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Abstract
Akkermansia muciniphila is a common member of the human gut microbiota and belongs to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum. Decreased levels of A. muciniphila have been associated with many diseases, and thus it is considered to be a beneficial resident of the intestinal mucus layer. Surface-exposed molecules produced by this organism likely play important roles in colonization and communication with other microbes and the host, but the protein composition of the outer membrane (OM) has not been characterized thus far. Herein we set out to identify and characterize A. muciniphila proteins using an integrated approach of proteomics and computational analysis. Sarkosyl extraction and sucrose density-gradient centrifugation methods were used to enrich and fractionate the OM proteome of A. muciniphila. Proteins from these fractions were identified by LC-MS/MS and candidates for OM proteins derived from the experimental approach were subjected to computational screening to verify their location in the cell. In total we identified 79 putative OM and membrane-associated extracellular proteins, and 23 of those were found to differ in abundance between cells of A. muciniphila grown on the natural substrate, mucin, and those grown on the non-mucus sugar, glucose. The identified OM proteins included highly abundant proteins involved in secretion and transport, as well as proteins predicted to take part in formation of the pili-like structures observed in A. muciniphila. The most abundant OM protein was a 95-kD protein, termed PilQ, annotated as a type IV pili secretin and predicted to be involved in the production of pili in A. muciniphila. To verify its location we purified the His-Tag labeled N-terminal domain of PilQ and generated rabbit polyclonal antibodies. Immunoelectron microscopy of thin sections immunolabeled with these antibodies demonstrated the OM localization of PilQ, testifying for its predicted function as a type IV pili secretin in A. muciniphila. As pili structures are known to be involved in the modulation of host immune responses, this provides support for the involvement of OM proteins in the host interaction of A. muciniphila. In conclusion, the characterization of A. muciniphila OM proteome provides valuable information that can be used for further functional and immunological studies.
Highlights
Akkermansia muciniphila is a Gram-negative, anaerobic bacterium, which colonizes the mucus layer of the human gastrointestinal (GI) tract (Derrien et al, 2004)
Akkermansia muciniphila outer membrane (OM) proteins were isolated using two different methods, based on either selective sarkosyl extraction or sedimentation gradient centrifugation of membrane proteins, derived from cells grown with mucin or glucose as major carbon and energy sources (Figure 1)
An over 20-fold enrichment of predicted OM proteins was achieved in the sarkosyl-treated membranes in comparison with the whole proteome and intracellular fraction of both mucin and glucose-grown cells of A. muciniphila (Figures 1A,B)
Summary
Akkermansia muciniphila is a Gram-negative, anaerobic bacterium, which colonizes the mucus layer of the human gastrointestinal (GI) tract (Derrien et al, 2004). Extracellular vesicles from A. muciniphila were shown to have protective effects on the development of dextran sulfate sodium (DSS) induced colitis in mice (Kang et al, 2013). A. muciniphila has been shown to adhere to intestinal epithelium and improve enterocyte monolayer integrity of Caco-2 cells (Reunanen et al, 2015). These findings suggest important host-bacteria interactions, the mechanisms of which are yet to be discovered (see Derrien et al, 2016 for a recent review)
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