Characterization of two putative cytochrome c peroxidases of Campylobacter jejuni involved in promoting commensal colonization of poultry

Abstract

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans throughout the world, but infection of animals, especially poultry, results in a commensal colonization of the intestines. We previously found that a mutant lacking docA, which encodes a putative cytochrome c peroxidase (CCP), demonstrates up to a 10(5)-fold reduction in colonization of the chick cecum compared to wild-type C. jejuni strain 81-176. Predictions from genomic sequences identified CJJ0382 as a second locus in C. jejuni encoding a CCP, making the bacterium unusual in having two putative CCPs. To understand what advantages are imparted by having two putative CCPs, we compared the colonization requirements of C. jejuni mutants lacking DocA or Cjj0382. Unlike the DeltadocA mutant, a DeltaCJJ0382 mutant demonstrates a maximal 50-fold colonization defect that is dependent on the inoculum dose. The colonization differences of mutants lacking DocA or Cjj0382 suggest that the two predicted CCPs are unlikely to perform redundant functions during in vivo growth. In the characterizations of DocA and Cjj0382, we found that they are stable periplasmic proteins with an apparent heme-dependent peroxidase activity, which are characteristics of bacterial CCPs. However, the peroxidase activities of the proteins do not appear to contribute to resistance to hydrogen peroxide. Instead, we found that resistance to hydrogen peroxide in C. jejuni is mostly attributed to the cytoplasmic catalase KatA. Our data suggest that DocA and Cjj0382 have characteristics of CCPs but likely perform different physiological functions for the bacterium in colonization that are not related to resisting oxidative stress.

FIG. 1.

The ΔCJJ0382 mutant of C. jejuni displays a less severe colonization defect than the ΔdocA mutant. One-day-old chicks were orally infected with 100 μl of C. jejuni 81-176 Sm^r (DRH212), 81-176 Sm^r ΔdocA (DRH1169), 81-176 Sm^r ΔCJJ0382 (LKB151), or 81-176 Sm^r ΔdocA ΔCJJ0382 (LKB177) with inocula of approximately 10⁶ (A), 10⁴ (B), or 10² (C) bacteria. Chicks were sacrificed 7 days postinfection to determine the cecal colonization capacity of each C. jejuni strain. Each filled symbol represents the amount of C. jejuni recovered from the cecum of a single chick, reported as the number of CFU per gram of cecal content. Each open symbol represents chicks in which the level of C. jejuni colonization was below the limit of detection (<100 CFU per gram of cecal content). The geometric mean of the bacterial loads from each set of chicks is denoted by a short horizontal line. Statistical analysis was performed using the Mann-Whitney U test (P < 0.05). *, significant difference between wild-type and mutant strains; **, significant difference between the 81-176 Sm^r ΔdocA mutant and the 81-176 Sm^r ΔdocA ΔCJJ0382 mutant. The actual inoculum doses ranged from 5 × 10⁵ to 1.7 × 10⁶ CFU (A), 4.4 × 10³ to 2.19 × 10⁴ CFU (B), and 58 to 208 CFU (C).

FIG. 2.

DocA and Cjj0382 are periplasmic proteins in C. jejuni. Proteins were separated by 10% SDS-PAGE. Anti-DocA and anti-Cjj0382 antisera were used for detection of the respective proteins. (A) Analysis of DocA localization. Strains used include C. jejuni 81-176 Sm^r (DRH212), ΔdocA (DRH1169), ΔdocA/pRY112 (LKB307), and ΔdocA/pRY112::docA (LKB313). (B) Analysis of Cjj0382 localization. Strains used include C. jejuni 81-176 Sm^r (DRH212), ΔCJJ0382 (LKB151), ΔCJJ0382 /pRY112 (LKB310), and ΔCJJ0382/pRY112::CJJ0382 (LKB277). For both panels A and B, fractions analyzed included those of the WCL, outer membrane, inner membrane, periplasm, and cytoplasm. Anti-FlgP, anti-AtpF, and anti-RpoA were used to verify fractionation procedures for the outer membrane, inner membrane, and cytoplasm, respectively. Proteins from WCL representing 200 μl of bacterial culture and proteins from the other fractions representing 400 μl of bacterial culture were used for immunoblotting.

FIG. 3.

DocA and Cjj0382 have heme-associated peroxidase activities. A heme stain assay was performed on proteins isolated from the periplasm. (A) Analysis of DocA heme-binding ability. Strains used included C. jejuni 81-176 Sm^r (DRH212), ΔdocA (DRH1169), ΔdocA/pRY112 (LKB307), and ΔdocA/pRY112::docA (LKB313). (B) Analysis of Cjj0382 heme-binding ability. Strains used include C. jejuni 81-176 Sm^r (DRH212), ΔCJJ0382 (LKB151), ΔCJJ0382/pRY112 (LKB310), and ΔCJJ0382 /pRY112::CJJ0382 (LKB277). The upper band represents Cjj0382, the faint lower band is DocA, and the middle band is an uncharacterized heme-bound protein of C. jejuni.

FIG. 4.

DocA and Cjj0382 do not promote significant resistance to hydrogen peroxide in vitro. C. jejuni strains were treated with 0.5 mM hydrogen peroxide for 30 min at 37°C under microaerophilic conditions. (A) The percent survival of wild-type bacteria was set at 100%, and all other strains were normalized to this value. Strains used include C. jejuni 81-176 Sm^r (DRH212), ΔdocA (DRH1169), ΔCJJ0382 (LKB151), ΔdocA ΔCJJ0382 (LKB177), and ΔkatA (LKB246). (B) The percent survival of the ΔkatA mutant was set at 100%, and all other strains were normalized to this value. Strains used include C. jejuni Sm^r ΔkatA (LKB246), ΔkatA ΔdocA (LKB181), ΔkatA ΔCJJ0382 (LKB231), and ΔkatA ΔdocA ΔCJJ0382 (LKB226). For both panels A and B, each diamond represents the percent survival of a bacterial sample after exposure to hydrogen peroxide.

FIG. 5.

A ΔkatA mutant of C. jejuni has a moderate defect for cecal colonization in chicks. One-day-old chicks were orally infected with 100 μl of C. jejuni 81-176 Sm^r (DRH212) or 81-176 Sm^r ΔkatA (LKB246) with inocula of approximately 10⁶, 10⁴, or 10² bacteria. Chicks were sacrificed 7 days postinfection to determine the cecal colonization capacity of each C. jejuni strain. Each filled symbol represents the amount of C. jejuni recovered from the cecum of a single chick, reported as the number of CFU per gram of cecal content. The geometric mean of the bacterial loads from each set of chicks is denoted by a short horizontal line. Statistical analysis was performed using the Mann-Whitney U test (P < 0.05). *, significant difference between wild-type and mutant strains. The actual inoculum doses ranged from 1.36 × 10⁶ to 2.38 × 10⁶ CFU (left), 5.9 × 10³ to 8.2 × 10³ CFU (middle), and 58 to 59 CFU (right). The colonization assay of wild-type C. jejuni shown in this figure is from the same experiments shown in Fig. 1A to C.