Cytolethal distending toxin (CDT)-negative Campylobacter jejuni strains and anti-CDT neutralizing antibodies are induced during human infection but not during colonization in chickens

Abstract

The cytolethal distending toxin (CDT) of Campylobacter jejuni was detectable, using an in vitro assay, in most but not all of 24 strains tested. The reason for the absence of toxin activity in these naturally occurring CDT-negative C. jejuni strains was then investigated at the genetic level. CDT is encoded by three highly conserved genes, cdtA, -B, and -C. In the CDT-negative strains, two types of mutation were identified. The CDT activities of C. jejuni strains possessing both types of mutation were successfully complemented with the functional genes of C. jejuni 11168. The first type of mutation comprised a 667-bp deletion across cdtA and cdtB and considerable degeneration in the remainder of the cdt locus. Using a PCR technique to screen for this deletion, this mutation occurred in fewer than 3% of 147 human, veterinary, and environmental strains tested. The second type of mutation involved at least four nonsynonymous nucleotide changes, but only the replacement of proline with serine at CdtB position 95 was considered important for CDT activity. This was confirmed by site-directed mutagenesis. This type of mutation also occurred in fewer than 3% of strains as determined using a LightCycler biprobe assay. The detection of two CDT-negative clinical isolates raised questions about the role of CDT in some cases of human campylobacteriosis. To determine if anti-CDT antibodies are produced in human infection, a toxin neutralization assay was developed and validated using rabbit antisera. Pooled human sera from infected patients neutralized the toxin, indicating expression and immunogenicity during infection. However, no neutralizing antibodies were detected in colonized chickens despite the expression of CDT in the avian gut as indicated by reverse transcription-PCR.

FIG. 1.

Amplified products of cdtA, -B, and -C of selected strains with known in vitro CDT activity, using primers Cdt-F and Cdt-R.

FIG. 2.

Schematic diagram of cdtABC of C. jejuni 81-176, illustrating the locations of the 667- and 50-bp deletions in strains C37596, C37533, and C35926. Arrows indicate direction of transcription.

FIG. 3.

Alignment of the CdtB amino acid sequences of C. jejuni strains EF and 11168 and other bacterial species. Shaded areas highlight amino acids identical to those in C. jejuni 11168. Accession numbers are as follows: C. jejuni EF, AY445094; C. jejuni 11168 CdtB, CAB72564; C. upsaliensis CdtB, AAF98364; A. actinmyetemcomitians CdtB, AAC70898; H. ducreyi CdtB, AAB57726; H. hepaticus CdtB, AAF19158; and E. coli CdtB, 2010282B. Asterisks indicate point mutations found in C. jejuni EF. The amino acids are numbered continuously on the left.

FIG. 4.

RT-PCR to detect the expression of CDT in C. jejuni EF and 11168, using primers DS15 and DS18 to amplify a 450-bp region overlapping cdtA and cdtB (A), and in strains 99/373, S58, and 99/12 (B). Lanes A, reverse-transcribed RNA sample; lanes B, RT-negative controls; lanes C, DNA controls; M., 1-kb ladder.

FIG. 5.

Melting curve analyses by LightCycler for 20 selected C. jejuni strains, including EF, for the detection of polymorphism at CdtB-95. A negative control is included. Vertical lines represent the presence of proline (codon CCT) (blue) (as represented by strain 81116), serine (codon TCT) (green) (as represented by strains EF, S58, and 99/373), or more than 1-bp mismatch (orange) (as represented by strain 99/12) at CdtB-95. A no-template control (NTC) was used in each run.

FIG. 6.

CDT activities of C. jejuni strains 99/68 and EF, complemented with a 2.4-kb region from C. jejuni 11168 containing the cdt genes, to give 99/68 pCDT and EF pCDT, and the same strains in which site-directed mutagenesis was performed to replace proline at CdtB-95 with serine, generating 99/68 pCDT^P95S and EF pCDT^P95S. Assays were performed in triplicate, and C. jejuni 11168 was used as a positive control. Error bars indicate standard deviations.

FIG. 7.

RT-PCR of total RNA extracted from the cecal contents of chickens colonized with C. jejuni strain C289/6 (CDT positive) and from campylobacter-free birds, using primers DS15 and DS18 to detect the expression of cdtA and cdtB. C. jejuni 11168 grown in vitro was used as the positive control. Lanes A, reverse-transcribed RNA sample; lanes B, RT-negative controls; lanes C, DNA controls; lanes M., 1-kb ladder.

FIG. 8.

Lysates of C. jejuni strains 81116, 81-176, and 11168 were treated with rabbit antisera directed against strain 81116 whole cells (R12) (), strain EF (R43) (▪), pooled campylobacteriosis patient sera (), or pooled experimentally colonized chicken sera (∥) prior to CDT activity assays. Sera from pooled normal human blood donors (▧) as well as sera from preimmunized rabbits (§) and uncolonized chickens (data not shown) were used as controls. The lysates were tested for CDT activity and the percent neutralization determined by comparison with untreated lysates. Error bars indicate standard deviations.

FIG. 9.

Western blots showing reactivities of sera from human blood donors (lanes A), human campylobacteriosis patients (lanes B), uncolonized chickens (lanes C), and experimentally C. jejuni 81116-colonized chickens (lanes D) to total protein profiles of C. jejuni 11168 and 81-176. Molecular mass markers (M) are shown on right.