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. 2018 Jan 17;13(1):e0190613.
doi: 10.1371/journal.pone.0190613. eCollection 2018.

The microbiota metabolite indole inhibits Salmonella virulence: Involvement of the PhoPQ two-component system

Nandita Kohli  1 , Zeni Crisp  2 , Rebekah Riordan  2 , Michael Li  1 , Robert C Alaniz  2 , Arul Jayaraman  1   2
Affiliations

The microbiota metabolite indole inhibits Salmonella virulence: Involvement of the PhoPQ two-component system

Nandita Kohli et al. PLoS One. .

Abstract

The microbial community present in the gastrointestinal tract is an important component of the host defense against pathogen infections. We previously demonstrated that indole, a microbial metabolite of tryptophan, reduces enterohemorrhagic Escherichia coli O157:H7 attachment to intestinal epithelial cells and biofilm formation, suggesting that indole may be an effector/attenuator of colonization for a number of enteric pathogens. Here, we report that indole attenuates Salmonella Typhimurium (Salmonella) virulence and invasion as well as increases resistance to colonization in host cells. Indole-exposed Salmonella colonized mice less effectively compared to solvent-treated controls, as evident by competitive index values less than 1 in multiple organs. Indole-exposed Salmonella demonstrated 160-fold less invasion of HeLa epithelial cells and 2-fold less invasion of J774A. 1 macrophages compared to solvent-treated controls. However, indole did not affect Salmonella intracellular survival in J774A. 1 macrophages suggesting that indole primarily affects Salmonella invasion. The decrease in invasion was corroborated by a decrease in expression of multiple Salmonella Pathogenicity Island-1 (SPI-1) genes VSports手机版. We also identified that the effect of indole was mediated by both PhoPQ-dependent and independent mechanisms. Indole also synergistically enhanced the inhibitory effect of a short chain fatty acid cocktail on SPI-1 gene expression. Lastly, indole-treated HeLa cells were 70% more resistant to Salmonella invasion suggesting that indole also increases resistance of epithelial cells to colonization. Our results demonstrate that indole is an important microbiota metabolite that has direct anti-infective effects on Salmonella and host cells, revealing novel mechanisms of pathogen colonization resistance. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. In vivo competition assays in C57BL/6 mice with indole treated Salmonella.
Competitive index (CI) values for the indole treated Salmonella versus the control in different organs harvested from infected mice (n = 5) at days 1 and 3 post inoculation. Two inoculum doses were tested- low dose (LD; ~5 × 107 cfu) and high dose (HD; ~5 × 108 cfu) and several organs—cecum, Peyer’s patches, spleen, liver and mesenteric lymph nodes—were harvested. Feces were collected prior to euthanization. The organs were homogenized and serial dilutions plated to obtain cfu counts that were used to calculate the CI values. Each symbol (circle, square, upright triangle and downward triangle) on the plot represents a mouse from the respective group (LD day 1, LD day 3, HD day 1 and HD day 3, respectively). Lack of symbol indicates that no colonies were observed with that sample. For organs where indole treated Salmonella were absent but solvent treated Salmonella were present, CI was calculated assuming a cfu of 1 for the indole treated Salmonella. The horizontal bar represents the median of the observed CI values. * denotes significantly lower (p < 0.05) recovery of indole-treated Salmonella relative to solvent-treated Salmonella, as represented by the plotted CI values, using the Wilcoxon matched pair test.
Fig 2
Fig 2. Effect of indole on Salmonella swimming motility at 37°C.
(A) Representative photographs of the swimming motility agar plates spotted with WT Salmonella. (B) Measured halo diameters for the different test conditions. Diameters were measured using Vernier calipers, 8 hours post spotting. ΔmotA was spotted on swimming motility agar plates as a negative control for motility. * denotes statistical significance relative to the solvent control at p < 0.05 using the Student’s t-test. Column bars depict mean (n = 4) and error bars represent standard deviation (SD).
Fig 3
Fig 3. Invasion of epithelial cells and invasion and intracellular survival within macrophages with indole-treated Salmonella.
Invasion in HeLa epithelial cell line (A) with Salmonella treated with or without 1mM indole. Invasion (B) and intracellular survival (C) in J774A.1 cells. Infection with the ΔSPI-1 and ΔSPI-2 strains were used as controls. A MOI of 50:1 was used for HeLa cells and a MOI of 10:1 was used for J774A.1 macrophages. Data shown are intracellular bacteria recovered and fold changes in survival (at 4 and 8 h post invasion) relative to the invasion. * denotes statistical significance relative to the solvent control at p < 0.05 using the Student’s t-test. Column bars depict mean (n = 3) and error bars represent standard deviation (SD).
Fig 4
Fig 4. SPI-1 virulence gene expression change in WT Salmonella upon treatment with 1 mM indole.
SPI-1 reporter strains for hilA, prgH, invF and sipC were treated overnight with and without 1 mM indole and the ß-gal activity was measured in exponential phase cultures after dilution. Data shown are the mean fold decrease in expression (n = 3) with indole-treatment relative to the solvent-treated control at a significance level of p < 0.05 using the Student’s t-test. Error bars represent SD.
Fig 5
Fig 5. Role of phoPQ in indole mediated down-regulation of virulence.
(A) SPI-1 virulence gene expression using ß-gal assay. The ΔphoPQ mutation was generated in the four SPI-1 reporter strains for hilA, prgH, invF and sipC. The WT and the ΔphoPQ reporter strains were treated overnight with and without 1 mM indole and the ß-gal activity was measured in exponential phase cultures after dilution. Data shown are the fold decrease in expression with indole-treatment relative to the solvent-treated control. * denotes statistical significance relative to the WT strain at p < 0.05 using the Student’s t-test. (B) Invasion in HeLa epithelial cell line with Salmonella WT and ΔphoPQ strain treated with or without 1mM indole. A MOI of 100:1 was used and the data shown is the indole-treated Salmonella invasion normalized to the control of the respective strain. * denotes statistical significance with respect to the solvent control at p < 0.05 using the Student’s t-test. Column bars depict mean (n = 3) and error bars represent standard deviation (SD).
Fig 6
Fig 6. Effect of indole in combination with cecal SCFAs on hilA expression.
SPI-1 reporter strain for hilA was treated overnight with and without indole (100 μM and 250 μM) in the presence of 200 mM cecal SCFAs or 200 mM NaCl, and the ß-gal activity was measured in exponential phase cultures after dilution. Data shown are the mean fold decrease (n = 3) in expression of hilA with treatment relative to the control: hilA expression in presence of 200 mM NaCl. * denotes statistical significance relative to cecal SCFAs alone at p < 0.05 using the Student’s t-test. Error bars represent SD.
Fig 7
Fig 7. Effect of indole on colonization resistance in HeLa epithelial cells.
HeLa cells were seeded in a 24 well plate and conditioned with 1 mM indole for 24 h prior to infection. A MOI of 10:1 was used for infection. Data shown are intracellular bacteria recovered from infected HeLa monolayers with indole treatment or control (solvent treatment). * denotes statistical significance relative to the solvent control at p < 0.05 using the Student’s t-test. Column bars depict mean (n = 3) and error bars represent SD.
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