Gamma-glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni

Abstract

The contribution of gamma-glutamyl transpeptidase (GGT) to Campylobacter jejuni virulence and colonization of the avian gut has been investigated. The presence of the ggt gene in C. jejuni strains directly correlated with the expression of GGT activity as measured by cleavage and transfer of the gamma-glutamyl moiety. Inactivation of the monocistronic ggt gene in C. jejuni strain 81116 resulted in isogenic mutants with undetectable GGT activity; nevertheless, these mutants grew normally in vitro. However, the mutants had increased motility, a 5.4-fold higher invasion efficiency into INT407 cells in vitro and increased resistance to hydrogen peroxide stress. Moreover, the apoptosis-inducing activity of the ggt mutant was significantly lower than that of the parental strain. In vivo studies showed that, although GGT activity was not required for initial colonization of 1-day-old chicks, the enzyme was required for persistent colonization of the avian gut.

Fig. 1

The arrangement of ggt and adjacent genes in strains 81116 and 81-176. Arrows denote predicted direction of transcription.

Fig. 2

Comparison of the capacity of the ggt mutant to invade INT407 cells compared to the wild-type strain 81116. Invasion is represented as the total number of bacteria surviving after the gentamicin treatment. The MOI at 200 was compared for the mutant and wild-type strain. Symbols: ▲, ggt mutant; ■, wild-type.

Fig. 3

Susceptibility of the ggt mutant to hydrogen peroxide compared to the wild-type strain 81116. This was determined as the numbers of bacteria (log₁₀ cfu ml⁻¹) that survived exposure to 0.5 mM H₂O₂ over a period of 120 min. The assay was performed three times and the results from one assay using triplicate samples is shown. Symbols: ▲, ggt mutant; ■, wild-type.

Fig. 4

(a) DAPI assay for C. jejuni infection induced apoptosis and (b) the relative number of adherent colonic epithelial (CCD 841 CoN ) cells. The assay was performed three times. Symbols: ▲, ggt mutant; ■, wild-type. Phase-contrast microscopy analysis of colonic epithelial (CCD 841 CoN) cells: (c) control uninfected cells, (d) infection with wild-type strain and (e) ggt mutant. DAPI stained epithelial nuclei: (f) control uninfected cells, (g) infection with wild-type strain and (h) ggt mutant.

Fig. 5

Chick caecal colonization at (a) 5 days post-challenge and (b) three weeks post-challenge by the ggt mutant compared to the wild-type strain 81116. Colonization was determined as the number of bacteria recovered from the caecum, expressed as cfu per gram of caecal contents. The geometric mean level of colonization for each group is shown by a+. The dotted line represents the limit of detection of 100 cfu. Both wild-type and mutant were given to the chicks at a dose of ~1 × 10⁴ cfu per 100 μl 0.1 M PBS pH 7.2. Symbols: ▲, ggt mutant; ■, wild-type.