Outer Membrane Vesicles Derived From Hypervirulent Klebsiella Pneumoniae Stimulatr The Inflammatory Response

Background: Hypervirulent Klebsiella pneumoniae(hv KP)is a clinically increasingly threatening bacterial pathogens,not only can cause severe liver abscess,still can cause many complications,which brought serious challenges to clinical therapy.Most of gram-negative bacteria can secrete Outer membrane vesicles(OMVs).Many studies have shown that OMVs are considered as one of the important carrier of molecules between bacterial and target cells.However,the components of OMVs secreted by hv KP and their role in bacterial pathogenicity have not been determined.Based on these findings,we propose a scientific hypothesis: hv KP exerts immune effects to the host may be mediated by OMVs.Objective: This subject focuses on the hv KP infection of the public health problems,from the angle of interaction between bacteria and host,with OMVs regulation of host cell immune response as the breakthrough point,to study OMVs mediated immune regulation and provide new ideas for hv KP infection in clinical treatment.Methods:1.First of all,we obtained OMVs using ultracentrifugation from hv KP in vitro.In addition,proteomic analysis of the strains and OMVs was performed to evaluate the protein types contained in both strains.2.In vitro,liver cells(L02),hepatic stellate cells(LX2),liver cancer cells(hep G2),bronchial lung epithelial cells(BEAS-2 B)and lung cancer cells(A549)were incubated with OMVs.The Cell proliferation activity detection Kit(Cell Counting Kit-8,CCK8)was performed to test the prolifiration of cells.In addition,we take on the culture supernatant and the inflammatory factor including Interleukin 6(IL-6),Interleukin 8(IL-8),and Interleukin 1β(IL-1β)were quantitatively analyzed by ELISA.3.Wild mouse models were established.Different concentrations of OMVs were injected into mice via trachea.The same volume of PBS was used as control.The pro-inflammatory factors containing IL-6,IL-8 and tumor necrosis factor α(TNF-α)in Bronchoalveolar lavage fluid(BAL)was measured by ELISA.Results:1.We observed the OMVs typical tea light sample structure under transmission electron microscope.Second,the particle size analysis shown most of its diameter distribution at around 100 nm,confirming it has good uniformity.In addition,sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)can see clear bands,demonstrating OMVs may contain a variety of proteins.Proteomic analysis confirmed that hv KP OMVS contained numerous proteins,some of which were derived from thallus.2.Co-incubated with cells,hv KP OMVs showed different cytotoxic effects on different types of cells in vitro.Furthermore,it induced the expression of pro-inflammatory cytokines in host cells,including IL-6 and IL-8.3.hv KP OMVs injected by trachea in wild type mice can cause inflammatory responses in mice,stimulating the expression of proinflammatory cytokines including IL-6,IL-8 and TNF-α in BAL,which was consistent with the experiment in vitro.However,hv KP OMVs was not enough to kill the mice.Conclusion: in the process of hv KP infection of the body,OMVs can be as medium causing the body’s inflammatory response.Therefore,clinical treatment of hv KP infection can turn OMVs as the breakthrough point to develop a kind of innovative treatment options.