Fig 1.
Crystal structures of chitinases ChiA and ChiB.
(A, C) Schematic overview of protein domain organization and overall structures of ChiA and ChiB, both consisting of an N-terminal glycosyl hydrolase 18 (GH18) Trios-phosphate Isomerase (TIM) barrel followed by a putative carbohydrate binding domain (CBD). (B, D) Comparison of overall protein structures of ChiA and ChiB with those of previously studied chitinases (Chromobacterium violaceum and Serratia marcescens). (E,F) Surface representation of the protein crystal structures of ChiA and ChiB identifying the glycosyl hydrolase 18 domain (purple), carbohydrate binding domain (orange) and their open and tunnel shaped binding cleft, respectively, containing the DxDxE binding motif. The transparent purple volume in the cartoon representation of the crystal structures describes the ligand-binding pocket. (G,H, I, J, K, L) Ligand interactions of ChiA or ChiB with substrates: chitin, chitosan or bisdionin C. Ligand OMIT maps are shown as blue mesh representing the difference electron density Fourier coefficient mFo-DFc contoured at + 3 σ carved 2A around the ligands. Maps were generated by performing reciprocal space coordinate refinement after randomizing all atoms on average 0.4 Å in the absence of the ligands.
Fig 2.
Binding of the inactive chitinases ChiA or ChiB towards (A) crystalline chitin, colloidal chitin or GlcNAc-covered beads or (B) crude chicken intestinal mucus.
(A) In solution binding assay: The amount of bound and unbound protein to the substrate was visualised using SDS-PAGE and subsequent Coomassie staining. B = bound fraction, S = supernatants representing the unbound protein fraction. Intensity of the bands was quantified as OD/mm2. The bound protein fraction was calculated by dividing the intensity of bound protein by total protein for each substrate. Assay was performed twice, one representative figure is shown. (B) Dotblot: Either porcine mucus type II (1), porcine mucus type III (2) or crude chicken mucus (3–8; 6 biological replicates) were spotted onto the membrane. Membranes were incubated with either recombinant ChiA or ChiB. The amount of bound enzyme was visualized using anti-HIS-antibody. Assay was performed twice, one representative figure is shown.
Table 1.
Kinetic parameters of the fitted models to the enzymatic activity data of ChiA or ChiB towards the pseudo-chitin substrates 4-MU-GlcNAc2, 4-MU-GlcNAc3 or colloidal chitin.
Kcat = catalytic constant, Km = substrate concentration given that the reaction rate reaches ½ Vmax, Kcat/Km = Specificity constant, ND: kinetic parameters not determined due to unambiguous model fitting for ChiB using 4-MU-(GlcNAc)3. BDL: “Below detection limit”.
Fig 3.
Enzymatic activity of chitinases ChiA and ChiB towards (A) pseudo-chitin substrates (effect temperature and pH) or (B) crude chicken mucus.
(A) Effect of temperature and pH on chitinase activity towards pseudo-chitin substrates (4-MU-GlcNAc2 and 4-MU-GlcNAc3). The enzymatic activity of ChiA (left) or ChiB (right) at different temperatures was assessed at a constant pH of 5 (upper panels), whereas the effect of the pH was monitored at a constant temperature of 42°C (lower panels) (no replicates). (B) Chitinase activity towards chicken mucus using turbidity assay. Biological replicates of crude intestinal chicken mucus (150 μg) were incubated with either recombinant ChiA, ChiB (15 μg) or PBS. After a one hour incubation period at 37°C, the turbidity of the mixture was measured at an OD-value of 450 nm. The relative index was calculated by dividing the OD of the chitinase-treated mucus by the OD of the untreated mucus sample for each mucus sample. Lines indicate the means with their respective standard deviations. Significant differences are indicated with ‘*’ (p≤0.05). ‘**’ (p≤0.01) and ‘***’ (p≤0.001).
Fig 4.
Effect of mucus on the growth of C. perfringens.
(A) Growth analysis of C. perfringens CP56 in media supplemented with 5% chicken intestinal mucus pre-treated with either PBS (= untreated), 15μg of chitinase ChiA or ChiB for one hour. Growth rate is defined as the slope during exponential rate. Bars indicate the means with their respective standard deviations. (B) Growth analysis of wild-type and chitinase mutant strains in different media. Wild type CP56 (Black), CP56ΔchiA1 (light blue), CP56ΔchiA2 (dark blue), CP56ΔchiB1 (light orange) or CP56ΔchiB2 (dark orange) were grown in either nutrient rich medium, nutrient poor medium or nutrient poor medium supplemented with 5% crude chicken mucus. Growth rate is defined as the slope during the exponential phase. Bars indicate the means with their respective standard deviations. Significant differences are indicated with ‘*’ (p≤0.05). ‘**’ (p≤0.01). ‘***’ (p≤0.001) and ‘****’ (p≤0.0001).
Fig 5.
Effect of mucus on colonisation C. perfringens.
(A) Competition assay of C. perfringens wild-type strain CP56 and mutant strains CP56ΔchiA1 or CP56ΔchiB1 in either in vitro or in vivo growth conditions. In vitro: An equal mix of wild-type and mutant strain was grown in either nutrient rich medium or nutrient poor medium supplemented with 5% chicken intestinal mucus. Samples were taken at the exponential growth phase and at saturation after overnight incubation. In vivo: 18-days old broiler chickens were inoculated with an equal mix of wild-type and CP56ΔchiA1 mutant strain. After 24 hours, samples were taken from the intestinal content (jejunum or ileum). The amount of wild-type or mutant strain in the samples was determined using dPCR. The competition index is defined as the ratio of the mutant on wild-type strain, divided by the respective ratio in the inoculum. Lines indicate the means. (B) Mucus binding assay of wild-type CP56 C. perfringens strain in media supplemented with recombinant chitinases, either ChiA or ChiB. Washed C. perfringens overnight culture was added to the wells containing a mucus agar layer, supplemented with either 50 μg of recombinant enzyme (ChiA or ChiB) or an equal volume of PBS as a negative control. Wells were anaerobically incubated for 90 min at 37°C after which the bound bacteria were quantified through titration. The binding ratio is defined as the percentage of bacteria bound to the mucus in supplemented media as compared to non-supplemented conditions. Bars indicate the means with their respective standard deviations.
Fig 6.
Necrotic enteritis in vivo trial.
Predisposed broilers (six replicate pens per C. perfringens strain, 15 birds/pen) were challenged with C. perfringens (either wild type CP56 or mutant CP56ΔchiA1) on days 18 and 19 after which the severity of necrotic lesions was determined on day 20; score 0: no gross lesions; score 2: focal necrosis and ulceration (1–5 foci); score 3: focal necrosis and ulceration (6–15 foci); score 4: focal necrosis and ulceration (16 or more foci); score 5: patches of necrosis 2 to 3 cm long and score 6: diffuse necrosis. Overall boxplots per C. perfringens strain (A) are shown, as well as individual boxplots per pen (B). The grey dots indicate individual scores per bird. Statistical difference in disease severity was assessed using a cumulative link mixed model, with disease severity score (ordinal factor 0–6) as the response variable and C. perfringens strain (CP56 or CP56ΔchiA1) as a predictor variable, thereby accounting for non-independence of birds housed within the same pen by specifying “pen” as a random effect.
Fig 7.
Graphic summary of the activity of pathogen-specific chitinases during pathogenesis.
Virulent C. perfringens type G strains are in close proximity to the intestinal mucus layer. The production of chitinases results in the breakdown of the intestinal mucus layer that could result in nutrient acquisition, the exposure of additional binding sites for the bacteria and the exposure of target sites for toxins or other virulence factors (or a simultaneous combination). This aids additional C. perfringens proliferation and bacterial colonization, key aspects of early pathogenesis.
Table 2.
Primer sets used to amplify and clone chitinase genes (chiA or chiB) into the pBAD TOPO TA expression vector. Additional nucleotides (underlined) were added to incorporate an additional stop-codon (TGA), ribosome binding site (AGGA) and start-codon (ATG). Mutagenic primers used to preform site-directed mutagenesis at the active site of the chitinase genes chiA or chiB. Flanking primers are equal to the primers used to produce the enzymatically active enzymes. Nucleotide substitution of the mutant primers is indicated in the box.