P0055 Impact of Indole-3-Propionic Acid on Bacterial Invasion Potential of Klebsiella Strains Isolated from Pediatric Ulcerative Colitis Patients
Authors: Arjomand Fard N , Aujla H , Stuart L , C Cheng C , Elhenawy W , Perry T , Wine E 
Published: January 2025

Abstract

Background
Indole-3-propionic acid (IPA), a tryptophan metabolite produced by microbes, has demonstrated beneficial effects, including reversing dysbiosis, balancing bacterial populations, and reducing inflammation.1,2,3 However, the mechanisms behind these effects and their impact on pathogenic microbes remain unclear. This study aimed to evaluate the effects of IPA on bacterial invasion in vitro, using potential pathobionts (Klebsiella variicola and Klebsiella quasipneumoniae) isolated from non-inflamed sections of the colon in pediatric ulcerative colitis (UC) patients. We hypothesized that IPA could reduce virulence traits, promoting a more homeostatic environment, which could lower inflammation in pediatric UC patients.

Methods
Caco-2 cells (colonic epithelial cell line) were infected with Klebsiella strains, with or without 0.5 mM IPA. Bacterial invasion was assessed via gentamicin protection assay, and qPCR measured Interleukin-8, IL-10, and TNF-α expression in infected cells. Transepithelial electrical resistance (TEER) assay evaluated barrier integrity, and qPCR analyzed the expression of bacterial virulence genes, including entB (siderophores), manC (capsule), and hcp1 (type VI secretion) in IPA-treated and untreated cultures. Additionally, siderophore production was quantified using a Chrome-Azurol Sulfonate (CAS) assay, and Anthony’s stain visualized the bacterial capsule.

Results
IPA-treated Caco-2 cells showed significantly reduced bacterial invasion, with further reductions when bacteria were also exposed to IPA. qPCR revealed a significant decrease in IL-8 expression in IPA-treated cells during infection. IL-10 levels decreased non-significantly, and TNF-α expression showed variable results. A trend toward improved tight junction integrity was observed in the TEER assay with IPA treatment, although no significant differences were detected. IPA treatment increased the expression of entB, manC, and hcp1 genes, except for K. variicola, where manC expression remained unchanged. IPA significantly reduced the capsule thickness of K. variicola and resulted in a significant reduction in K. quasipneumoniae siderophore production.

Conclusion
IPA reduces bacterial invasion and modulates immune responses in Caco-2 cells infected with Klebsiella pathobiont strains. It reduces the virulence of K. quasipneumoniae by targeting siderophore production and diminishes the virulence of K. variicola by reducing capsule formation, suggesting its potential as an anti-virulence agent against these pathobionts. These findings highlight IPA’s potential as an immunomodulatory agent for managing IBD. However, further research is needed into other Klebsiella virulence pathways and host-microbiota interactions.

References
1. Ghiboub M, Verburgt CM, Sovran B, Benninga MA, de Jonge WJ, Van Limbergen JE. Nutritional therapy to modulate tryptophan metabolism and aryl hydrocarbon-receptor signaling activation in human diseases. Nutrients. 2020 Sep 17;12(9):2846.
2. Flannigan KL, Nieves KM, Szczepanski HE, Serra A, Lee JW, Alston LA, Ramay H, Mani S, Hirota SA. The pregnane X receptor and indole-3-propionic acid shape the intestinal mesenchyme to restrain inflammation and fibrosis. Cellular and molecular gastroenterology and hepatology. 2023 Jan 1;15(3):765-95.
3. Fu Y, Gao H, Hou X, Chen Y, Xu K. Pretreatment with IPA ameliorates colitis in mice: Colon transcriptome and fecal 16S amplicon profiling. Frontiers in Immunology. 2022 Sep 8;13:1014881.