Using a human colonoid-derived monolayer to study bacteriophage translocation

ABSTRACT

Bacteriophages (phages) are estimated to be the most abundant microorganisms on Earth. Their presence in human blood suggests that they can translocate from non-sterile sites such as the gastrointestinal tract where they are concentrated. To examine phage translocation ex vivo, we adapted a primary colonoid monolayer model possessing cell diversity and architecture, and a thick layer of mucus akin to the colonic environment in vivo. We show that the colonoid monolayer is superior to the Caco-2 cell-line model, possessing intact and organized tight junctions and generating a physiologically relevant mucus layer. We showed, using two different phages, that translocation across the colonoid monolayer was largely absent in differentiated monolayers that express mucus, unlike Caco-2 cultures that expressed little to no mucus. By stimulating mucus production or removing mucus, we further demonstrated the importance of colonic mucus in preventing phage translocation. Finally, we used etiological drivers of gut permeability (alcohol, fat, and inflammatory cytokines) to measure their effects on phage translocation, demonstrating that all three stimuli have the capacity to amplify phage translocation. These findings suggest that phage translocation does occur in vivo but may be largely dependent on colonic mucus, an important insight to consider in future phage applications.

KEYWORDS:

• Bacteriophage
• phage therapy
• colonoid
• intestinal permeability
• translocation

Acknowledgments

We would like to acknowledge the Pathogen Genomics Group at Westmead Hospital for phage genome sequencing as well as the Cell Imaging and Histology core facilities at the Westmead Institute for Medical Research and Westmead Precinct Hub. In particular, we would like to acknowledge Hong Yu and Hui Zhang for fluorescent microscopy expertise, Emma Kettle for performing electron microscopy and Li Ma and Virginia James for their histology expertise. This project was supported by the Ainsworth Bequest to the School of Medicine of Western Sydney University and the Robert W. Storr Bequest to the Sydney Medical Foundation of the University of Sydney.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2024.2331520

Additional information

Funding

The work was supported by the National Health and Medical Research Council Thyne Reid Foundation Ainsworth Bequest Robert W. Storr Bequest Early-Mid Career Phage Therapy grants (Office for Health and Medical Research; Ministry of Health, New South Wales; Australia MRFF Frontiers Stage 1).