Epithelial Cell Contact is Critical for Probiotic Action

Arun K. Bhunia and Rishi Drolia

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The transient nature of probiotic bacteria in the gut prevents them from exerting their full health benefits. Persistence and stable colonization can happen when a probiotic bacterium finds a receptor on the host cells to cling on. Surface layer protein or other surface molecules is known to provide an advantage for some Lactobacilli to interact with host cell receptors such as toll-like receptors1.  Such natural ligand-receptor interaction is inconsistent and is often ineffective. Moreover, probiotics are claimed to compete with pathogens for adhesion sites2, however, there is no direct mechanistic evidence of such a phenomenon1. Therefore, an engineering approach to modify a probiotic to express proteins that specifically target a host cell receptor may be a solution to improve probiotic functionality.

We cloned and expressed a well-characterized Listeria adhesion protein (LAP)3,4 from a nonpathogenic Listeria on the surface of Lactobacillus casei, a probiotic bacterium5. LAP binds to the host cell receptor, heat shock protein 60 (Hsp60), a chaperone protein, and its cell membrane expression is linked to stress or diseased condition6,7.  The resulting bioengineered Lactobacillus casei probiotic (BLP) strain expressing LAP forms a biofilm-like structure that restricts Listeria interaction with the gut epithelial cells (Figure 1). 

Figure 1. Bioengineered Lactobacillus probiotic (BLP) forms a biofilm-like structure that restricts Listeria. BLP strain colonizes and forms biofilm-like structure (brown-colored biofilm) on top of villous epithelial cells (left panel) of mouse colon and prevents Listeria monocytogenes interaction (red arrows, left panel). In contrast, parental Lactobacillus casei (LbcWT) do not restrict Listeria interaction with villus epithelial cells thus invaded Listeria are found in the deeper tissues (right panel, pink arrows).  

This demonstrates the ability of BLP to pass through the layers of gut microbiota and the loosely and tightly adherent mucus to interact directly with epithelial cells. The BLP restricts Listeria by interacting with Hsp60, the cognate epithelial surface receptor of LAP (Figure 2). 

Figure 2. BLP Interacts with surface Hsp60 to restrict Listeria. Images showing the interaction of BLP (a, green, yellow arrows) with surface Hsp60 (white) expressed at the cell-periphery (red, ZO-1), in human Caco-2 cell line. Images showing the interaction of BLP (b, red) with surface Hsp60 (white, yellow arrows), blocking Listeria monocytogenes (green, white arrows). 

Intimate probiotic contacts with the epithelial cells not only prevent the interaction of Listeria monocytogenes with the epithelial Hsp60 receptor and lethality (92% survival rate) but also augments immunomodulatory function and maintain epithelial barrier integrity (Figure 3). In contrast, parental Lactobacillus strain is ineffective.

Figure 3. Schematics showing the mechanism of BLP-mediated protection against listeriosis. The BLP prevents Lm infection by three mechanisms (i) competitive exclusion, (ii) improved intestinal barrier function and (iii) contact-dependent immunomodulation.  

 This study thus offers direct evidence that rational engineering of probiotic strains could outcompete and diminish the colonization of the pathogens by competing for the receptor binding adhesion sites. Because of its specific interaction with Hsp60, this bioengineered probiotic strain has the potential to exert immunomodulatory action to alleviate symptoms associated with a variety of chronic inflammatory diseases of the gut where Hsp60 expression is very high6.  

You can read about all details of our study in the full paper here.

References

  1. Plaza-Diaz, J., Ruiz-Ojeda, F.J., Gil-Campos, M. & Gil, A. Mechanisms of Action of Probiotics. Adv. Nutr. 10, S49-S66 (2019).
  2. Amalaradjou, M.A.R. & Bhunia, A.K. Modern approaches in probiotics research to control foodborne pathogens. Adv. Food Nutr. Res. 67, 185-239 (2012).
  3. Drolia, R., Tenguria, S., Durkes, A.C., Turner, J.R. & Bhunia, A.K. Listeria adhesion protein induces intestinal epithelial barrier dysfunction for bacterial translocation. Cell Host & Microbe 23, 470-484 (2018).
  4. Drolia, R., & Bhunia, A. K. Crossing the intestinal barrier via Listeria adhesion protein and internalin A. Trends in Microbiology27 (5), 408-425 (2019).
  5. Drolia, R., Amalaradjou, M.A.R., Ryan, V. et al. Receptor-targeted engineered probiotics mitigate lethal Listeria infection. Nat Commun 11, 6344 (2020). https://doi.org/10.1038/s41467-020-20200-5. 
  6. Cappello, F. et al. Hsp60 as a Novel Target in IBD Management: A Prospect. Front. Pharmacol. 10 (2019).
  7. Burkholder, K.M. & Bhunia, A.K. Listeria monocytogenes uses Listeria adhesion protein (LAP) to promote bacterial transepithelial translocation, and induces expression of LAP receptor Hsp60. Infect. Immun. 78, 5062-5073 (2010).

 

 

Arun K. Bhunia

Prof, Purdue University