Figures & data
Figure 1. Role of lon in Adhesion and Invasion of HeLa and HepG2 and macrophage survival.
(A) Adhesion and Invasion of HeLa, HepG2, and Caco-2 monolayer were observed post-infection with JOL 401, JOL 909, and JOL 909::lon (MOI = 20). The data represent the adhesion and invasion (Log CFU/mL) ± the SD from at least three independent experiments performed in duplicates. P values of <0.05 were considered significantly different, and bars with the same lowercase letter are not significantly different from one another. (B) Fluorescent microscopy images of RAW cells infected with Salmonella (JOL 401, JOL 909, and JOL 909::lon) containing pHLJ65::gfp plasmid to visualize the live (green fluorescent) bacteria within the cells. (C) Intracellular survival of Salmonella wild type, lon mutant, and complemented strain in RAW cells.
Figure 2. Actin polymerization is a distinctive attribute of individual host cells.
Microscopic images show membrane ruffling and lamellipodia formation. (A) HeLa cells (B) HepG2 cells (C) Caco-2 cells were infected with stress exposed Salmonella (JOL 401, JOL 909, and JOL 909::lon; MOI = 20) for 30 min and stained with phalloidin (for F-actin) and DAPI (nuclear stain);1-actin condensation,2-lamellipodia. These observations demonstrate that Salmonella enters the HeLa cells vigorously rearranging the host actin filaments via a trigger mechanism. The interaction with HepG2 cells results in a zipper-type of actin rearrangement. The interaction with Caco-2 cells results in a minimum ruffling of the membrane.
Figure 3. Endogenous Hydroxyl radical (OH·) formation during stress treatments in the lon mutant.
JOL 401, JOL 909 and JOL 909::lon were exposed to different abiotic stress (cold, oxidative, osmotic, and acidic) for 5 h and their endogenous hydroxyl radical levels were measured and the values were compared with the bacterial cells grown at 37ºC without any stress inducers. The data was represented as relative OH• formation ± the SD from at least three independent experiments performed in duplicates. Means were compared by Tukey’s multiple comparison test. Similar lowercase letters indicate the non-significant difference of means (p > .05). Significant difference (*p < .05, ***p < .0001) compared to the wild type control.
Figure 4. Expression of bacterial cell surface protein genes during stress treatments.
JOL 401, JOL 909 and JOL 909::lon were subjected to abiotic (oxidative-1 mM H2O2; acidic-3.0 pH;osmotic- 5% NaCl and cold-4ºC) stress treatment for 5 h. The regulation of the ompF, ompD, fepA, and csgB were evaluated post-stress treatments. The results are expressed as mean fold change in the transcription levels of ompF, ompD, fepA, and csgB genes compared to unstimulated bacterial cells. The data was represented as relative mean fold change ± the SD from at least three independent experiments performed in duplicates. Means were compared by Tukey’s multiple comparison test. Similar lowercase letters indicate the non-significant difference of means (p > .05). Significant difference (*p < .05, **p < .01, ***p < .0001) compared to the wild type control. ompD- Outer membrane protein D; ompF- Outer membrane protein F; fepA- Ferric Enterobactin Transporter; csgB- Minor Curlin subunit.
Figure 5. Expression of regulatory genes that participate in iron uptake and sequestration during stress exposure.
Post-exposure to the abiotic stress factors the transcription levels of sitA, dps, and fur were evaluated in JOL 401, JOL 909, JOL 909::lon, JOL 2768, and JOL 2769. The results are expressed as mean fold change in the transcription levels of sitA, dps, and fur genes compared to unstimulated bacterial cells. The data was represented as relative mean fold change ± the SD from at least three independent experiments performed 2compared by Tukey’s multiple comparison test. Similar lowercase letters indicate the non-significant difference of means (p > .05). Significant difference (*p < .05, **p < .01) compared to the wild type control.
Figure 6. Regulation of Magnesium uptake associated genes.
The mRNA expression of Magnesium uptake genes was studied after the bacterial cells were subjected to stress treatments for 5 h. The mean fold change in the levels of corA, mgtA, and mgtB in JOL 401, JOL 909, and JOL 909::lon were recorded. The data was represented as relative mean fold change ± the SD from at least three independent experiments performed in duplicates. Means were compared by Tukey’s multiple comparison test. Similar lowercase letters indicate the non-significant difference of means (p > .05). Significant difference (*p < .05, **p < .01) compared to the wild type control.
Figure 7. Expression of a virulence-associated gene from SPI 1 and 2 gene clusters.
mRNA levels of (A) SPI1 genes-invF and hilC and (B)SPI2 genes- sifA and sseJ were recorded as relative mean fold changes using rrsG as the endogenous control. The data was represented as relative mean fold change ± the SD from at least three independent experiments performed in duplicates. Means were compared by Tukey’s multiple comparison test. Similar lowercase letters demonstrate the non-significant difference of means (p > .05). Significant difference (*p < .05, **p < .01, ***p < .001) or no significant difference (nsp>.05) compared to the wild type control.
Figure 8. Bacterial recovery and survival to study the involvement of lon in bacterial longevity.
(A) In vitro bacterial survival post-stress exposure was evaluated by serial dilution and plating of stress endured JOL 401, JOL 909, and JOL 909::lon on BGA plate. (B) The bacterial recovery was calculated by plating the serially diluted homogenized spleen (1 g) on BGA plates. (C) The spleens were collected aseptically from mice (n = 3; infected with JOL 401, JOL 909, and JOL 909::lon) on days 1, 6, 9, and 12 post-infection for evaluation of gross pathological lesions including splenomegaly. The data was represented as log CFU/mL± the SD from at least three independent experiments performed in duplicates. Means were compared by Tukey’s multiple comparison test. Significant difference (*p < .05, **p < .01, ***p < .001) compared to the wild-type control.
Figure 9. The kinetics of cytokine gene expression in the phagocytic and non-phagocytic epithelial cells.
The IL-2, IL-4, IFN-γ, and IL-10 mRNA expression in JOL 401, JOL 909, and JOL 909::lon infected HeLa, HepG2, and RAW cells were evaluated at various time points post-infection. The data was represented as relative mean fold change ± the SD from at least three independent experiments performed in duplicates. The experiment was performed using GAPDH as the endogenous control. IL- Interleukin; IFN- Interferon. Means were compared by Tukey’s multiple comparison test. Similar lowercase letters indicate the non-significant difference of means (p > .05). Significant difference (*p < .05, **p < .01, ***p < .001) compared to the wild-type control.
Table 1. List of bacterial strains and plasmids used in this study.
Table 2. List of primers used in this study.
Supplemental material
Supplemental Material
Download Zip (382.1 KB)Data availability statement
The fluorescent microscopy imaging conducted for Salmonella mediated actin cytoskeleton rearrangement compiled and has been deposited in the Mendeley Data repository and can be accessed using the following link. Lee, John Hwa (2020), “Salmonella mediated actin condensation upon entry into HeLa and HepG2 cells”, Mendeley Data, V1, doi: 10.17632/czng5n4fdk.1.