Influence of excess branched-chain amino acid uptake by Streptococcus mutans in human host cells

Influence of excess branched-chain amino acid uptake by Streptococcus mutans in human host cells Abstract Oral streptococci, including cariogenic bacterium Streptococcus mutans, comprise a large percentage of human supragingival plaque, which contacts both tooth surfaces and gingiva. Eukaryotic cells are able to take up macromolecules and particles, including bacteria, by endocytosis. Increasing evidence indicates endocytosis may be used as an entry process by bacteria. We hypothesized that some endocytosed bacteria might survive and obtain nutrients, such as amino acids, until they are killed. To verify this hypothesis, we focused on bacterial utilization of branched-chain amino acids (BCAAs; isoleucine, leucine and valine) in host cells. A branched-chain aminotransferase, IlvE (EC 2.6.1.42), has been suggested to play an important role in internal synthesis of BCAAs in S. mutans UA159. Therefore, we constructed an ilvE-deficient S. mutans 109c strain and confirmed that it had similar growth behavior as reported previously. 14C radioactive leucine uptake assays showed that ilvE-deficient S. mutans took up more leucine both inside and outside of host cells. We further clarified that a relative decrease of BCAAs in host cells caused enhanced endocytic and autophagic activity. In conclusion, S. mutans is endocytosed by host cells and may survive and obtain nutrients, such as BCAAs, inside the cells, which might affect cellular functions of host cells. Streptococcus mutans, branched-chain amino acids, radioactive transport, autophagic activity, endocytosis, bacterial invasion INTRODUCTION Dental plaque comprises the community of oral microorganisms contained within biofilms on a tooth surface, which has been implicated as the major cause of dental caries and periodontal disease. Streptococcus mutans, a well-established causative agent of human dental caries (Mitchell 2003), is usually seen in dental plaque but can also be found in blood, on heart valves in subacute endocarditis and in atheromatous plaques (Vose et al.1987; Moreillon and Que 2004; Nakano et al.2006). A number of PCR-based analyses using S. mutans-specific 16S rRNA gene-amplifying primers have demonstrated the existence of S. mutans in lesions. In addition, a report in which living S. mutans strains were isolated from the blood or heart valve of a patient with infective endocarditis (IE) strongly supports the relationship between S. mutans and systemic diseases (Nomura et al.2006). Moreover, experimental invasion of S. mutans into human umbilical vein endothelial cells and human aortic endothelial cells was reported (Abranches et al.2009; Nagata, de Toledo and Oho 2011). These findings suggest that S. mutans has the ability to invade and survive in host cells. Therefore, this bacterium may take up and utilize host-derived nutrients, specifically amino acids, inside host cells. However, to our knowledge, amino acid utilization by intracellular S. mutans has not yet been investigated. Branched-chain amino acids (BCAAs; isoleucine, leucine and valine) are typically found in the core of globular proteins and are a quantitatively important group of amino acids in bacterial proteins (Neidhart and Umbarger 1996; Patek 2007). BCAAs are synthesized by a series of sequential reactions, and aminotransferase IlvE (EC 2.6.1.42) catalyzes the final step (Ganesan and Weimer 2004). IlvE has been shown to play an important role in the growth of S. mutans UA159 when no exogenous BCAAs were available (Santiago et al.2012) and for the optimal growth of S. thermophils in milk (Garault et al.2000). In addition, the function of ilvC in BCAA synthesis in S. pneumoniae has been suggested to be essential for virulence factor of respiratory infections (Kim et al.2017). BCAAs not only act as building blocks for tissue proteins, but also have other metabolic functions in human host cells. BCAAs are known to exert several signaling responses mainly via activation of the mammalian target of rapamycin (mTORC1), which can result in hypertrophy, proliferation and insulin resistance (Newgard et al.2009; Liu et al.2014; Neishabouri, Hutson and Davoodi 2015). It is well known that mTORC1 activity is inhibited by nutrient starvation, which results in autophagy induction (Noda and Ohsumi 1998; Scott, Schuldiner and Neufeld 2004). Autophagy is a lysosome- or vacuole-mediated degradation system that responds to reductions in available nutrients, especially amino acids (Zoncu et al.2011; Chen et al.2014). In many studies, cellular functions related to amino acids levels were analyzed by increasing or decreasing amino acid concentrations outside of host cells (Kakazu et al.2007; Zoncu et al.2011; Wubetu et al.2014; Zhenyukh et al.2017). However, changes in relative BCAA concentrations inside host cells have not been focused on by researchers. Moreover, pathogenic effects of decreased intracellular BCAA concentrations caused by invasive bacteria have not been elucidated. Therefore, in this study, we constructed an ilvE-deficient S. mutans 109c strain to evaluate BCAA utilization in host cells and clarify the biological effect of decreasing BCAA levels in host cells. MATERIALS AND METHODS Bacterial strains and culture conditions Streptococcus mutans 109c (wild type; serotype c) (Sato, Yamamoto and Kizaki 1997) and its isogenic mutant constructed in this study were anaerobically maintained (80% N2, 10% H2 and 10% CO2) at 37°C in brain heart infusion broth (BHI; Difco Laboratories, Detroit, MI, USA). Bacterial growth was measured using BCAA-free Roswell Park Memorial Institute (RPMI) medium (RPMI medium without BCAA and phenol red, IFP, Yamagata, Japan) or RPMI medium (without phenol red). RPMI medium was prepared by supplementation with adequate BCAAs (L-isoleucine: 50 mg L−1, L-leucine: 50 mg L−1 and L-valine: 20 mg L−1, Wako, Tokyo, Japan) to BCAA-free RPMI medium. BCAA-free medium was also used for exogenous radioactive leucine uptake assays. When required, 50 μg mL−1 tetracycline (Wako) was added to the medium. Cell culture HSC-2 (RCB1945), human cell line derived from oral squamous cell carcinoma, was purchased from the Cell Engineering Division of RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). Cells were cultured in Eagle's minimum essential medium (E-MEM) (Wako, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum, 100 U mL−1 penicillin and 100 μg mL−1 streptomycin (complete E-MEM) at an initial density of 2.0 × 105 cells/well on 6-well plates (Iwaki, Tokyo, Japan) for 24 h at 37°C in a 5% CO2 humidified incubator. Construction of an ilvE-deficient S. mutans 109c strain Genomic DNA from S. mutans 109c was extracted using a GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA). An ilvE-deficient mutant strain of S. mutans 109c was constructed by PCR-based gene replacement with an antibiotic resistance gene cassette as described previously (Kunii et al.2014). In this experiment, the ilvE gene of S. mutans was replaced with the tetracycline-resistance gene (Tcr). PCR primers used to construct the mutant were designed with reference to the S. mutans UA159 genome sequence (Ajdic et al.2002) (GenBank accession number AE014133). A fragment containing the upstream flanking region and 5΄-end of ilvE was amplified using primers ilvEP1-F (5΄-CCACTGTAGGTGAACAAGGACAAG-3΄) and ilvEP2-R (5΄-AACTCCAATATTAATAATTTTCATTTTAATACCTCTTTCCTCG-3΄). A fragment containing the downstream flanking region and 3΄-end portion of the target gene was amplified using primer pair ilvEP3-F (5΄-CAATAAAATAACTTAGATAATTAGCGAGCTTGC-3΄) and ilvEP4-R (5΄-TTCGCTACCCTTCCAGTGACAACA-3΄). Tcr was amplified from pUCTet (Arimoto and Igarashi 2008) using primer pair ilvEP2-F (5΄-CGAGGAAAGAGGTATTAAAATGAAAATTATTAATATTGGAGTT-3΄) and ilvEP3-R (5΄-GCAAGCTCGCTAATTATCTAAGTTATTTTATTG-3΄). Reverse primer ilvEP2-R and forward primer ilvEP3-F were synthesized with additional nucleotides complementary to the 5΄- and 3΄-terminal regions of Tcr, respectively. The antibiotic resistance gene was amplified using pUCTet as a template. All three fragments were annealed in one reaction and amplified by PCR using primer pair ilvEP1-F/ilvEP4-R. The resultant amplicon was then used to transform S. mutans 109c cells (Perry and Slade 1962). Replacement mutants of the ilvE gene were selected on BHI agar plates containing 50 μg mL−1 tetracycline. Replacement of the antibiotic resistance gene cassette was verified by sequence analysis. Growth of S. mutans with or without BCAAs Growth of S. mutans cells was examined in culture medium with or without BCAAs. Streptococcus mutans cells in exponential phase were collected by centrifugation at 5000× g for 10 min at room temperature. Harvested cells were washed three times with phosphate buffered saline (PBS) and then adjusted to an optical density (OD) at 600 nm of 1.0 with BCAA-free medium (RPMI medium without leucine, isoleucine and valine). Adjusted bacterial cell suspensions were diluted 10-fold in 2 mL of RPMI or BCAA-free medium and incubated at 37°C in an anaerobic chamber. Bacterial growth was determined by measuring the OD600 of cultures at the time indicated. Environmental and intracellular radioactive leucine uptake assays A previously described protocol (Trip, Mulder and Lolkema 2013) was adapted to measure the uptake of environmental 14C-labeled leucine (PerkinElmer, Shelton, CT, USA) by S. mutans. Briefly, bacteria were grown to OD600 = 0.5, harvested by centrifugation, washed twice with PBS and adjusted to OD600 = 1.0 with PBS before resuspension in BCAA-free medium. 14C-labeled leucine was added to cells at a final concentration of 1 μM (=2 mCi μmol−1, 23 kBq) and incubated for 1 h at 37°C in a CO2-humidified incubator. Cells were then filtered through 0.22-μm membrane filters and immediately washed with 0.1 M LiCl2 at room temperature. Filters were dried and placed in scintillation vials containing 8 mL of Ultima Gold scintillation cocktail (PerkinElmer, Boston, MA, USA). 14C radioactivity was measured using TRI-CARB 2900TR Liquid Scintillation Analyzer (PerkinElmer, Shelton, CT, USA). Bacterial uptake of 14C-labeled leucine in HSC-2 cells was also measured. Prepared HSC-2 cells were incubated with 165 nM 14C-labeled leucine (=300 μCi μmol−1, 3.7 kBq) for 1 h. Before S. mutans infection, HSC-2 cells were washed three times with HBSS (-) and incubated with HBSS (-) containing 100 nM bafilomycin A1 for 1 h. An overnight culture of S. mutans grown in BHI was diluted 20-fold in 3 mL of fresh BHI and then anaerobically incubated at 37°C to OD600 = 1.0. The culture was harvested by centrifugation at 5000× g for 10 min at room temperature. Harvested cells were then washed three times with 50 mM 4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid (HEPES) buffer, adjusted to OD600 = 1.0 with HEPES, and anaerobically incubated at 37°C for 2 h prior to assay. The heat-killed ilvE mutant was prepared by heating the bacterial suspension at 100°C for 1 h. After 2-h incubation, S. mutans cells were harvested, resuspended in HBSS (-) and added at a multiplicity of infection (MOI) of 50 to HSC-2 cells. Infected HSC-2 cells were incubated at 37°C for 1 h in a 5% CO2-humidified incubator. Infected monolayers were washed three times with HBSS (-) to remove unbound S. mutans cells and lysed with 0.3% Triton X-100 (Wako). Cell lysates were separated as filtrated and flow-through samples with 0.22-μm membrane filters. The filtrated sample was washed, dried and measured for 14C radioactivity as described above. The flow-through fraction of 14C radioactivity was measured by direct counting (i.e. addition to a scintillation cocktail). Bacterial invasion assay Bacterial samples were prepared as described above except an MOI of 20 was used to infect HSC-2 cells. After 2-h starvation, bacteria were added to HSC-2 cells and incubated at 37°C for 2 h in a 5% CO2-humidified incubator. Infected monolayers were washed three times with HBSS (-) to remove unbound S. mutans cells. Washed cells were resuspended and incubated at 37°C for 1 h with fresh HBSS (-) containing 400 μg mL−1 gentamicin to kill residual extracellular S. mutans. Monolayers were again washed three times with HBSS (-) and lysed with 0.3% Triton X-100 to release intracellular S. mutans. Serial dilutions of lysates were plated onto BHI agar plates and incubated anaerobically at 37°C for 48 h. The numbers of S. mutans colony-forming units (CFUs) grown on the plates were determined. Cell viability of infected and non-infected HSC-2 cells was evaluated by trypan blue assay (Fardini et al.2011) using a TC20 automated cell counter (Bio-Rad, Hercules, CA, USA). Western blotting HSC-2 cells were washed three times with HBSS (-) and lysed with RIPA buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% NP40, 0.5% sodium deoxy cholate and 0.1% sodium dodecyl sulfate) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The protein concentration of the lysate was adjusted to 1 mg mL−1, and proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with tris-based saline (TBS) with 0.1% Triton X-100 (TBST) containing 2% skim milk overnight at 4°C. The next day, the blocked membrane was incubated for 3 h at room temperature with rabbit anti-LC3 (1:1000; BML, Saitama, Japan) and diluted in blocking solution. The membrane was washed three times with TBST, incubated for 1 h at room temperature with Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:20000 dilution; GE Healthcare, Buckinghamshire, UK) in blocking solution, and washed four times with TBST. Immunoreactive bands were then detected using ImmunoStar®LD (Wako) and C-DiGit western blot imaging system (MS TechnoSystems Inc., Osaka, Japan). The same membrane was incubated with WB Stripping solution (Nacalai Tesque, Kyoto, Japan), and reprobed with mouse anti-β-actin (1:1000; Abcam, Cambridge, England), followed by HRP-conjugated anti-mouse IgG antibody (1:20000 dilution; GE Healthcare). Immunofluorescence microscopy To visualize the biological effect of endocytosed S mutans cells, microtubule-associated protein light chain 3 (LC3) localization was determined by immunofluorescence microscopy. HSC-2 cells infected with S. mutans at an MOI of 20 for 1 h were fixed with 4% paraformaldehyde phosphate buffer solution (Wako) and permeabilized with 0.3% Triton X-100 in PBS. Non-specific binding was subsequently blocked by incubation for 30 min with Blocking buffer (Nacalai Tesque). Cells were then stained using rabbit anti-LC3 antibody (1:1000), followed by incubation with Cy3-conjugated anti-mouse secondary antibodies (1:1000; Jackson ImmunoResearch, West Grove, PA, USA), for 1 h at 37°C. Triplicate samples of >50 HSC-2 cells were investigated for LC3 accumulation. Statistical analysis Growth data are shown as representative results from three independent experiments with similar results. Data are presented as mean ± standard deviations (SD) of three samples. Results for ODs were compared using a two-tailed Student's t-test with the multiple comparison procedure as necessary. P values < 0.05 were considered statistically significant. RESULTS AND DISCUSSION Growth of S. mutans with or without BCAAs IlvE has been implied to play an important role in intracellular synthesis of BCAAs in S. mutans, as growth of the ilvE-deficient S. mutans UA159 strain was impaired in chemically defined minimal medium lacking BCAA (Santiago et al.2012). Therefore, we hypothesized that an ilvE-deficient mutant strain of S. mutans would require more exogenous BCAA than the wild-type strain, which emphasized the biological effect caused by decreased BCAA concentrations in host cells. We initially confirmed whether the constructed ilvE-deficient S. mutans 109c strain showed similar growth behavior in the absence of BCAA as wild-type S. mutans UA159. In this experiment, BCAA-free RPMI medium was used instead of minimal medium without BCAA. Both the wild-type and ilvE mutant strains grew in RPMI medium. However, while the wild-type strain grew in BCAA-free medium, the ilvE mutant strain did not grow at all (Fig. 1). This result differed from that of a previous report on S. mutans UA159, in which significant, but not complete, growth impairment was noted in the absence of BCAA (Santiago et al.2012). The reason for the discrepant results may be explained by differences in growth medium and strain used. Nonetheless, our finding demonstrated that IlvE plays a critical role in BCAA biosynthesis in S. mutans 109c. Figure 1. View largeDownload slide Growth behavior of S. mutans in RPMI or BCAA-free RPMI medium. Bacterial growth was determined by measuring the optical density at 600 nm (OD600). Closed circles, wild type; open circles, ilvE mutant. Solid lines and dotted lines represent growth in RPMI medium and BCAA-free medium, respectively. Growth data shown are representative of three independent experiments with similar results. OD results were compared using a two-tailed Student's t-test. Significant differences (P < 0.01) between the growth behavior of wild-type and ΔilvE mutant strains in BCAA-free medium are indicated by asterisks. Figure 1. View largeDownload slide Growth behavior of S. mutans in RPMI or BCAA-free RPMI medium. Bacterial growth was determined by measuring the optical density at 600 nm (OD600). Closed circles, wild type; open circles, ilvE mutant. Solid lines and dotted lines represent growth in RPMI medium and BCAA-free medium, respectively. Growth data shown are representative of three independent experiments with similar results. OD results were compared using a two-tailed Student's t-test. Significant differences (P < 0.01) between the growth behavior of wild-type and ΔilvE mutant strains in BCAA-free medium are indicated by asterisks. 14C leucine uptake by S. mutans Since S. mutans was found in human host cells, we performed radioactive leucine assays to clarify whether S. mutans took up host-derived leucine in HSC-2 cells. First, extracellular leucine uptake by S. mutans was evaluated. As shown in Fig. 2a, exogenous 14C leucine was clearly taken up by both S. mutans strains (around 0.1 nmol by the wild-type strain, around 0.15 nmol by the ilvE mutant strain). This result suggests that S. mutans takes up environmental leucine, and the ilvE mutant strain requires more exogenous leucine than the wild-type strain. Figure 2. View largeDownload slide 14C-labeled leucine uptake by S. mutans. (A) Exogenous 14C leucine uptake by S. mutans. Bacterial cells were harvested from cultures grown to mid-exponential phase in BHI. 14C leucine uptake was measured at 1 h. As a negative control, radioactivity of a filter cup was measured. As a positive control, 0.3 nmol 14C leucine was directly added to scintillation cocktails. (B) Uptake of host cell-derived 14C leucine by S. mutans. HSC-2 cells were pre-incubated with 14C leucine for 1 h followed by 1 h starvation in HBSS (-) buffer before bacterial infection. Cells were infected with bacteria, prepared as described above, at an MOI of 50. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.05; **P < 0.01. Figure 2. View largeDownload slide 14C-labeled leucine uptake by S. mutans. (A) Exogenous 14C leucine uptake by S. mutans. Bacterial cells were harvested from cultures grown to mid-exponential phase in BHI. 14C leucine uptake was measured at 1 h. As a negative control, radioactivity of a filter cup was measured. As a positive control, 0.3 nmol 14C leucine was directly added to scintillation cocktails. (B) Uptake of host cell-derived 14C leucine by S. mutans. HSC-2 cells were pre-incubated with 14C leucine for 1 h followed by 1 h starvation in HBSS (-) buffer before bacterial infection. Cells were infected with bacteria, prepared as described above, at an MOI of 50. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.05; **P < 0.01. Entry of non-invasive bacteria into non-phagocytic host cells is generally thought to depend on an endocytic mechanism. Bacteria taken up by endocytosis are finally digested by lysosomes. Preliminary experiments showed that a portion of bacteria taken into HSC-2 cells was digested, which resulted in increased 14C leucine concentration from the cytosolic fraction of infected cells compared to that of non-infected control cells (data not shown). Therefore, we used bafilomycin A1, a strong inhibitor of vacuolar-type H+-ATPase, to protect internal S. mutans from lysosomal degradation. No significant difference in 14C radioactivity was found from either a filtered sample (S. mutans collected from inside host cells) or a cytosolic fraction compared to that of negative control cells. However, 14C radioactivity of collected ilvE mutant cells was increased compared to the filtered sample of negative control cells. Moreover, 14C radioactivity of the cytosolic fraction from ilvE-mutant infected cells was decreased compared to that of negative control cells. Notably, differences observed between the bacterial fraction and cytosolic fraction from infected HSC-2 cells were diminished when the ilvE mutant was dead, indicating that the decrease in cytosolic 14C leucine was caused by utilization by the ilvE mutant strain. Although it was difficult to determine whether the wild-type S. mutans strain took up 14C leucine in HSC-2 cells, the utilization of leucine by the ilvE mutant strain in HSC-2 cells was clearly demonstrated. Taken together, S. mutans (at least the ilvE mutant strain) is taken up by HSC-2 cells and utilizes host-derived leucine in the cells. Invasion assay of HSC-2 cells with S. mutans The invasive properties of S. mutans 109c were examined by antibiotic protection assay. The number of HSC-2 cells was not significantly different between the control condition (incubated in nutrient-rich media), starvation and S. mutans infection (Fig. 3a). The number of S. mutans recovered from the intracellular compartment of HSC-2 cells was about 4.8 × 104 CFU for the wild-type strain and 6 × 104 CFU for the ilvE mutant strain. These results suggest that S. mutans was taken up by HSC-2 cells and persisted, and the relative decrease in BCAAs provoked by the ilvE mutant strain may induce bacterial endocytosis of HSC-2 cells. Figure 3. View largeDownload slide Invasion assay of S. mutans in HSC-2 cells. (A) Cell viability of infected and non-infected HSC-2 cells. The number of live cells was counted by trypan blue assay as described in Materials and Methods. (B) Invasive properties of S. mutans wild-type and ilvE mutant strains. The number of S. mutans CFUs recovered from the intracellular compartment of HSC-2 cells after 2-h infection is shown. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.05; **P < 0.01. Figure 3. View largeDownload slide Invasion assay of S. mutans in HSC-2 cells. (A) Cell viability of infected and non-infected HSC-2 cells. The number of live cells was counted by trypan blue assay as described in Materials and Methods. (B) Invasive properties of S. mutans wild-type and ilvE mutant strains. The number of S. mutans CFUs recovered from the intracellular compartment of HSC-2 cells after 2-h infection is shown. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.05; **P < 0.01. Invasion of S. mutans has often been discussed from the perspective of serotypes (Nomura et al.2006; Nakano et al.2007a,b; Abranches et al.2009). PCR methods by which serotype-specific nucleotide sequences are detected revealed that serotype e was the most prevalent, followed by serotype c (Nakano et al.2007a,b). Nomura et al. (2006) reported that only S. mutans serotype c strains were isolated from the heart valve of an IE patient. The detection frequency of S. mutans serotype c seems very high. However, invasion experiments performed by Abranches et al. showed that serotype e strain B14 and serotype f strain OMZ175 were invasive, whereas a serotype c strain was not invasive in human coronary artery endothelial cells (HCAEC). Under the conditions tested, the invasion efficiency of wild-type S. mutans 109c in HSC-2 cells was about 0.24% at an MOI of 20. This invasive efficiency seems higher than that of S. mutans serotype c strain B14 in HCAEC at an MOI of 100 (0.05%) (Abranches et al.2009) and that of S. mutans serotype c strain Xc in HCAEC at an MOI of 1 (0.11%) (Nagata, de Toledo and Oho 2011). Thus, the relationship between S. mutans serotype and invasive efficiency in host cells remains unclear. We also demonstrated that the number of collected bacteria from HSC-2 cells infected with the wild-type or ilvE mutant strain increased by bafilomycin A1 treatment (Fig. 3B). In addition, the degradation of the wild type was higher than of the mutant without bafilomycin A1; however, the intracellular number of ilvE mutant was comparable with that of the wild type in the presence of bafilomycin A1. These results suggest a relationship between lysosomal degradation and S. mutans invasion, and that the ilvE mutant might suppress the lysosomal degradation. Degradation of intracellular bacteria seems to start within 2 h. Evaluation of autophagosome formation activity of HSC-2 cells Invading bacteria are specific targets for autophagy (also called xenophagy) by which their growth is restricted (Mizushima et al.2008). Autophagy is an intracellular degradation system that comprises a unique organelle, called autophagosome, formation and lysosomal degradation (Mizushima, Yoshimori and Levine 2010). As we used bafilomycin A1, lysosome inhibitor, in this study, we focused on the influence of bacterial invasion to the autophagosome formation. LC3 is commonly used as a marker of autophagosomes. In the autophagosome formation, LC3-I is converted to LC3-II via the addition of a phosphatidylethanolamine group to the C-terminus to synthesize autophagosome. Converted LC3-II was detected by western blotting (Fig. 4A) and the relative intensity of stained LC3-II normalized to β-actin was shown in Fig. 4B. Western blot analysis showed that LC3 lipidation was not observed in non-stimulated control HSC-2 cells, whereas LC3-II formation was induced in HSC-2 cells treated with Torin 1, an inhibitor of mTORC1. Apparent LC3-II conversion was observed in HSC-2 cells infected with the ilvE mutant strain for 2 h. Conversely, 2-h infection with the wild-type strain did not induce conversion from LC3-I to LC3-II in HSC-2 cells. However, when HSC-2 cells were infected with S. mutans for 3 and 4 h, infection with both wild-type and ilvE mutant strains strongly stimulated the conversion of LC3-II in HSC-2 cells, with no significant difference observed. Figure 4. View largeDownload slide Evaluation of autophagic activity of HSC-2 cells. (A) Western blot analysis for the detection of LC3 (top) and β-actin (internal control, bottom). Total cell lysates obtained from infected and non-infected HSC-2 cells treated with bafilomycin A1 were analyzed by western blot analysis. Data shown are representative of three replicate studies. Lane 1, negative control (HSC-2 cells incubated in E-MEM); lane 2, Torin 1 treatment (positive control); lane 3, wild-type infection for 2 h; lane 4, ΔilvE infection for 2 h; lane 5, wild-type infection for 3 h; lane 6, ΔilvE infection for 3 h; lane 7, wild-type infection for 4 h; lane 8, ΔilvE infection for 4 h. (B) Relative quantitative evaluation of immunoreactive LC3-II and β-actin in HSC-2 cells. Band intensity was calculated by Image Studio Digits software. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.01. (C) Immunofluorescence microscopy analysis of LC3 localization in HSC-2 cells. Stimulation conditions for HSC-2 cells were the same as those for western blotting. Immunostained LC3 in HSC-2 cells is shown. Scale bars, 20 μm. (D) The number of LC3 puncta in HSC-2 cells. The number of LC3 puncta was counted from three fields of view (with a minimum of 10 cells per field) for each sample. Data are presented as mean ± SD. Significant differences were determined by Student's unpaired t-test. *P < 0.01. Figure 4. View largeDownload slide Evaluation of autophagic activity of HSC-2 cells. (A) Western blot analysis for the detection of LC3 (top) and β-actin (internal control, bottom). Total cell lysates obtained from infected and non-infected HSC-2 cells treated with bafilomycin A1 were analyzed by western blot analysis. Data shown are representative of three replicate studies. Lane 1, negative control (HSC-2 cells incubated in E-MEM); lane 2, Torin 1 treatment (positive control); lane 3, wild-type infection for 2 h; lane 4, ΔilvE infection for 2 h; lane 5, wild-type infection for 3 h; lane 6, ΔilvE infection for 3 h; lane 7, wild-type infection for 4 h; lane 8, ΔilvE infection for 4 h. (B) Relative quantitative evaluation of immunoreactive LC3-II and β-actin in HSC-2 cells. Band intensity was calculated by Image Studio Digits software. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.01. (C) Immunofluorescence microscopy analysis of LC3 localization in HSC-2 cells. Stimulation conditions for HSC-2 cells were the same as those for western blotting. Immunostained LC3 in HSC-2 cells is shown. Scale bars, 20 μm. (D) The number of LC3 puncta in HSC-2 cells. The number of LC3 puncta was counted from three fields of view (with a minimum of 10 cells per field) for each sample. Data are presented as mean ± SD. Significant differences were determined by Student's unpaired t-test. *P < 0.01. LC3 localization in HSC-2 cells was visualized by immunofluorescence microscopy. LC3 was diffusely distributed in the cytosol of HSC-2 cells incubated with nutrient-rich media without infection (negative control). However, numerous LC3-positive puncta were observed in starved HSC-2 cells (Fig. 4C). HSC-2 cells infected by the wild-type strain showed slight LC3 accumulation; however, HSC-2 cells infected by the ilvE mutant strain displayed clear LC3-positive puncta. The results of western blotting and immunofluorescence microscopy analysis suggest that the excess intracellular utilization of BCAA by the ilvE mutant strain induced autophagic activity through inactivation of mTORC1. To elucidate the relationship between intracellular decrease of BCAA and mTORC1 function, another mTORC1-related function, such as hypertrophy or insulin resistance, should be investigated. From findings presented here, we concluded that invading S. mutans took up and utilized host-derived BCAAs until they were digested by lysosomes. Although bacteria survived for a short time, the relative BCAA concentration may be altered, which affects cellular functions such as bacterial degradation. Consequently, reduced bacterial digestion may increase bacterial survival. Therefore, excess consumption of intracellular BCAAs by bacteria inside host cells could be an important pathogenic property. To our knowledge, the present study is the first to demonstrate that invading S. mutans takes up and utilizes host-derived BCAAs (at least leucine), which affects the cellular function of host cells. FUNDING This work was supported by the Private University High Technology Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [grant number S1001010]. Conflict of interest. None declared. REFERENCES Abranches J , Zeng L, Belanger Met al.   Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175. Oral Microbiol Immunol  2009; 24: 141– 5. 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Influence of excess branched-chain amino acid uptake by Streptococcus mutans in human host cells

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Abstract

Abstract Oral streptococci, including cariogenic bacterium Streptococcus mutans, comprise a large percentage of human supragingival plaque, which contacts both tooth surfaces and gingiva. Eukaryotic cells are able to take up macromolecules and particles, including bacteria, by endocytosis. Increasing evidence indicates endocytosis may be used as an entry process by bacteria. We hypothesized that some endocytosed bacteria might survive and obtain nutrients, such as amino acids, until they are killed. To verify this hypothesis, we focused on bacterial utilization of branched-chain amino acids (BCAAs; isoleucine, leucine and valine) in host cells. A branched-chain aminotransferase, IlvE (EC 2.6.1.42), has been suggested to play an important role in internal synthesis of BCAAs in S. mutans UA159. Therefore, we constructed an ilvE-deficient S. mutans 109c strain and confirmed that it had similar growth behavior as reported previously. 14C radioactive leucine uptake assays showed that ilvE-deficient S. mutans took up more leucine both inside and outside of host cells. We further clarified that a relative decrease of BCAAs in host cells caused enhanced endocytic and autophagic activity. In conclusion, S. mutans is endocytosed by host cells and may survive and obtain nutrients, such as BCAAs, inside the cells, which might affect cellular functions of host cells. Streptococcus mutans, branched-chain amino acids, radioactive transport, autophagic activity, endocytosis, bacterial invasion INTRODUCTION Dental plaque comprises the community of oral microorganisms contained within biofilms on a tooth surface, which has been implicated as the major cause of dental caries and periodontal disease. Streptococcus mutans, a well-established causative agent of human dental caries (Mitchell 2003), is usually seen in dental plaque but can also be found in blood, on heart valves in subacute endocarditis and in atheromatous plaques (Vose et al.1987; Moreillon and Que 2004; Nakano et al.2006). A number of PCR-based analyses using S. mutans-specific 16S rRNA gene-amplifying primers have demonstrated the existence of S. mutans in lesions. In addition, a report in which living S. mutans strains were isolated from the blood or heart valve of a patient with infective endocarditis (IE) strongly supports the relationship between S. mutans and systemic diseases (Nomura et al.2006). Moreover, experimental invasion of S. mutans into human umbilical vein endothelial cells and human aortic endothelial cells was reported (Abranches et al.2009; Nagata, de Toledo and Oho 2011). These findings suggest that S. mutans has the ability to invade and survive in host cells. Therefore, this bacterium may take up and utilize host-derived nutrients, specifically amino acids, inside host cells. However, to our knowledge, amino acid utilization by intracellular S. mutans has not yet been investigated. Branched-chain amino acids (BCAAs; isoleucine, leucine and valine) are typically found in the core of globular proteins and are a quantitatively important group of amino acids in bacterial proteins (Neidhart and Umbarger 1996; Patek 2007). BCAAs are synthesized by a series of sequential reactions, and aminotransferase IlvE (EC 2.6.1.42) catalyzes the final step (Ganesan and Weimer 2004). IlvE has been shown to play an important role in the growth of S. mutans UA159 when no exogenous BCAAs were available (Santiago et al.2012) and for the optimal growth of S. thermophils in milk (Garault et al.2000). In addition, the function of ilvC in BCAA synthesis in S. pneumoniae has been suggested to be essential for virulence factor of respiratory infections (Kim et al.2017). BCAAs not only act as building blocks for tissue proteins, but also have other metabolic functions in human host cells. BCAAs are known to exert several signaling responses mainly via activation of the mammalian target of rapamycin (mTORC1), which can result in hypertrophy, proliferation and insulin resistance (Newgard et al.2009; Liu et al.2014; Neishabouri, Hutson and Davoodi 2015). It is well known that mTORC1 activity is inhibited by nutrient starvation, which results in autophagy induction (Noda and Ohsumi 1998; Scott, Schuldiner and Neufeld 2004). Autophagy is a lysosome- or vacuole-mediated degradation system that responds to reductions in available nutrients, especially amino acids (Zoncu et al.2011; Chen et al.2014). In many studies, cellular functions related to amino acids levels were analyzed by increasing or decreasing amino acid concentrations outside of host cells (Kakazu et al.2007; Zoncu et al.2011; Wubetu et al.2014; Zhenyukh et al.2017). However, changes in relative BCAA concentrations inside host cells have not been focused on by researchers. Moreover, pathogenic effects of decreased intracellular BCAA concentrations caused by invasive bacteria have not been elucidated. Therefore, in this study, we constructed an ilvE-deficient S. mutans 109c strain to evaluate BCAA utilization in host cells and clarify the biological effect of decreasing BCAA levels in host cells. MATERIALS AND METHODS Bacterial strains and culture conditions Streptococcus mutans 109c (wild type; serotype c) (Sato, Yamamoto and Kizaki 1997) and its isogenic mutant constructed in this study were anaerobically maintained (80% N2, 10% H2 and 10% CO2) at 37°C in brain heart infusion broth (BHI; Difco Laboratories, Detroit, MI, USA). Bacterial growth was measured using BCAA-free Roswell Park Memorial Institute (RPMI) medium (RPMI medium without BCAA and phenol red, IFP, Yamagata, Japan) or RPMI medium (without phenol red). RPMI medium was prepared by supplementation with adequate BCAAs (L-isoleucine: 50 mg L−1, L-leucine: 50 mg L−1 and L-valine: 20 mg L−1, Wako, Tokyo, Japan) to BCAA-free RPMI medium. BCAA-free medium was also used for exogenous radioactive leucine uptake assays. When required, 50 μg mL−1 tetracycline (Wako) was added to the medium. Cell culture HSC-2 (RCB1945), human cell line derived from oral squamous cell carcinoma, was purchased from the Cell Engineering Division of RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). Cells were cultured in Eagle's minimum essential medium (E-MEM) (Wako, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum, 100 U mL−1 penicillin and 100 μg mL−1 streptomycin (complete E-MEM) at an initial density of 2.0 × 105 cells/well on 6-well plates (Iwaki, Tokyo, Japan) for 24 h at 37°C in a 5% CO2 humidified incubator. Construction of an ilvE-deficient S. mutans 109c strain Genomic DNA from S. mutans 109c was extracted using a GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA). An ilvE-deficient mutant strain of S. mutans 109c was constructed by PCR-based gene replacement with an antibiotic resistance gene cassette as described previously (Kunii et al.2014). In this experiment, the ilvE gene of S. mutans was replaced with the tetracycline-resistance gene (Tcr). PCR primers used to construct the mutant were designed with reference to the S. mutans UA159 genome sequence (Ajdic et al.2002) (GenBank accession number AE014133). A fragment containing the upstream flanking region and 5΄-end of ilvE was amplified using primers ilvEP1-F (5΄-CCACTGTAGGTGAACAAGGACAAG-3΄) and ilvEP2-R (5΄-AACTCCAATATTAATAATTTTCATTTTAATACCTCTTTCCTCG-3΄). A fragment containing the downstream flanking region and 3΄-end portion of the target gene was amplified using primer pair ilvEP3-F (5΄-CAATAAAATAACTTAGATAATTAGCGAGCTTGC-3΄) and ilvEP4-R (5΄-TTCGCTACCCTTCCAGTGACAACA-3΄). Tcr was amplified from pUCTet (Arimoto and Igarashi 2008) using primer pair ilvEP2-F (5΄-CGAGGAAAGAGGTATTAAAATGAAAATTATTAATATTGGAGTT-3΄) and ilvEP3-R (5΄-GCAAGCTCGCTAATTATCTAAGTTATTTTATTG-3΄). Reverse primer ilvEP2-R and forward primer ilvEP3-F were synthesized with additional nucleotides complementary to the 5΄- and 3΄-terminal regions of Tcr, respectively. The antibiotic resistance gene was amplified using pUCTet as a template. All three fragments were annealed in one reaction and amplified by PCR using primer pair ilvEP1-F/ilvEP4-R. The resultant amplicon was then used to transform S. mutans 109c cells (Perry and Slade 1962). Replacement mutants of the ilvE gene were selected on BHI agar plates containing 50 μg mL−1 tetracycline. Replacement of the antibiotic resistance gene cassette was verified by sequence analysis. Growth of S. mutans with or without BCAAs Growth of S. mutans cells was examined in culture medium with or without BCAAs. Streptococcus mutans cells in exponential phase were collected by centrifugation at 5000× g for 10 min at room temperature. Harvested cells were washed three times with phosphate buffered saline (PBS) and then adjusted to an optical density (OD) at 600 nm of 1.0 with BCAA-free medium (RPMI medium without leucine, isoleucine and valine). Adjusted bacterial cell suspensions were diluted 10-fold in 2 mL of RPMI or BCAA-free medium and incubated at 37°C in an anaerobic chamber. Bacterial growth was determined by measuring the OD600 of cultures at the time indicated. Environmental and intracellular radioactive leucine uptake assays A previously described protocol (Trip, Mulder and Lolkema 2013) was adapted to measure the uptake of environmental 14C-labeled leucine (PerkinElmer, Shelton, CT, USA) by S. mutans. Briefly, bacteria were grown to OD600 = 0.5, harvested by centrifugation, washed twice with PBS and adjusted to OD600 = 1.0 with PBS before resuspension in BCAA-free medium. 14C-labeled leucine was added to cells at a final concentration of 1 μM (=2 mCi μmol−1, 23 kBq) and incubated for 1 h at 37°C in a CO2-humidified incubator. Cells were then filtered through 0.22-μm membrane filters and immediately washed with 0.1 M LiCl2 at room temperature. Filters were dried and placed in scintillation vials containing 8 mL of Ultima Gold scintillation cocktail (PerkinElmer, Boston, MA, USA). 14C radioactivity was measured using TRI-CARB 2900TR Liquid Scintillation Analyzer (PerkinElmer, Shelton, CT, USA). Bacterial uptake of 14C-labeled leucine in HSC-2 cells was also measured. Prepared HSC-2 cells were incubated with 165 nM 14C-labeled leucine (=300 μCi μmol−1, 3.7 kBq) for 1 h. Before S. mutans infection, HSC-2 cells were washed three times with HBSS (-) and incubated with HBSS (-) containing 100 nM bafilomycin A1 for 1 h. An overnight culture of S. mutans grown in BHI was diluted 20-fold in 3 mL of fresh BHI and then anaerobically incubated at 37°C to OD600 = 1.0. The culture was harvested by centrifugation at 5000× g for 10 min at room temperature. Harvested cells were then washed three times with 50 mM 4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid (HEPES) buffer, adjusted to OD600 = 1.0 with HEPES, and anaerobically incubated at 37°C for 2 h prior to assay. The heat-killed ilvE mutant was prepared by heating the bacterial suspension at 100°C for 1 h. After 2-h incubation, S. mutans cells were harvested, resuspended in HBSS (-) and added at a multiplicity of infection (MOI) of 50 to HSC-2 cells. Infected HSC-2 cells were incubated at 37°C for 1 h in a 5% CO2-humidified incubator. Infected monolayers were washed three times with HBSS (-) to remove unbound S. mutans cells and lysed with 0.3% Triton X-100 (Wako). Cell lysates were separated as filtrated and flow-through samples with 0.22-μm membrane filters. The filtrated sample was washed, dried and measured for 14C radioactivity as described above. The flow-through fraction of 14C radioactivity was measured by direct counting (i.e. addition to a scintillation cocktail). Bacterial invasion assay Bacterial samples were prepared as described above except an MOI of 20 was used to infect HSC-2 cells. After 2-h starvation, bacteria were added to HSC-2 cells and incubated at 37°C for 2 h in a 5% CO2-humidified incubator. Infected monolayers were washed three times with HBSS (-) to remove unbound S. mutans cells. Washed cells were resuspended and incubated at 37°C for 1 h with fresh HBSS (-) containing 400 μg mL−1 gentamicin to kill residual extracellular S. mutans. Monolayers were again washed three times with HBSS (-) and lysed with 0.3% Triton X-100 to release intracellular S. mutans. Serial dilutions of lysates were plated onto BHI agar plates and incubated anaerobically at 37°C for 48 h. The numbers of S. mutans colony-forming units (CFUs) grown on the plates were determined. Cell viability of infected and non-infected HSC-2 cells was evaluated by trypan blue assay (Fardini et al.2011) using a TC20 automated cell counter (Bio-Rad, Hercules, CA, USA). Western blotting HSC-2 cells were washed three times with HBSS (-) and lysed with RIPA buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% NP40, 0.5% sodium deoxy cholate and 0.1% sodium dodecyl sulfate) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The protein concentration of the lysate was adjusted to 1 mg mL−1, and proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with tris-based saline (TBS) with 0.1% Triton X-100 (TBST) containing 2% skim milk overnight at 4°C. The next day, the blocked membrane was incubated for 3 h at room temperature with rabbit anti-LC3 (1:1000; BML, Saitama, Japan) and diluted in blocking solution. The membrane was washed three times with TBST, incubated for 1 h at room temperature with Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:20000 dilution; GE Healthcare, Buckinghamshire, UK) in blocking solution, and washed four times with TBST. Immunoreactive bands were then detected using ImmunoStar®LD (Wako) and C-DiGit western blot imaging system (MS TechnoSystems Inc., Osaka, Japan). The same membrane was incubated with WB Stripping solution (Nacalai Tesque, Kyoto, Japan), and reprobed with mouse anti-β-actin (1:1000; Abcam, Cambridge, England), followed by HRP-conjugated anti-mouse IgG antibody (1:20000 dilution; GE Healthcare). Immunofluorescence microscopy To visualize the biological effect of endocytosed S mutans cells, microtubule-associated protein light chain 3 (LC3) localization was determined by immunofluorescence microscopy. HSC-2 cells infected with S. mutans at an MOI of 20 for 1 h were fixed with 4% paraformaldehyde phosphate buffer solution (Wako) and permeabilized with 0.3% Triton X-100 in PBS. Non-specific binding was subsequently blocked by incubation for 30 min with Blocking buffer (Nacalai Tesque). Cells were then stained using rabbit anti-LC3 antibody (1:1000), followed by incubation with Cy3-conjugated anti-mouse secondary antibodies (1:1000; Jackson ImmunoResearch, West Grove, PA, USA), for 1 h at 37°C. Triplicate samples of >50 HSC-2 cells were investigated for LC3 accumulation. Statistical analysis Growth data are shown as representative results from three independent experiments with similar results. Data are presented as mean ± standard deviations (SD) of three samples. Results for ODs were compared using a two-tailed Student's t-test with the multiple comparison procedure as necessary. P values < 0.05 were considered statistically significant. RESULTS AND DISCUSSION Growth of S. mutans with or without BCAAs IlvE has been implied to play an important role in intracellular synthesis of BCAAs in S. mutans, as growth of the ilvE-deficient S. mutans UA159 strain was impaired in chemically defined minimal medium lacking BCAA (Santiago et al.2012). Therefore, we hypothesized that an ilvE-deficient mutant strain of S. mutans would require more exogenous BCAA than the wild-type strain, which emphasized the biological effect caused by decreased BCAA concentrations in host cells. We initially confirmed whether the constructed ilvE-deficient S. mutans 109c strain showed similar growth behavior in the absence of BCAA as wild-type S. mutans UA159. In this experiment, BCAA-free RPMI medium was used instead of minimal medium without BCAA. Both the wild-type and ilvE mutant strains grew in RPMI medium. However, while the wild-type strain grew in BCAA-free medium, the ilvE mutant strain did not grow at all (Fig. 1). This result differed from that of a previous report on S. mutans UA159, in which significant, but not complete, growth impairment was noted in the absence of BCAA (Santiago et al.2012). The reason for the discrepant results may be explained by differences in growth medium and strain used. Nonetheless, our finding demonstrated that IlvE plays a critical role in BCAA biosynthesis in S. mutans 109c. Figure 1. View largeDownload slide Growth behavior of S. mutans in RPMI or BCAA-free RPMI medium. Bacterial growth was determined by measuring the optical density at 600 nm (OD600). Closed circles, wild type; open circles, ilvE mutant. Solid lines and dotted lines represent growth in RPMI medium and BCAA-free medium, respectively. Growth data shown are representative of three independent experiments with similar results. OD results were compared using a two-tailed Student's t-test. Significant differences (P < 0.01) between the growth behavior of wild-type and ΔilvE mutant strains in BCAA-free medium are indicated by asterisks. Figure 1. View largeDownload slide Growth behavior of S. mutans in RPMI or BCAA-free RPMI medium. Bacterial growth was determined by measuring the optical density at 600 nm (OD600). Closed circles, wild type; open circles, ilvE mutant. Solid lines and dotted lines represent growth in RPMI medium and BCAA-free medium, respectively. Growth data shown are representative of three independent experiments with similar results. OD results were compared using a two-tailed Student's t-test. Significant differences (P < 0.01) between the growth behavior of wild-type and ΔilvE mutant strains in BCAA-free medium are indicated by asterisks. 14C leucine uptake by S. mutans Since S. mutans was found in human host cells, we performed radioactive leucine assays to clarify whether S. mutans took up host-derived leucine in HSC-2 cells. First, extracellular leucine uptake by S. mutans was evaluated. As shown in Fig. 2a, exogenous 14C leucine was clearly taken up by both S. mutans strains (around 0.1 nmol by the wild-type strain, around 0.15 nmol by the ilvE mutant strain). This result suggests that S. mutans takes up environmental leucine, and the ilvE mutant strain requires more exogenous leucine than the wild-type strain. Figure 2. View largeDownload slide 14C-labeled leucine uptake by S. mutans. (A) Exogenous 14C leucine uptake by S. mutans. Bacterial cells were harvested from cultures grown to mid-exponential phase in BHI. 14C leucine uptake was measured at 1 h. As a negative control, radioactivity of a filter cup was measured. As a positive control, 0.3 nmol 14C leucine was directly added to scintillation cocktails. (B) Uptake of host cell-derived 14C leucine by S. mutans. HSC-2 cells were pre-incubated with 14C leucine for 1 h followed by 1 h starvation in HBSS (-) buffer before bacterial infection. Cells were infected with bacteria, prepared as described above, at an MOI of 50. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.05; **P < 0.01. Figure 2. View largeDownload slide 14C-labeled leucine uptake by S. mutans. (A) Exogenous 14C leucine uptake by S. mutans. Bacterial cells were harvested from cultures grown to mid-exponential phase in BHI. 14C leucine uptake was measured at 1 h. As a negative control, radioactivity of a filter cup was measured. As a positive control, 0.3 nmol 14C leucine was directly added to scintillation cocktails. (B) Uptake of host cell-derived 14C leucine by S. mutans. HSC-2 cells were pre-incubated with 14C leucine for 1 h followed by 1 h starvation in HBSS (-) buffer before bacterial infection. Cells were infected with bacteria, prepared as described above, at an MOI of 50. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.05; **P < 0.01. Entry of non-invasive bacteria into non-phagocytic host cells is generally thought to depend on an endocytic mechanism. Bacteria taken up by endocytosis are finally digested by lysosomes. Preliminary experiments showed that a portion of bacteria taken into HSC-2 cells was digested, which resulted in increased 14C leucine concentration from the cytosolic fraction of infected cells compared to that of non-infected control cells (data not shown). Therefore, we used bafilomycin A1, a strong inhibitor of vacuolar-type H+-ATPase, to protect internal S. mutans from lysosomal degradation. No significant difference in 14C radioactivity was found from either a filtered sample (S. mutans collected from inside host cells) or a cytosolic fraction compared to that of negative control cells. However, 14C radioactivity of collected ilvE mutant cells was increased compared to the filtered sample of negative control cells. Moreover, 14C radioactivity of the cytosolic fraction from ilvE-mutant infected cells was decreased compared to that of negative control cells. Notably, differences observed between the bacterial fraction and cytosolic fraction from infected HSC-2 cells were diminished when the ilvE mutant was dead, indicating that the decrease in cytosolic 14C leucine was caused by utilization by the ilvE mutant strain. Although it was difficult to determine whether the wild-type S. mutans strain took up 14C leucine in HSC-2 cells, the utilization of leucine by the ilvE mutant strain in HSC-2 cells was clearly demonstrated. Taken together, S. mutans (at least the ilvE mutant strain) is taken up by HSC-2 cells and utilizes host-derived leucine in the cells. Invasion assay of HSC-2 cells with S. mutans The invasive properties of S. mutans 109c were examined by antibiotic protection assay. The number of HSC-2 cells was not significantly different between the control condition (incubated in nutrient-rich media), starvation and S. mutans infection (Fig. 3a). The number of S. mutans recovered from the intracellular compartment of HSC-2 cells was about 4.8 × 104 CFU for the wild-type strain and 6 × 104 CFU for the ilvE mutant strain. These results suggest that S. mutans was taken up by HSC-2 cells and persisted, and the relative decrease in BCAAs provoked by the ilvE mutant strain may induce bacterial endocytosis of HSC-2 cells. Figure 3. View largeDownload slide Invasion assay of S. mutans in HSC-2 cells. (A) Cell viability of infected and non-infected HSC-2 cells. The number of live cells was counted by trypan blue assay as described in Materials and Methods. (B) Invasive properties of S. mutans wild-type and ilvE mutant strains. The number of S. mutans CFUs recovered from the intracellular compartment of HSC-2 cells after 2-h infection is shown. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.05; **P < 0.01. Figure 3. View largeDownload slide Invasion assay of S. mutans in HSC-2 cells. (A) Cell viability of infected and non-infected HSC-2 cells. The number of live cells was counted by trypan blue assay as described in Materials and Methods. (B) Invasive properties of S. mutans wild-type and ilvE mutant strains. The number of S. mutans CFUs recovered from the intracellular compartment of HSC-2 cells after 2-h infection is shown. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.05; **P < 0.01. Invasion of S. mutans has often been discussed from the perspective of serotypes (Nomura et al.2006; Nakano et al.2007a,b; Abranches et al.2009). PCR methods by which serotype-specific nucleotide sequences are detected revealed that serotype e was the most prevalent, followed by serotype c (Nakano et al.2007a,b). Nomura et al. (2006) reported that only S. mutans serotype c strains were isolated from the heart valve of an IE patient. The detection frequency of S. mutans serotype c seems very high. However, invasion experiments performed by Abranches et al. showed that serotype e strain B14 and serotype f strain OMZ175 were invasive, whereas a serotype c strain was not invasive in human coronary artery endothelial cells (HCAEC). Under the conditions tested, the invasion efficiency of wild-type S. mutans 109c in HSC-2 cells was about 0.24% at an MOI of 20. This invasive efficiency seems higher than that of S. mutans serotype c strain B14 in HCAEC at an MOI of 100 (0.05%) (Abranches et al.2009) and that of S. mutans serotype c strain Xc in HCAEC at an MOI of 1 (0.11%) (Nagata, de Toledo and Oho 2011). Thus, the relationship between S. mutans serotype and invasive efficiency in host cells remains unclear. We also demonstrated that the number of collected bacteria from HSC-2 cells infected with the wild-type or ilvE mutant strain increased by bafilomycin A1 treatment (Fig. 3B). In addition, the degradation of the wild type was higher than of the mutant without bafilomycin A1; however, the intracellular number of ilvE mutant was comparable with that of the wild type in the presence of bafilomycin A1. These results suggest a relationship between lysosomal degradation and S. mutans invasion, and that the ilvE mutant might suppress the lysosomal degradation. Degradation of intracellular bacteria seems to start within 2 h. Evaluation of autophagosome formation activity of HSC-2 cells Invading bacteria are specific targets for autophagy (also called xenophagy) by which their growth is restricted (Mizushima et al.2008). Autophagy is an intracellular degradation system that comprises a unique organelle, called autophagosome, formation and lysosomal degradation (Mizushima, Yoshimori and Levine 2010). As we used bafilomycin A1, lysosome inhibitor, in this study, we focused on the influence of bacterial invasion to the autophagosome formation. LC3 is commonly used as a marker of autophagosomes. In the autophagosome formation, LC3-I is converted to LC3-II via the addition of a phosphatidylethanolamine group to the C-terminus to synthesize autophagosome. Converted LC3-II was detected by western blotting (Fig. 4A) and the relative intensity of stained LC3-II normalized to β-actin was shown in Fig. 4B. Western blot analysis showed that LC3 lipidation was not observed in non-stimulated control HSC-2 cells, whereas LC3-II formation was induced in HSC-2 cells treated with Torin 1, an inhibitor of mTORC1. Apparent LC3-II conversion was observed in HSC-2 cells infected with the ilvE mutant strain for 2 h. Conversely, 2-h infection with the wild-type strain did not induce conversion from LC3-I to LC3-II in HSC-2 cells. However, when HSC-2 cells were infected with S. mutans for 3 and 4 h, infection with both wild-type and ilvE mutant strains strongly stimulated the conversion of LC3-II in HSC-2 cells, with no significant difference observed. Figure 4. View largeDownload slide Evaluation of autophagic activity of HSC-2 cells. (A) Western blot analysis for the detection of LC3 (top) and β-actin (internal control, bottom). Total cell lysates obtained from infected and non-infected HSC-2 cells treated with bafilomycin A1 were analyzed by western blot analysis. Data shown are representative of three replicate studies. Lane 1, negative control (HSC-2 cells incubated in E-MEM); lane 2, Torin 1 treatment (positive control); lane 3, wild-type infection for 2 h; lane 4, ΔilvE infection for 2 h; lane 5, wild-type infection for 3 h; lane 6, ΔilvE infection for 3 h; lane 7, wild-type infection for 4 h; lane 8, ΔilvE infection for 4 h. (B) Relative quantitative evaluation of immunoreactive LC3-II and β-actin in HSC-2 cells. Band intensity was calculated by Image Studio Digits software. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.01. (C) Immunofluorescence microscopy analysis of LC3 localization in HSC-2 cells. Stimulation conditions for HSC-2 cells were the same as those for western blotting. Immunostained LC3 in HSC-2 cells is shown. Scale bars, 20 μm. (D) The number of LC3 puncta in HSC-2 cells. The number of LC3 puncta was counted from three fields of view (with a minimum of 10 cells per field) for each sample. Data are presented as mean ± SD. Significant differences were determined by Student's unpaired t-test. *P < 0.01. Figure 4. View largeDownload slide Evaluation of autophagic activity of HSC-2 cells. (A) Western blot analysis for the detection of LC3 (top) and β-actin (internal control, bottom). Total cell lysates obtained from infected and non-infected HSC-2 cells treated with bafilomycin A1 were analyzed by western blot analysis. Data shown are representative of three replicate studies. Lane 1, negative control (HSC-2 cells incubated in E-MEM); lane 2, Torin 1 treatment (positive control); lane 3, wild-type infection for 2 h; lane 4, ΔilvE infection for 2 h; lane 5, wild-type infection for 3 h; lane 6, ΔilvE infection for 3 h; lane 7, wild-type infection for 4 h; lane 8, ΔilvE infection for 4 h. (B) Relative quantitative evaluation of immunoreactive LC3-II and β-actin in HSC-2 cells. Band intensity was calculated by Image Studio Digits software. Data are presented as mean ± SD and representative of three independent experiments with similar results. Significant differences were determined by Student's unpaired t-test. *P < 0.01. (C) Immunofluorescence microscopy analysis of LC3 localization in HSC-2 cells. Stimulation conditions for HSC-2 cells were the same as those for western blotting. Immunostained LC3 in HSC-2 cells is shown. Scale bars, 20 μm. (D) The number of LC3 puncta in HSC-2 cells. The number of LC3 puncta was counted from three fields of view (with a minimum of 10 cells per field) for each sample. Data are presented as mean ± SD. Significant differences were determined by Student's unpaired t-test. *P < 0.01. LC3 localization in HSC-2 cells was visualized by immunofluorescence microscopy. LC3 was diffusely distributed in the cytosol of HSC-2 cells incubated with nutrient-rich media without infection (negative control). However, numerous LC3-positive puncta were observed in starved HSC-2 cells (Fig. 4C). HSC-2 cells infected by the wild-type strain showed slight LC3 accumulation; however, HSC-2 cells infected by the ilvE mutant strain displayed clear LC3-positive puncta. The results of western blotting and immunofluorescence microscopy analysis suggest that the excess intracellular utilization of BCAA by the ilvE mutant strain induced autophagic activity through inactivation of mTORC1. To elucidate the relationship between intracellular decrease of BCAA and mTORC1 function, another mTORC1-related function, such as hypertrophy or insulin resistance, should be investigated. From findings presented here, we concluded that invading S. mutans took up and utilized host-derived BCAAs until they were digested by lysosomes. Although bacteria survived for a short time, the relative BCAA concentration may be altered, which affects cellular functions such as bacterial degradation. Consequently, reduced bacterial digestion may increase bacterial survival. Therefore, excess consumption of intracellular BCAAs by bacteria inside host cells could be an important pathogenic property. To our knowledge, the present study is the first to demonstrate that invading S. mutans takes up and utilizes host-derived BCAAs (at least leucine), which affects the cellular function of host cells. FUNDING This work was supported by the Private University High Technology Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [grant number S1001010]. Conflict of interest. None declared. REFERENCES Abranches J , Zeng L, Belanger Met al.   Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175. Oral Microbiol Immunol  2009; 24: 141– 5. 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FEMS Microbiology LettersOxford University Press

Published: Feb 1, 2018

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