| Bioaerosol,a subset of atmospheric particles composed of bacteria,fungi,viruses and their products,are commonly released into the atmosphere from soil,water and vegetation,as well as animals(including humans),composting,sewage treatment plants,landfills,farms and healthcare sites,posing a very high public health risk.The occasional epidemiological spread of bioaerosol components can be highly disruptive to societies and economies,as demonstrated by the current COVID-19 global pandemic.There are various pathogenic bacteria in the air that cause respiratory infections,and most of them are transmitted in the form of"aerosols".Inhalation and deposition of pathogenic bacteria in the respiratory system area can cause allergic reactions or toxic reactions in humans and animals and even cause human diseases.Respiratory inhalation,the main way of exposure to the pathogenic bacteria,makes respiratory epithelial cells vulnerable to harmful effects of inhaled pathogenic bacteria.Despite this,little is known about the toxic effects of the exposure of pathogenic bacteria to respiratory cells.In addition,the deposition of pathogenic bacteria in the respiratory tract depends on the microbial properties,airway morphology and respiratory characteristics.However,the research on the deposition sites and toxicity trend of pathogenic bacteria exposed to respiratory cells is also limited.In addition,the toxic mechanism of pathogen exposure to respiratory cells has not been extensively elucidated.Therefore,in this thesis,a typical opportunistic pathogen pseudomonas aeruginosa(P.aeruginosa)was employed as the experimental bacteria,and respiratory epithelial cell lines(nasal mucosal cells HNEp C,nasopharyngeal cells NP69,bronchial cells 16HBE,and lung epithelial cells Beas-2B)were selected as the exposed objects.Based on the three exposure and infection processes of pathogenic bacteria to respiratory tract cells,the toxic tendency of typical pathogenic bacteria to respiratory tract cells was discussed,and then the toxic mechanism of typical pathogenic bacteria to respiratory tract cells was clarified.The main research contents and results of this thesis are described below:First,the toxicity trend of P.aeruginosa was studied based on the exposure,adhesion and invasion performance of P.aeruginosa to the respiratory epithelial cell lines,the recognition mechanism of P.aeruginosa to different respiratory epithelial cell lines and the toxic effect of P.aeruginosa on different respiratory epithelial cell lines.The results showed that,10-10~5CFU/m L P.aeruginosa tended to adhere to HNEp C and NP69 cells,and tended to invade HNEp C and 16HBE cells during the process of exposure to respiratory tract.Both adhesion and invasion processes were positively and negatively regulated at the gene level.The tendency of adhesion and invasion of P.aeruginosa to respiratory tract cells was the final result of intracellular equilibrium.In addition,after exposing for 6 h,nucleotide oligomeric structure domain(NOD)like receptor(NLRs receptor)genes(NOD1 and NOD2)were found to be upregulated by 1.1-26.5 times in NP69 cells,and the cells specifically recognized the cell wall of P.aeruginosa and activated downstream signaling.The downstream toxicity showed that P.aeruginosa inhibited the proliferation activity of respiratory tract cells in the range of 3.9%-109.5%at 10~3-10~5 CFU/m L.Combined with statistical analysis,P.aeruginosa was found to be more likely to induce the decrease of mitochondrial membrane potential(MMP)by 47.1%-90.3%of the NP69 cells,thus resulting in mitochondrial dysfunction,promoting the secretion of Interleukin-6(IL-6)and Interleukin-1?(IL-1?)to induce inflammation,and then inducing the chemokine Interleukin-8(IL-8)with a relative increase of 17.1%to 385.7%to recruit immune leukocytes to resist bacterial infection.At the same time,the regulation genes of inflammation and chemotaxis were upregulated by 1.1-271.8times in the downstream intermediate domain and terminal of NLRs in NP69 cells,confirming that P.aeruginosa induced inflammatory effects by activating NOD signaling pathway.Subsequently,the up-regulation of genes regulating mesenchymal transformation markers in epithelial cells indicated that the inflammatory effect induced by P.aeruginosa may be further developed into diseases such as organ fibrosis,cancer occurrence and metastasis,asthma and bronchial remodeling.Second,lower respiratory tract 16HBE cells were selected as the research object,and the exposure time was further extended to study the toxicity mechanism of pathogenic bacteria on respiratory tract cells from two aspects:the toxic effect of cells and the promoting toxic effect of P.aeruginosa.The results showed that:P.aeruginosa exposure for 8 h induced a decrease in the activity by 4.0%-62.8%of 16HBE cells,which targeted mitochondrial damage by promoting the decrease of MMP and stimulated the excessive release of reactive oxygen species(ROS)in mitochondria with an increase of 3.6%-30.7%,which induced oxidative stress effect,thereby activating catalase(CAT)and superoxide dismutase(SOD)and initiating the oxidative stress compensation mechanism.Intracellular REDOX imbalance induced the production of IL-1?and interferon-?(IFN-?)with the highest growth rate of 83.4%and24.8%,respectively,leading to inflammatory immunity.On the other hand,P.aeruginosa adhered to the cells to form a dense biofilm,and partially invaded cells.Meanwhile,virulence factors(rhamnolipid,protease,pyocyanine and pyoverdin)were produced to promote biofilm formation and bacterial invasion,weaken the ability of the cells to remove ROS,and uptake of iron in cell fluid,thus synergistically promoting cytotoxicity.Two matrix correlation analysis showed that pyoverdin was the dominant cytotoxicity factor.Further dual transcriptome sequencing analysis showed that P.aeruginosa up-regulated genes related to cellular iron accumulation and lipid peroxidation,thus promoting ROSs accumulation to induce ferroptosis in weakly tolerant cells,and up-regulated genes involved in antimicrobial peptides,tissue remodeling,inflammation and chemokines,thus activating the IL-17inflammatory signaling pathway of strongly tolerant cells.These revealed mechanisms of cellular death and defense.In addition,the upregulation of genes involved in type?secretion system,Rhl system and the synthesis of pel polysaccharide of P.aeruginosa activated the biofilm formation signaling pathway,revealing the promoting toxicity mechanism of P.aeruginosa.In conclusion,this thesis revealed the toxicity trend and mechanism of P.aeruginosa to different respiratory tract cells.It was found that bacteria tended to adhere to HNEp C and NP69 cells,and tended to invade HNEp C cells,and then were more easily recognized by NP69cells,which further targeted mitochondrial damage,and induce downstream inflammatory effects through NOD signaling pathway,thus causing the possibility of disease.In addition,P.aeruginosa enhanced cytotoxicity by forming biofilms and secreting virulence factors,inducing 16HBE cells to be in a state of high oxidative stress,which led to cell damage and death and activated inflammatory defense.In this thesis,we first screened the colonization sites of pathogens in the respiratory tract and revealed the microscopic mechanisms of cellular death and defense as well as promoting toxicity of pathogenic bacteria.These results provide basic research on the exposure,infection process and toxic effects of pathogenic microorganisms,resulted in valuable insights on the exposure risk of pathogenic bacteria to human respiratory cells,and molecular biological references for the health hazards and mechanisms of bioaerosols.It can be used as a reference to self-protection of the population and exposure detection of pathogenic microorganisms in epidemic period. |
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