- Research article
- Open Access
- Open Peer Review
Characterization of a potential ABC-type bacteriocin exporter protein from Treponema denticola
© The Author(s). 2016
- Received: 11 March 2016
- Accepted: 2 July 2016
- Published: 16 July 2016
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.
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.
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.
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.
- ABC transporter
- Antimicrobial agent susceptibility
- Treponema denticola
Treponema denticola is a spiral-shaped motile rod that is frequently isolated from the periodontal pockets of subjects with chronic periodontitis [1, 2] and that possesses several potential virulence factors . It is often co-isolated with Porphyromnas gingivalis and Tannerella forsythia from the dental plaque biofilms of chronic periodontitis patients  and is involved in the development of periodontitis [5, 6].
Interactions among bacterial species that reside in dental plaque biofilms influence the composition of the biofilms [7–9]. Microorganisms in biofilms develop their niche by symbiosis with other microorganisms and suppression of competitors by secreting antagonistic factors [10, 11]. To suppress the growth of a competitor, various microorganisms produce antimicrobials such as bacteriocins and H2O2 . These antimicrobial factors play an important role in the survival strategy of the microorganisms in the biofilms. Similarly, in dental plaque biofilms, many species of microorganisms have been reported to produce bacteriocins or bacteriocin-like substances [13–18]. In the bacteriocin-producing bacteria, genes encoding a bacteriocin, a bacteriocin ABC transporter, and bacteriocin immunity proteins are involved in bacteriocin production . Bacteriocin immunity proteins protect the microbes against the effects of their own bacteriocins . Bacteriocin ABC transporters have two functional domains: a peptidase C39 domain, which is involved in the processing of bacteriocin precursors at the double glycine, and an ABC transporter domain, which is involved in the export of the bacteriocins . A synergistic effect between T. denticola and P. gingivalis has been reported , while growth of T. denticola was inhibited by plaque-associated Streptococcus mutans . The antagonistic effects produced by bacteriocins have been genetically characterized in oral streptococci [24–26], while those in periodontopathic bacteria remain to be established. Suppressing the growth of and avoiding inhibition by competitors would benefit T. denticola in the colonization of the subgingival plaque and the development of periodontopathic biofilms.
In the present study, we intended to characterize bacteriocin-associated proteins from T. denticola. By screening of T. denticola genomic DNA using S. mutans bacteriocin immunity protein, ABC-type bacteriocin exporter-like proteins were detected and the function of the exporter was investigated.
Bacterial strains and culture conditions
Sequence homology-based screening
The whole genome sequence of T. denticola ATCC 35405 in the Los Alamos oral pathogen database (http://www.oralgen.org) was screened for homologous sequences with the S. mutans bacteriocin immunity protein (ImmA/Bip) sequence  using the protein blast program. The obtained homologous sequences were further compared against the database of National Center for Biotechnology Information (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi). The DNA sequences coding for the homologous proteins in T. denticola were designated as tepA1, A2, and A3 as described in the Results section, and they were characterized with Genetyx-MAC v. 17.0.6 (Genetyx Corporation, Tokyo, Japan).
List of gene-specific primers used in this study
Primers and probes
Construction of a tepA2 mutant
As TepA2 showed similarity to ImmA, a tepA2-deficient mutant of T. denticola ATCC 35405 was constructed by allelic exchange mutation to investigate the role of tepA2. Briefly, two fragments flanking the tepA2 gene were amplified with primer pairs 718D/719U and 719D/720U (listed in Table 2), respectively. The ermF-ermAM cassette was amplified with the primers EMD2 and EMU2 and the fragment was inserted between the upstream and downstream fragments using the PCR-based overlap-extension method . The constructed fragments were introduced by electroporation and transformants were isolated on TYGVS agar plates containing 40 μg/ml erythromycin as described previously . Inactivation of the gene in mutant KT-3 was confirmed by Southern blot and PCR analyses.
Antibiotic susceptibility testing
The effect of tepA2 inactivation on the susceptibility of T. denticola to antibiotics was investigated. Chloramphenicol, ofloxacin, and kanamycin, to which T. denticola showed low susceptibility in our preliminary results, were selected. T. denticola ATCC 35405 and KT-3 were cultured as described above for 4 days. The cells were adjusted to an optical density at 660 nm (OD660) of 0.1 with TYGVS medium using a spectrophotometer (UV-2550, Shimadzu, Kyoto, Japan), and 100 μl of the cell suspension was added to TYGVS containing 0.5–1 μg/ml of chloramphenicol, 16–64 μg/ml of kanamycin, or 8–32 μg/ml of ofloxacin. After incubation for 7 days under anaerobic conditions, cell growth was measured at OD660 with the spectrophotometer.
DNA microarray analysis
T. denticola ATCC 35405 and KT-3 were cultured as described above for 2 days. To investigate the relation between inactivation of tepA2 and increase of chloramphenicol resistance, exponentially growing cells (OD660 ~ 0.2) were incubated with chloramphenicol (1 μg/ml) for 4 h. The cells were harvested immediately after chloramphenicol treatment and total RNA was extracted using Trizol (Life Technologies). DNase treatment was carried out using a TURBO DNA-free kit Life Technologies). cDNA was synthesized using a SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen). DNA microarray gene expression analysis was carried out using Roche NimbleGen custom arrays (2006-07-27_TI243275_60mer; Roche, Indianapolis, IN) according to the standard NimbleGen procedure (NimbleGen arrays user’s guide: gene expression analysis, v6.0). Briefly, cDNA (0.5–1 μg) was labeled using a NimbleGen one-color labeling kit, in which Cy3 was randomly incorporated into the newly synthesized DNA by the Klenow fragment. Labeled cDNA (3 μg) derived from each RNA sample was hybridized with each array for 16 to 18 h. The slides were washed, spun dry, and scanned with an Agilent Microarray Scanner with a resolution of 5 μm. Normalization was carried out with the NimbleScan 184.108.40.206 built-in normalization function. Target genes were confirmed by real-time PCR analysis using primers listed in Table 2.
Quantitative reverse transcription (qRT) PCR expression analysis of tepA1-B1, tepA3-B3, and TDE_0820
To investigate the relationships among tepA1, A2 and A3, expression of tepA1 and A3, tepB1 and B3, and TDE_0820 in the wild-type strain and KT-3 were evaluated. T. denticola ATCC 35405 and KT-3 were cultured as described above. Cells at mid-log phase (OD660 of 0.4–0.6) were harvested and total RNA was extracted using Trizol (Life Technologies). DNase treatment was carried out using a TURBO DNA-free kit (Life Technologies) and cDNA was synthesized using ReverTra Ace (Toyobo, Osaka, Japan). Gene expression was measured with real-time PCR using primers and the Taqman probe (Life Technologies) listed in Table 2 on a 7500 Real-Time PCR System (Life Technologies). Expression of each gene was normalized to the level of 16S rRNA as an internal control and was expressed as a fold modulation relative to the wild-type strain grown without chloramphenicol.
Comparisons of gene expression and susceptibility to antibiotics were carried out using Student’s t-test. One-way ANOVA followed by Tukey’s multiple comparison test was used for comparisons of gene expression among the two strains grown with or without chloramphenicol. All tests were carried out using Prism v. 5f (GraphPad software, San Diego, CA). The level of significance for all statistical tests was set at P < 0.05.
Screening of homologous sequences for bacterial immunity proteins in T. denticola
TDE_0720, which is located immediately downstream of TDE_0719, displayed similarity to the multidrug resistance efflux pump of Spirochaeta africana DSM 8902 (22 % identity in a 369-amino-acid overlap) and the HlyD family secretion protein of Desulfosporosinus sp. OT (27 % identity in a 459 amino-acid overlap). The region of TDE_0720 spanning residues 240 to 350 had similarity to a domain of a HlyD family secretion protein (pfam13437) that is reported to be part of an accessory protein for ABC exporters of gram-negative bacteria for translocating proteins across the outer membrane . Downstream of tepA1 and tepA3, TDE_0426 and TDE2430 were detected, and both also showed similarity to sequences coding HlyD family secretion proteins. The presence of the C39B peptidase domain, a conserved ATP-binding motif, a membrane-spanning domain, and a nearby accessory protein facilitating export is consistent with the properties of a bacterial ABC exporter. Therefore, we designated TDE_0426, TDE_0720, and TDE2430 as Treponema exporter proteins B1, B2, and B3 (tepB1, tepB2, and tepB3), respectively.
Bacteriocin transporters are reported to cleave the double-glycine leader peptides from the precursors of bacteriocins for their secretion . A search of the flanking regions of tepA1, A2, and A3 in the genome sequence of T. denticola ATCC 35405 revealed that proteins coded by three open reading frames (TDE_0416, TDE_0422, and TDE_0423) upstream of tepA1 have double-glycine bacteriocin-type signal domains. However, no double glycine-containing protein-coding sequence exists near tepA2 and A3. Of the three proteins, those coded by TDE_0422 and TDE_0424 showed high overall similarity (92 %). The protein coded by TDE_0416 showed high sequence identity with TDE_0422 and TDE_0424 (98 % and 84 %, respectively); however, the sequence was truncated at residue 167. TDE_0422 and TDE_0424 showed only weak similarity to penicillin-binding proteins of Bacillus cereus VD148 (37 % identity in a 89-amino-acid overlap).
Prevalence of tepA1, A2, and A3 in T. denticola strains
Antimicrobial sensitivity of the tepA2-deficient mutant
DNA microarray analysis of the tepA2-deficient mutant
Genes with increased expression in the tepA2-deficient mutant in the presence of chloramphenicol
Gene expression fold change (KT-3 versus wild type)
TDE_0499 hypothetical protein
TDE_2748 acetyltransferase, GNAT family
TDE_0337 glucosamine-6-phosphate deaminase
TDE_2214 hypothetical protein
TDE_0561 hypothetical protein
TDE_0614 precorrin-4 C11-methyltransferase
TDE_0506 DNA-damage-inducible protein J, putative
TDE_1848 hypothetical protein
TDE_0307 hypothetical protein
TDE_2378 ABC transporter, ATP-binding protein, putative
TDE_0259 transcriptional regulator, MarR family
TDE_1599 ABC transporter, ATP-binding/permease protein
TDE_0551 hypothetical protein
TDE_0820 transcriptional regulator, TetR family
TDE_1517 hypothetical protein
TDE_1692 hypothetical protein
TDE_0528 hypothetical protein
TDE_0475 ABC transporter, ATP-binding protein
TDE_2519 hypothetical protein
TDE_0231 DNA polymerase III, beta subunit
TDE_0382 hypothetical protein
TDE_2638 hypothetical protein
TDE_0375 ABC transporter, ATP-binding protein
TDE_1977 hypothetical protein
TDE_0748 iron compound ABC transporter, periplasmic iron compound-binding protein, putative
TDE_0426 bacteriocin ABC transporter, ATP-binding/permease protein, putative
TDE_2431 bacteriocin ABC transporter, ATP-binding/permease protein, putative
Genes with decreased expression in the tepA2-deficient mutant in the presence of chloramphenicol
Gene expression fold change (KT-3 versus wild type)
TDE_0719 bacteriocin ABC transporter, ATP-binding/permease protein, putative
TDE_1057 hypothetical protein
TDE_1181 methyltransferase domain protein
TDE_2761 hypothetical protein
TDE_0953 branched-chain amino acid ABC transporter, permease protein
TDE_1883 hypothetical protein
TDE_1066 hypothetical protein
TDE_2582 GGDEF domain protein
TDE_1058 hypothetical protein
TDE_0720 bacteriocin ABC transporter, bacteriocin-binding protein, putative
TDE_0998 hypothetical protein
TDE_1930 hypothetical protein
TDE_0625 ABC transporter, ATP-binding protein
TDE_0175 pyrrolidone-carboxylate peptidase
TDE_1921 hypothetical protein
TDE_0485 hypothetical protein
TDE_0849 hypothetical protein
TDE_1446 hypothetical protein
TDE_0894 hypothetical protein
TDE_2497 hypothetical protein
TDE_0912 hypothetical protein
TDE_0243 ABC transporter, ATP-binding protein
TDE_2785 hypothetical protein
TDE_1975 hypothetical protein
TDE_0485 hypothetical protein
Expression of tepA1-B1, tepA3-B3, and TDE_0820 in the tepA2 mutant
TepA2 shows 42–44 % identity with the bacteriocin exporters of S. africana and C. lentocellum as well as with two orthologs, TepA1 and TepA3, of T. denticola ATCC 35405. The putative active-site residues for bacteriocin peptidases, membrane-spanning domains, and ATP-binding sites were conserved in all of the orthologs. Downstream of the orthologs, sequences for accessory proteins (TepBs), which showed similarity to the functional domain of hlyd-like domains, were found. Although bacteriocin production has not been reported in T. denticola, putative proteins with double-glycine signal peptides, TDE_0416, TDE_0422, and TDE_0424 were located upstream of tepA1. These results tentatively suggest that tepA-tepB may code for exporter proteins for these proteins.
The Southern blot analysis results indicated that the number of orthologs of tepA differs among T. denticola strains. Similarly, diversity among T. denticola strains has been reported for Msp (major sheath protein) . However, diversity in the number of orthologs has not yet been documented. Msps are 53–63 kDa and the identity of the amino acid sequences between T. denticola strains ATCC 35405 and OKT is 43 % . The amino acid identity among tepAs (40 %) was similar to that observed among Msps. Only tepA3 was detected in all tested strains of T. denticola. The genes coding proteins with a bacteriocin-like leader peptide were detected only upstream of tepA. It is possible that tepA and tepB, but not the bacteriocin-like genes, were duplicated during the evolution of T. denticola although further analysis is required to clarify the duplication events.
In the tepA2-deficient mutant, the expression of tepA3-B3 increased significantly while that of tepA1-B1 increased only slightly. In T. denticola, regulation of an ABC transporter has been reported only for a thiamine pyrophosphate transporter, which is regulated by a TPP-binding riboswitch . TepA2-B2 and tepA3-B3 have similarity to the functional motif for bacteriocin ABC transporters but they are not proximal to bacteriocin-like genes. Only tepA1-B1 has a bacteriocin-like protein directly upstream. A recent report indicated that peptides secreted through a bacteriocin export system could have signaling functions . It is possible that a reduction in the export of a signaling molecule by tepA2 may affect the expression of tepA3-B3. The expression of tepA3-B3 increased in the tepA2 mutant, while that of tepA1-B1 did not. These results suggest an interaction between regulation of expression of tepA2-B2 and that of tepA3-B3, and that regulation of tepA1-B1 is independent of tepA2-B2 and tepA3-B3.
Interestingly, resistance to chloramphenicol and ofloxacin was increased while resistance to kanamycin was reduced in the tep2-deficient mutant under conditions that induced increased tepA3 expression. Bacterial efflux pumps, including ABC transporters, are involved in drug resistance in several bacteria . In T. denticola, resistance to antimicrobial agents such as human β-defensin 2 and 3, and rifampicin has been reported [38, 39], and an ABC transporter was suggested to be involved in resistance to β-defensin 3. In the microarray analysis, several genes encoding ABC transporters and potential transcriptional regulators including TDE_0820 showed increased expression in the tepA2 mutant as compared to the wild-type strain. In addition, an acetyltransferase of the GNAT family exhibited significantly increased expression in the tepA2 mutant. These changes can potentially affect the susceptibility against chloramphenicol. However, the substrate of the enzyme group was reported to be kanamycin ; thus, the involvement of the enzyme in chloramphenicol resistance seems unlikely. The expression of tepA3-B3 in the KT-3 mutant was higher than that in the wild-type strain, although the level was low when compared to that in the mutant without chloramphenicol. It is possible that the changes in the expression of tepA3-B3 are required for sensitivity to chloramphenicol. Obviously, further analysis is required to define the role of these proteins with putative ABC transporter functions in relation to sensitivity to the three antibiotics tested in this study.
T. denticola ATCC 35405 has three potential bacteriocin export proteins and the presence of these genes differs among the Treponema strains. Furthermore, TepA3-B3 of the proteins may be involved in resistance to chloramphenicol. The changes in susceptibility in T. denticola may contribute to our knowledge of the use of chemotherapy for chronic periodontitis.
ABC transporters, ATP-binding cassette transporters; ImmA, bacteriocin immuniry protein; NCBI, National Center for Biotechnology Information; Tep, Treponema exporter protein
The authors thank Tomomi Kita for her technical assistance.
This work was partially supported by Grant 24592778 (K.I.) and 15 K11023 (K.I.) from the Ministry of Education, Science, Sport, Culture and Technology of Japan.
Availability of data and materials
The datasets except the array data supporting the conclusions of this article are included within the article. The array data in this report have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number GSE83445 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE83445).
HKK, MY, and KI designed the research. KT-K, YS-K, and HKK carried out the screening of bacteriocin-associated genes from T. denticola and in silico analysis of the genes. KT-K, YK, and YS-K investigated the prevalence of bacteriocin-associated genes in the strains of T. denticola. KT-K, YK, and SS performed the gene expression analysis. KT-K, MY, and KI wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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