Application of biotin-streptavidin labeling technology for pathogen tracing, bacteria-containing phagosome purification, and immune evasion mechanisms of intracellular pathogens

Based on the specific binding and signal amplification effects of the biotin-streptavidin system, a hypothesis is proposed that the constructed biotin-streptavidin labeling technology can achieve tracing of pathogenic bacteria and isolation of bacteria-containing phagosomes without affecting the biological characteristics and infectivity of pathogenic bacteria, and can be preliminarily applied to elucidate the molecular mechanisms of immune evasion by intracellular pathogenic bacteria. This study aims to verify this hypothesis and provide technical support for research on pathogen-host interactions.

① Burkholderia pseudomallei (B. p), Salmonella typhimurium (S. t) and Shigella flexneri (S. f) were used as research objects, and J774A.1 macrophages were employed to construct a bacteria-host cell infection interaction model. Biotin-streptavidin-labeled and unlabeled groups were established. Through covalent conjugation of NHS-biotin to amino groups on the bacterial membrane, a tracing system was constructed by coupling with streptavidin-FITC, or with streptavidin magnetic beads for magnetic sorting and isolation. ② Immunofluorescence assay was utilized to detect labeling efficiency and intracellular distribution. After the cells were infected at a multiplicity of infection (MOI) of 10 for 1, 3, and 5 h respectively, cell infection rates, intracellular bacterial load per cell, and intracellular survival were compared. ③ ELISA was applied to detect the secretion levels of IL-1β, IL-12 and TNF-α in cell supernatants. ④ Furthermore, the cells were infected with magnetic bead-labeled bacteria, and bacteria-containing phagosomes were isolated by magnetic separation at 1 and 2 h or 3 and 5 h. Western blotting was performed to detect the expression of phagosome markers Rab5, Rab7, and LAMP-2, as well as the cytosolic internal reference GAPDH, to identify separation purity. ⑤ This technology was also applied to perform proteomic analysis of bacteria-containing phagosomes from wild-type B. p and bopA knockout strain (ΔbopA).

The labeling efficiency of this labeling system for B. p, S. t, and S. f all reached above 90%, with uniform fluorescence signals and no non-specific adsorption. There were no significant differences between the labeled group and the unlabeled group in terms of cell infection rate, intracellular bacterial load per cell, intracellular survival trend and intracellular distribution patterns at 1, 3 and 5 h after infection (P>0.05). ELISA showed no statistically significant differences in the secretion levels of IL-1β, IL-12 and TNF-α between the 2 groups (P>0.05). In bacteria-containing phagosomes obtained by magnetic separation, the protein levels of Rab5, Rab7, and LAMP-2 were significantly higher than those in the cytosolic fractions (P<0.05), the level of cytoplasmic GAPDH contamination in phagosomes was extremely low. Proteomic analysis revealed that host proteins related to vesicle transport, including VAMP7, EEA1, RABEP1, and RAB3GAP2, were significantly upregulated in bacteria-containing phagosomes of the ΔbopA strain compared with the wild-type strain.

The biotin-streptavidin labeling and magnetic sorting system established in this study does not change the biological characteristics of pathogens, host cell infectivity, or the secretion of inflammatory factors. This system can accomplish fluorescent tracing of pathogenic bacteria and isolate high-purity bacteria-containing phagosomes, and be applied to preliminary elucidation of the potential mechanisms by which pathogen-related effector proteins mediate host defense evasion research.