Genome organization and pathogenicity of Corynebacterium diphtheriae C7(-) and PW8 strains

Abstract

Corynebacterium diphtheriae is the causative agent of diphtheria. In 2003, the complete genomic nucleotide sequence of an isolate (NCTC13129) from a large outbreak in the former Soviet Union was published, in which the presence of 13 putative pathogenicity islands (PAIs) was demonstrated. In contrast, earlier work on diphtheria mainly employed the C7(-) strain for genetic analysis; therefore, current knowledge of the molecular genetics of the bacterium is limited to that strain. However, genomic information on the NCTC13129 strain has scarcely been compared to strain C7(-). Another important C. diphtheriae strain is Park-Williams no. 8 (PW8), which has been the only major strain used in toxoid vaccine production and for which genomic information also is not available. Here, we show by comparative genomic hybridization that at least 37 regions from the reference genome, including 11 of the 13 PAIs, are considered to be absent in the C7(-) genome. Despite this, the C7(-) strain still retained signs of pathogenicity, showing a degree of adhesion to Detroit 562 cells, as well as the formation of and persistence in abscesses in animal skin comparable to that of the NCTC13129 strain. In contrast, the PW8 strain, suggested to lack 14 genomic regions, including 3 PAIs, exhibited more reduced signs of pathogenicity. These results, together with great diversity in the presence of the 37 genomic regions among various C. diphtheriae strains shown by PCR analyses, suggest great heterogeneity of this pathogen, not only in genome organization, but also in pathogenicity.

FIG. 1.

Comparison of genomes by CGH. Genomic DNAs of C. diphtheriae C7(−) or PW8 (test) and the reference strain (reference) were subjected to comparative genomic hybridization with NimbleGen-type tiling arrays covering the entire genome of the C. diphtheriae reference strain NCTC13129. By comparing the hybridization signals with reference DNA and with test DNA, regions present in the reference genome but lacking in the test genome were identified. (A) Summary of results for the corynephage region (PAI 1). (B and C) Summary of results from the whole C7(−) and PW8 genomes, respectively. Rows 1, CDSs in the NCTC13129 genome; rows 2, regions in which a difference between signals from the two strains was suggested; rows 3, regions suggested to be absent in the test genome; rows 4, ratio of signal intensity (reference/test, expressed as log₂); rows 5, signal intensity from the test genome; rows 6, signal intensity from the reference genome. The red bar in panel A indicates the span of the corynephage region.

FIG. 2.

Circle representation of genome regions proposed to be absent in the C7(−) and PW8 genomes. The solid circle represents the whole genome of C. diphtheriae NCTC13129 (reference strain). The putative PAIs are 13 PAIs reported in NCTC13129 by Cerdeño-Tarrága et al. (9). The numbers correspond to PAI numbers in the text and Fig. 3. The artwork was prepared using PlasMapper Web software (14).

FIG. 3.

PCR analysis of selected CDSs in C. diphtheriae clinical isolates and laboratory strains. Amplification of DNA fragments corresponding to selected CDSs located in the regions proposed to be absent in the C7(−) genome was performed with template DNA from C7(−), the reference strain, ATCC 11951 (C4B), the vaccine strain PW8, and 10 Japanese clinical isolates, with primers listed in Table S1 in the supplemental material. Pink shading indicates that amplification was successful. Yellow shading indicates that weak bands were observed at positions identical to those of the band observed for the reference strain. Green shading indicates one or more bands observed at a different position(s) from that observed for the reference strain. These bands probably represent nonspecific amplification, because raising the annealing temperature from 54°C to 56°C eliminated such bands as far as we tested. No amplification was observed for the white squares. *, not determined.

FIG. 4.

Adhesion of C. diphtheriae to Detroit 562 cells. Adhesion of the C. diphtheriae reference strain, C7(−), and PW8 was assayed as described in Materials and Methods. The inoculum sizes were 1.2 × 10⁷ (reference strain), 3.2 × 10⁷ [C7(−)], and 7.1 × 10⁶ (PW8). The bars indicate standard errors from quadruplicate assays. Assays were repeated 6 times, and representative results from one of the assays are shown. P = 0.004 [reference versus C7(−)], 0.004 (reference versus PW8), and 0.010 [C7(−) versus PW8] by Student's t test.

FIG. 5.

Mouse intradermal challenge. The backs of female ICR mice (6 weeks of age) were depilated and then intradermally inoculated with C. diphtheriae C7(−), the reference strain, and PW8. The mice were sacrificed 3 days after inoculation, and the skins were removed. (A) Internal views of abscesses formed by 7.0 × 10⁶ CFU (a, site 1) and 7.0 × 10⁷ CFU (b, site 1) C. diphtheriae C7(−); 2.0 × 10⁶ CFU (a, site 2) and 2.0 × 10⁷ CFU (b, site 2) C. diphtheriae PW8; 6.4 × 10⁶ CFU (a, site 3) and 6.4 × 10⁷ (b, site 3) CFU C. diphtheriae reference strain; and 0.05 ml of saline (a and b, sites 4). Three mice were used for each of the experiments (a and b), and representative results from each are shown. (B) Recovery of viable cells from abscesses formed by ca. 10⁶ CFU of bacterial cells. Abscesses from tissues (shown in panel A, a) were homogenized, and the numbers of viable bacteria in homogenates were measured as described in Materials and Methods. Assays were done in triplicate. P = 0.56 [reference strain versus C7(−)]. The error bar [C7(−)] indicates the standard error. The standard error could not be calculated for the reference strain because a set of three data could not be obtained (from one of three mice, viable bacteria were not recovered). The actual numbers of CFU recovered were 1.2 × 10⁵ (reference strain) and 5.2 × 10⁵ [C7(−)]. PW8 was not recovered from any of three mice. (C) Recovery of viable cells from abscesses formed by ca. 10⁷ CFU of bacterial cells. Recovery from abscesses from tissues (shown in panel A, b) are shown. P = 0.046 [reference strain versus C7(−)], 0.059 (reference strain versus PW8), and 0.018 [C7(−) versus PW8]. The actual numbers of CFU recovered were 5.4 × 10⁶ (reference strain), 8.9 × 10⁶ [C7(−)]), and 7.9 × 10⁴ (PW8). The mouse assays were repeated twice with consistent results.

FIG. 6.

Electron microscopic appearance of abscess contents. Mice were inoculated with C. diphtheriae C7(−) (9.5 × 10⁷ CFU), and the abscesses were removed and prepared for electron microscopy as described in Materials and Methods 3 days after inoculation. (A) Mouse phagocytic cells containing C. diphtheriae C7(−). The arrows indicate bacteria enclosed in a vacuole-like structure. The arrowheads indicate bacteria in the cytosol. The asterisks indicate lysosomes. Original magnification, ×5,000; bar, 1 μm. (B) Disintegrating mouse phagocytic cells containing C. diphtheriae C7(−). The arrow indicates a disrupted cytoplasmic membrane and vacuole-like structure from which bacteria could escape into the abscess milieu. The arrowhead shows a dividing bacterial cell in the cytosol. Original magnification, ×3,000; bar, 2 μm. (C) Dividing C. diphtheriae C7(−) cells in vacuole-like structures in a mouse phagocytic cell. The arrow indicates a disrupted membrane. The arrowheads show septa, typical of dividing C. diphtheriae cells. Original magnification, ×10,000; bar, 500 nm.

FIG. 7.

Hemagglutinating and hemolytic activities of C. diphtheriae. Dilutions of the C. diphtheriae reference strain, C7(−), and a human erythrocyte suspension were prepared as described in Materials and Methods. The trypsin and neuraminidase treatments are also described in the text. The bacterial suspensions were mixed with erythrocyte suspensions in a 96-well microtiter plate; after incubation at room temperature for 16 h, hemagglutination was scored macroscopically and hemolysis was measured by absorbance at 545 nm. (A) Hemagglutination by the reference strain. −/−, untreated erythrocytes; T/−, treated with trypsin; −/N, treated with neuraminidase; T/N, treated with both trypsin and neuraminidase; no bacteria, control without bacterial suspension. (B) Measurement of hemolytic activity by C7(−). OD₆₀₀ of bacterial suspension is the dilution of C7(−) suspension expressed in OD units; absorbance at 545 nm is the absorbance of the supernatant of the mixture after incubation for 16 h. −/−, untreated erythrocytes; −/N, treated with neuraminidase; T/−, treated with trypsin; T/N, treated with both trypsin and neuraminidase.