Campylobacter jejuni serine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration

Abstract

Campylobacter jejuni is an important pathogen of foodborne illness. Transmigration across the intestinal epithelial barrier and invasion are considered as primary reasons for tissue damage triggered by C. jejuni. Using knockout mutants, it was shown that the serine protease HtrA may be important for stress tolerance and physiology of C. jejuni. HtrA is also secreted in the extra-cellular environment, where it can cleave junctional host cell proteins such as E-cadherin. Aim of the present study was to establish a genetic complementation system in two C. jejuni strains in order to introduce the wild-type htrA gene in trans, test known htrA phenotypes, and provide the basis to perform further mutagenesis. We confirm that reexpression of the htrA wild-type gene in ΔhtrA mutants restored the following phenotypes: 1) C. jejuni growth at high temperature (44 °C), 2) growth under high oxygen stress conditions, 3) expression of proteolytically active HtrA oligomers, 4) secretion of HtrA into the supernatant, 5) cell attachment and invasion, and 6) transmigration across polarized epithelial cells. These results establish a genetic complementation system for htrA in C. jejuni, exclude polar effects in the ΔhtrA mutants, confirm important HtrA properties, and permit the discovery and dissection of new functions.

Keywords: E-cadherin; HtrA; cellular invasion; chaperone; flagellum; molecular pathogenesis; paracellular; secretion; signaling; stress response; transwell; virulence.

Fig. 1.

Western blotting confirms genetic complementation of wild-type (wt) htrA gene in C. jejuni ΔhtrA deletion mutants. The wt htrA gene with its own promoter was complemented in the ΔhtrA deletion mutants of C. jejuni strains 81–176 (a) and NCTC11168 (b). Bands show presence of HtrA protein expression. As loading controls, the α-CiaB and α-CadF blots confirmed that equal amounts of protein were present in each sample

Fig. 2.

Genetic complementation confirms the importance of HtrA in heat tolerance and oxygen stress resistance by C. jejuni. Serial dilutions of C. jejuni strains [81–176 wild-type (wt), 81–176ΔhtrA, and complemented 81–176ΔhtrA/htrA] were spotted onto MH agar plates as indicated. The plates were incubated for 3 days in jars under microaerobic conditions at 37 °C (a), 42 °C (b), and 44 °C (c) or at 42 °C in the presence of 17–18% O₂ (d). Representative sections of the agar plates from three independent experiments are presented. The results show that reintroduction of wt htrA gene in the ΔhtrA mutant strain restored C. jejuni growth under the indicated conditions. Experiments were done in triplicates

Fig. 3.

Western blotting showing presence of HtrA protein in bacterial pellets (top) and culture supernatants (bottom). Wild-type (wt), isogenic ΔhtrA deletion mutants, and complemented ΔhtrA/htrA strains of C. jejuni 81–176 (a, b) and NCTC11168 (c, d) were grown in BHI medium with 10% FCS for 12 h at 37 °C, and then fractionated. Equal amounts of protein in each bacterial pellet and absence of bacterial lysis in the supernatants were confirmed by probing with α-MOMP antibody

Fig. 4.

Secreted and cell-associated HtrA of the complemented C. jejuni strains generate proteolytically active oligomers. Wild-type (wt), isogenic ΔhtrA deletion mutants, and complemented ΔhtrA/htrA strains of C. jejuni 81–176 and NCTC11168 were grown in BHI medium with 10% FCS for 12 h at 37 °C. Bacterial pellets (a) and culture supernatants (b) were prepared and subjected to investigation of protease activity by casein zymography. The position of proteolytically active oligomeric HtrA proteins is indicated with arrows. Asterisks label the position of two other proteolytically active protein bands at ~65 kDa and ~42 kDa, respectively

Fig. 5.

Genetic complementation of htrA restores binding of C. jejuni to human intestinal epithelial cells. INT-407 cells infected for 6 h with wild-type (wt), isogenic ΔhtrA deletion mutants, and complemented ΔhtrA/htrA strains of C. jejuni 81–176 (a) or NCTC11168 (b). Cell adhesion of the C. jejuni strains was analyzed by gentamicin protection assay. The α-GAPDH blot served as loading control and confirmed that equal amounts of protein are present in each sample. Bars represent averages and standard deviations of three independent experiments

Fig. 6.

Genetic complementation of htrA restores invasive properties of C. jejuni in human intestinal epithelial cells. INT-407 cells were infected for 6 h with wild-type (wt), isogenic ΔhtrA deletion mutants, and complemented ΔhtrA/htrA strains of C. jejuni 81–176 (a) or NCTC11168 (b). Intracellular C. jejuni were quantified by gentamicin protection assay. The α-FAK blot served as loading control and confirmed that equal amounts of protein are present in each sample. Bars represent averages and standard deviations of three independent experiments

Fig. 7.

Transmigration of C. jejuni across polarized epithelial cells is restored by genetic complementation of htrA. Differentiated MKN-28 epithelial cells were grown in a transwell filter system for 14 days to reach a confluent monolayer. The cells were infected in the apical chamber. A time course of infection with wild-type (wt), isogenic ΔhtrA deletion mutants, and complemented ΔhtrA/htrA strains of C. jejuni 81–176 (a–c) or NCTC11168 (d) is shown. Transmigrated bacteria were harvested from the bottom chambers, grown on MH agar plates, and CFUs were determined in triplicates (a, c, d). The transepithelial resistance (TER) was measured before and after infection and did not change significantly during the indicated time course (b)