antigen of the B.pseudomallei BPC006 in China is classified as type A.Moreover,type A was found to be the predominant serotype of B.pseudomallei in our laboratory,up to 91.2%.（2）The genes wbiI and wbiG in the O-antigen biosynthetic gene cluster of B.pseudomallei,were predicted to encode specific sugar epimerase.It was observed thatΔwbiI andΔwbiG（strains with knocked-out wbiI and wbiG genes）lacked O-antigen in their lipopolysaccharides.Moreover,6-dexo-Tal did not be detected by monosaccharide composition analysis.These findings suggest that both wbiI and wbiG likely play a critical role in the isomerization process that converts dTDP-L-Rha to dTDP-L-6d Tal.（3）The cell morphological change and the cell wall structural damage of B.pseudomallei were observed when the O-antigen structure was absent,these changes increased the permeability and surface hydrophobicity of the cell,weakened its ability to biofilm formation,and caused more sensitive to vancomycin and ceftazidime,which target the cell wall structure.（4）ΔwbiI andΔwbiG exhibited stronger adhesion and invasion to host cells,compared with BPC006.However,their intracellular survival and replication ability was weakened over time,while their cytotoxicity increased.Also,the mice infected withΔwbiI andΔwbiG had shorter survival times,higher bacterial loads in the spleens and lungs,and higher endotoxin in the serum compared to those infected with the wild strain.Moreover,these infected mice showed higher levels of inflammatory factors（such as IL-6,IL-17,IL-12p70）and chemokines（such as TARC,MCP-5,RANTES）,as well as severe organ tissue damage with massive infiltration of inflammatory cells.These findings suggest that the virulence of B.pseudomallei increased significantly in the absence of an O-antigen structure.【Conclusions】（1）Both wbiI and wbiG are essential for the biosynthesis of type A O-antigen in B.pseudomallei by facilitating the conversion from dTDP-L-Rha to dTDP-L-6d Tal.（2）When the O-antigen synthesis pathway in B.pseudomallei is blocked,the bacterial lipopolysaccharide structure becomes incomplete,the bacterial physiological and biological function were influenced.（3）The O-antigen of B.pseudomallei may play a role that reduce cytotoxicity and inhibit excessive host inflammatory responses,evade the host immune clearance through O antigen modification of LPS structures.It is benefit to the bacterial survival longer after initial exposure.【Significance】This study revealed key genes and their functions in the O-antigen synthesis process of B.pseudomallei,and explored the role of O-antigen of B.pseudomallei in its infectionand immune.It provided a theoretical basis for developing new strategies for preventing and controling of melioidosis.