Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract

Background:

The mammalian gut is a complex environment populated by a diverse range of bacteria, the microbiota, which in normal settings provide myriad benefits to the host. Elimination of intestinal pathogens therefore ideally would rely on precise approaches that avoid complete disruption of the microbiota. Interest in phage therapy has renewed internationally in recent years, as phages may naturally show or be engineered to have exquisite specificity for particular bacterial strains. However, the gut environment is highly complex, with the diverse microbes, endogenous resident phages, and food- and host-derived molecules that create distinct spatial and physico-chemical niches that must be accessed for effective phage therapy. In this preprint, Green et al identify a phage strain that overcomes mucin-based inhibition to increase antibacterial killing in the gut environment.

 

Key Findings:

• Mucin inhibits anti-bacterial phages in the gut.

The authors initially examined the efficacy of the previously-characterized HP3 phage against ExPEC (extraintestinal pathogenic E. coli) in the gastrointestinal tract, rather than in systemic bacteremia. However, the phage were ineffective at reducing ExPEC in the gut. In vitro experiments with cecal contents, fractionation, and chemical treatments revealed that mucin was the inhibitory component, limiting efficacy of HP3 in the intestinal environment.

• Discovery of a mucin-enhanced phage, ES17.

The observation that mucin inhibited HP3 (and other phages) prompted the authors to search their phage collection for phage that were effective at killing in mucin-containing media. They identified one phage, ES17, which not only was effective in mucin-containing media, but was actually enhanced by mucin and was ineffective in media alone.

• ES17 targets the host glycan heparin sulfate to localize in the gut.

The authors examined the tail fiber protein of ES17 and showed that it binds mammalian heparin sulfate glycans with high specificity. This enabled them to bind to human intestinal enteroids in a heparin sulfate-dependent manner. Importantly, these potential enhancements manifested as increased efficacy in treatment of intestinal ExPEC infection in mice.

 

Importance:

Previous research had illustrated the potential association of phages with mucus in the gut to provide a host-autonomous form of anti-bacterial immunity, as well as a role for Ig-domain proteins in mediating this association (Barr JJ et al, PNAS 2013). This preprint presents alternative phage mechanisms, including an alternative potential phage receptor (a non-Ig-domain tail fiber protein) and the “bridge receptor” model. These differences, and the screening paradigm presented, highlight the need to consider the precise microenvironment in which a potential phage therapy will function. This study also highlights the potential of the collective “phage genosphere” for identification of phages with incredibly diverse and useful properties, such as mucin-based enhancement of killing.

 

Moving Forward:

1)             What is the inhibitory mechanism of mucin on other phages, such as HP3? Is this mechanism the same as the mechanism by which ES17 is enhanced by mucin? Is the 10 min pre-incubation (as in the adsorption assay) sufficient for gene expression changes in the ExPEC, or is the effect of mucin (both inhibitory/enhancing, for the different phage) most likely due to physical association with the surface?

2)             It will be interesting to understand the ecological consequences of interactions with the mammalian host for phage during “natural” colonization, and how those vary according to mucin-binding mechanism (the “BAM” model vs the bridge-receptor model). How does the balance of phage localization to different layers of the gut (lumen, different levels of mucus, and directly at epithelial cells) impact accessibility of the bacterial “prey” during periods of homeostasis and infection? How are these types of binding activities represented in natural human phage communities (and are they hard to identify in samples like fecal or sewage collections which might be biased *away* from tightly host-associated phages), and what are their relative fitness advantages or disadvantages?

 

doi: https://doi.org/10.1242/prelights.24272

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