Gut immunity in a protochordate involves a secreted immunoglobulin-type mediator binding host chitin and bacteria

Abstract

Protochordate variable region-containing chitin-binding proteins (VCBPs) consist of immunoglobulin-type V domains and a chitin-binding domain (CBD). VCBP V domains facilitate phagocytosis of bacteria by granulocytic amoebocytes; the function of the CBD is not understood. Here we show that the gut mucosa of Ciona intestinalis contains an extensive matrix of chitin fibrils to which VCBPs bind early in gut development, before feeding. Later in development, VCBPs and bacteria colocalize to chitin-rich mucus along the intestinal wall. VCBP-C influences biofilm formation in vitro and, collectively, the findings of this study suggest that VCBP-C may influence the overall settlement and colonization of bacteria in the Ciona gut. Basic relationships between soluble immunoglobulin-type molecules, endogenous chitin and bacteria arose early in chordate evolution and are integral to the overall function of the gut barrier.

Figure 1. Two types of epithelium-associated mucus line the Ciona gut.

A thin layer of mucus (a) covers the epithelium of the stomach in the adult gut, while thicker mucus (b,c) is found in the mid- to distal-gut epithelium. (a–c) Alcian blue staining suggests abundant acid mucopolysaccharides. (d) Staining with Alcian blue/periodic acid-Schiff identified neutral polysaccharides that were confined mostly to the intracellular vacuoles of the secretory epithelial cells forming the gut walls. Sections (a–c) were counterstained with nuclear fast red. Scale bars (a,c), 50 μm and (b,d) 25 μm. Arrows indicate two types of mucus: dense and layered (b) and loosely associated (c). E, epithelium; L, lumen.

Figure 2. Detection of chitin within Ciona gut mucus.

(a) Chitin was detected initially (arrow) within the mucus throughout the gut by staining with Fc-CBD-C DyLight 488 (green) and (b) subsequently was depleted by treatment with chitinase. The absence of Fc-CBD signal also is achieved by pre-treating tissue sections with chitinase. (c) Calcofluor white staining confirmed the presence of chitin at the epithelial surface (arrow); copious amounts of loose chitin-rich mucus often are detectable in the lumen (d). Identical chitin staining patterns were detected with Alexa Fluor 488-coupled chitin-binding protein (New England BioLabs; Supplementary Fig. 1f). Staining with isotype and secondary antibody controls was negative. Scale bars (a–c), 25 μm and (d) 100 μm). E, epithelium; L, lumen; S, stool.

Figure 3. VCBP-C colocalizes with chitin-rich mucus at the surface of the stomach epithelium near the midgut and is visualized with confocal microscopy.

(a) Chitin staining (arrow) detected by Fc-CBD-C DyLight 488 (green), (b) VCBP-C staining (arrow) detected by Alexa Fluor 594 (red) at the surface mucus as well as within granules of the epithelium, (c) Hoechst staining of DNA and (d) merged (arrow; overlay indicated in yellow). Staining with isotype and secondary antibody controls at varying concentrations was negative. Scale bars, 50 μm. E, epithelium; L, lumen.

Figure 4. Detection of VCBP-C in both dense and loosely associated mucus.

(a,b) Colocalization (yellow-merged signal) of chitin (Fc-CBD-C DyLight 488, green) and VCBP-C (Alexa Fluor 594, red). Dense mucus (glycocalyx-like) of the midgut can form (a) ribbon-like structures (arrow) as opposed to (b) less dense mucus (arrow) seen in the distal gut. (c) Microbiota-sized particles (arrow) seen in the mucus detected by Hoechst staining of DNA were confirmed as bacteria by 16S FISH (arrow) (d). Staining is negative with isotype and secondary antibody controls. Scale bars,10 μm. E, epithelium; L, lumen.

Figure 5. Chitin is expressed endogenously and its colocalization with VCBP-C is independent of microbial exposure.

(a) Signals for VCBP-C (Alexa Fluor 594, red) and chitin (Fc-CBD-C DyLight 488, green) are colocalized (yellow, arrow) in the intestinal region of the gut primordium of late rotation stage juveniles maintained under germ-free conditions and persists throughout development. Gut development is complete by stage 5/7, with the immediate onset of feeding. (b) Intestinal mucus is chitin-rich (in green, arrow). (c) Magnified view of gut (from b) in which VCBP-C (in red, arrow) is distributed primarily at the edges of the gut tissue surfaces. Chitin-rich pellets are seen in the distal gut and are purged into environment before feeding. (d) Magnified view of the stomach demonstrates chitin-rich mucus and VCBP-C in the epithelium, red. (e) Chitin (green) is prominent and restricted to the stomach and midgut epithelium 2–3 weeks post fertilization; chitin-rich mucus cannot be detected in the oesophagus or branchial basket. Intense signal is evident at the outer edges and are more prominent on the ventral side (arrows) of the stomach and midgut; a chitin signal also is prominent in fecal pellets (not visible in e) but cannot be detected in the hindgut epithelium. (f) Chitin is prominent throughout the gut to the anus in the whole-mount staining of young adults (second ascidian stage and onwards); images of both sides of the stomach are included to emphasize enhanced signal in ridges of the epithelial grooves (inset). Bright field overlays are shown in a,b,e. Scale bars (a,c), 50 μm; (b,e), 100 μm; (d) 25 μm; and (f) 200 μm. BB, branchial basket; E, epithelium; es, oesophagus; hg, hindgut; L, stomach lumen; mg, midgut; st, stomach.

Figure 6. VCBP-C affects in vitro biofilm formation differentially in five gut bacterial isolates recovered from C. intestinalis.

(a) Stationary cultures of a Bacillus sp. isolated from the Ciona gut forms biofilms within 3–5 days. Biofilm formation is enhanced in the presence of recombinant VCBP-C (6.5 μg ml⁻¹); a similar effect is noted for gut isolates of (b) Shewanella sp. and (c) Pseudoalteromonas sp. (d) Vibrio isolate 6251 (laboratory-assigned number) exhibits a significant increase only with the addition of VCBP-C plus chitin; (e) no significant difference is seen with Vibrio isolate 6269. (f) E. coli biofilms are increased in the presence of SIgA; VCBP-C at similar concentrations does not influence E. coli biofilms. Biofilms were stained with crystal violet, dried, re-dissolved in acetic acid and absorbance (Abs) was read at OD₅₅₀. Increased absorbance reflects the increased surface biofilm. Each experiment was performed in triplicate a minimum of four separate times; results shown represent one triplicate experiment. s.d. is shown by black error bars. Significant differences from control samples were calculated by using analysis of variance with post hoc Dunnett's test. *P<0.05; **P<0.01; CTL, control; VCBP, VCBP-C; V/C, VCBP-C plus hydrolysed chitin.

Figure 7. VCBP-C associates with bacteria in a Shewanella sp. biofilm.

Shewanella were stationary-cultured in the presence of VCBP-C. Immunofluorescent staining detected VCBP-C bound to the bacteria of the biofilms using anti-VCBP-C (Alexa Fluor 594, red). (a) Control stain (no addition of VCBP-C) using primary and secondary antibodies, (b) VCBP-C-positive biofilm. SIgA influences biofilm formation in a non-pathogenic E. coli strain. (c) Control stain (no addition of SIgA) using fluorescently tagged anti-IgA antibodies and (d) SIgA-positive biofilm. Scale bars, 20 μm.