Cutting through mucus with the influenza virus neuraminidase

influenza virusNeuraminidase is one of three different viral proteins embedded in the lipid membrane of influenza virus (NA is blue in the illustration at left). This enzyme has a clear and proven role in virus release from cells. NA is also believed to be important during virus entry, by degrading the mucus barrier of the respiratory tract and allowing virus to reach cells. This role is supported by the finding that treatment of mucus-covered human airway epithelial cells with the NA inhibitor Tamiflu substantially suppresses the initiation of infection.  Further evidence comes from the recent finding that influenza virus binds to sialic acids in mucus and that NA cleaves these sugars to allow infection.

The mucus layer of the respiratory tract has a defensive role because it contains soluble glycoproteins that are rich in sialic acids, which are cell receptors for some viruses. When influenza virions enter the respiratory tract, they are thought to be trapped in mucus where they bind sialic acids, preventing infection of the underlying cells. The role of NA in penetration of the mucus layer was studied in frozen sections of human tracheal and bronchial tissues, in which the mucus layer is preserved. Influenza A virions bind to this mucus layer; the interaction is blocked when the tissues are first treated with a bacterial neuramindase, which removes sialic acids from glycoproteins. These observations indicate that the mucus layer of human airway epithelial cells contain sialic acid-containing decoys that bind influenza A viruses.

Human salivary mucins, which approximate the mucus of the human respiratory tract, can protect cultured cells from influenza virus infection. The protective effect can be achieved by simply adding the mucins to cells. The inhibitory effect is dependent on the sialic acid content of the mucins: fewer cells are infected when higher concentrations are used. In contrast, porcine salivary mucins do not substantially reduce influenza virus infection. The type of sialic acids and the virus strain also determine the extent of protection.

To determine the role of the viral NA in mucin-mediated inhibition, Tamiflu was mixed with virus before infection of mucus-coated cells. The presence of Tamiflu increased the inhibition of infection caused by mucins, indicating that the sialic acid-cleaving (sialidase) activity of NA is needed to overcome inhibition by human salivary mucins. When influenza virions are incubated with human salivary mucins linked to beads, sialic acids are cleaved from mucins, and this enzymatic activity is inhibited by Tamiflu. Human salivary mucins inhibit NA by binding to the active site of the enzyme.

These studies establish a clear role for the influenza viral NA in bypassing the defenses of the mucosal barrier. An important message is that not all strains of influenza virus are equally inhibited by mucus; presumably this property is one of many that determines viral virulence. The balance between how tightly the viral HA binds to sialic acids, and how well the NA cleaves them, is probably one predictor of how well we our protected by our mucus.

 

Viruses might provide mucosal immunity

T4 HocThe mucosal membranes that line our respiratory, alimentary, and urogenital tracts and the outer surface of the eyes are portals of entry for microbes. The cells at these surfaces have functions that require that they are exposed to the environment – for example, gaseous exchange in the lung between inspired air and the blood. Mucus, pH extremes, enzymes, and immune cells are some of the antimicrobial defenses that are present at various mucosal surfaces. It now appears that bacteriophages – viruses that infect bacteria – might also be part of the mucosal antimicrobial defense system.

A sampling of the ratio of bacteriophages to bacteria in a variety of mucosal surfaces (sea anemone, hard and soft coral, polychaete, teleost, human gum, and mouse intestine) revealed higher ratios when compared to non-mucosal samples (e.g. neighboring sea water or saliva). A model bacteriophage of E. coli, T4, was used to show that phage specifically attach to mucus: adherence to cultured cells was reduced when mucus was not produced or removed by chemical treatment.

The principal macromolecules in mucus are mucin glycoproteins, which consist of a polypeptide chain linked to hundreds of variable, branched sugar molecules. Mucins are continuously produced at mucosal surfaces which gives rise to a thick protective layer. Phage T4 was found to attach specifically to mucins and not other components of mucus such as protein or DNA. The attachment of phage T4 to mucus-producing cultured cells reduced the number of bacteria that could attach to and kill the cells. This antimicrobial effect was substantially reduced when a strain of phage T4 was used that cannot lyse its bacterial host. Therefore phages bound in mucus protect cells by infecting and lysing bacterial invaders.

Phage T4 attaches to mucins through immunoglobulin-like (Ig-like) proteins present in the viral capsid. First discovered in antibody molecules, the Ig domain has since been found in hundreds of different proteins with various functions and appear to be encoded in ~25% of dsDNA phages. The Ig domain, typically 80 amino acids in length, is often involved in interactions with other proteins or ligands. For example, the Ig domains of antibodies interact with antigens, and the poliovirus receptor interacts with poliovirus via an Ig domain on the receptor molecule. The capsid of phage T4 contains 155 copies of an Ig-like protein called Hoc (colored yellow in the image). Deletion of the phage T4 hoc gene reduced binding of the virus to mucin, showing that adherence to mucin requires Ig-like protein domains.

These results demonstrate that a model bacteriophage, T4, attaches to mucus via an interaction between viral Ig-like capsid proteins and mucins. The ability of phages to attach to mucin clearly helps protect cultured cells from bacterial attachment and killing. Bacteriophages may be part of a previously unrecognized mucosal immune defense system. This suggests a symbiotic relationship between phages and metazoan hosts: the phages provide protection to mucosal surfaces, and in turn are provided hosts (bacteria) in which to reproduce. However, additional experiments are required to prove the authors’ conclusion of a “key role of the world’s most abundant biological entities in the metazoan immune system”. It will be necessary to directly demonstrate that phages attaching to mucins in mucus can protect an animal (e.g. mice) from bacterial invasion. This will not be an easy experiment because the phage and bacteria composition of mucus is likely to be complex and continuously changing as mucus is sloughed from cells and new mucins are produced.

The finding that phages play roles in mucosal immunity would have far-reaching consequences for human health. Some fascinating questions that come to mind include: do phage populations play roles in human diseases? Are they altered in human diseases and can we correct these diseases by restoring phage populations? Are the phage populations altered by antimicrobial therapy that alters bacterial populations? Do phages contribute to development of the immune system by modulating bacterial populations? Might mucus-bound phages stabilize the microbiome?

Update: Michael Schmidt and I discussed these remarkable findings on episode #59 of the science show This Week in Microbiology.