| Naturally occurring biological nanoparticles have been widely used in many fields due to their characteristics of self-assembly,easy modification,and easy preparation.Virus and bacterial membrane vesicles(BMVs),as important parts of natural biological nanoparticles,have attracted great attention.Viruses are a kind of non-cellular biological particles composed of nucleic acids and proteins,with particle size ranging between 17 and 350 nm.The coronavirus disease-19(COVID-19)pandemic caused by severe acute respiratory syndrome coronavirus 2(SARSCoV-2)has affected more than 130 million people globally since December 2019.However,with the deepening of the research on viruses,viruses are not only regarded as pathogens,but also widely used as vaccine,gene therapy vectors and alternative drugs to antibiotics.Therefore,viral analysis is important in basic research,public health protection,and biomedical technology.Bacterial membrane vesicles(BMVs)are nanostructures shed naturally by most bacterial species,which particles size ranging between 20 and 250 nm.BMVs carry nucleic acids,proteins,especially virulence factors and immune regulatory factors for the inter and intra-species communication.Many bacterial physiological processes are closely related to BMVs.Therefore,study of the biological function of BMVs is particularly important for understanding the relationship between bacteria and their hosts.Although many researchers have made great efforts in developing single-particle analysis technology for nanoparticle characterization,many problems exist,such as insufficient sensitivity,low throughput and time-consuming process.In our laboratory,employing strategies for single molecule fluorescence detection in a sheathed flow,we have recently developed nano-flow cytometry(nFCM)that enables light scattering detection of single viruses and extracellular vesicles as small as 27 nm and 40 nm in diameter,respectively.The fluorescence detection is 2-3 order of magnitudes more sensitive than traditional FCMs.Combined with nucleic acid labeling or immunofluorescence labeling,quantitative analysis and characterization of viruses and BMVs at the single-particle level can be achieved.This dissertation consists of the following sections.In the first chapter,an overview of the recent technologies for advanced characterization of viruses and BMVs is given.The biological functions of BMVs and recent research progresses are discussed.It is hoped that nFCM can provides a new tool for solving the problems associated with the analysis of viruses and BMVs.The research plan and main contents of this dissertation are put forward in the end.Chapter two describes the development of a high-throughput single-particle method to enumerate intact viral particles by ultrasensitive flow virometry.The nucleic acid dye SYTO 82 was used to stain the viral(or vector)genome,and a laboratory-built nFCM was employed to simultaneously detect the side-scatter and fluorescence signals of individual viral particles.Successful measurement of the physical virus titer and purity was demonstrated for recombinant adenoviruses,which could be used for gene delivery,therapeutic products derived from phage cocktails,and vaccine.Other methods for virus quantification,like plaque counting and qPCR,quantify a particular component of the virus of interest(infectious virus or viral nucleic acid,respectively)and result in derived titers versus direct quantification.The nFCM-based assay measures physical virus titers in a simple,rapid,and accurate manner and can serve as an efficient tool for the routine use in the quality control of virus preparations.In chapter three,based on the unique advantages of the laboratory-built nFCM in the sensitive and rapid analysis of individual nanoparticles,the purity,concentration,and size distribution of BMV preparations upon densitity gradient ultracentrifigation were measured.It was identified that BMVs secreted by Gram-positive bacteria S.aureus and Gram-negative bacteria E.coli O157:H7 were significantly different in density and size.The experimental results demonstrated that the components in each density layer could be detected by nFCM to obtain BMVs samples with a high purity.nFCM was expected to provide powerful technical support for the biological function study of BMVs.Chapter four describes the application of nFCM for the in-depth study of the interaction between bacteriophages and BMVs at the single-particle level.Salmonella enterica serovar Typhimurium(S.Typhimurium)BMVs and phages P22 were used as the model system.Simultaneous side scatter and fluorescence detection was conducted upon protein and nucleic acid labeling.The interaction between bacteriophages P22 and BMVs were investigated:1.P22 can adsorb on BMVs by recognizing LPS;2.P22 adsorp to BMVs more readily than they adsorp to their host bacteria.The difference may be due to the different LPS structures on the surface of BMVs and bacteria.This suggests that BMVs play a important role in decoying phages;3.P22 releases its DNA upon contact with LPS expressed on BMVs.Different subpopulations of BMVs have different effects on P22 DNA.OMVs contain nonspecific nucleases from the periplasmic space,P22 DNA injected into OMVs can be degraded accordingly.In the case of OIMVs,nonspecific nucleases exist between double bilayered membranes.P22 DNA injected into the entocoele of OIMVs is not in contact with the nucleases and thus can be retained.For EOMVs,it has nucleic acid inside,so we assume that it doesn’t have nuclease.P22 DNA injected into EOMV can also be retained.Present study reveals that as an innate bacterial defense mechanism,BMVs play important roles in inhibiting bacteriophage infection.In chapter five,by using ampicillin resistant bacteria E.coli O157:H7(RP4)as the model system,nFCM,qPCR,and Western blot were combined to study the relationship between BMVs and bacterial antibiotic resistance.The research data implicate three mechanisms in which BMVs help bacteria develop resistance to antibiotics,i.e.BMVs contain antibiotic resistance genes,BMVs contain antibiotic hydrolysis proteins,and BMVs absorb antibiotics from the environment through porin.In addition,it was found that inhibiting the secretion of BMVs could increase the sensitivity of bacteria to ampicillin.Thus,it is anticipated that BMVs inhibitors can be used in combination with antibiotics to reduce the usage of antibiotics,reduce the probability of bacteria developing resistance,and promote the problem solving of bacterial antibiotic resistance.In chapter six,the study results as well as their limitations are summarized,and the future investigation is prospected. |
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