The Impact Of Iron On The Cytotoxicity Of Outer Membrane Vesicles Secreted By Klebsiella Pneumoniae On Human Lung Epithelial Cells

Background:Klebsiella pneumoniae（K.pneumoniae）is an opportunistic pathogen that can cause both hospital-acquired and community-acquired infections and is of great public health concern.Outer membrane vesicles（OMV）are nanoscale spherical liposomes that are naturally secreted by Gram-negative bacteria and released into the external environment.OMV is closely related to bacterial pathogenesis and can act as a novel secretion system for targeted delivery of bioactive molecules such as bacterial toxins to host cells to mediate bacterial-host cell interactions.There are few reports on the cytotoxicity of OMV secreted by K.pneumoniae,and further studies are needed to determine whether environmental iron affects the characteristics and cytotoxicity of K.pneumoniae OMV.Objective:To investigate the cytotoxicity of K.pneumoniae OMV on human lung epithelial cells,and also to explore the effect of environmental iron on the characteristics and cytotoxicity of K.pneumoniae OMV.Methods:（1）K.pneumoniae ATCC 10031 was cultured in iron-limited and iron-supplemented media and growth curves were plotted,OMV secreted by K.pneumoniae cultured in iron-limited and iron-supplemented media was extracted by ultracentrifugal method,and OMV morphology and size were identified by transmission electron microscopy and dynamic light scattering methods,and OMV yield was analyzed by BCA protein assay combined with plate colony counting,and OMV protein fractions were analyzed by SDS-PAGE.（2）OMV secreted by K.pneumoniae cultured in iron-limited and iron-supplemented media were co-cultured with human lung epithelial cells BEAS-2B cells,respectively.Cell morphology was observed by inverted microscopy,cell proliferation was measured by CCK-8 assay,the release of LDH from cells was measured by LDH release assay,and the expression levels of pro-inflammatory cytokines IL-8,IL-6 and IL-1βwere measured by RT-qPCR and ELISA,and apoptosis was analyzed by Annexin V-FITC/PI double-staining combined with Caspase-3 activity assay,changes in mitochondrial membrane potential were detected by JC-1 fluorescent probe,and intracellular reactive oxygen species（ROS）release was detected by DCFA-DA fluorescent probe.Results:（1）Increased environmental iron availability promoted the growth of K.pneumoniae;OMV secreted by K.pneumoniae in iron-limited and iron-supplemented media were successfully extracted and were typical spherical nanostructures of varying sizes.The mean particle size of OMV secreted by K.pneumoniae in iron-limited medium was 112.07 nm and the OMV yield was 5.026×10^-10μg/CFU.The mean particle size of OMV secreted in iron-supplemented medium was 78.90 nm and the OMV yield was 3.589×10^-10μg/CFU.OMV secreted by K.pneumoniae in iron-limited medium was larger in particle size（P<0.05）and the OMV yield was greater（P<0.05）.OMV secreted by K.pneumoniae in iron-limited medium showed distinctive protein bands.（2）BEAS-2B cells showed altered morphology,decreased cell survival（P<0.05）,increased LDH release（P<0.05）,increased expression of pro-inflammatory cytokines IL-8,IL-6 and IL-1β（P<0.05）,increased percentage of apoptotic cells（P<0.05）,increased Caspase-3 activity（P<0.05）,decreased cellular mitochondrial membrane potential decreased（P<0.05）,and increased intracellular ROS release（P<0.05）.（3）BEAS-2B cells co-cultured with OMV secreted by K.pneumoniae in iron-supplemented medium showed higher expression of pro-inflammatory cytokines（P<0.05）,more significant decrease in mitochondrial membrane potential（P<0.05）,and more ROS release（P<0.05）.Conclusions:（1）OMV secreted by K.pneumoniae under both iron-limited and iron-supplemented conditions exhibited cytotoxicity on human lung epithelial cells,altering normal cell morphology,inhibiting cell proliferation,inducing cellular LDH release,inducing inflammatory responses,inducing apoptosis,and inducing a decrease in mitochondrial membrane potential and ROS release.（2）Environmental iron affects the yield,morphological size,protein fraction of OMV secreted by K.pneumoniae and cytotoxicity of OMV on human lung epithelial cells.11 figures,4 tables,78 references...