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.

To explore the action mechanism of tauroursodeoxycholic acid (TUDCA) promoting intracellular clearance of Burkholderia pseudomallei (B. pseudomallei) in RAW264.7 macrophages.

After TUDCA of different concentrations were used to treat RAW264.7 cells pre-infected with B. pseudomallei for 8 h or not, flow cytometry was applied to detect the apoptosis of the infected and control cells. In addition, another endoplasmic reticulum stress (ERS) inhibitor 4-PBA was used to detect the apoptosis and proliferation of host cells after B. pseudomallei infection with Annexin-V/PI double staining and MTT cell proliferation assay. Furthermore, after transfected with CHOP siRNA, Western blotting and flow cytometry were employed to detect the effect of TUDCA on the expression levels of Caspase-3 and Caspase-12 and the changes in apoptotic rate after B. pseudomallei infection, respectively. Finally, the effect of TUDCA on intracellular multiplication of infected RAW264.7 cells were observed to estimate the CFU value in the presence and absence of CHOP siRNA.

Under different concentrations of TUDCA, 100 or 200 μmol/L TUDCA significantly reduced B. pseudomallei-induced apoptosis in RAW264.7 cells (P < 0.05). Meanwhile, both TUDCA and 4-PBA treatment could decrease the apoptosis induced by B. pseudomallei infection by ERS (P < 0.05). Further, the expression levels of Caspase-3 and Caspase-12 were obviously increased after B. pseudomallei infection compared with uninfected groups, but their expression levels in the siCHOP group was significantly lower than that in the siC group. Besides, flow cytometry also showed that TUDCA could reduce apoptosis induced by B. pseudomallei infection (P < 0.05), but no significant effect of TUDCA on apoptosis was observed under CHOP knockdown. Finally, intracellular CFU assay indicated that TUDCA treatment promoted the host cell clearance of B. pseudomallei (P < 0.05), but no such effect was observed in siCHOP group.

In B. pseudomallei infected RAW264.7 cells, TUDCA promotes the intracellular clearance of the bacteria by inhibiting ERS-induced apoptosis.