Dubaissi E et al. (2018), Functional characterization of the mucus barrie...

XB-ART-54451

Proc Natl Acad Sci U S A 2018 Jan 23;1154:726-731. doi: 10.1073/pnas.1713539115.

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Functional characterization of the mucus barrier on the Xenopus tropicalis skin surface.

Dubaissi E , Rousseau K , Hughes GW , Ridley C , Grencis RK , Roberts IS , Thornton DJ .

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Mucosal surfaces represent critical routes for entry and exit of pathogens. As such, animals have evolved strategies to combat infection at these sites, in particular the production of mucus to prevent attachment and to promote subsequent movement of the mucus/microbe away from the underlying epithelial surface. Using biochemical, biophysical, and infection studies, we have investigated the host protective properties of the skin mucus barrier of the Xenopus tropicalis tadpole. Specifically, we have characterized the major structural component of the barrier and shown that it is a mucin glycoprotein (Otogelin-like or Otogl) with similar sequence, domain organization, and structural properties to human gel-forming mucins. This mucin forms the structural basis of a surface barrier (∼6 μm thick), which is depleted through knockdown of Otogl. Crucially, Otogl knockdown leads to susceptibility to infection by the opportunistic pathogen Aeromonas hydrophila To more accurately reflect its structure, tissue localization, and function, we have renamed Otogl as Xenopus Skin Mucin, or MucXS. Our findings characterize an accessible and tractable model system to define mucus barrier function and host-microbe interactions.

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Species referenced: Xenopus tropicalis
Genes referenced: mrc1 muc5ac muc5b ncor2 otog otogl2 pts vwf
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	Fig. 1. Otogl is a large protein with mucin-like domains. (A) Model of Otogl transcript showing sites corresponding to EST clones TNeu027a13 and THdA045k18, as well as 5′ and 3′ UTRs, start (ATG) and stop (TAA) codons, and primer sites (F1 and R1). (B) RNA in situ hybridization expression patterns of TNeu027a13 and THdA045k18. (Scale bar: 100 μm.) (C) PCR using F1-R1 primers on X. tropicalis cDNA. “M” is marker DNA ladder. (D) Predicted domains of Otogl. VWD is von Willebrand factor type D domain. The mucin domain shows the 39 Cys-rich and 34 PTS-rich subdomains. CK, cysteine knot domain. The size of the domains in numbers of amino acids (AA) is shown.
	Fig. 2. Otogl is a multimeric O-glycosylated glycoprotein. (A) Rate zonal centrifugation of nonreduced and reduced tadpole skin secretions probed with anti-Otogl antibody. (B) TEM of CsCl density gradient purified Otogl shows long-chain–like networks (arrow). (Scale bar: 200 nm.) (C) Coprobing of a Western blot of tadpole lysate with anti-Otogl antibody and PNA; a merged image is also shown. CG, cement gland mucin; OG, glycosylated form of Otogl; OP, precursor form of Otogl. (D) Treatment of tadpole lysate with O-glycosidase (2 h, lane 2, and 4 h, lane 3) and PNGase F (lane 4) compared with control (lane 1), probed with anti-Otogl and PNA; a merged image is also shown. Dashed lines represent the position of bands for control OG and OP. (E) Treatment of tadpole lysate with sialidase alone (lane 2) and sialidase + O-glycosidase (lane 3) compared with control (lane 1); a merged image is also shown. Dashed lines represent the position of bands for control OG and OP. In D and E, the signal for PNA from the cement gland mucin shows the approximately equivalent loading between lanes. (F) Image of section from fixed whole-mount tadpole skin with immunofluorescence for anti-Otogl and anti-GFP [labeling membrane-GFP (memGFP) to identify membranes] together with lectin histochemistry (PNA) shows colocalization of Otogl and PNA staining. The boxed area highlights two adjacent SSCs. (Scale bar: 50 μm.) (G) Zoomed-in image of boxed area from F shows Otogl and PNA colocalize within the vesicles of SSCs. ab, antibody. (Scale bar: 10 μm.)
	Fig. 3. Otogl forms a host-protective barrier on the epidermal surface. (A) Western blot of lysate from MOC- and Otogl MO-injected tadpoles, coprobing with the anti-Otogl antibody (green) and PNA (red), shows loss of Otogl protein upon knockdown. Glycosylated (OG) and precursor (OP) forms of Otogl are highlighted, while the similar intensity of signal for the cement gland mucin (CG) in the two lanes indicates equivalent loading of lysate. (B) Representative examples of sections from snap-frozen MOC- and Otogl MO-injected tadpoles stained with anti-Otogl antibody and DAPI. (Scale bar: 25 μm.) (C) Representative examples of sections from snap-frozen control MOC- and Otogl MO-injected tadpoles stained with PNA and DAPI. (Scale bar: 40 μm.) (D) Representative Cryo-TEM images of sections of snap-frozen MOC- and Otogl MO-injected tadpoles. Double-headed arrows show size of surface barrier. (Scale bar: 2 μm.) (E) Representative images of sections of MOC- and Otogl MO-injected tadpoles following exposure to GFP-expressing DH5α E. coli bacteria. White lines on images to the Left represent the apical surface membrane from brightfield images (Right). (Scale bar: 25 μm.)
	Fig. 4. Otogl morphants are sensitive to infection with A. hydrophila. (A) Survival time course of MOC-injected and Otogl MO-injected tadpoles in 0.01× Marc’s Modified Ringer’s (MMR). (B) Survival time course of MOC-injected and Otogl MO-injected tadpoles in 0.01× MMR containing 1.5 × 108 cfu/mL of A. hydrophila (at time point 0 h). Individual points represent mean survival levels from three independent experiments, and error bars represent the SEM. (C) Bar chart comparing the frequency of GFP-expressing A. hydrophila bacteria located within the tadpole in MOC- and Otogl MO-injected tadpoles fixed and sectioned at the 34-h time point. Bars represent mean number of bacteria found within MOC (n = 3 tadpoles)- and Otogl MO (n = 5 tadpoles)-injected tadpoles. Error bars represent SEM and P = 0.0179 (one-tailed Mann–Whitney U test). (D) Representative images of sections of MOC- and Otogl MO-injected tadpoles following exposure to GFP-expressing A. hydrophila bacteria at the 34-h time point. The white line on the Left image represents the apical surface membrane from brightfield images (Right). (Scale bar: 10 μm.)
	Fig. S1. Full Otogl amino acid sequence translated from SMRT with the different domains highlighted in the colors shown in the key. Highlighted in yellow are the peptides identified by mass spectrometry of purified secreted Otogl. Underlined is the peptide (repeated four times) used to generate an antibody to Otogl.
	Fig. S2. Alignment of Otogl with human gel-forming mucins (MUC2, MUC5AC, MUC5B, and MUC6) shows conservation of protein sequence in D2, D′, D3, and CK domains. Clustal Omega analysis indicates 30.57% identity of Otogl with human MUC5B in the region from D2 to the end of D3 [44% similarity (positives)]. There is 30% identity of human MUC5B and Otogl in the CK domain (45% similarity), with complete conservation of the position of cysteine residues. D2, D′, D3, and CK domains are annotated. Conserved cysteine residues are highlighted in yellow, black boxes are identical amino acids, and gray boxes represent similar amino acids.
	Fig. S3. Alignment of repeats in the mucin domain. Alignment of sequence repeats in the Otogl mucin domain. Proline, threonine, and serine (PTS) residues are highlighted in blue. Cysteine residues are highlighted in red. The consensus sequence (see right-aligned sequences) CCxxxxC forms the basis for the alignment, while three other cysteine residues are also well conserved. These are named Cys-rich regions and are highlighted with double asterisks (*). Through this alignment, PTS-rich regions are also evident and highlighted with a single asterisk (). Overall, there are 39 Cys-rich regions and 34 PTS-rich regions in the mucin domain. Peptides identified in the mucin domain by mass spectrometry are underlined, with the vast majority identified in the Cys-rich regions.
	Fig. S4. Otogl morphants have a depleted mucus surface layer. Representative environmental scanning electron micrographs of the skin surfaces of live MOCand Otogl MO-injected tadpoles. The boxes highlight globular features that are more abundant in MOC- than in Otogl MO-injected tadpoles. (Scale bar: 50 μm.)
	Fig. S5. Infection studies with heat-treated A. hydrophila and live A. hydrophila-GFP. (A) Survival time course of MOC- and Otogl MO-injected tadpoles in 0.01× MMR containing 1.5 × 108 cfu/mL of A. hydrophila, heat-killed at 65 °C for 30 min before infection (at time point 0 h). (B) Survival time course of MOCand Otogl MO-injected tadpoles in 0.01× MMR containing 1.5 × 108 cfu/mL of GFP-expressing A. hydrophila (at time point 0 h). (C) Chart showing frequency of bacteria located inside MOC- and Otogl MO-injected tadpoles at time point 18 h 30 min. Bars represent mean number of bacteria found within MOC (n = 3 tadpoles)- and Otogl MO (n = 3 tadpoles)-injected tadpoles. Error bars represent SEM. (D) Representative image of an Otogl morphant tadpole section at 34-h time point showing internally localized GFP-expressing A. hydrophila bacteria. White line on Left image represents the apical surface membrane from brightfield images (Right). Note the presence of a highly pigmented region (white arrow). (Scale bar: 10 μm.)

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Ambort, Function of the CysD domain of the gel-forming MUC2 mucin. 2011, Pubmed