Characterization of a potential ABC-type bacteriocin exporter protein from Treponema denticola

Abstract

Background: Treponema denticola is strongly associated with the development of periodontal disease. Both synergistic and antagonistic effects are observed among bacterial species in the process of biofilm formation. Bacteriocin-related genes have not yet been fully characterized in periodontopathic bacteria. The aim of this study was to detect and characterize bacteriocin-associated proteins in T. denticola.

Methods: The whole genome sequence of T. denticola ATCC 35405 was screened with a Streptococcus mutans bacteriocin immunity protein (ImmA/Bip) sequence. The prevalence of homologous genes in T. denticola strains was then investigated by Southern blotting. Expression of the genes was evaluated by qRT-PCR.

Results: In the genome sequence of T. denticola, an amino acid sequence coded by the open reading frame TDE_0719 showed 26 % identity with the S. mutans ImmA. Furthermore, two protein sequences encoded by TDE_0425 and TDE_2431 in T. denticola ATCC 35405 showed ~40 % identity with that coded by TDE_0719. Therefore, TDE_0425, TDE_0719, and TDE_2431 were designated as tepA1, A2, and A3, respectively. Open reading frames showing similarity to the HlyD family of secretion proteins were detected downstream of tepA1, A2, and A3. They were designated as tepB1, B2, and B3, respectively. A gene harboring a bacteriocin-like signal sequence was detected upstream of tepA1. The prevalence of tepA1 and A2 differed among Treponema species. Susceptibility to chloramphenicol and ofloxacin was slightly decreased in a tepA2 mutant while that to kanamycin was increased. Expression of tepA3-B3 was increased in the tepA2 mutant.

Conclusion: These results indicate that T. denticola ATCC 35405 has three potential bacteriocin export proteins and that the presence of these genes differs among the Treponema strains. TepA3-B3 of the corresponding proteins may be involved in resistance to chloramphenicol.

Keywords: ABC transporter; Antimicrobial agent susceptibility; Bacteriocin; Treponema denticola.

Fig. 1

Homology between the S. mutans bacteriocin immunity protein ImmA and the deduced amino acid sequence of TDE_0719 in T. denticola ATCC35405

Fig. 2

Multiple sequence alignments of bacteriocin ABC transporters. Cysteine and histidine, which are part of the putative active site of the peptidase family C39B, as well as glutamine, which contributes to the oxyanion pore in other cysteine protease families, are marked with * and indicated in yellow. The ATP-binding site and ABC transporter signature motifs are indicated in yellow and marked with † and #, respectively. The alignment was carried out using the program Genetyx-Mac 16.0.9. C. divergens: ATP-dependent transporter of Carnobacterium divergens, L. lactis: Lactococcin-A transport/processing ATP-binding protein LcnC of Lactococcus lactis subsp. lactis, L. mesenteroides: Mesentericin-Y105 transport/processing ATP-binding protein MesD of Leuconostoc mesenteroides, P. acidilactici: Pediocin PA-1 transport/processing ATP-binding protein PedD of Pediococcus acidilactici, TDE_0425: tepA1, TDE_0719: tepA2, TDE_2431: tepA3

Fig. 3

Southern blot analysis of tepA1 (a), tepA2 (b), and tepA3 (c). Genomic DNA from T. denticola strains was digested with HindIII. 1: genomic DNA from ATCC 33520, 2: genomic DNA from ATCC 33521, genomic DNA from ATCC 35404, 4: genomic DNA from ATCC 35405, 5: genomic DNA from GM1

Fig. 4

Effect of inactivation of tepA2 on the growth of T. denticola in medium containing chloramphenicol (a), kanamycin (b), or ofloxacin (c) in the wild-type and tepA2-deficient KT-3 strains. T. denticola was adjusted to OD₆₆₀ = 0.1 and inoculated into TYGVS medium containing antibiotics. Growth of T. denticola was evaluated by measuring the OD₆₆₀. The experiments were performed twice in quadruplicate. Data are presented as the mean ± SD (n = 8). *P < 0.05 vs. ATCC 35405 under the same concentration of antibiotics

Fig. 5

Expression of tep and TDE_0820 in the presence or absence of chloramphenicol. Expression of tepA1 (a), tepB1 (b), tepA3 (c), tepB3 (d), TDE_0820 (e) in the presence or absence of chloramphenicol in the wild-type and tepA2-deficient mutant KT-3. Expression levels of each gene were normalized using 16S rRNA levels as internal controls and are expressed as a fold modulation relative to the wild-type strain grown without chloramphenicol. Experiments were performed three times in triplicate. Data are presented as the mean ± SD (n = 9). *P < 0.05 vs. ATCC 35405 without chloramphenicol, † P < 0.05 vs. ATCC 35405 with chloramphenicol, § P < 0.05 vs. KT-3 without chloramphenicol