Curcumin increases the pathogenicity of Salmonella enterica serovar Typhimurium in murine model

Abstract

Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Contrary to this, our study reveals that it regulates the defense pathways of Salmonella enterica serovar Typhimurium (S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in Peyer's patches, mesenteric lymph node, spleen and liver, when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. This increased tolerance might be attributed to the up-regulation of genes involved in resistance against antimicrobial peptides--pmrD and pmrHFIJKLM and genes with antioxidant function--mntH, sodA and sitA. We implicate that iron chelation property of curcumin have a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ regulatory system. Curcumin downregulates SPI1 genes, required for entry into epithelial cells and upregulates SPI2 genes required to intracellular survival. Since it is known that the SPI1 and SPI2 system can be regulated by the PhoPQ system, this common regulator could explain curcumin's mode of action. This data urges us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks.

Figure 1. Fold proliferation (2 h to 16 h) of S. Typhimurium.

The cells (RAW 264.7, Intestine 407 & Caco-2) infected with solvent (S), curcumin (C, 20 µM) treated or untreated (UT) S. Typhimurium were lysed at 2 h & 16 h post infection and fold replication of the bacteria was calculated. *** P<0.001.

Figure 2. Curcumin treated S. Typhimurium showed enhanced virulence in murine model of typhoid fever.

A. Bacterial load in different organs of the mice infected with solvent (S) curcumin (C, 20 µM) treated or untreated (UT) S. Typhimurium. 3-days post-infection, different organs of infected mice were aseptically isolated, weighed, homogenized and plated to get the CFU/gm. Cecum weight was plotted for mice infected intragastrically. B. Survival of mice after infection with either curcumin treated or untreated S. Typhimurium. ** 0.001≤P<0.01 and * 0.01≤P<0.05.

Figure 3. Modulation of SPI2 genes by curcumin.

A. Promoter assay for spiC gene using a reporter strain (WT harbouring lacZ fusion of spiC promoter in pHG86 plasmid) and immunoblot for SseB protein. β-galactosidase assay was done at different time points for bacteria, subcultured (1∶33) from overnight culture in F-media (pH 5) or isolated from RAW 264.7 cells. B. Fold proliferation of WT and phoP^- S. Typhimurium. RAW 264.7 infected with curcumin (C, 20 µM) treated or untreated (UT) S. Typhimurium were lysed after 2 h & 16 h and fold replication of the bacteria was calculated.

Figure 4. Regulation of antioxidant genes by curcumin to improve resistance of bacteria against oxidative stress.

Survival of S. Typhimurium grown in presence or absence of curcumin (20 µM) and iron (32 µg/ml FeCl₃ or 20 µM FeSO₄) against1 mM H₂O₂ (A, D) or NaNO₂ (B, E). C. Effect of curcumin on the transcriptional activities of mntH, sitA, sodA genes. β-galactosidase assay was performed 6 h post-incubation of S. Typhimurium harboring either sitA or sodA-lacZ construct in presence or absence of curcumin (20 µM) and iron (32 µg/ml FeCl₃ or 20 µM FeSO₄) in LB. F & G. Intracellular proliferation of curcumin treated and untreated bacteria in RAW 264.7 and NRAMP^+/+ BMDM respectively. *** P<0.001, ** 0.001≤P<0.01 and * 0.01≤P<0.05. UT- untreated, C- curcumin treated, ΔsitA – sitA knockout and sitA^c – sitA complement in ΔsitA.

Figure 5. Sensitivity of S. Typhimurium to antimicrobial peptides.

Survival of S. Typhimurium grown in presence (C) or absence (UT) of curcumin (20 µM) and iron (32 µg/ml FeCl₃ or 20 µM FeSO₄) against A. Polymyxin B (1 µg/ml) and B. Protamine (50 µg/ml). C. Membrane potential assay. The polymyxinB or protamine treated S. Typhimurium were stained with DiBAC₄ (3) and analysed in FACS scanner.

Figure 6. Regulation of pmr genes by curcumin to improve resistance of bacteria against AMPs.

A. Effect of curcumin on the transcriptional activities of pmrD and pmrHFIJKLM genes. β-galactosidase assay was performed 3 h post-incubation of S. Typhimurium harboring either pmrD or pmrHFIJKLM -lacZ construct in presence or absence of curcumin (20 µM) and iron (32 µg/ml FeCl₃ or 20 µM FeSO₄) in LB. B. Susceptibilty of phoP^- strain to polymyxin B. C. Intracellular survival assay of phoP^- strain in Intestine 407 cells. The Intestine 407 cells infected with solvent (S), curcumin (C, 20 µM) treated or untreated (UT) S. Typhimurium were lysed at 2 h & 16 h post infection and fold replication of the bacteria was calculated. *** P<0.001, ** 0.001≤P<0.01 and * 0.01≤P<0.05.

Figure 7. Curcumin treated bacteria show reduced entry in-vitro but not in-vivo.

A. The entry of curcumin (C, 20 µM) treated and untreated (UT) S. Typhimurium in Intestine 407 cells. The entry in Intestine 407 cells was determined by lysing the infected cells 30 min post-infection. B. CFU per gram weight of Peyer's patch of mice infected intragastrically with curcumin-treated and untreated S. Typhimurium. 1 h post-infection Peyer's patch were aseptically isolated, processed to get CFU/gm. C. Effect of curcumin on the transcriptional activity of hilA and sopD promoter. β-galactosidase activity was performed using S. Typhimurium harboring either hilA or sopD -lacZ construct grown in presence or absence of curcumin to know the transcriptional activity. D. RT-PCR analysis to check the expression of hilA in curcumin-treated and untreated S. Typhimurium. RT-PCR with the mRNA isolated from curcumin treated and untreated S. Typhimurium was performed. E. Percentage survival of bacteria in the mucus from small intestine of mice. Curcumin-treated and untreated bacteria were inoculated in the mucus for 1 h and their survival calculated. *** P<0.001 and * 0.01≤P<0.05.

Figure 8. Schematic summary representing the mode of action of curcumin.

Iron chelation caused by curcumin might increase the expression of different genes involved in defense of S. Typhimurium against host immune response.