The cell cycle-specific growth-inhibitory factor produced by Actinobacillus actinomycetemcomitans is a cytolethal distending toxin

Abstract

Actinobacillus actinomycetemcomitans has been shown to produce a soluble cytotoxic factor(s) distinct from leukotoxin. We have identified in A. actinomycetemcomitans Y4 a cluster of genes encoding a cytolethal distending toxin (CDT). This new member of the CDT family is similar to the CDT produced by Haemophilus ducreyi. The CDT from A. actinomycetemcomitans was produced in Escherichia coli and was able to induce cell distension, growth arrest in G2/M phase, nucleus swelling, and chromatin fragmentation in HeLa cells. The three proteins, CDTA, -B and -C, encoded by the cdt locus were all required for toxin activity. Antiserum raised against recombinant CDTC completely inhibited the cytotoxic activity of culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. These results strongly suggest that the CDT is responsible for the cytotoxic activity present in the culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. This CDT is a new putative virulence factor of A. actinomycetemcomitans and may play a role in the pathogenesis of periodontal diseases.

FIG. 1

Effect of A. actinomycetemcomitans Y4 sonic lysate on cultured HeLa cells. (A) HeLa cells 3 days after incubation with (b) or without (a) 4 CD₅₀ of sterile sonic lysate of A. actinomycetemcomitans Y4. Magnification, ×150. (B) Cell cycle pattern of HeLa cells incubated with 4 CD₅₀ of sterile sonic lysate of A. actinomycetemcomitans Y4 (Aa lysate) for the indicated times. Control, cells without treatment.

FIG. 2

Restriction map of subclones from A. actinomycetemcomitans DNA that contains cdtABC. Arrows represent ORFs and directions of transcription. CDT activity was measured by incubating sterile sonic lysates of recombinant strains with HeLa cell cultures for 3 days. Clones expressing CDT activity are indicated at the right. The restriction sites of relevant endonucleases are indicated: HIII, HindIII; HII, HincII; E, EcoRI; A, AccI; S, SmaI. MCS, multicloning site for cloning vector.

FIG. 3

DNA sequence analysis in the region of the HincII-EcoRI site of the cloned DNA fragment. ORF1, ORF2, and three complete ORFs defining cdtA, -B, and -C were found. Putative ribosome binding (Shine-Dalgarno [SD]) sites for cdt are boxed. Putative signal peptide cleavage sites are indicated (▴). Oligonucleotide primers used are labelled and are indicated by arrows above the relevant sequences. A DNA sequence similar to that of the integrating plasmid of H. influenzae is underlined.

FIG. 3

DNA sequence analysis in the region of the HincII-EcoRI site of the cloned DNA fragment. ORF1, ORF2, and three complete ORFs defining cdtA, -B, and -C were found. Putative ribosome binding (Shine-Dalgarno [SD]) sites for cdt are boxed. Putative signal peptide cleavage sites are indicated (▴). Oligonucleotide primers used are labelled and are indicated by arrows above the relevant sequences. A DNA sequence similar to that of the integrating plasmid of H. influenzae is underlined.

FIG. 4

Autoradiograph of an SDS–12% polyacrylamide gel containing [³⁵S]methionine-labelled products of in vitro transcription-translation. Lanes: 1, pGEM-T Easy; 2, pTK3251, containing the A. actinomycetemcomitans cdtA gene; 3, pTK3252, containing the A. actinomycetemcomitans cdtB gene; 4, pTK3253, containing the A. actinomycetemcomitans cdtC gene; 5, pTK3022, containing the entire cdtABC gene cluster; 6, pUC19. The CDT activities of sterile sonic lysates from the recombinant strains are indicated at the bottom. Radiolabelled bands marked by #, ∗, and + are putative gene products of cdtA, -B, and -C, respectively.

FIG. 5

Effect of A. actinomycetemcomitans Y4 CDT on morphology of cultured HeLa cells. Phase-contrast microscopy (A) and PI and Hoechst 33342 staining (B) of HeLa cells incubated with 20 CD₅₀ of a sterile sonic lysate of E. coli(pTK3022) for 3 days (b) or 4 days (c) are shown. (a) Control HeLa cells treated with a lysate of E. coli XL-1 Blue (5) for 4 days. Magnification, ×200.

FIG. 6

Effect of A. actinomycetemcomitans Y4 (Aa) CDT on cell cycle pattern of HeLa cells. HeLa cells were incubated with 20 CD₅₀ of a sterile sonic lysate of E. coli(pTK3022) for the indicated times.

FIG. 7

Immunological detection of CDT in culture supernatant or sonic lysate of A. actinomycetemcomitans or recombinant E. coli carrying pTK3022. Western blotting was performed as described previously (49). Immunodetection was performed with antiserum against His-tagged CDTA (A), His-tagged CDTB (B), or His-tagged CDTC (C) or with control nonimmune serum (D). Lanes: 1, sonic lysate of E. coli XL-1 Blue(pTK3022); 2, culture supernatant of E. coli XL-1 Blue(pTK3022); 3, sonic lysate of A. actinomycetemcomitans Y4; 4, culture supernatant of A. actinomycetemcomitans Y4; 5, sonic lysate of E. coli XL-1 Blue(pUC19); 6, culture supernatant of E. coli XL-1 Blue(pUC19).

FIG. 8

Effect of anti-CDT serum on cytodistending activity of sterile sonic lysate of A. actinomycetemcomitans Y4. (A) HeLa cells were incubated with a sterile sonic lysate of A. actinomycetemcomitans Y4 (17 CD₅₀) in the presence of the indicated dilutions of anti-CDTA (bars 1), anti-CDTB (bars 2), and anti-CDTC (bars 3) for 4 days. The percentage of distended cells was calculated by counting the number of distended cells per 200 cells. (B) Phase-contrast microscopy of HeLa cells incubated with a sterile sonic lysate of A. actinomycetemcomitans Y4 (17 CD₅₀) in the presence of a 300-fold dilution of indicated antiserum for 4 days. Control, cells without treatment. Magnification, ×150.