Bacteriophage adhering to mucus provide a non-host-derived immunity

Abstract

Mucosal surfaces are a main entry point for pathogens and the principal sites of defense against infection. Both bacteria and phage are associated with this mucus. Here we show that phage-to-bacteria ratios were increased, relative to the adjacent environment, on all mucosal surfaces sampled, ranging from cnidarians to humans. In vitro studies of tissue culture cells with and without surface mucus demonstrated that this increase in phage abundance is mucus dependent and protects the underlying epithelium from bacterial infection. Enrichment of phage in mucus occurs via binding interactions between mucin glycoproteins and Ig-like protein domains exposed on phage capsids. In particular, phage Ig-like domains bind variable glycan residues that coat the mucin glycoprotein component of mucus. Metagenomic analysis found these Ig-like proteins present in the phages sampled from many environments, particularly from locations adjacent to mucosal surfaces. Based on these observations, we present the bacteriophage adherence to mucus model that provides a ubiquitous, but non-host-derived, immunity applicable to mucosal surfaces. The model suggests that metazoan mucosal surfaces and phage coevolve to maintain phage adherence. This benefits the metazoan host by limiting mucosal bacteria, and benefits the phage through more frequent interactions with bacterial hosts. The relationships shown here suggest a symbiotic relationship between phage and metazoan hosts that provides a previously unrecognized antimicrobial defense that actively protects mucosal surfaces.

Keywords: host-pathogen; immune system; immunoglobulin; symbiosis; virus.

Fig. 1.

Phage adhere to cell-associated mucus layers and mucin glycoprotein. (A) PBR for diverse mucosal surfaces and the adjacent environment. On average, PBRs for mucosal surfaces were 4.4-fold greater than for the adjacent environment (n = 9, t = 4.719, ***P = 0.0002, unpaired t test). (B) Phage adherence to TC cell monolayers, with and without surface mucus (unpaired t tests). (Left) Non–mucus-producing Huh-7 liver hepatocyte cells and mucus-producing T84 colon epithelial cells (n > 18, t = 8.366, ****P < 0.0001). (Center) Mucus-producing A549 lung epithelial cells with and without treatment with NAC, a mucolytic agent (n > 40, t = 9.561, ****P < 0.0001). (Right) Mucus-producing shRNA control A549 cells (shControl) and mucus knockdown (MUC^–) A549 cells (n > 37, t = 7.673, ****P < 0.0001). (C) Phage adherence to uncoated agar plates and agar coated with mucin, DNA, or protein (n = 12, t = 5.306, ****P < 0.0001, unpaired t test).

Fig. 2.

Effect of phage adsorption on subsequent bacterial infection of epithelial cells. (A) Bacterial attachment to mucus-producing (T84 and A549) and non–mucus-producing (Huh-7, MUC^–) TC cells, with and without phage pretreatment. T4 phage pretreatment significantly decreased subsequent bacterial adherence to mucus-producing TC cell lines (T84: n > 30, t = 32.05, ****P < 0.0001; A549: n > 30, t = 36.85, ****P < 0.0001; unpaired t tests). Less dramatic shifts were seen for non–mucus-producing cells (Huh-7: n > 30, t = 2.72, **P = 0.0098; MUC^–: n > 30, t = 3.52, ***P = 0.0007; unpaired t tests). (B) Mucus-producing A549 cells were pretreated with T4 am43^–44^– phage (Materials and Methods) and then incubated for 4 h with either wild-type (wt) or amber-suppressor (SupD) E. coli. Phage replication in the SupD E. coli strain significantly reduced bacterial colony-forming units (CFU) in the mucus (n = 8, ****P < 0.0001, Tukey’s two-way ANOVA) and increased phage-forming units (PFU) relative to the no-phage replication wt E. coli (n = 8, *P = 0.0227). (C) Mortality of mucus-producing (A549) and mucus knockdown (MUC^–) A549 lung epithelial cells following overnight incubation with E. coli. Phage pretreatment completely protected mucus-producing A549 cells from bacterial challenge (n = 12, ****P < 0.0001, Tukey’s one-way ANOVA); protection of MUC^– cells was 3.1-fold less (n = 12, *P = 0.0181). ns, not significant.

Fig. 3.

Effect of Hoc protein on phage–mucin interactions. (A) Adherence of hoc⁺ and hoc^– T4 phage to agar coated with mucin, DNA, or protein reported as an increase relative to plain agar controls (n > 11, t = 3.977, ***P = 0.0007, unpaired t test). (B) Competitive effect of mucin on phage adherence when hoc⁺ and hoc^– T4 phage in 0–5% (wt/vol) mucin solution (1× PBS) were washed over mucus-producing A549 cells (n = 25 per sample). (C) Diffusion of fluorescence-labeled hoc⁺ (Left) and hoc^– (Right) T4 phage in buffer and 1% mucin as determined by MPT. Mucin hindered diffusion of hoc⁺ T4 phage but not hoc^– phage (10 analyses per sample, trajectories of n > 100 particles for each analysis; error bars represent SE).

Fig. 4.

Hoc-mediated glycan binding and Hoc-related phylogeny. (A) Phylogenetic tree of sequences from viral metagenomes with high-sequence homology to Ig-like domains. Many of the identified homologs are from mucus-associated environments (e.g., human feces, sputum). Also included are the Hoc protein of T4 phage and the hypervariable Ig-like domains previously obtained by deep sequencing of phage DNA from the human gut (44). The scale bar represents an estimated 0.5 amino acid substitutions per site. See SI Materials and Methods for methods. (B) Binding of fluorescence-stained hoc⁺ and hoc^– T4 phage to a microarray of 610 mammalian glycans. Normalized relative fluorescence units (RFU) were calculated from mean fluorescence minus background binding.

Fig. 5.

The BAM model. (1) Mucus is produced and secreted by the underlying epithelium. (2) Phage bind variable glycan residues displayed on mucin glycoproteins via variable capsid proteins (e.g., Ig-like domains). (3) Phage adherence creates an antimicrobial layer that reduces bacterial attachment to and colonization of the mucus, which in turn lessens epithelial cell death. (4) Mucus-adherent phage are more likely to encounter bacterial hosts, thus are under positive selection for capsid proteins that enable them to remain in the mucus layer. (5) Continual sloughing of the outer mucus provides a dynamic mucosal environment.