Distinct effects of mucin on phage-host interactions in model systems of beneficial and pathogenic bacteria

Abstract

Phage-host interactions that occur in host-associated microbiomes are influenced by a plethora of environmental factors. Mucins are glycoproteins that represent the main component of mucus, which is found in the animal digestive tract and on the surface of certain organs, serving as the first line of defense against toxins and pathogens. Previous studies have shown that lytic phages have an important influence on the microbial composition in mucosal areas. Our study expands this knowledge to interactions between previously untested lytic phages targeting probiotic and pathogenic bacteria, as well as temperate phages targeting probiotic bacteria. These interactions could be important in shaping microbial communities and affecting the well-being of their host. This study demonstrates that mucins enhance the adherence of Vibrio anguillarum lytic phages and Bacillus subtilis lytic and temperate phages, as well as B. subtilis and V. anguillarum cells, to solid surfaces. Our results also show that mucins positively affect the attachment of B. subtilis cells even in the presence of phages. This positive effect was not observed in the case of V. anguillarum. This suggests that mucin may shield certain bacteria from phage infections. We also found that mucin influenced the metabolic activity of the two tested bacterial species differently, with strong positive effects on V. anguillarum but not on B. subtilis. This work supports previous findings that phages adhere efficiently to mucus and extends these studies to include other beneficial and pathogenic bacterial species. It also reveals that mucins have different effects on phage-host interactions in different phage-host systems, which may have implications for phage therapies or probiotic treatment strategies.

Fig. 1

Attachment of phages Nf, SP10, SPꞵ, and KVP40 to mucin. (A) Attachment of phage Nf to mucin compared to control surface (LB agar) (n = 18). (B) Attachment of phage SP10 to mucin compared to control surface (LB agar) (n = 10). (C) Attachment of phage SPꞵ to mucin compared to control surfaces (LB agar and BSA) (n = 12). (D) Attachment of phage KVP40 to mucin compared to control surfaces (LB agar and BSA) (n = 4). Values are normalized to the maximum within a single experiment. Statistical analysis was performed using ANOVA with Tukey’s post hoc test (*, p < 0.05; ***, p < 0.001).

Fig. 2

Number of B. subtilis and V. anguillarum cells attached to different surfaces, including an uncoated agar surface, a PBS-coated surface, a BSA-coated surface, and a mucin-coated surface. Statistical significance was assessed using ANOVA with Tukey’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (A) Counting of B. subtilis cells by flow cytometry after 6 hours of incubation on the plates (n = 3). (B) Counting of V. anguillarum cells by flow cytometry after 6 hours of incubation on the plates (n = 6). (C) Fluorescence, DIC, and merged microscopy images of B. subtilis cells attached to different surfaces (uncoated 1.5% agar, BSA-coated surface, and mucin-coated surface). Top, fluorescence microscopy images showing GFP-expressing cells (TL); middle, DIC; bottom, merged image. Arrows indicate the adhered cells where the fluorescent signal was obstructed. The scale bar in the upper left corner represents 50 µm. (D) Counting of B. subtilis by microscopy after 1 hour of incubation on the plates (n = 15 fields of view).

Fig. 3

Oxygen consumption rate and regrowth of B. subtilis and V. anguillarum cells attached to three surfaces: an uncoated agar surface, a PBS-coated surface, and a mucin-coated surface. Statistical significance was assessed using ANOVA with Tukey’s post hoc test. **, p < 0.01; ***, p < 0.001. (A) Graphical representation of the experiment. (B) Activity of B. subtilis cells that were allowed to attach for 6 hours (n = 4). (C) Activity of V. anguillarum cells that were allowed to attach for 6 hours (n = 8). (D) Number of regrown B. subtilis cells from cells attached to different surfaces (n = 8). (E) Number of regrown V. anguillarum cells from cells attached to different surfaces (n = 8).

Fig. 4

Number of B. subtilis and V. anguillarum cells attached to surfaces coated with phages only or with mucin + phages, and measurement of their metabolic activity. Statistical significance was assessed using the Tukey test. One star (*) represents statistically significant differences with p < 0.05. (A) Counting of B. subtilis cells by flow cytometry after 6 hours of incubation on the plates (n = 3). (B) Counting of V. anguillarum cells by flow cytometry after 6 hours of incubation on the plates (n = 6). (C) Oxygen consumption rate of B. subtilis cells that were attached to phage-coated or mucin+phage-coated surfaces for 6 hours (n = 4). (D) Oxygen consumption rate of V. anguillarum cells that were attached to phage-coated or mucin+phage-coated surfaces for 6 hours (n = 8)