Intracellular Survival and Pathogenicity Modulation of Salmonella Lon, CpxR, and RfaL Mutants Used as Live Bacterial Vectors under Abiotic Stress, Unveiling the Link between Stress Response and Virulence in Epithelial Cells

Abstract

In the current study, two Salmonella Typhimurium strains, JOL 912 and JOL 1800, were engineered from the wild-type JOL 401 strain through in-frame deletions of the lon and cpxR genes, with JOL 1800 also lacking rfaL. These deletions significantly attenuated the strains, impairing their intracellular survival and creating unique immunological profiles. This study investigates the response of these strains to various abiotic stress conditions commonly experienced in vivo, including temperature, acidity, osmotic, and oxidative stress. Notably, cold stress induced a non-significant trend towards increased invasion by Salmonella compared to other stressors. Despite the observed attenuation, no significant alterations in entry mechanisms (trigger vs. zipper) were noted between these strains, although variations were evident depending on the host cell type. Both strains effectively localized within the cytoplasm, demonstrating their ability to invade and interact with the intracellular environment. Immunologically, JOL 912 elicited a robust response, marked by substantial activation of nuclear factor kappa B (NF-kB), and chemokines, interleukin 8 (CXCL 8) and interleukin 10 (CXCL 10), comparable to the wild-type JOL 401 (over a fourfold increase compared to JOL 1800). In contrast, JOL 1800 exhibited a minimal immune response. Additionally, these attenuations influenced the expression of cyclins D1 and B1 and caspases 3 and 7, indicating cell cycle arrest at the G2/M phase and promotion of the G0/G1 to S phase transition, alongside apoptosis in infected cells. These findings provide valuable insights into the mechanisms governing the association, internalization, and survival of Salmonella mutants, enhancing our understanding of their regulatory effects on host cell physiology.

Keywords: NF-kB; Salmonella Typhimurium; abiotic stress; cell cycle arrest; chemokines; intracellular survival.

Figure 1

Invasion and intracellular replication in HeLa and HepG2 cells by Salmonella Typhimurium, JOL 401, JOL 912, and JOL 1800. Cells were infected with Salmonella WT, and mutants subjected to cold, oxidative, osmotic, and acidic stress conditions at an MOI of 5, and afterward, medium without gentamicin was added for 1 h. Next, this medium was replaced by a medium containing gentamicin at 10 µg/mL. Intracellular replication was calculated by taking the ratio of CFU recovered 8 h post-infection (hpi) to 1 hpi. Mean values and standard deviations (SD) obtained from three independent experiments are demonstrated. The level of significance was indicated as * p < 0.05; ** p < 0.01.

Figure 2

Kinetics of Fenton reaction in Salmonella Typhimurium-, JOL 401-, JOL 912-, and JOL 1800-stimulated HeLa and HepG2 Cells. Results are expressed RLUs in ST-stimulated Cells compared to unstimulated cells. Cells treated with 10 µM CuCl₂ and 100 µM H₂O₂ at 37 °C for 1 h were served as a positive control, whereas non-treated cells served as a negative control. Mean values and standard deviations (SDs) obtained from three independent experiments are demonstrated. The levels of significance are indicated as ** p < 0.05. “hpi” stands for hours post-infection, “NS” stands for non-significant.

Figure 3

Trigger mechanisms used by Salmonella to HeLa cells. HeLa cells were infected with WT JOL 401 and mutant strains JOL 912 and JOL 1800, subjected to various stress conditions for 90 min, fixed, and labeled with phalloidin to visualize F-actin (green in merged images), and DAPI was used to label the nucleus (blue color). Images are micrographs of representative infected cells. 1. Accumulation of polymerized actin. 2. Membrane rearrangement and cellular protrusions are depicted.

Figure 4

Zipper mechanisms used by Salmonella to HepG2 cells. HepG2 cells were infected with WT JOL 401 and mutant strains JOL 912 and JOL 1800, subjected to various stress conditions for 90 min, fixed, and labeled with phalloidin to visualize F-actin (green in merged images), and DAPI was used to label the nucleus (blue color). Images are micrographs of representative infected cells. 1. Accumulation of polymerized actin. 2. Sites of milder actin remodeling. Yellow arrows indicate intracellular Salmonella.

Figure 5

Localization of Salmonella in HepG2 cells. The mechanisms that direct the invading bacterium to follow the cytosolic or intra-vacuolar “pathway” remain poorly understood. In vitro studies show a predominance of either the cytosolic or the intra-vacuolar population depending on the host cell type invaded by the pathogen. 1. Sites of bacterial accumulations. White arrows indicate their spread within the cytoplasm, either scattered or in clusters.

Figure 6

Modulation of cytokines and chemokines owing to Salmonella WT and mutant infection. Expression of NF-κB, IFN-γ, CXCL 8, and CXCL 10 messenger RNA (mRNA) expression in Salmonella Typhimurium-, JOL 401-, JOL 912-, and JOL 1800-stimulated HepG2 Cells after 12 h incubation. Results are expressed as mean fold change in cytokine transcripts in ST-stimulated cells to unstimulated cells. Mean values and standard deviations (SDs) obtained from two biological replicates are demonstrated. The level of significance is indicated as * p < 0.05.

Figure 7

Expression of cyclin D1, cyclin D2, cyclin B1, and cyclin B2 messenger RNA (mRNA) expression in Salmonella Typhimurium- and JOL 401-stimulated (A) HeLa and (B) HepG2 Cells. Results are expressed as mean fold change in cytokine transcripts in ST-stimulated cells at various hpi compared to unstimulated cells. Mean values and standard deviations (SDs) obtained from two biological replicates are demonstrated. The level of significance is determined at p < 0.05.

Figure 8

Expression of caspase 3 and caspase 7 messenger RNA (mRNA) expression in Salmonella Typhimurium- and JOL 401-stimulated (A) HeLa cells and (B) HepG2 Cells. Results are expressed as mean fold change in gene transcripts in ST-stimulated cells compared to unstimulated cells. Mean values and standard deviations (SD) obtained from three independent experiments are demonstrated. The level of significance was determined at p < 0.05.

Figure 9

Cell cycle analysis by propidium iodide staining. (A) HepG2 and HeLa cells were infected with JOL 401, JOL 912, and JOL 1800. The effect of infection on the cell cycle was investigated using the PI staining method. Cells were gated to eliminate the dead cell population, and the remaining cells were assessed for red fluorescence. A histogram was prepared and the effect against each treatment relative to G0/G1, S, and G2/M phases. Encaged section demarcates induced apoptotic cell population. (B) Proportions of each G0/G1, S, and G2/M cell population were quantified and presented as a bar graph. Mean values and standard deviations (SDs) obtained from two biological replicates are demonstrated. The level of significance is determined at p < 0.05.