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
:1. Introduction
2. Results
2.1. ST Mutants (JOL 912 and JOL 1800) Exposed to Abiotic Stress Remain Invasive in Both Eukaryotic Cell Lines
2.2. Fenton Reaction: Free Radical Production Is Dependent on Gene Knockout and pH
2.3. Invasion of HeLa and HepG2 by ST Causes Actin Rearrangement
2.4. ST Localization and Intracellular Spread
2.5. Expression of Cytokine, Cyclin, and Caspase Transcripts
2.6. Effect on Cell Cycle Kinetics Demarcates G2/M Arrest
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains
4.2. Cell Culture
4.3. Bacterial Growth under Stress Conditions
4.4. Bacterial Invasion and Intracellular Replication
4.5. Hydroxyl Radical (OH·) Measurement
4.6. Effect on Actin during ST Interaction for Invasion
4.7. Localization of ST in the HeLa and HepG2 Cells
4.8. Expression of Cytokines, Cyclin, and Apoptosis Genes
4.9. Real-Time Quantitative PCR (qPCR) Analysis
4.10. Cell Cycle Analysis by Flow Cytometer
4.11. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Bacteria | Features |
---|---|
JOL 401 | Salmonella Typhimurium wild type, SPI-1 invAE+, hilA+, avr+; SPI-2, amino acid permease; SPI-3, mgtC+; SPI4, ABC transporter; SPI5, pipB+ |
JOL 911 | JOL 401 ∆lon, ∆cpxR; smooth ST strain |
JOL 912 | JOL 401 ∆lon, ∆cpxR, Δasd; smooth ST strain |
JOL 1800 | JOL 912 ∆rfaL, O-antigen deficient strain, improved bacterial delivery vector; rough ST strain |
Gene | Primer | Sequence (5′ → 3′) |
---|---|---|
Nuclear factor-κB | Forward | TGGGACCAGCAAAGGTTATT |
Reverse | GATCCCATCCTCACAGTGTTT | |
Interferon-γ | Forward | ATGTCCAACGCAAAGCAATAC |
Reverse | ACCTCGAAACAGCATCTGAC | |
Cyclin D1 | Forward | CATCTACACCGACAACTCCATC |
Reverse | TCTGGCATTTTGGAGAGGAAG | |
Cyclin B1 | Forward | GGCTTTCTCTGATGTAATTCTTGC |
Reverse | GTATTTTGGTCTGACTGCTTGC | |
Cyclin B2 | Forward | CCTCCCTTTTCAGTCCGC |
Reverse | CTCCTGTGTCAATATTCTCCAAATC | |
Caspase 3 | Forward | AATGGACCTGTTGACCTGAAA |
Reverse | CACGGCAGGCCTGAATAA | |
Caspase 7 | Forward | CTGACTTCCTCTTCGCCTATTC |
Reverse | TCTGCATGATTTCCAGGTCTT | |
CXCL 8 | Forward | GAGAGTGATTGAGAGTGGACCAC |
Reverse | CACAACCCTCTGCACCCAGTTT | |
CXCL 10 | Forward | GGTGAGAAGAGATGTCTGAATCC |
Reverse | GTCCATCCTTGGAAGCACTGCA | |
GAPDH | Forward | CCCTTCATTGACCTCAACTACA |
Reverse | ATGACAAGCTTCCCGTTCTC |
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Kirthika, P.; Senevirathne, A.; Park, S.; Aganja, R.P.; Kim, I.-S.; Tae, H.-J.; Lee, J.H. 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. Int. J. Mol. Sci. 2024, 25, 9056. https://doi.org/10.3390/ijms25169056
Kirthika P, Senevirathne A, Park S, Aganja RP, Kim I-S, Tae H-J, Lee JH. 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. International Journal of Molecular Sciences. 2024; 25(16):9056. https://doi.org/10.3390/ijms25169056
Chicago/Turabian StyleKirthika, Perumalraja, Amal Senevirathne, Sungwoo Park, Ram Prasad Aganja, In-Shik Kim, Hyun-Jin Tae, and John Hwa Lee. 2024. "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" International Journal of Molecular Sciences 25, no. 16: 9056. https://doi.org/10.3390/ijms25169056