Contribution of conserved ATP-dependent proteases of Campylobacter jejuni to stress tolerance and virulence

Abstract

In prokaryotic cells the ATP-dependent proteases Lon and ClpP (Clp proteolytic subunit) are involved in the turnover of misfolded proteins and the degradation of regulatory proteins, and depending on the organism, these proteases contribute variably to stress tolerance. We constructed mutants in the lon and clpP genes of the food-borne human pathogen Campylobacter jejuni and found that the growth of both mutants was impaired at high temperature, a condition known to increase the level of misfolded protein. Moreover, the amounts of misfolded protein aggregates were increased when both proteases were absent, and we propose that both ClpP and Lon are involved in eliminating misfolded proteins in C. jejuni. In order to bind misfolded protein, ClpP has to associate with one of several Clp ATPases. Following inactivation of the ATPase genes clpA and clpX, only the clpX mutant displayed the same heat sensitivity as the clpP mutant, indicating that the ClpXP proteolytic complex is responsible for the degradation of heat-damaged proteins in C. jejuni. Notably, ClpP and ClpX are required for growth at 42 degrees C, which is the temperature of the intestinal tract of poultry, one of the primary carriers of C. jejuni. Thus, ClpP and ClpX may be suitable targets of new intervention strategies aimed at reducing C. jejuni in poultry production. Further characterization of the clpP and lon mutants revealed other altered phenotypes, such as reduced motility, less autoagglutination, and lower levels of invasion of INT407 epithelial cells, suggesting that the proteases may contribute to the virulence of C. jejuni.

FIG. 1.

Effects of puromycin on growth of C. jejuni clpP, lon, and clpP lon mutants on solid surfaces. Tenfold serial dilutions of C. jejuni NCTC11168 (wt), LB1277 (clpP), MTA21 (lon), and LB1313 (clpP lon) cultures at an OD₆₀₀ of 0.1 were spotted in volumes of 10 μl onto a base II agar plate containing 6 μg/ml puromycin. The plates were incubated for 5 days at 37°C under microaerobic conditions provided by CampyGen (Oxoid). As a control, the same dilutions were spotted onto base II agar plates. One representative of three experiments is presented.

FIG. 2.

Effects of the clpP lon mutations on the amounts of aggregated proteins. C. jejuni NCTC11168 (wt) and LB1313 (clpP lon) were grown at 37°C. Aggregated proteins were isolated according to the method of Tomoyasu et al. (58). Fractions corresponding to identical cell numbers were analyzed on a NuPage 4 to 12% N,N-methylenebisacrylamide-Tris gel (Invitrogen), followed by Coomassie staining using Safe Stain (Invitrogen).

FIG. 3.

Effects of temperature on the growth of C. jejuni wild type (wt) and clpP, lon, and clpP lon mutants (A) and the wild type and clpP, clpX, and clpA mutants (B) on solid surfaces. Tenfold serial dilutions of C. jejuni NCTC11168 (wt), LB1277 (clpP), MTA21 (lon), LB1313 (clpP lon), MTA11 (clpA), and LB1263 (clpX) cultures at an OD₆₀₀ of 0.1 were spotted in volumes of 10 μl onto base II agar plates, which were incubated for 3 days at 37, 42, or 44°C under microaerophilic conditions provided by CampyGen (Oxoid). One representative of three experiments is presented.

FIG. 4.

Effects of clpP and lon mutations on the ability of C. jejuni to move in a soft-agar medium based on heart infusion broth containing 0.25% agar. Plates were inoculated in the center with C. jejuni NCTC11168 (wt) or the LB1277 (clpP) or MTA21 (lon) mutant and incubated for 48 h at 37°C under microaerobic conditions using CampyGen (Oxoid). One representative of five experiments is presented.

FIG. 5.

The abilities to autoagglutinate of the clpP, clpA, and clpX mutants after incubation for 24 h at 25°C in PBS. C. jejuni NCTC111168 (wt), LB1277 (clpP), MTA11 (clpA), and LB1263 (clpX) were suspended in PBS to an OD₆₀₀ of 1 and incubated at 25°C for 24 h.

FIG. 6.

Effects of lon, clpP, clpP lon, clpX, and clpA mutations on the ability of C. jejuni to adhere to and enter cultured intestine cells (INT407). Cells adhered to (A) and invaded (B) are expressed as percentages of the number of cells inoculated. The bars represent the mean values, while the error bars represent the standard deviations of four wells. One representative of three experiments is presented.