The Interactions Between Pathogens And Their Host Cells As Studied By Single-Particle/ Single-molecule Tracking

Pathogens are great threats to human health and result in huge economic losses because they would induce the outbreaks of the infectious diseases, and some pathogens would even cause cancers. Therefore, understanding the mechanisms of the interactions between pathogens and their host cells will contribute to design and develop the targeted drugs for prevention and treatment of pathogenic diseases. The journey of pathogens in the living cells is continuous and complicated, however, the conventional methods based on fixed cells could only provide static and average information, resulting in the neglect of the transient but important processes. Herein, taking advantage of the single-particle/ single-molecule tracking techniques (SPT/SMT), we took pseudorabies virus (PrV) and anthrax toxin as two examples of pathogens and investigated the mechanisms of the interactions between them and their host cells in real time.To visualize the entire life cycle of virus in living cells, we have established a universal strategy for site-specifically labeling viral capsid, realizing the labeling of parental and progeny viruses with different fluorescent dyes, respectively. Due to the rapid, irreversible and covalent reaction between HaloTag protein expressed on the viral capsid and its fluorescently labeled ligand, the viral capsid could be specifically labeled with fluorescent dyes. The labeled replication-competent recombinant PrV harvested from medium can be applied directly in SPT experiments without any further modifications. Thus, virus intactness and infectivity are retained to the largest extent. In addition, the diversity of HaloTag ligands enables us to temporal-controllably label parental and progeny viruses with fluorescent dyes with different emission wavelengths. The established strategy provides us with the opportunity to reveal the mechanisms and dynamic picture of the life cycle of PrV in living cells.On the basis of the proposed strategy and the confocal microscope-based SPT technique, the entire course of virus infection and replication can be visualized continuously, including (i) virus attachment and capsid entry, (ii) transportation of viral capsid to the nucleus along microtubules in the cytoplasm, (iii) docking of viral capsids on the nucleus, (iv) endonuclear diffusion of assembled progeny viral capsids, and (v) the egress of progeny viruses. Moreover, with combination of the strategy for labeling of viral envelope, we have realized the simultaneous labeling of viral capsid and envelope, and have investigated the distinct entry pathway of pseudorabies virus in Vero cells and HeLa cells. In the case of Vero cells, viral envelope fused with the plasma membrane, and viral capsid containing viral genome is released to the cytoplasm for further infection. As for HeLa cells, pseudorabies virus would hijack the cellular endocytosis for its cell entry.In the last work, taking anthrax toxin as an example, we have studied the interaction mechanism between bacterium and its host cells. The mutant of protective antigen (PA), containing a single cysteine residue was labeled with a single fluorescent dye molecule modified with maleimide. Taking advantage of the SMT technique, we have visualized the oligomerization of PA on the cell surface for the first time. We found that the oligomerization process is cholesterol-dependent. Subsequently, the PA oligomers would in turn recruit the specific sphingolipid (such as ganglioside GM1) and glycophosphatidylinositol anchored protein (such as urokinase type plasminogen activator receptor) to form raft lipid, which would further stabilize the oligomers. During these processes, PA oligomers would recruit actin related protein Arp3 and its activator N-WASP, inducing the actin polymerization at the binding site of PA oligomers. The assembled actin would further confine the diffusion of PA oligomers on the cell surface, preparing for the cell entry.