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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Anaerobe. 2020 Jun 27;64:102231. doi: 10.1016/j.anaerobe.2020.102231

Multiple Enzymes Can Make Hydrogen Sulfide From Cysteine in Treponema denticola

Linda Phillips a,c, Lianrui Chu b,d, David Kolodrubetz a,*
PMCID: PMC7484134  NIHMSID: NIHMS1610202  PMID: 32603680

Abstract

Treponema denticola is a spirochete that is involved in causing periodontal diseases. This bacterium can produce H2S from thiol compounds found in the gingival crevicular fluid. Determining how H2S is made by oral bacteria is important since this molecule is present at high levels in periodontally-diseased pockets and the biological effects of H2S can explain some of the pathologies seen in periodontitis. Thus, it is of interest to identify the enzyme, or enzymes, involved in the synthesis of H2S by T. denticola. We, and others, have previously identified and characterized a T. denticola cystalysin, called HlyA, which hydrolyzes cysteine into H2S (and pyruvate and ammonia). However, there have been no studies to show that HlyA is, or is not, the only pathway that T. denticola can use to make H2S. To address this question, allelic replacement mutagenesis was used to make a deletion mutant (ΔhlyA) in the gene encoding HlyA. The mutant produces the same amount of H2S from cysteine as do wild type spirochetes, indicating that T. denticola has at least one other enzyme that can generate H2S from cysteine. To identify candidates for this other enzyme, a BLASTp search of T. denticola strain 33520 was done. There was one gene that encoded an HlyA homolog so we named it HlyB. Recombinant His-tagged HlyB was expressed in E. coli and partially purified. This enzyme was able to make H2S from cysteine in vitro. To test the role of HlyB in vivo, an HlyB deletion mutant (ΔhlyB) was constructed in T. denticola. This mutant still made normal levels of H2S from cysteine, but a strain mutated in both hly genes (ΔhlyA ΔhlyB) synthesizes significantly less H2S from cysteine. We conclude that the HlyA and HlyB enzymes perform redundant functions in vivo and are the major contributors to H2S production in T. denticola. However, at least one other enzyme can still convert cysteine to H2S in the ΔhlyA ΔhlyB mutant. An in silico analysis that identifies candidate genes for this other enzyme is presented.

Keywords: Treponema denticola, Hydrogen sulfide synthesis, Periodontal disease, Cystathionine-β-lyase, Cysteine catabolism

Introduction

The anaerobic spirochete Treponema denticola is considered to be an etiologic agent of periodontitis since it is present at higher levels in the subgingival microbiome of periodontally-diseased patients than in healthy individuals [1-3]. In addition, this organism can cause disease pathology (soft tissue destruction and alveolar bone loss) in animal models [4-6]. Numerous virulence factors have been described in this bacterium [7,8] including metabolic end-products that might affect T. denticola’s pathogenic potential [8]. One end product that could play several roles in T. denticola virulence is H2S. This thiol compound is of particular interest because it is present at high levels in periodontally-diseased pockets [9-12] and its biological effects are congruous with some pathologies seen in periodontitis. For example, H2S can induce apoptosis in human periodontal epithelial, ligament and fibroblast cells [13-15]. This suggests that the production of H2S by T. denticola could be important in inducing the increased numbers of apoptotic cells seen in gingival tissue in periodontitis patients [16-18]. H2S made by T. denticola can cause hemoxidation and hemolysis of human red blood cells [19]. Lai et al [20] have shown that when T. denticola generates high levels of H2S, this anaerobic spirochete can grow aerobically. Thus, the production of this reducing agent by T. denticola could play a role in creating the low oxygen tension found in diseased periodontal pockets [21] to enhance the growth of other anaerobic periodontal pathogens [1,22]. Finally, bacterially produced H2S can make the organisms more resistant to antibiotics by reducing bacterial oxidative stress [23] and there is evidence that this could affect periodontal disease [24]. Taken together, this literature argues that H2S synthesized by T. denticola, and other periodontal pathogens [25,26], will play a critical role in the initiation of periodontitis and the tissue/bone damage seen in diseased patients.

In light of the potential disease impact of H2S synthesized by periodontal pathogens, we, and others, have identified, purified and characterized cystalysin from T. denticola [27-33]. This enzyme, also called HlyA because it can hemolyze red blood cells, hydrolyzes cysteine into ammonia, pyruvate and H2S. Interestingly, HlyA is the third enzyme in a three-step-pathway (GTSP) for glutathione metabolism in T. denticola [34]. Initially, glutathione is cleaved into Cys-Gly and glutamate by gamma-glutamyltransferase (GGT) [34,35]. Then, Cys-Gly is degraded into glycine and cysteine by cysteinylglycinase [36]. Finally, HlyA acts on cysteine to release ammonia, pyruvate and H2S. The disease relevance of H2S production by the T. denticola GTSP is evidenced by the fact that a GGT deletion mutant, which does not produce H2S from glutathione, forms significantly smaller lesions than wild type T. denticola in a mouse model of soft tissue destruction, a major symptom of periodontal diseases [37]. Since the ability of T. denticola to make H2S would appear to be one key virulence attribute of this spirochete, we are interested in defining the enzyme or enzymes involved in making H2S from cysteine in T. denticola. Thus, we have constructed an hlyA-deletion strain and demonstrated that this mutant makes normal amounts of H2S from cysteine. BLASTP analysis was used to identify an HlyA homolog, HlyB, in T. denticola. Recombinant HlyB hydrolyzes cysteine to H2S. A deletion mutant in hlyB still makes wild type levels of H2S but a double mutant, ΔhlyA ΔhlyB, synthesizes significantly less H2S from cysteine. Thus, although the HlyA and HlyB enzymes are the major contributors to H2S production in T. denticola, there is at least one other enzyme that can partially compensate for their loss in this spirochete. Three candidates for the other enzyme that might convert cysteine into H2S in the ΔhlyA ΔhlyB mutant were identified by an in silico analysis.

Materials and Methods

Construction of hly mutants in T. denticola.

HlyA deletion mutants (ΔhlyA), hlyB deletion mutants (ΔhlyB), and double mutants (ΔhlyA ΔhlyB) were made in Treponema denticola strain ATCC 33520 using an allelic replacement protocol [38].

HlyA Mutants.

PCR and standard recombinant DNA procedures were used to construct plasmid pDK967, in which an erythromycin resistance cassette (ermres) encoding ermFAM [39] replaced part of the hlyA coding region. Genomic DNA from T. denticola strain ATCC 33520 was used as a template in PCR, with the primers listed in Table 1, to amplify regions upstream (Tdp101/Tdp102) and downstream (Tdp109/Tdp110) of the hlyA coding region. In plasmid pDK967, a 1.07 kb fragment of DNA with 0.98 kb of sequence that is normally upstream of the hlyA coding region plus 90 base pairs of the start of the hlyA coding region is cloned in front of the ermres cassette. The other side of the ermres cassette is flanked by a 1.01 kb fragment of DNA that contains the last two-thirds of the hlyA ORF and 0.22 kb of sequences that are normally downstream of the hlyA gene. These inserts were sequenced and are identical to the appropriate regions of the T. denticola 33520 genome [http://www.homd.org/index.php] [40]. Plasmid pDK967 was linearized by PstI, which cuts at one of the junctions between the insert DNA and vector, and electroporated into T. denticola [38]. After electroporation, the cells were incubated overnight in 2 mL GM-1 broth [38] without erythromycin at 37°C in a Coy anaerobic chamber (5% CO2, 10% H2, 85% N2) and then plated on tryptone-yeast extract-gelatin-volatile fatty acids-serum (TYGVS) [41] containing 0.85% sea plaque agarose with 40 μg/mL erythromycin. Antibiotic resistant colonies, which appeared after 7–12 days, were tested by PCR for the presence of the ermres cassette in the hlyA locus. One mutant, Tdm101, was used in subsequent experiments.

Table 1.

Oligonucleotides used to construct plasmids to make T. denticola mutants.

Oligonucleotides in hlyA region
Name Sequencea Position relative
to start of
hlyA ORFb 		Orientationc Restriction site
added to 5’
end
Tdp101 5’-gagctcCAGCAATCATAACAGGTTCA-3’ −980 for SacI
Tdp102 5’-ggatccTCCAACCTCAGGATTTTGTG-3’ +90 rc BamHI
Tdp109 5’-ggatccGTAAAATCATAGAATGTG-3’ +403 for BamHI
Tdp110 5'-ctgcagAGCTTATGACAATAAAGACA-3' +1403 rc PstI
Oligonucleotides in hlyB region
Name Sequencea Position relative
to start of
hlyB ORFb 		Orientationc Restriction site
added to 5’
end
Tdp135 5'-gagctcAAACTCTTCCCCATTATGAA-3' −1053 for SacI
Tdp147 5'-ggatccATCTTTATGTATTACTTAGC-3' −165 rc BamHI
Tdp137 5'-ggatccCATTTTGGAAATGCTCGGTGAT-3' +1144 for BamHI
Tdp138 5'-ctgcagATTGAAAACACGGCAGTAATC-3' +2116 rc PstI
Oligonucleotides in ermFAM region
Name Sequencea Position relative
to start of
ermF ORFb 		Orientationc Restriction site
added to 5’
end
Tdp105 5'-ggatccAACATCATAGAAATTGC-3' −244 for BamHI
Tdp106 5'-ggatccCCGAAGCTGTCAGTAGTATAC-3' +1874 rc BamHI
Oligonucleotides in AAD(9) [specres] region
Name Sequencea Position relative
to start of
AAD(9) ORFb 		Orientationc Restriction site
added to 5’
end
Spc7 5'-ggatccGAAATGTTGCCCTCACGAGTT-3' −162 for BamHI
Spc8 5'-ggatccCTTATTCGGCGGCTGTAGACT-3' +808 rc BamHI
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a)

The non-capitalized bases are sequences that add a restriction endonuclease site to the PCR product. They are not complementary to any of the templates used in the PCRs.

b)

The position indicated is for the 5’end of each primer. The first base of the translation initiation codon in each open reading frame (ORF) is assigned position +1.

c)

Orientation of the oligonucleotide, where a forward (for) sequence is in the same orientation as the gene(s) in the operon and a sequence that is in the reverse complement orientation is indicated by “rc”.

To make a spectinomycin resistant deletion in hlyA, plasmid (pDK965) with a specres-disrupted hlyA gene was made using standard cloning procedures. This plasmid is identical to hlyA-deletion plasmid pDK967 above, except pDK965 has a spectinomycin resistance cassette (specres) [42] instead of the ermres cassette. The specres cassette, from Enterococcus faecalis, encodes a spectinomycin adenyltransferase protein [42]. This cassette was chosen for use as another selectable marker in T. denticola because it is expressed in and confers spectinomycin resistance to a wide range of organisms, including Aggregatibacter actinomycetemcomitans [43], Bacillus subtilis [44], Campylobacter rectus [45], and Streptococcus pneumoniae [46]. Plasmid pDK965 was linearized by PstI, which cuts at one of the junctions between the insert DNA and vector, and electroporated into T. denticola ATCC 33520. After electroporation, the cells were incubated anaerobically overnight in 2 mL TYGVS broth without spectinomycin and then plated on TYGVS containing 0.85% sea plaque agarose with 100 μg/mL spectinomycin. Antibiotic resistant colonies were tested by PCR for the presence of the specres sequence in the hlyA locus. Three ΔhlyA::specres mutants were tested for the ability to make H2S from cysteine. HlyB Mutant. PCR and standard recombinant DNA procedures were used to construct plasmid pDK972, in which an erythromycin resistance cassette (ermres) encoding ermFAM [39] replaced part of the hlyB coding region. Genomic DNA from T. denticola strain ATCC 33520 was used as a template in PCR, with the primers listed in Table 1, to amplify regions upstream (Tdp135/Tdp147) and downstream (Tdp137/Tdp138) of the hlyB coding region. In plasmid pDK972, a 0.89 kb fragment of DNA that contains sequences that are −165 to −1053 bp upstream of the hlyB coding region is cloned in front of the ermres cassette. The other side of the ermres cassette is flanked by a 0.97 kb fragment of DNA that contains the last 40 bp of the hlyB ORF and 0.93 kb of sequences that are normally immediately downstream of HlyB. These inserts were sequenced and are identical to the appropriate regions of the T. denticola 33520 genome. Plasmid pDK972 was linearized by PstI, which cuts at one of the junctions between the insert DNA and vector, and electroporated into T. denticola [38]. After electroporation, erythromycin resistant colonies were selected as described above and PCR was used to show that the ermres cassette had recombined into the hlyB locus. Mutant Tdm109 was used in the ensuing studies.

HlyA HlyB Double Mutant.

A plasmid (pDK973) with a specres-disrupted hlyB gene was made using standard cloning procedures. This plasmid is identical to hlyB-deletion plasmid pDK972, except pDK973 has a spectinomycin resistance cassette (described above) [42] instead of the ermres cassette. Plasmid pDK973 was linearized by PstI, which cuts at one of the junctions between the insert DNA and vector, and electroporated into the T. denticola ΔhlyA::ermres strain Tdm101. After electroporation, the cells were incubated anaerobically overnight in 2 mL TYGVS broth without spectinomycin and then plated on TYGVS containing 0.85% sea plaque agarose with 100 μg/mL spectinomycin. Antibiotic resistant colonies were tested by PCR for the presence of the specres sequence in the hlyB locus and then PCR was used to confirm that the ermres cassette still disrupted the hlyA gene. One mutant, Tdm111, was used in subsequent experiments.

Determination of H2S production from cysteine by T. denticola.

T. denticola cells, wild type or mutant (Tdm101, Tdm109, Tdm111), were grown in TYGVS broth overnight at 37°C in a Coy anaerobic chamber (5% CO2, 10% H2, 85% N2). Cell concentrations in the cultures were measured by their optical density at 620nm and equivalent numbers of cells (equal to the number of cells in 1 mL of a culture at OD620nm) were removed from each sample into centrifuge tubes. The cells were pelleted by centrifugation, resuspended in 1 mL PBS, centrifuged again and the final cell pellet was resuspended in 1 mL PBS. A 0.5 mL aliquot was removed from each sample into a new tube, 10 μL of freshly prepared L-cysteine•HCl (100 mM) was added and the samples were covered with parafilm and incubated aerobically for 2 hr in a water bath at 37°C. Then the H2S produced by the various T. denticola strains was assayed as described by Siegel [47] with some modifications. Briefly, 50 μL of 0.02 M N,N-dimethyl-p-phenylenediamine sulfate in 7.2 N HCl and 50 μL of 0.3 M FeCl3 in 1.2 N HCl were added sequentially to each tube. After color development for 30 min at room temperature, the samples were centrifuged briefly and the absorbance of each supernatant was measured at 620 nm. The sulfide concentration was determined from a standard curve with Na2S as the substrate. The three T. denticola mutants and wild type spirochetes were grown together and assessed for H2S production on four different days. The data H2S concentrations are expressed as means ± standard deviations. The statistical significance was evaluated for four independent experiments using the Tukey’s multiple comparison test. Differences between samples were considered statistically significant if the p value was <0.05.

Expression and purification of recombinant HlyB.

Based upon the genomic DNA sequence of T. denticola strain 33520 [http://www.homd.org/index.php], two primers were designed to amplify the entire SEQF1865_00956 open reading frame, which we will refer to as hlyB, and to put non-genome-encoded BamHI and EcoRI sites at opposite ends of the resulting PCR product. With T. denticola 33520 genomic DNA as template, a 1.187 kb fragment was amplified by PCR and ligated into the BamHI/EcoRI sites of the expression vector pRSET-B (ThermoFisher Scientific). The insert of the resulting plasmid, pDK981, was sequenced and is identical to the hlyB gene of the T. denticola 33520 genomic sequence. The plasmid was transformed into E. coli BL21(DE3)pLysS for expression of recombinant HlyB (rHlyB). An overnight culture of E. coli cells, grown in LB with chloramphenicol (35 μg/mL) and ampicillin (100 μg/mL), was used to inoculate a fresh batch of the same media (5 mL or 250 mL ), the cells were grown to an OD600 of ~0.45 and IPTG, final concentration 1 mM, was added. After 3.5 hr, the bacterial culture was centrifuged and the cell pellet was frozen at −20°C. Since the recombinant HlyB has a 6-His tag, it was purified on a 2.5 mL Ni-NTA gel column using the QIAexpress Ni-NTA Fast Start Kit (QIAGEN, Germantown, MD). Briefly, the cell pellet was resuspended in 10 mL of the kit’s native Lysis Buffer and cellular debris from the lysed cells was removed by centrifugation. The cleared lysate was loaded onto the Ni-NTA Fast Start column and the flow-thru material was collected and saved. The column was washed twice, each time with 4 mL native Wash Buffer. Finally, the rHlyB was eluted from the column with 1 mL native Elution Buffer. This step was repeated to get a second elution fraction. Aliquots of each of the cleared lysate (load), wash and elution samples were removed to fresh tubes, PBS was added to bring the final volume to 0.5 mL and cystalysin activity, the ability to convert cysteine to H2S, was assayed as described above for whole cells.

Western blot analysis.

Aliquots from each column sample were electrophoresed on duplicate SDS-polyacrylamide gels. The proteins in one set of gels were visualized by staining with Coomassie brilliant blue. The duplicate gel was used for Western blot analysis using antibody which recognizes the 6-histidine-tag that is part of the recombinant HlyB protein. Briefly, the proteins in the duplicate gel were electroblotted onto an immobilon-P membrane. After washing two times in TBS [Tris-buffered saline (20 mM Tris, 0.5 M NaCl, pH 7.5)], the membrane was blocked with 1.5% non-fat milk (Borden) and 1% Tween in TBS. The membrane was then incubated for 3 hr in a 1:5000 dilution of mouse anti-His-Tag HRP-conjugated monoclonal antibody (Invitrogen/ThermoFisher Scientific) in TBS. After two short washes with TBS the membrane was soaked in luminol and autoradiography was used to detect the resulting chemiluminescence.

BLAST analyses.

All searches for protein homologs used the BLASTP program (ver 2.2.25) on the Human Oral Microbiome Database site [http://www.homd.org/index.php] [40]. The site default parameters were used, which included an Expect Value threshold of 10, the BLOSUM-62 substitution scoring matrix, and the low complexity filter. Various amino acid sequences were used as query sequences in a BLASTP search of the annotated proteins from the seventeen strains of T. denticola whose sequences are in the Human Oral Microbiome Database. The strains in the database are: AL-2/FO461, ASLM/FO460, ATCC 33520, ATCC 33521, ATCC 35404, ATC 35405/DSM14222, FO402, H-22/FO457, H1-T/FO456, MYR-T/FO458, OTK/FO454, SP23/FO463, SP32/FO464, SP33/FO465, SP37/FO455, SP44/FO466, US-Trep/FO459. The proteins used individually as query sequences were: HlyA and HlyB from T. denticola strain ATCC 33520, the cystathionin-β-synthases from Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus anthracis [23], cystathionine-γ-lyase (MccB) from Staphylococcus aureus [48], methionine-γ-lyase (MegL) from E. coli [49], the cysteine desulfhydrase TnaA from E. coli, [50], and the three E. coli cysteine desulfurases, CsdA, CsdB, and IgcS [51-53]. Matches with Expect values <0.001 are reported as possible homologs.

Results and Discussion

T. denticola hlyA deletion mutants make normal levels of H2S from cysteine.

In order to evaluate the biological role of the production of H2S by T. denticola, it would be useful to have a mutant that does not make H2S from various sulfide donors. T. denticola cystalysin, which converts cysteine into ammonia, pyruvate and H2S [29], is the final enzyme in the three-step-pathway for glutathione metabolism [34], so a mutant that no longer makes cystalysin should be unable to produce H2S from two of the human host’s major non-protein sulfide donors, glutathione and cysteine [54,55]. Since the gene encoding cystalysin has been identified, named hly in Chu et al [28] but called hlyA here, we made a mutant in the hlyA gene of T. denticola by allelic replacement mutagenesis [38]. Several strains were made independently in which either an erythromycin-resistance cassette (ermres) or a spectinomycin-resistance gene (specres) had replaced amino acids 29 through 134 (out of 397) of the hlyA open reading frame. Importantly, the amino acids removed include Tyr64 and Tyr123, which have been shown to be critical for the activity of the T. denticola enzyme [32,56]. Primers complementary to the regions upstream or downstream of the hlyA gene were paired with primers specific for the ermres cassette or specres gene and used in diagnostic PCR with DNA from the T. denticola mutants. The presence and sizes of the resulting PCR products (data not shown) proved that the expected allelic replacement events had occurred at the T. denticola hlyA locus in five mutants.

Cystalysin activity was measured in two ΔhlyA:ermres mutants and in three ΔhlyA:specres strains. Surprisingly, all of the mutants produced wild type or close to wild type levels of H2S from cysteine (data not shown). One ermres deletion strain, Tdm101, was tested multiple times along with parental wild type T. denticola spirochetes. The results (Fig. 1) show that the hlyA deletion has wild type levels of cystalysin activity. Thus, either there is a second cystalysin gene in T. denticola or the organism has an alternate pathway to convert cysteine to H2S.

Fig. 1. Cystalysin activity levels in wild type and mutant T. denticola.

Fig. 1.

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The indicated wild type and mutant strains of T. denticola were assessed for their ability to convert cysteine to H2S. After overnight growth, cells were washed in PBS and then cysteine was added to equivalent numbers of cells in PBS, except for the Cells w/o Cys samples to which cysteine was not added. The Media Alone control contained buffer with no bacteria. After 2 hr incubation at 37°C, the amount of H2S produced by each sample was measured as detailed in Materials and Methods. The level of H2S produced was converted to the concentration of H2S shown in the figure by comparison to a standard curve with Na2S as the substrate. Each experiment was done four times. The bars indicate standard deviations of the mean. The brackets comparing the double mutant to wild type or single mutant cells mark the samples with significant p-values (* p ≤ 0.005), per Tukey’s test analysis.

The T. denticola genome has a homolog to the HlyA cystalysin.

To investigate the possibility that there was a second hly gene in T. denticola, the HlyA protein sequence was used in a BLASTp search. The query was done against all of the proteins in T. denticola strain ATCC 33520, as annotated in the Human Oral Microbiome Database [http://www.homd.org/index.php]. There was one significant hit: predicted protein SEQF1865_00956 had an expect-value of 10−128. The next best “hit” had an e-value of 6x10−3. The match between SEQF1865_00956, which we will call HlyB, and HlyA was along the entire length of each protein with only one gap of one amino acid (Fig. 2). The two proteins had identical amino acids at 209 positions (53%) and functionally similar amino acids were found at another 78 amino acids. The T. denticola HlyA protein has been crystallized and mutations in a few of the active site residues have been constructed and shown to be important for HlyA enzymatic activity [32,56,57]. Eleven of the thirteen active site residues in HlyA are also in HlyB, including all five that are directly involved in catalysis (Fig. 2). Finally, cystalysin is a pyridoxal 5’phosphate (PLP) dependent enzyme and both HlyA and HlyB have a lysine at position 238, the amino acid to which PLP attaches [28,32]. Thus, this in silico analysis indicates that T. denticola has a second cystalysin gene, hlyB.

Fig. 2. T. denticola has a homolog to its HlyA protein.

Fig. 2.

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The top line in each row shows the predicted amino acid sequence for HlyA [28]. The second line in each row shows the predicted amino acid sequence encoded by gene hlyB, which is called SEQF1865_00956 in the genomic DNA sequence of T. denticola strain 33520 [http://www.homd.org/index.php]. The asterisks represent amino acids that are identical in the two proteins. The five filled rectangles (▮) mark the residues that have a direct role in the catalysis of cysteine to H2S in HlyA [32,56,57]. Triangles (▼) mark the other eight residues that are also in the active site of T. denticola HlyA [32].

HlyB can make H2S from cysteine.

To prove that HlyB has cystalysin activity, the hlyB gene was cloned into the E. coli expression vector pRSET-B. This construct should express recombinant HlyB (rHlyB) with a polyhistidine (6xHis) tag and twenty-one other vector encoded amino acids, including the T7 gene 10 leader region to enhance translation and the eight amino acid Xpress™ epitope, fused to the amino terminus of HlyB. When E. coli cells with the rHlyB expression vector were induced with IPTG to express rHlyB, the cells made 2.0 μmoles of H2S when incubated with cysteine for two hours, which was significantly more than the 0.24 μmoles of H2S made from cysteine by E. coli cells without vector. The fact that E. coli cells alone had cystalysin activity was not surprising since a BLASTp search showed that E. coli has at least one homolog to T. denticola Hly (data not shown; 35% amino acid sequence identity with an expect value of 8x10−77). Thus, in order to distinguish between innate E. coli cystalysin activity and rHlyB activity, small batches of cells, with or without the rHlyB plasmid, were lysed and put over Ni-NTA columns in order to partially purify the His-tagged rHlyB. E. coli cells without plasmid produced H2S from cysteine, but almost all of this activity was in the column flo-thru or wash fractions (Fig. 3). By contrast, the cells with the rHlyB plasmid had significant amounts of activity in the elution fractions containing, as evidenced by Western blot analysis, His-tagged rHlyB (Fig. 3). However, no proteins were seen in the elution fractions of the Coomassie stained gel. Thus, a larger volume of cells was induced and the rHlyB in the cleared lysate was purified on a Ni-NTA column. The results showed that a protein of the size expected for rHlyB (48.9 kDa) was enriched in the column elution fractions (Fig. 4, bottom). Importantly, these fractions produced significant amounts of H2S when incubated with cysteine. Thus, we conclude that HlyB is the second cystalysin in T. denticola.

Fig. 3. Cystalysin activity of E. coli that is or is not expressing recombinant T. denticola HlyB.

Fig. 3.

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E. coli cells (5 mL cultures) were induced with IPTG. Equal amounts of cell lysates from E. coli that did or did not contain the HlyB plasmid, as indicated, were passed over a Ni-NTA column, which binds to the His-Tag on recombinant HlyB. The column was washed twice, each time with native Wash Buffer. Then recombinant HlyB was eluted from the column with native Elution Buffer. This step was repeated to get elution fraction two. Aliquots (10 μL) from each column sample were used in duplicate SDS-polyacrylamide gels which were either stained with Coomassie Brilliant blue (bottom panel) or used for a Western blot with anti-His-Tag monoclonal antibody (top panel). The numbers on the left show the positions of molecular size markers in kDa. Aliquots (0.1 mL) of each of the cleared lysate (Load), Flo-thru, Wash and Elution samples were assayed for cystalysin activity (the conversion of cysteine to H2S). The amount of H2S produced by each sample after 2 hr incubation at 37°C was converted to the concentration of H2S, as shown in the figure, by comparison to a standard curve with Na2S as the substrate.

Fig. 4. Partial purification of recombinant T. denticola HlyB expressed in E. coli.

Fig. 4.

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A 250 mL culture of E. coli cells containing the HlyB plasmid was induced with IPTG. A cleared cell lysate (Load) from those cells was put over a Ni-NTA column, which binds to the His-Tag on recombinant HlyB (rHlyB). The column was washed twice, each time with native Wash Buffer, and rHlyB was eluted from the column with native Elution Buffer. The elution step was repeated to get elution fraction two. Aliquots (10 μL) from each column sample were used in duplicate SDS-polyacrylamide gels which were either stained with Coomassie Brilliant blue (bottom panel) or used for a Western blot with anti-His-Tag monoclonal antibody (top panel). The numbers on the left show the positions of molecular size markers in kDa. The arrow marks the position of rHlyB in the stained gel. Aliquots of each of the Load (20 μL), Flo-thru (50 μL), Wash (50 μL) and Elution (50 μL) samples were assayed for cystalysin activity. The amount of H2S produced by each sample after 2 hr incubation at 37°C was converted to the concentration of H2S, as shown in the figure, by comparison to a standard curve with Na2S as the substrate.

T. denticola hlyA hlyB double mutant makes significantly less H2S from cysteine.

If HlyB functions as a cystalysin in T. denticola, and not just when expressed as a recombinant protein in E. coli, an hlyB mutant should have less cystalysin activity in the absence of HlyA. To examine the in vivo role of HlyB, we used allelic replacement mutagenesis [38] to make an hlyB deletion (ΔhlyB) in a wild type background. Two independently-derived strains were made in which an ermres cassette had replaced amino acids 1 through 381 (out of 395) of the hlyB open reading frame plus 165 bases in its promoter region. The same approach was used to construct a double mutant (ΔhlyA ΔhlyB), by putting an hlyB deletion with a specres gene into the ΔhlyA:ermres strain (Tdm101) used for our HlyA deletion analysis above. Diagnostic PCR with DNA from the T. denticola mutants, using primers complementary to the regions upstream or downstream of the hlyB gene paired with primers specific for the ermres cassette or specres gene (data not shown), proved that the allelic replacement events had occurred at the T. denticola hlyB locus as expected.

Cystalysin activity was measured in the ΔhlyB mutant, in the double mutant (ΔhlyA ΔhlyB), and in the wild type T. denticola parent strain (Fig. 1). The ΔhlyB single mutant made wild type levels of H2S from cysteine since the strain still has a normal hlyA gene. As expected, the ΔhlyA ΔhlyB cells produced significantly less H2S than did wild type or single mutant spirochetes. These results prove that HlyA and HlyB both have cystalysin activity in T. denticola and that both are expressed in normal growth conditions. Surprisingly, the double mutant can still convert some cysteine into H2S (Fig. 1). Although an ANOVA with post-hoc Tukey HSD test indicates that the amount of H2S produced is not statistically significantly more than that made in control tubes (media alone or T. denticola without added cysteine), the differences are significant when evaluated by a Student’s t-test (p = 0.01). Thus, we conclude that T. denticola either has a third enzyme that can function as a cystalysin or the spirochete has an alternate pathway to convert cysteine to H2S

Other genes in T. denticola that might make H2S from cysteine.

An in silico approach was used to identify a T. denticola gene (or genes) encoding another enzyme that could be the one making H2S from cysteine in the ΔhlyA ΔhlyB mutant cells. There are five sub-classes of enzymes, aside from HlyA/HlyB which are cystathionin-β-lyases, that have been shown in other organisms to be able to make H2S from cysteine. To see if T. denticola might encode homologs of these enzymes, protein sequences from canonical enzymes representing the five sub-classes were used in BLASTP searches against all of the proteins encoded by T. denticola 33520. Cystathionine-γ-lyase, whose normal role in bacteria is in the biosynthesis of cysteine from cystathionine [48], is one of the sub-classes of proteins that can convert cysteine to H2S (plus pyruvate and NH3). A BLASTp search of T. denticola 33520 with the Staphylococcus aureus cystathionine-γ-lyase (MccB) [48] found a homolog; SEQF1865_01135 (expect score = 3 x 10−82). T. denticola SEQF1865_01135 is also a homolog (expect score = 1 x 10−125) of a second sub-class of proteins whose prototypical enzyme is E. coli methionine-γ-lyase (MegL) [49]. This enzyme is involved in helping maintain the levels of sulfur containing compounds in bacteria and although it has a preference for methionine and homocysteine as substrates, it can catalyze the breakdown of cysteine and make H2S [49,58]. Clearly, T. denticola protein 1135, whether its normal substrate turns out to be cystathionine or methionine, could be an enzyme that makes H2S from cysteine in our ΔhlyA ΔhlyB mutant. A third sub-class of proteins that can synthesize H2S from cysteine are cysteine desulfhydrases. These enzymes can play a role in cysteine homeostasis, by catabolizing cysteine into pyruvate, H2S and NH3, but they all function in the cell in other pathways as well. For example, the canonical cysteine desulfhydrase from E. coli, TnaA, is a tryptophanase with important roles in breaking down tryptophan and producing indole, a signaling molecule [50,59,60]. T. denticola 33520 has a TnaA homolog, SEQF1865_00322 (expect score = 1 x 10−123). Protein 322 most likely serves mainly as a tryptophanase in T. denticola, but it could also function as a cysteine desulfhydrase in the absence of HlyA and HlyB. The BLASTp search with E. coli TnaA produced a second homolog in T. denticola, SEQF1865_02121 (expect score = 1 x 10−113). Protein 2121 is named in HOMD as a tyrosine phenol-lyase but it almost certainly will be able to also function as a weak cysteine desulfhydrase that can produce H2S from cysteine since E. coli’s tyrosine phenol-lyase has cysteine desulfhydrase activity [61]. T. denticola 33520 has four homologs to the three E. coli cysteine desulfurases, CsdA, CsdB and IgcS [62], which are members of the fourth sub-class of enzymes that can make H2S from cysteine. The expect scores for the matches with the T. denticola proteins SEQF1865_00483, SEQF1865_00899, SEQF1865_01264, and SEQF1865_02531 ranged from 8 x 10−24 to 1 x 10−92. Cysteine desulfurases do not typically release H2S during their normal functions in the cell, which is to provide sulfur to various sulfur-containing molecules (biotin, lipoic acid, iron-sulfur clusters, etc.) [63]. Instead, the sulfur from cysteine is covalently bound to the enzyme as a persulfide intermediate before the sulfur gets incorporated directly into the appropriate sulfur-containing compound. However, cysteine can react with the persulfide intermediate in some cysteine desulfurases to release H2S [64,65]. Thus, the T. denticola cysteine desulfurases might also produce some level of H2S from cysteine in our ΔhlyA ΔhlyB mutant. Finally, there is no homolog in T. denticola for the fifth class of enzymes, cystathionin-β-synthases [23,66]. In sum, T. denticola 33520 has genes which encode homologs for the canonical enzymes from four of the five sub-classes of proteins that can convert cysteine to H2S. Any one, or all, of these proteins could contribute to the remaining cystalysin-like activity in the T. denticola ΔhlyA ΔhlyB cells. Determining which non-HlyA/B enzymes are actually involved in converting cysteine to H2S in T. denticola would require making additional mutants in the background of the double mutant cells.

The Hly genes are also duplicated in most other strains of T. denticola.

If cystalysin has an important function for T. denticola in general, then other strains of this species should also have duplicate hly genes. In fact, this is the case. The amino acid sequences of HlyA and HlyB from T. denticola strain ATCC 33520 were used in BLASTP searches of the annotated proteins from the sixteen other strains of T. denticola whose sequences are in the Human Oral Microbiome Database [http://www.homd.org/index.php]. Fourteen of sixteen strains had two genes that encoded proteins whose sequences were basically identical to HlyA and HlyB from strain 33520. Two of the strains each had an HlyB homolog but not a match for HlyA. The possible lack of HlyA in strains F0464 and F0465 may be due to incomplete sequence analysis; these two strains still had their genomic sequences spread across 4 and 6 contigs, respectively, instead of having one full length genome sequence (one contig). One strain, F0454, had a third Hly homolog, with an expect value of 6 x 10−66, but it only shared 35% identity with HlyA whereas the other HlyA and HlyB homologs were 100% and 95% identical to HlyA and HlyB from strain 33520.

Conclusion.

The construction of a double deletion mutant (ΔhlyA ΔhlyB) in T. denticola has allowed us to show that the two cystalysins, HlyA and HlyB, perform redundant functions in vivo; both genes must be deleted in order for spirochetes to make less H2S from cysteine. The fact that almost all other T. denticola strains also have duplicate hly genes suggests very strongly that cystalysin has a critical function in this spirochete as the enzyme responsible for the third step of the glutathione three-step pathway (GTSP) [34]. The GTSP, which metabolizes glutathione into H2S and pyruvate, appears to have evolved in this periodontal pathogen to take advantage of the high levels of glutathione in healthy gingival crevicular fluid [67,68]. T. denticola uses its GTSP, and the HlyA and HlyB enzymes, to produce pyruvate, which enhances the growth of T. denticola [34], and H2S, which plays several roles in the pathogenic potential of this spirochete [19,20,37].

Highlights.

  • T. denticola (ΔhlyA) lacking the cystalysin HlyA still make H2S from cysteine

  • T. denticola encodes one homologue of HlyA, named HlyB

  • Recombinant HlyB has cystalysin activity in vitro

  • An ΔhlyA ΔhlyB double mutant has 25% of wild type T. denticola cystalysin activity

  • T. denticola encodes more than two enzymes that can produce H2S from cysteine

Acknowledgments

Funding

This work was supported by a National Institutes of Health grant (1RO1DE023532) from the NIH/NIDCR to L.C.

Footnotes

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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