Construction Of A Fluorescent Localized Reporter Strain Of Salmonella Typhimurium And Its Tracer Application In Cells

Salmonella is a gram-negative bacterium that can cause intestinal infections and is an important zoonotic pathogen.It is mainly transmitted through contaminated food or water.Once it enters the intestinal tract,it will continue to multiply and adhere to the epithelial cells of the intestinal mucosa,and then invade the lamina propria,causing symptoms such as enteritis and sepsis.However,the molecular mechanism of Salmonella dissemination in the host has not been elucidated.Current research on Salmonella is mainly focused on the regulation of the expression of its pathogenic factors,the generation of specific antibodies against virulence factors and the search for new drug targets.To elucidate the physiological and pathological requirements of pathogenic bacteria,these studies involve interactions between pathogenic bacteria and their hosts.Therefore,to study the regulatory mechanisms of pathogenic factor expression networks during Salmonella-host interactions,effective methods are needed to continuously monitor bacterial behavior in the host.Fluorescent protein labeling systems are widely used in cell biology as a molecular labeling method to visualize pathogen-host interactions.The purpose of this study was to:（1）construction of Salmonella typhimurium expressing enhanced green fluorescence and characterization of the standard strain of Salmonella typhimurium 14028s（Sal-14028）using the enhanced green fluorescence protein（EGFP）gene;（2）preliminary investigation of its biological properties has proved that the introduction of the EGFP gene and the kanamycin（Km）resistance gene（aminoglycoside antibiotics）has no significant effect on the biological properties of the strain and will not affect the performance of subsequent experiments;（3）In vitro cell experiments were performed after infection of porcine alveolar macrophages（3D4/21）and human promyelocytic acute leukemia cells（HL60）to verify the co-localization of EGFP-expressing Salmonella typhimurium with 3D4/21 stage markers LAMP1 and HL60 extracellular trap NETs.This study aims to provide a visual tool to further explore its infection and pathogenesis.1.Construction of Salmonella typhimurium Sal-pPpagC-EGFP stably expressing enhanced green fluorescent proteinThe gene for the enhanced green fluorescent protein（EGFP）was transferred into Sal-14028 by the electric shock method.After screening and sequencing on kanamycin-resistant medium,a positive strain with strong green fluorescent signal was obtained,named Sal-pPpagC-EGFP,and its fluorescence stability was tested.The results showed that the fluorescent Salmonella typhimurium Sal-pPpagC-EGFP was successfully constructed.Under fluorescence microscopy,it was observed that the strain expressed strong green fluorescence signals and could stably express green fluorescence under both anti-and non-anti conditions.2.Comparison of the biological properties of Sal-pPpagC-EGFP and Sal-14028Phenotype,growth curves,LD₅₀,drug resistance,intracellular proliferation and other biological properties were determined in identical conditions.The results showed that under the same culture conditions,there were no significant changes in growth curve,virulence and strain morphology of Sal-pPpagC-EGFP and Sal-14028;drug sensitivity experiments have shown that Sal-pPpagC-EGFP has high sensitivity to aminoglycoside antibiotics and studies have shown that such antibiotics have no effect on intracellular proliferation and survival of Salmonella;There was no significant difference in the ability of Sal-pPpagC-EGFP and Sal-14028 to proliferate in host cells based on flow cytometric analysis of fluorescence intensity and bacterial proliferation experiments.This indicates that introducing plasmid carrying EGFP and km resistance gene has no significant effect on the biological characteristics of Salmonella and will not affect the following experiments..3.Preliminary investigation of the localizing and tracking role of Sal-pPpagC-EGFP in phagocytesSal-pPpagC-EGFP and Sal-14028 invaded porcine alveolar macrophages 3D4/21.Indirect immunofluorescence method was used to observe the co-localization of Sal-pPpagC-EGFP and Sal-14028 with lysosomes and stage marker LAMP1 of porcine alveolar macrophages.The results showed that Sal-pPpagC-EGFP and LAMP1,a late marker protein of phagocytic lysosomes in macrophages,were co-localized by fluorescence microscopy;Sal-pPpagC-EGFP invaded into human acute promyelocytic leukemia cell line HL60,and then the indirect immunofluorescence method was used to observe the co-localization of Sal-pPpagC-EGFP and Sal-14028 with extracellular trapping nets（NETs）of human acute promyelocytic leukemia cells.The results showed that Sal-pPpagC-EGFP and NETs elastase co-localized under fluorescence microscopy.In conclusion,this study constructed a Salmonella typhimurium Sal-pPpagC-EGFP stably expressing enhanced green fluorescent protein;introducing EGFP and Km resistance genes did not significantly affect its biological characteristics;Sal-pPpagC-EGFP co-localized with marker proteins in phagocytes and played a tracer role.The results of the research provide biomaterials for the further investigation of the pathogenic mechanism of Salmonella.