Campylobacter jejuni gene cj0511 encodes a serine peptidase essential for colonisation

Abstract

According to MEROPS peptidase database, Campylobacter species encode 64 predicted peptidases. However, proteolytic properties of only a few of these proteins have been confirmed experimentally. In this study we identified and characterised a Campylobacter jejuni gene cj0511 encoding a novel peptidase. The proteolytic activity associated with this enzyme was demonstrated in cell lysates. Moreover, enzymatic studies conducted with a purified protein confirmed a prediction of it being a serine peptidase. Furthermore, cj0511 mutant was found to be severely attenuated in chicken colonisation model, suggesting a role of the Cj0511 protein in infection.

Keywords: Campylobacter; Colonisation; Peptidases; Protease inhibitors; Proteases; Proteolysis.

Fig. 1

Identification of peptidase activities of C. jejuni strain 11168H using zymography. (A) 1, size markers (Mark 12, Invitrogen); 2, 11168H. (B) 1–3, 11168H/cj1228::kan^r mutant (three clonal isolates). (C) 1, 11168H; 2, 11168H/cj0511::kan^r; 3, 11168H/cj0511::kan^r/cj0511+ (complement). (D) 1, 11168H; 2, purified Cj0511His protein.

Fig. 2

Cj0511His activity is affected by serine and serine/cystein but not metallo peptidase specific inhibitors. (A) Effect of various inhibitors on enzymatic activity of Cj0511His. The error bars denote standard deviations. (B) Statistical analysis of changes using one-way Anova. The experiments were repeated three times, with three technical replicates in each test. Text in bold denotes statically valid difference (P < 0.01) compared to control (no inhibitors). (C) Conservation of amino acid residues required for enzymatic activity of serine peptidase D1P (highlighted in bold) in Cj0511 and related proteins (see text). The identical amino acid residues are denoted by asterisks. The Swissprot amino acid sequence database accession numbers are: spO04073.1 (D1P), spP23865 (TspA) and spQ4L6D0.2 (CtpA).

Fig. 3

Attenuation of mutant 11168H/cj0511::kan^r in chicken colonisation model of infection. Post-mortem cecal contents counts of Campylobacter strains used in this study. Counts were determined at 7 days post-infection and are displayed as the Log 10 of the cfu/g. Values of 0 represent undetectable levels of Campylobacter <100 cfu/g. Open circles indicate each individual bird. The bars represent average values. Cj0511 denotes 11168H/cj0511::kan^r mutant.