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. 2019 Nov 2;10(1):910–924. doi: 10.1080/21505594.2019.1685640

Figure 4.

Figure 4.

Mutation of grx increased the effciency of bacterial intracellular growth and cell-to-cell spreading via upregulating the internalins, InlA and InlB.

(a) Intracellular growth of L. monocytogenes in murine-derived J774 macrophages. Gentamycin (50 μg/mL) was added 30 min post infection. The J774 cells infected with L. monocytogenes wild-type EGD-e and grx mutant were lysed at the indicated time points (2, 6, and 12 h), and viable bacteria serially plated on BHI plates. The number of recovered bacteria able to invade cells and survive are expressed as means ± SDs for each strain. (b) The transcriptional level changes of the virulence-associated factors (prfA, plcA, plcB, hly, mpl, inlA and inlB) of L. monocytogenes wild-type EGD-e and grx mutant, which were identified by the transcript analysis. Expression of InlB in the wild-type and mutant strains were assayed by Western blotting. (c) Adhesion and invasion of L. monocytogenes in human epithelial cells, Caco-2. Cells infected with L. monocytogenes wild-type EGD-e, and gene deletion and complementation mutants (Δgrx and CΔgrx) at the indicated time points were lysed and viable bacteria serially plated on BHI plates. The number of recovered bacteria able to invade cells and survive are expressed as means ± SDs for each strain. (d) Intracellular multiplication of L. monocytogenes in Caco-2 cells 6 h post infection. Bacteria were detected with anti-Lm (green), and bacteria actin tails and host actin were detected using phalloidin (red), while the cell nucleus was labeled with DAPI (blue). The scale bar is 10 μm. The high-magnification images displayed at the bottom of each image show F-actin (red), bacteria (green), and nuclei (blue). (e) Plaque assay performed on the L929 fibroblast monolayers infected by L. monocytogenes wild-type EGD-e, and gene deletion and complementation mutants (Δgrx and CΔgrx). The plaque numbers of the mutant strains were indicated as a percentage of those formed by the wild-type strain. The mutant strain hly, which is completely unable to spread during cell infection, was taken as a reference negative control. *P<0.05; **P<0.01; ns means no significance. Data are expressed as means ± SDs.