Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae

Abstract

Ecotin, first described in Escherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen, Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.

Fig 1. Sequence comparison and secondary structural analysis of ecotins from Campylobacter spp.

(A) A multiple sequence alignment was generated with ClustalW and BOXshade; http://www.ch.embnet.org/ with ecotin sequences from C. rectus, WP_004320172.1; C. showae, WP_002949877.1; C. curvus, WP_011991897.1; C. concisus, WP_107709881.1, C. hominis, WP_012108817.1, C. gracilis, WP_005871804.1; C. ureolyticus, WP_016646581.1; E. coli (WP_137532711.1) and other species (P. aeruginosa, WP_132540651.1, S. marcescens, WP_015671699.1. Black shading indicates >50% amino acid identity. Grey shading is >50% similarity in amino acid charge. In red, the primary protease binding sites (including the P1 residue (Ec-Met84) and residues 51–54; in yellow, the secondary protease binding sites (Ec-residues 67 to 70 and 108 to 113); green letters, conserved cysteine residues; in blue, dimerization interface (Ec-residues 133 to 142) [–61]; highlighted in green, signal peptide according to SignalP [47] (cut-off 0.5, except for C. ureolyticus, here no signal peptide was predicted even with a cut-off of 0.3). (B) In silico structural analysis of ecotins using the Protein Homology/analogY Recognition Engine V2.0 (PHYRE2) is shown. Ecotin proteins from E. coli (Ec), C. rectus (Cr), C. showae, (Cs), C. ureolyticus (Cu), C. concisus (Cco), C. gracilis (Cg), C. curvus (Ccu) and C. hominis (Ch) display very similar structures despite the low % of amino acid conservation between the E. coli and the Campylobacter homologs (i.e. ecotins from C. rectus, C. showae, C. ureolyticus, C. curvus, C. gracilis, C. concisus, C. hominis share 27%, 33%, 37%, 34%, 31%, 25% and 39% amino acid identity with the E. coli ecotin, respectively).

Fig 2. Overexpression and purification of Campylobacter ecotin in E. coli.

(A) Western blot with hexa-histidine-specific antibodies of whole cell lysates to follow the expression of Campylobacter ecotins in E. coli BL21 after 2, 4 and 24 h of induction with IPTG is shown, protein samples before induction (-) were included as controls. The signal migrating at ~18 kDa represents the ecotin-His₆ protein from the indicated species, Ec, E. coli; Cr, C. rectus; Csh, C. showae. A full, top to bottom scan of the membrane is provided in the supplement, S1 Fig. (B) SDS-PAGE (15%, Coomassie stained) of ecotin proteins from the indicated species after overexpression and purification from whole cell lysates of E. coli BL21. Molecular weight markers (Mw, in kDa) are indicated on the left.

Fig 3. Ecotins inhibit trypsin-mediated proteolysis.

SDS-PAGE (15%, Coomassie stained) of trypsin protease protection assays by ecotin proteins carried out at: (A) 37°C and (B) 45°C are shown. Samples contained the protease substrate CmeA-His₆ (10 nM), trypsin (10 nM) and ecotin (15 nM) with the indicated strain. (—) indicates the absence of ecotin from the assay. Aliquots were taken at t = 0, and after 1 h and 3 h of incubation. The signals migrating at ~18 kDa represent the ecotin-His₆ proteins; the signals migrating at ~42 kDa represent CmeA-His₆. Molecular weight markers (in kDa) are indicated on the left. Full scans of the gels are available in the supplement (S2 Fig).

Fig 4. Ecotins inhibit factor Xa-mediated FRET peptide cleavage.

(A) Illustration of the in vitro FRET assay. FRET peptides were incubated with purified ecotin homologs in 96-well plates with or without factor Xa. When the peptide is cleaved by the protease, fluorescence is produced. The factor Xa cut-site (IDGR) in the FRET peptide is highlighted in red. (B) The FRET peptide was incubated with factor Xa (5 pmol) and ecotin (15 nM) from E. coli (open circles), C. rectus (filled squares) and C. showae (filled diamonds) over a time frame of 60 min in 96 well plates. The control wells contained FRET peptide substrate only (no ecotin, filled triangles), the basal fluorescence level (no ecotin, no factor Xa) is indicated by a dashed line. Arbitrary fluorescent units (y-axis) were determined using a microplate reader with a filter set of Ex/Em = 355/530 nm. Standard deviations are indicated by error bars.

Fig 5. E. coli and Campylobacter ecotins possess similar IC₅₀ values for neutrophil elastase.

Experimentally determined IC₅₀ values (nM) for E. coli (circles, 4.64 ± 0.23), C. rectus (squares, 4.49 ± 0.25) and C. showae (triangles, 4.78 ± 0.31) ecotins used at increasing concentrations (nM) to inhibit NE. Each data point represents the mean from three independent measurements. Relative florescence (in %, where 100% indicates fully digested FRET-peptide and 0% indicates fully inhibited protease) was determined after measuring the samples in a microplate reader with a filter set of Ex/Em = 355/530 nm. Separate graphs for each IC₅₀ determination were included in the supplementary information (S5 Fig).

Fig 6. Campylobacter ecotins rescue neutrophil-mediated killing of ecotin-deficient E. coli and protect E. coli cells from killing by live and purified NETs.

(A) The results of a time-dependent neutrophil killing assay are shown. E. coli BL21 WT (Ec-WT), the corresponding E. coli BL21 ecotin mutant (Ec eco::kan), and Ec eco::kan complemented with either the E. coli (+ Ec ecotin), C. rectus (+ Cr ecotin) or C. showae ecotin (+ Csh ecotin) were incubated with human neutrophils and bacterial survival was determined using a microplate-based bacterial growth assay. Remaining bacteria (expressed in % survival, based on colony forming units CFU in each sample (100% = 1 x 10⁷ bacteria) were calculated based on a CFU per OD₆₀₀ standard curve that was created for each strain (not shown). Error bars represent the standard deviation for a dataset obtained from 3 biological replicates (each done in triplicate) using neutrophils from different human donors. (B) Bar graph of colony counts determined from LB agar plates after spotting 10 μl of 10-fold serial dilution series of cells of the indicated E. coli strains after 30 min of incubation with PSB (control) or NETs (the original plate pictures from 3 biological replicates are shown in S7 Fig). The insert depicts the values for the NET samples at a different scale; strain designations are identical to (A). Error bars depict the standard error of the mean (SEM). Statistically significant differences (paired t-test, p<0.005) are indicated by an asterisk.

Fig 7. Expression of C. rectus and C. showae ecotins partially rescue the protease sensitive phenotype of a C. jejuni N-glycosylation mutant.

CFU of the C. jejuni wildtype and the C. jejuni pglB mutant expressing native ecotins from C. rectus (Cr) or C. showae (Csh) were determined in media supplemented with chicken cecal contents (CCC). CFU determined in the absence of CCCs and heat inactivated CCCs were used as controls. Bars represent the mean from 3 biological replicates carried out as triplicates; standard deviations are indicated by error bars, statistical differences (one-way ANOVA) between the control (MH) and the experimental samples (Dunnett's test) as well as in between samples (paired t-test) are indicated (*p<0.05, **p<0.005).