Protein Truncating Variants of colA in Clostridium perfringens Type G Strains

Abstract

Extracellular matrix (ECM) degrading enzymes produced by Clostridium perfringens may play an important role during the initial phases of avian necrotic enteritis by facilitating toxin entry in the intestinal mucosa and destruction of the tissue. C. perfringens is known to produce several ECM-degrading proteases, such as kappa toxin, an extracellular collagenase that is encoded by the colA gene. In this study, the colA gene sequence of a collection of 48 C. perfringens strains, including pathogenic (i.e. toxinotype G) and commensal (i.e. toxinotype A) chicken derived strains and strains originating from other host species, was analyzed. Although the colA gene showed a high level of conservation (>96% nucleotide sequence identity), several gene variants carrying different nonsense mutations in the colA gene were identified, leading to the definition of four truncated collagenase variant types (I-IV). Collagenase variant types I, III and IV have a (nearly) complete collagenase unit but lack parts of the C-terminal recruitment domains, whereas collagenase variant types II misses the N-terminal part of collagenase unit. Gene fragments encoding a truncated collagenase were mainly linked with necrotic enteritis associated C. perfringens type G strains with collagenase variant types I and II being the most prevalent types. Gelatin zymography revealed that both recombinant full-length and variant type I collagenase have active auto-cleavage products. Moreover, both recombinant fragments were capable of degrading type I as well as type IV collagen, although variant type I collagenase showed a higher relative activity against collagen type IV as compared to full-length collagenase. Consequently, these smaller truncated collagenases might be able to break down collagen type IV in the epithelial basement membrane of the intestinal villi and so contribute to the initiation of the pathological process leading to necrotic enteritis.

Keywords: Clostridium perfringens; collagenase; kappa toxin; necrotic enteritis disease; nonsense mutation.

Figure 1

Primers used in this study and their relative position in colA. Positions are based on C. perfringens strain EHE-NE18 colA sequence.

Figure 2

Gelatinolytic activities expressed by different C. perfringens strains. Supernatant samples of 2 C. perfringens type A strains (JIR4857, CP24) and 2 C. perfringens type G strains (EHE-NE18, CP56) were analyzed for proteolytic activity by gelatin zymography. Lane L, Protein Ladder (Precision Plus Protein All Blue Pre-stained Protein Standards, Biorad).

Figure 3

Schematic representation and distribution of collagenase variants. Schematic representation of the domain architecture of full-length and truncated collagenase types and their distribution among chicken (type G and type A strains) and non-chicken derived C. perfringens strains (respectively n=14, n=11 and n=23). PRE = signal sequence, PRO = pro-domain, M9N = activator domain, M9 = peptidase domain, HELPER = peptidase helper domain, PKD = polycystic kidney disease-like domain and CBD = collagen binding domains. Collagenase variant types are encoded by gene variants carrying a premature stop mutation in the colA gene which results in the occurrence of 2 protein-coding regions. Predicted active fragment containing the HEXXH motif is represented in dark grey, while the other fragment which is expected to be inactive is represented in light grey. *PRE & PRO domains are removed upon maturation. Molecular weight of the predicted mature active fragment is given.

Figure 4

colA gene based dendrogram reflecting the similarity for 48 C. perfringens strains. A UPGMA phylogenetic tree was constructed based on the colA gene sequences (i.e. full gene sequences - from original start to original stop codon - including both fragments when nonsense mutations leading to premature stop codons are present). Sequence similarity percentages (top bar) are shown. Colored branches depict collagenase variant types (see Figure 3 , Green, full-length; Red, collagenase type I variant; Blue, collagenase type II variant; Light blue, collagenase type III variant; Orange, collagenase type IV variant). Branch labels include the C. perfringens strains from which the colA gene sequence was derived.

Figure 5

Analysis of proteolytic activity at different stages of growth of C. perfringens strains encoding for 3 different collagenase types. Growth was analyzed by measuring optical density at 600 nm at 8 timepoints (0, 2, 3, 4, 5, 6, 7 and 24 h post inoculation). At each timepoint supernatant samples were collected and analyzed by zymography. The observed bands were measured by Quantity One to determine the relative gelatinolytic activity. AU = arbitrary units. One representative image per collagenase variant type is shown (full-length collagenase: JIR4857, type I: CP56 and type II: D8).

Figure 6

Recombinant full-length and variant type I collagenase have active auto-cleavage products. (A) SDS page and (B) Gelatin zymography of the recombinantly expressed collagenase fragments. F = full-length collagenase, expected fragment 120 kDa; T = Type I variant collagenase, expected fragment 94 kDa; M1= Pageruler plus pre-stained, M2 = Precision Plus protein All blue Pre-stained Protein standard. (C) Substrate-degrading activity of both the full-length collagenase and variant type I collagenase towards collagen type I or collagen type IV was measured using the EnzChek collagenase assay. The bar shows the activity of the variant type I collagenase relative to the full-length collagenase towards each of the tested substrates. Data represents mean ± standard deviations (n=3). One-sample t-tests were performed to evaluate whether the ratios differed significantly from 1, i.e. the ratio value that corresponds with an equal activity of both collagenase variant I and full-length collagenase. **p ≤ 0,01.