Rapid screening method for detecting highly pathogenic Streptococcus intermedius strains carrying a mutation in the lacR gene

Rapid screening method for detecting highly pathogenic Streptococcus intermedius strains carrying... Abstract Streptococcus intermedius is a member of the normal human commensal flora and secretes a human-specific cytolysin intermedilysin (ILY) as a major virulence factor. Expression of ily is repressed by LacR and loss-of-function mutations of LacR are observed in many ILY high-producing strains isolated from deep-seated abscesses, suggesting that high ILY production is necessary for increased virulence. However, because ILY exhibits no β-hemolysis on animal blood agar plates, differentiating ILY high- and low-producing strains using conventional laboratory methods is not possible. Interestingly, S. intermedius also produces glycosidases, including MsgA and NanA, which exhibit N-acetyl-β-d-glucosaminidase and neuraminidase activities, respectively. Moreover, MsgA expression, but not NanA, is negatively regulated by LacR. Here we measured the activities of MsgA, NanA and ILY in strains isolated from clinical specimens and dental plaque to determine the correlation between these glycosidase activities and ILY hemolytic activity. Hemolytic activity showed a strong positive correlation with MsgA and a weak negative correlation with NanA activities. Therefore, we calculated the ratio of MsgA and NanA activity (M/N ratio). This value showed a stronger positive correlation (r = 0.81) with ILY hemolytic activity and many strains with high M/N ratios (>2) were ILY-high producers with loss-of-function mutations in LacR. Streptococcus intermedius, intermedilysin, screening, MsgA, NanA, LacR INTRODUCTION Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that belongs to the anginosus group of streptococci (Whiley et al.1990; Jensen, Hoshino and Kilian 2013). This pathogen is associated with oral infections, including periodontal disease and recurrent tonsillitis, and with deep-seated purulent infections such as brain and lung abscesses (Whiley et al.1992; Jerng et al.1997; Tanner et al.1997; Gray 2005; Takayanagi et al.2010; Weaver, Nguyen and Brooks 2010; Saito et al.2012; Noguchi et al.2015; Yost et al.2015). Streptococcus intermedius produces a human-specific cytotoxin intermedilysin (ILY) of the cholesterol-dependent cytolysin (CDC) family, encoded by the ily gene (Nagamune et al.1996). ILY is believed to be a crucial virulence factor for infectivity and toxicity to human cells because knockout mutations of ily or inactivation of ILY using an anti-ILY antibody result in greatly decreased cytotoxicity (Sukeno et al.2005). In contrast to other CDC family members, ILY does not use cholesterol as a primary binding receptor and can specifically recognize a glycosylphosphatidylinositol-linked human cell membrane protein, CD59 (Giddings et al.2004). Therefore, S. intermedius is considered to be a strictly human-specific pathogen. Two transcriptional repressors that can regulate ily expression have been reported thus far: catabolite control protein (CcpA) and lactose phosphotransferase system repressor (LacR) (Tomoyasu et al.2010, 2013). LacR can repress transcription of the ily operon by binding to the ily promoter (Tomoyasu et al.2013). Disruption of lacR in S. intermedius has been shown to cause constitutive overproduction of ILY and increased toxicity to the human hepatoma cell line HepG2 (Tomoyasu et al.2013). Because a loss-of-function mutation in LacR is observed in almost all ILY high-producing strains isolated from deep-seated abscesses, high production of ILY seems to be necessary for increased virulence of this bacterium (Tomoyasu et al.2013). Streptococcus intermedius produces glycosidases such as MsgA and NanA (Takao, Nagamune and Maeda 2010; Imaki et al.2014). MsgA has four glycosidase activities (β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase) (Imaki et al.2014) that are stably conserved across all S. intermedius strains reported to date (Jensen, Hoshino and Kilian 2013; Imaki et al.2014). Because msgA is localized in the lac operon, its expression, like that of ily, is regulated by LacR (Imaki et al.2014). In addition, S. intermedius is the only species among the anginosus group of streptococci reported to exhibit neuraminidase activity. This glycosidase activity (NanA) is stably conserved in all S. intermedius strains (Jensen, Hoshino and Kilian 2013). Because of the correlation between higher production of ILY and increased virulence of S. intermedius, an accurate and rapid method for detecting ILY high-producing strains is required. However, since human-specific ILY does not produce β-hemolysis on animal blood agar plates (Nagamune et al.1996), the detection of ILY high-producing strains and differentiation from ILY low-producing strains by conventional laboratory testing is not routinely possible. Therefore, we developed a rapid and simple method for discriminating these strain types by calculating the ratio between N-acetyl-β-d-glucosaminidase (MsgA) and neuraminidase (NanA) activities. MATERIALS AND METHODS Bacterial strains and growth conditions The bacterial strains and the positions of amino acid substitutions in LacR are listed in Table 1 (Takao, Nagamune and Maeda 2004; Tomoyasu et al.2013). Streptococcus intermedius was cultured at 37°C under anaerobic conditions (N2:H2:CO2 = 85:10:5). Brain-heart infusion (BHI) broth (Becton-Dickinson, Palo Alto, CA, USA) and 3-(N-morpholino)propanesulfonic acid-buffered BHI (MOPS-BHI) broth were used as the culture media (Tomoyasu et al.2010). Table 1. Streptococcus intermedius strains used for this study. Strains  Isolation source  Mutation  UNS38  Brain abscess  V21D  UNS46  Liver abscess  L48F  A4676a  Brain abscess  R37L  JICC 33616  Brain abscess  42Q_44Ldup  JICC 40138–2  Infective endocarditis  42Q_44Ldup  UNS32  Liver abscess  S117I  UNS45  Liver abscess  V30A  UNS35  Brain abscess  R50W  HW7  Brain abscess  S117N  JICC 32122  Brain abscess  –a  UNS40  Liver abscess  –  JICC 1063  Liver abscess  V30A  TK-I2  Unknown  R143Cb  NMH2  Brain abscess  V21D  PC7466  Dental plaque  –  DP101  Dental abscess  –  JICC 32135  Empyema, mediastinitis  –  F600  Abdominal abscess  –  HW13  Abdominal Umbilical  –  DP102  Dental plaque  –  P58  Gingivitis  –  TK-I15  Unknown  –  TK-I19  Unknown  –  AC4720  Dental plaque  –  CDC415/87  Brain abscess  –  GN472  Dental plaque  –  TU-C11  Dental abscess  –  2Q  Brain abscess  –  P88  Gingivitis  –  JICC 33405  Empyema, mediastinitis  –  JICC 689  Infective endocarditis  –  HW58  Brain abscess  –  TK-I16  Unknown  –  JICC 32132  Brain abscess  –  JICC 32100  Septicemia  –  NMH8  Unknown  –  P101  Gingivitis  –  JICC 53299  Suppurative arthritis  –  WS100s  Bite wound, hand  –  AC5803  Dental plaque  –  TK-I20  Unknown  –  PC574  Dental plaque  –  P22  Gingivitis  –  JICC 33404  Pelvic abscess  –  NCDO2227  Type strain  –  JICC 33620  Brain abscess  –  TU-C41  Dental abscess  –  JICC 674  Septicemia  –  JICC 32138  Mediastinitis  –  AC800  Dental plaque  –  JICC 32151  Empyema, mediastinitis  –  JICC 33412  Subcutaneous abscess  –  P68  Gingivitis  –  F458s  Abdominal mass  –  UNS42  Liver abscess  –  HW69  Brain abscess  –  TU-C43  Dental abscess  –  E691  Eye  –  JICC 33494  Brain abscess  –  JICC 33425  Subcutaneous abscess  –  TU-C46  Dental abscess  –  IW-I2  Unknown  –  TU-C45  Dental abscess  –  IW-I1  Unknown  –  Strains  Isolation source  Mutation  UNS38  Brain abscess  V21D  UNS46  Liver abscess  L48F  A4676a  Brain abscess  R37L  JICC 33616  Brain abscess  42Q_44Ldup  JICC 40138–2  Infective endocarditis  42Q_44Ldup  UNS32  Liver abscess  S117I  UNS45  Liver abscess  V30A  UNS35  Brain abscess  R50W  HW7  Brain abscess  S117N  JICC 32122  Brain abscess  –a  UNS40  Liver abscess  –  JICC 1063  Liver abscess  V30A  TK-I2  Unknown  R143Cb  NMH2  Brain abscess  V21D  PC7466  Dental plaque  –  DP101  Dental abscess  –  JICC 32135  Empyema, mediastinitis  –  F600  Abdominal abscess  –  HW13  Abdominal Umbilical  –  DP102  Dental plaque  –  P58  Gingivitis  –  TK-I15  Unknown  –  TK-I19  Unknown  –  AC4720  Dental plaque  –  CDC415/87  Brain abscess  –  GN472  Dental plaque  –  TU-C11  Dental abscess  –  2Q  Brain abscess  –  P88  Gingivitis  –  JICC 33405  Empyema, mediastinitis  –  JICC 689  Infective endocarditis  –  HW58  Brain abscess  –  TK-I16  Unknown  –  JICC 32132  Brain abscess  –  JICC 32100  Septicemia  –  NMH8  Unknown  –  P101  Gingivitis  –  JICC 53299  Suppurative arthritis  –  WS100s  Bite wound, hand  –  AC5803  Dental plaque  –  TK-I20  Unknown  –  PC574  Dental plaque  –  P22  Gingivitis  –  JICC 33404  Pelvic abscess  –  NCDO2227  Type strain  –  JICC 33620  Brain abscess  –  TU-C41  Dental abscess  –  JICC 674  Septicemia  –  JICC 32138  Mediastinitis  –  AC800  Dental plaque  –  JICC 32151  Empyema, mediastinitis  –  JICC 33412  Subcutaneous abscess  –  P68  Gingivitis  –  F458s  Abdominal mass  –  UNS42  Liver abscess  –  HW69  Brain abscess  –  TU-C43  Dental abscess  –  E691  Eye  –  JICC 33494  Brain abscess  –  JICC 33425  Subcutaneous abscess  –  TU-C46  Dental abscess  –  IW-I2  Unknown  –  TU-C45  Dental abscess  –  IW-I1  Unknown  –  a –: No loss-of-function mutation in LacR. b Function of LacR R143C was not determined. View Large Nucleotide sequences of lacR from Streptococcus intermedius clinical isolates Fragments of the lacR gene from strains TK-I2, TK-I15, TK-I19, TK-I16, TK-I20, TU-C11, TU-C41, TU-C43, TU-C45, TU-C46, IW-I1 and IW-I2 were amplified by PCR and sequenced as previously described (Tomoyasu et al.2013). Measurement of N-acetyl-β-d-glucosaminidase and neuraminidase activities Streptococcus intermedius strains were pre-cultured in 10 mL BHI broth at 37°C for 24 h. Subsequently, 100 μL of pre-culture was inoculated in 10 mL MOPS–BHI broth and cultured at 37°C for 24 h under anaerobic conditions. The optical density at 600 nm (OD600) of the bacterial culture was measured for the hemolysis assay, and then the culture supernatant and the cells were separated by centrifugation. The culture supernatant was used for the hemolysis assay and the cell pellet was used for the measurement of glycosidase activities as described below. The cell pellet was suspended in 20 mM Tris-HCl buffer (pH 7.5) to OD600 = 1.0. Cell-associated glycosidase activities were determined using chromogenic substrates. Assays were carried out in 96-well plates with a final volume of 100 μL/well. N-Acetyl-β-d-glucosaminidase activity (MsgA activity) was determined using 10 μL of cell suspension in 70 mM sodium citrate buffer (pH 5.5) containing 500 μM 4-nitrophenyl N-acetyl-β-d-glucosaminide. Neuraminidase activity (NanA activity) was determined using 50 or 15 μL of cell suspension in 70 mM sodium citrate buffer (pH 5.5) containing 200 μM 2-O-(p-Nitrophenyl)-α-d-N-acetylneuraminic acid. The 96-well plate was incubated in a PST-100HL plate shaker thermostat (Biosan, Riga, Latvia) at 50°C for 1 h with shaking at 1000 rpm. After the reaction, 100 μL of 0.5 M sodium carbonate buffer (pH 10.2) was added to terminate the enzymatic reaction and development of the color. Subsequently, the A405 of each well was measured in a Multiskan FC microplate photometer (Thermo Fisher Scientific Inc., MA, USA). MsgA and NanA activities relative to these activities observed with PC574 as a reference strain were calculated as follows: Relative activity = (A405 of the sample after incubation—A405 of the sample without incubation)/(A405 of PC574 after incubation—A405 of PC574 without incubation). The MsgA/NanA (M/N) ratio was calculated as follows: M/N ratio = Relative activity of MsgA/Relative activity of NanA. Hemolysis assay Hemolysis was assayed as described previously (Tomoyasu et al.2017). Relative hemolytic activity = (dilution rate of culture supernatant sample giving 50% of hemolysis/dilution rate of culture supernatant of reference strain UNS38 giving 50% of hemolysis) × 10. Statistics All experiments were performed in triplicate. Results were expressed as means ± standard deviations. Correlation between glycosidase activities or M/N ratios and hemolytic activities was evaluated by Pearson's product–moment correlation coefficient. RESULTS AND DISCUSSION Evidence has shown ILY expression to be a major virulence factor in Streptococcus intermedius. Furthermore, the majority of highly pathogenic S. intermedius strains isolated from deep-seated abscesses have a loss-of-function mutation in LacR that causes higher production of ILY than in the less pathogenic strains most frequently isolated from normal sites (Nagamune et al.2000; Tomoyasu et al.2017). Therefore, it is important to develop a rapid detection method for the presence of ILY high-producing strains. Streptococcus intermedius strains can be identified at the species level by rapid PCR methods that amplify ily or a ribosomal RNA coding region (Nagamune et al.2000; Takao, Nagamune and Maeda 2004). Whereas ILY acts specifically on human cells, S. intermedius does not exhibit β-hemolysis on animal blood agar (Nagamune et al.1996). Therefore, it is impossible to discriminate ILY high-producing strains from low-producing strains of this species by routine laboratory blood agar culture. Furthermore, the strategy of using nucleotide sequencing of lacR of all isolates to check for amino acid substitutions is not realistic, as this is costly and time consuming. In addition, amino acid substitutions alone cannot be used to estimate LacR activity. For example, a mutation from cysteine to tyrosine at position 135 (C135Y) in LacR is a neutral mutation (Tomoyasu et al.2013) and has been observed in 17/64 strains including both ILY low- and high-producing strains used in this study (data not shown). MsgA activity is increased by disruption of lacR because msgA localizes in the lac operon repressed by LacR (Imaki et al.2014). Therefore, we examined the relationship between MsgA activity and ILY mediated hemolytic activity within strains. MsgA and hemolytic activities were assayed in 56 strains isolated from clinical specimens (11 of which had a loss-of-function mutation in LacR), 7 strains isolated from dental plaque and the type strain (NCDO2227) (Table 1 and Table S1, Supporting Information). MsgA has two catalytic domains (LacZ and CH20); the LacZ domain confers β-d-galactosidase activity and the CH20 domain confers N-acetyl-β-d-glucosaminidase activity (Imaki et al.2014). Because N-acetyl-β-d-glucosaminidase activity had a higher kcat/Km value and higher thermostability than β-d-galactosidase (Imaki et al.2014), we measured N-acetyl-β-d-glucosaminidase activity as an index of MsgA activity at 50°C. The results obtained demonstrate that MsgA activities of strains showed a strong positive correlation with hemolytic activities (Pearson's product-moment correlation coefficient; r = 0.73) (Fig. 1a). Exceptions were only observed with three ILY high-producing strains (NMH2, UNS32 and UNS45) that had a loss-of-function mutation in LacR but showed lower MsgA activity than some ILY low-producing strains (Table S1). We determined hemolytic activity of the strains that showed more than 10% of the activity of UNS38 and thereby were classified as ILY high-producing strains. The majority of ILY low-producing strains have only weak hemolytic activity, these activity levels were sufficiently low to preclude accurate determinations and these were therefore scored as zero. Figure 1. View largeDownload slide (a) Correlation between MsgA activity and hemolytic activity. Vertical axis shows relative MsgA activity (PC574 set as 1) and horizontal axis represents relative hemolytic activity (UNS38 set as 10). (b) Correlation between NanA activity and hemolytic activity. Vertical axis shows relative NanA activity (PC574 set as 1) and horizontal axis represents relative hemolytic activity. Relative activities of each strain are represented as open circles. Relative hemolytic activity of ILY-low producing strains (less than 10% activity of UNS38) is plotted as 0 on the horizontal axis. Error bars show standard deviations from three independent experiments. Figure 1. View largeDownload slide (a) Correlation between MsgA activity and hemolytic activity. Vertical axis shows relative MsgA activity (PC574 set as 1) and horizontal axis represents relative hemolytic activity (UNS38 set as 10). (b) Correlation between NanA activity and hemolytic activity. Vertical axis shows relative NanA activity (PC574 set as 1) and horizontal axis represents relative hemolytic activity. Relative activities of each strain are represented as open circles. Relative hemolytic activity of ILY-low producing strains (less than 10% activity of UNS38) is plotted as 0 on the horizontal axis. Error bars show standard deviations from three independent experiments. Previously, we observed ∼50% reduction in NanA activity for a strain with disrupted lacR compared to a wild-type strain, although the reason for this is still unknown (Imaki et al.2014). Therefore, we examined the correlation between NanA activity and hemolytic activity (Fig. 1b; Table S2, Supporting Information). Because the optimal temperature for NanA activity is 52°C (unpublished data), this activity could be measured under the same conditions as for determining MsgA activity. A weak negative correlation (r = –0.28) was observed between NanA and hemolytic activities for all strains tested and with an increased negative correlation (r = –0.54) observed for the 16 ILY high-producing strains tested. Because MsgA and NanA activities showed positive and negative correlations with hemolytic activity respectively, we calculated the ratio between relative activity of MsgA and NanA (M/N ratio) (Fig. 2). The M/N ratio was strongly correlated with hemolytic activity (r = 0.81). Among 14 strains with a high M/N ratio (>2), 12 strains were high producers of ILY and 11 strains had loss-of-function mutations in LacR. No strain that had a loss-of-function mutations in LacR had an M/N ratio <2. ILY high-producing strain TK-I2 had a R143C mutation in LacR. Because arginine residue 143 of S. intermedius LacR is well conserved among the LacR homologs of streptococci (data not shown), this mutation seems to cause reduction in, or inactivation of, LacR function. On the other hand, in strain HW7 a loss-of-function mutation in LacR did not lead to overproduction of ILY (Tomoyasu et al.2013). Figure 2. View largeDownload slide Correlation between M/N ratio and hemolytic activity. All cells were grown in MOPS–BHI broth for 24 h at 37°C. Left vertical axis shows M/N ratio (PC574 set as 1), right vertical axis shows relative hemolytic activity (UNS38 set as 10) and strain name is shown on the horizontal axis. Black bars show M/N ratio of strains that have a loss-of-function mutation in LacR. Gray bar shows M/N ratio of strain TK-I2, which has an uncharacterized mutation in LacR. White bars show M/N ratio of strains with wild-type LacR. The hemolytic activity of each strain is shown with red circles. The hemolytic activity of the strains without red circle is <1. Figure 2. View largeDownload slide Correlation between M/N ratio and hemolytic activity. All cells were grown in MOPS–BHI broth for 24 h at 37°C. Left vertical axis shows M/N ratio (PC574 set as 1), right vertical axis shows relative hemolytic activity (UNS38 set as 10) and strain name is shown on the horizontal axis. Black bars show M/N ratio of strains that have a loss-of-function mutation in LacR. Gray bar shows M/N ratio of strain TK-I2, which has an uncharacterized mutation in LacR. White bars show M/N ratio of strains with wild-type LacR. The hemolytic activity of each strain is shown with red circles. The hemolytic activity of the strains without red circle is <1. Based on these results, we conclude that determining the M/N ratio is an effective index for discriminating between potentially clinically significant, highly toxigenic (ILY high-producing) strains with lacR mutations and ILY low-producing strains. Interestingly, some exceptions were observed: both ILY high-producing strain UNS40 and ILY low-producing strain JICC32122 had wild-type LacR but showed higher M/N ratios. Strains F600, JICC33405, JICC674 and UNS42 exhibited ILY high-producing phenotypes but had wild-type LacR and lower M/N ratios (<2). These data suggest that ily expression in these strains is activated by a mutation other than in the LacR gene. Further elucidation of the mechanisms underlying ILY expression levels in S. intermedius will contribute to a greater understanding of the variation in virulence observed for strains of this human pathogen. It has been shown that S. intermedius is more virulent in patients immunocompromised due to aging, diabetes, cirrhosis and cancer than in immunocompetent individuals (Murray et al.1978; Jacobs et al.1995; Jerng et al.1997; Bert, Bariou-Lancelin and Lambert-Zechovsky 1998; Takayanagi et al.2010; Noguchi et al.2015). It is thought that this is especially so for ILY high-producing strains that would pose a higher risk to immunocompromised individuals (Tomoyasu et al.2017). Therefore, an increasing number of serious infections with ILY high-producing strains can be anticipated in populations with increasing proportions of elderly people. Our screening method adopting the ratio of two glycosidase activities can be carried out in virtually identical test formats making testing simpler and therefore less prone to experimental error. This screening method will be useful for detecting the existence of ILY high-producing, potentially pathogenic strains of this increasingly recognized clinically important species in the normal flora and at sites of focal infections. SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. Acknowledgements We thank Miyako Ishizu for experimental assistance. FUNDING This work was supported by KAKENHI (Grants-in-Aid for Scientific Research (C) 26460528) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of the Japanese Government. Conflict of interest. None declared. REFERENCES Bert F , Bariou-Lancelin M, Lambert-Zechovsky N. Clinical significance of bacteremia involving the “Streptococcus milleri” group: 51 cases and review. Clin Infect Dis  1998; 27: 385– 37. Google Scholar CrossRef Search ADS PubMed  Giddings KS , Zhao J, Sims PJet al.   Human CD59 is a receptor for the cholesterol-dependent cytolysin intermedilysin. 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Rapid screening method for detecting highly pathogenic Streptococcus intermedius strains carrying a mutation in the lacR gene

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Abstract

Abstract Streptococcus intermedius is a member of the normal human commensal flora and secretes a human-specific cytolysin intermedilysin (ILY) as a major virulence factor. Expression of ily is repressed by LacR and loss-of-function mutations of LacR are observed in many ILY high-producing strains isolated from deep-seated abscesses, suggesting that high ILY production is necessary for increased virulence. However, because ILY exhibits no β-hemolysis on animal blood agar plates, differentiating ILY high- and low-producing strains using conventional laboratory methods is not possible. Interestingly, S. intermedius also produces glycosidases, including MsgA and NanA, which exhibit N-acetyl-β-d-glucosaminidase and neuraminidase activities, respectively. Moreover, MsgA expression, but not NanA, is negatively regulated by LacR. Here we measured the activities of MsgA, NanA and ILY in strains isolated from clinical specimens and dental plaque to determine the correlation between these glycosidase activities and ILY hemolytic activity. Hemolytic activity showed a strong positive correlation with MsgA and a weak negative correlation with NanA activities. Therefore, we calculated the ratio of MsgA and NanA activity (M/N ratio). This value showed a stronger positive correlation (r = 0.81) with ILY hemolytic activity and many strains with high M/N ratios (>2) were ILY-high producers with loss-of-function mutations in LacR. Streptococcus intermedius, intermedilysin, screening, MsgA, NanA, LacR INTRODUCTION Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that belongs to the anginosus group of streptococci (Whiley et al.1990; Jensen, Hoshino and Kilian 2013). This pathogen is associated with oral infections, including periodontal disease and recurrent tonsillitis, and with deep-seated purulent infections such as brain and lung abscesses (Whiley et al.1992; Jerng et al.1997; Tanner et al.1997; Gray 2005; Takayanagi et al.2010; Weaver, Nguyen and Brooks 2010; Saito et al.2012; Noguchi et al.2015; Yost et al.2015). Streptococcus intermedius produces a human-specific cytotoxin intermedilysin (ILY) of the cholesterol-dependent cytolysin (CDC) family, encoded by the ily gene (Nagamune et al.1996). ILY is believed to be a crucial virulence factor for infectivity and toxicity to human cells because knockout mutations of ily or inactivation of ILY using an anti-ILY antibody result in greatly decreased cytotoxicity (Sukeno et al.2005). In contrast to other CDC family members, ILY does not use cholesterol as a primary binding receptor and can specifically recognize a glycosylphosphatidylinositol-linked human cell membrane protein, CD59 (Giddings et al.2004). Therefore, S. intermedius is considered to be a strictly human-specific pathogen. Two transcriptional repressors that can regulate ily expression have been reported thus far: catabolite control protein (CcpA) and lactose phosphotransferase system repressor (LacR) (Tomoyasu et al.2010, 2013). LacR can repress transcription of the ily operon by binding to the ily promoter (Tomoyasu et al.2013). Disruption of lacR in S. intermedius has been shown to cause constitutive overproduction of ILY and increased toxicity to the human hepatoma cell line HepG2 (Tomoyasu et al.2013). Because a loss-of-function mutation in LacR is observed in almost all ILY high-producing strains isolated from deep-seated abscesses, high production of ILY seems to be necessary for increased virulence of this bacterium (Tomoyasu et al.2013). Streptococcus intermedius produces glycosidases such as MsgA and NanA (Takao, Nagamune and Maeda 2010; Imaki et al.2014). MsgA has four glycosidase activities (β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase) (Imaki et al.2014) that are stably conserved across all S. intermedius strains reported to date (Jensen, Hoshino and Kilian 2013; Imaki et al.2014). Because msgA is localized in the lac operon, its expression, like that of ily, is regulated by LacR (Imaki et al.2014). In addition, S. intermedius is the only species among the anginosus group of streptococci reported to exhibit neuraminidase activity. This glycosidase activity (NanA) is stably conserved in all S. intermedius strains (Jensen, Hoshino and Kilian 2013). Because of the correlation between higher production of ILY and increased virulence of S. intermedius, an accurate and rapid method for detecting ILY high-producing strains is required. However, since human-specific ILY does not produce β-hemolysis on animal blood agar plates (Nagamune et al.1996), the detection of ILY high-producing strains and differentiation from ILY low-producing strains by conventional laboratory testing is not routinely possible. Therefore, we developed a rapid and simple method for discriminating these strain types by calculating the ratio between N-acetyl-β-d-glucosaminidase (MsgA) and neuraminidase (NanA) activities. MATERIALS AND METHODS Bacterial strains and growth conditions The bacterial strains and the positions of amino acid substitutions in LacR are listed in Table 1 (Takao, Nagamune and Maeda 2004; Tomoyasu et al.2013). Streptococcus intermedius was cultured at 37°C under anaerobic conditions (N2:H2:CO2 = 85:10:5). Brain-heart infusion (BHI) broth (Becton-Dickinson, Palo Alto, CA, USA) and 3-(N-morpholino)propanesulfonic acid-buffered BHI (MOPS-BHI) broth were used as the culture media (Tomoyasu et al.2010). Table 1. Streptococcus intermedius strains used for this study. Strains  Isolation source  Mutation  UNS38  Brain abscess  V21D  UNS46  Liver abscess  L48F  A4676a  Brain abscess  R37L  JICC 33616  Brain abscess  42Q_44Ldup  JICC 40138–2  Infective endocarditis  42Q_44Ldup  UNS32  Liver abscess  S117I  UNS45  Liver abscess  V30A  UNS35  Brain abscess  R50W  HW7  Brain abscess  S117N  JICC 32122  Brain abscess  –a  UNS40  Liver abscess  –  JICC 1063  Liver abscess  V30A  TK-I2  Unknown  R143Cb  NMH2  Brain abscess  V21D  PC7466  Dental plaque  –  DP101  Dental abscess  –  JICC 32135  Empyema, mediastinitis  –  F600  Abdominal abscess  –  HW13  Abdominal Umbilical  –  DP102  Dental plaque  –  P58  Gingivitis  –  TK-I15  Unknown  –  TK-I19  Unknown  –  AC4720  Dental plaque  –  CDC415/87  Brain abscess  –  GN472  Dental plaque  –  TU-C11  Dental abscess  –  2Q  Brain abscess  –  P88  Gingivitis  –  JICC 33405  Empyema, mediastinitis  –  JICC 689  Infective endocarditis  –  HW58  Brain abscess  –  TK-I16  Unknown  –  JICC 32132  Brain abscess  –  JICC 32100  Septicemia  –  NMH8  Unknown  –  P101  Gingivitis  –  JICC 53299  Suppurative arthritis  –  WS100s  Bite wound, hand  –  AC5803  Dental plaque  –  TK-I20  Unknown  –  PC574  Dental plaque  –  P22  Gingivitis  –  JICC 33404  Pelvic abscess  –  NCDO2227  Type strain  –  JICC 33620  Brain abscess  –  TU-C41  Dental abscess  –  JICC 674  Septicemia  –  JICC 32138  Mediastinitis  –  AC800  Dental plaque  –  JICC 32151  Empyema, mediastinitis  –  JICC 33412  Subcutaneous abscess  –  P68  Gingivitis  –  F458s  Abdominal mass  –  UNS42  Liver abscess  –  HW69  Brain abscess  –  TU-C43  Dental abscess  –  E691  Eye  –  JICC 33494  Brain abscess  –  JICC 33425  Subcutaneous abscess  –  TU-C46  Dental abscess  –  IW-I2  Unknown  –  TU-C45  Dental abscess  –  IW-I1  Unknown  –  Strains  Isolation source  Mutation  UNS38  Brain abscess  V21D  UNS46  Liver abscess  L48F  A4676a  Brain abscess  R37L  JICC 33616  Brain abscess  42Q_44Ldup  JICC 40138–2  Infective endocarditis  42Q_44Ldup  UNS32  Liver abscess  S117I  UNS45  Liver abscess  V30A  UNS35  Brain abscess  R50W  HW7  Brain abscess  S117N  JICC 32122  Brain abscess  –a  UNS40  Liver abscess  –  JICC 1063  Liver abscess  V30A  TK-I2  Unknown  R143Cb  NMH2  Brain abscess  V21D  PC7466  Dental plaque  –  DP101  Dental abscess  –  JICC 32135  Empyema, mediastinitis  –  F600  Abdominal abscess  –  HW13  Abdominal Umbilical  –  DP102  Dental plaque  –  P58  Gingivitis  –  TK-I15  Unknown  –  TK-I19  Unknown  –  AC4720  Dental plaque  –  CDC415/87  Brain abscess  –  GN472  Dental plaque  –  TU-C11  Dental abscess  –  2Q  Brain abscess  –  P88  Gingivitis  –  JICC 33405  Empyema, mediastinitis  –  JICC 689  Infective endocarditis  –  HW58  Brain abscess  –  TK-I16  Unknown  –  JICC 32132  Brain abscess  –  JICC 32100  Septicemia  –  NMH8  Unknown  –  P101  Gingivitis  –  JICC 53299  Suppurative arthritis  –  WS100s  Bite wound, hand  –  AC5803  Dental plaque  –  TK-I20  Unknown  –  PC574  Dental plaque  –  P22  Gingivitis  –  JICC 33404  Pelvic abscess  –  NCDO2227  Type strain  –  JICC 33620  Brain abscess  –  TU-C41  Dental abscess  –  JICC 674  Septicemia  –  JICC 32138  Mediastinitis  –  AC800  Dental plaque  –  JICC 32151  Empyema, mediastinitis  –  JICC 33412  Subcutaneous abscess  –  P68  Gingivitis  –  F458s  Abdominal mass  –  UNS42  Liver abscess  –  HW69  Brain abscess  –  TU-C43  Dental abscess  –  E691  Eye  –  JICC 33494  Brain abscess  –  JICC 33425  Subcutaneous abscess  –  TU-C46  Dental abscess  –  IW-I2  Unknown  –  TU-C45  Dental abscess  –  IW-I1  Unknown  –  a –: No loss-of-function mutation in LacR. b Function of LacR R143C was not determined. View Large Nucleotide sequences of lacR from Streptococcus intermedius clinical isolates Fragments of the lacR gene from strains TK-I2, TK-I15, TK-I19, TK-I16, TK-I20, TU-C11, TU-C41, TU-C43, TU-C45, TU-C46, IW-I1 and IW-I2 were amplified by PCR and sequenced as previously described (Tomoyasu et al.2013). Measurement of N-acetyl-β-d-glucosaminidase and neuraminidase activities Streptococcus intermedius strains were pre-cultured in 10 mL BHI broth at 37°C for 24 h. Subsequently, 100 μL of pre-culture was inoculated in 10 mL MOPS–BHI broth and cultured at 37°C for 24 h under anaerobic conditions. The optical density at 600 nm (OD600) of the bacterial culture was measured for the hemolysis assay, and then the culture supernatant and the cells were separated by centrifugation. The culture supernatant was used for the hemolysis assay and the cell pellet was used for the measurement of glycosidase activities as described below. The cell pellet was suspended in 20 mM Tris-HCl buffer (pH 7.5) to OD600 = 1.0. Cell-associated glycosidase activities were determined using chromogenic substrates. Assays were carried out in 96-well plates with a final volume of 100 μL/well. N-Acetyl-β-d-glucosaminidase activity (MsgA activity) was determined using 10 μL of cell suspension in 70 mM sodium citrate buffer (pH 5.5) containing 500 μM 4-nitrophenyl N-acetyl-β-d-glucosaminide. Neuraminidase activity (NanA activity) was determined using 50 or 15 μL of cell suspension in 70 mM sodium citrate buffer (pH 5.5) containing 200 μM 2-O-(p-Nitrophenyl)-α-d-N-acetylneuraminic acid. The 96-well plate was incubated in a PST-100HL plate shaker thermostat (Biosan, Riga, Latvia) at 50°C for 1 h with shaking at 1000 rpm. After the reaction, 100 μL of 0.5 M sodium carbonate buffer (pH 10.2) was added to terminate the enzymatic reaction and development of the color. Subsequently, the A405 of each well was measured in a Multiskan FC microplate photometer (Thermo Fisher Scientific Inc., MA, USA). MsgA and NanA activities relative to these activities observed with PC574 as a reference strain were calculated as follows: Relative activity = (A405 of the sample after incubation—A405 of the sample without incubation)/(A405 of PC574 after incubation—A405 of PC574 without incubation). The MsgA/NanA (M/N) ratio was calculated as follows: M/N ratio = Relative activity of MsgA/Relative activity of NanA. Hemolysis assay Hemolysis was assayed as described previously (Tomoyasu et al.2017). Relative hemolytic activity = (dilution rate of culture supernatant sample giving 50% of hemolysis/dilution rate of culture supernatant of reference strain UNS38 giving 50% of hemolysis) × 10. Statistics All experiments were performed in triplicate. Results were expressed as means ± standard deviations. Correlation between glycosidase activities or M/N ratios and hemolytic activities was evaluated by Pearson's product–moment correlation coefficient. RESULTS AND DISCUSSION Evidence has shown ILY expression to be a major virulence factor in Streptococcus intermedius. Furthermore, the majority of highly pathogenic S. intermedius strains isolated from deep-seated abscesses have a loss-of-function mutation in LacR that causes higher production of ILY than in the less pathogenic strains most frequently isolated from normal sites (Nagamune et al.2000; Tomoyasu et al.2017). Therefore, it is important to develop a rapid detection method for the presence of ILY high-producing strains. Streptococcus intermedius strains can be identified at the species level by rapid PCR methods that amplify ily or a ribosomal RNA coding region (Nagamune et al.2000; Takao, Nagamune and Maeda 2004). Whereas ILY acts specifically on human cells, S. intermedius does not exhibit β-hemolysis on animal blood agar (Nagamune et al.1996). Therefore, it is impossible to discriminate ILY high-producing strains from low-producing strains of this species by routine laboratory blood agar culture. Furthermore, the strategy of using nucleotide sequencing of lacR of all isolates to check for amino acid substitutions is not realistic, as this is costly and time consuming. In addition, amino acid substitutions alone cannot be used to estimate LacR activity. For example, a mutation from cysteine to tyrosine at position 135 (C135Y) in LacR is a neutral mutation (Tomoyasu et al.2013) and has been observed in 17/64 strains including both ILY low- and high-producing strains used in this study (data not shown). MsgA activity is increased by disruption of lacR because msgA localizes in the lac operon repressed by LacR (Imaki et al.2014). Therefore, we examined the relationship between MsgA activity and ILY mediated hemolytic activity within strains. MsgA and hemolytic activities were assayed in 56 strains isolated from clinical specimens (11 of which had a loss-of-function mutation in LacR), 7 strains isolated from dental plaque and the type strain (NCDO2227) (Table 1 and Table S1, Supporting Information). MsgA has two catalytic domains (LacZ and CH20); the LacZ domain confers β-d-galactosidase activity and the CH20 domain confers N-acetyl-β-d-glucosaminidase activity (Imaki et al.2014). Because N-acetyl-β-d-glucosaminidase activity had a higher kcat/Km value and higher thermostability than β-d-galactosidase (Imaki et al.2014), we measured N-acetyl-β-d-glucosaminidase activity as an index of MsgA activity at 50°C. The results obtained demonstrate that MsgA activities of strains showed a strong positive correlation with hemolytic activities (Pearson's product-moment correlation coefficient; r = 0.73) (Fig. 1a). Exceptions were only observed with three ILY high-producing strains (NMH2, UNS32 and UNS45) that had a loss-of-function mutation in LacR but showed lower MsgA activity than some ILY low-producing strains (Table S1). We determined hemolytic activity of the strains that showed more than 10% of the activity of UNS38 and thereby were classified as ILY high-producing strains. The majority of ILY low-producing strains have only weak hemolytic activity, these activity levels were sufficiently low to preclude accurate determinations and these were therefore scored as zero. Figure 1. View largeDownload slide (a) Correlation between MsgA activity and hemolytic activity. Vertical axis shows relative MsgA activity (PC574 set as 1) and horizontal axis represents relative hemolytic activity (UNS38 set as 10). (b) Correlation between NanA activity and hemolytic activity. Vertical axis shows relative NanA activity (PC574 set as 1) and horizontal axis represents relative hemolytic activity. Relative activities of each strain are represented as open circles. Relative hemolytic activity of ILY-low producing strains (less than 10% activity of UNS38) is plotted as 0 on the horizontal axis. Error bars show standard deviations from three independent experiments. Figure 1. View largeDownload slide (a) Correlation between MsgA activity and hemolytic activity. Vertical axis shows relative MsgA activity (PC574 set as 1) and horizontal axis represents relative hemolytic activity (UNS38 set as 10). (b) Correlation between NanA activity and hemolytic activity. Vertical axis shows relative NanA activity (PC574 set as 1) and horizontal axis represents relative hemolytic activity. Relative activities of each strain are represented as open circles. Relative hemolytic activity of ILY-low producing strains (less than 10% activity of UNS38) is plotted as 0 on the horizontal axis. Error bars show standard deviations from three independent experiments. Previously, we observed ∼50% reduction in NanA activity for a strain with disrupted lacR compared to a wild-type strain, although the reason for this is still unknown (Imaki et al.2014). Therefore, we examined the correlation between NanA activity and hemolytic activity (Fig. 1b; Table S2, Supporting Information). Because the optimal temperature for NanA activity is 52°C (unpublished data), this activity could be measured under the same conditions as for determining MsgA activity. A weak negative correlation (r = –0.28) was observed between NanA and hemolytic activities for all strains tested and with an increased negative correlation (r = –0.54) observed for the 16 ILY high-producing strains tested. Because MsgA and NanA activities showed positive and negative correlations with hemolytic activity respectively, we calculated the ratio between relative activity of MsgA and NanA (M/N ratio) (Fig. 2). The M/N ratio was strongly correlated with hemolytic activity (r = 0.81). Among 14 strains with a high M/N ratio (>2), 12 strains were high producers of ILY and 11 strains had loss-of-function mutations in LacR. No strain that had a loss-of-function mutations in LacR had an M/N ratio <2. ILY high-producing strain TK-I2 had a R143C mutation in LacR. Because arginine residue 143 of S. intermedius LacR is well conserved among the LacR homologs of streptococci (data not shown), this mutation seems to cause reduction in, or inactivation of, LacR function. On the other hand, in strain HW7 a loss-of-function mutation in LacR did not lead to overproduction of ILY (Tomoyasu et al.2013). Figure 2. View largeDownload slide Correlation between M/N ratio and hemolytic activity. All cells were grown in MOPS–BHI broth for 24 h at 37°C. Left vertical axis shows M/N ratio (PC574 set as 1), right vertical axis shows relative hemolytic activity (UNS38 set as 10) and strain name is shown on the horizontal axis. Black bars show M/N ratio of strains that have a loss-of-function mutation in LacR. Gray bar shows M/N ratio of strain TK-I2, which has an uncharacterized mutation in LacR. White bars show M/N ratio of strains with wild-type LacR. The hemolytic activity of each strain is shown with red circles. The hemolytic activity of the strains without red circle is <1. Figure 2. View largeDownload slide Correlation between M/N ratio and hemolytic activity. All cells were grown in MOPS–BHI broth for 24 h at 37°C. Left vertical axis shows M/N ratio (PC574 set as 1), right vertical axis shows relative hemolytic activity (UNS38 set as 10) and strain name is shown on the horizontal axis. Black bars show M/N ratio of strains that have a loss-of-function mutation in LacR. Gray bar shows M/N ratio of strain TK-I2, which has an uncharacterized mutation in LacR. White bars show M/N ratio of strains with wild-type LacR. The hemolytic activity of each strain is shown with red circles. The hemolytic activity of the strains without red circle is <1. Based on these results, we conclude that determining the M/N ratio is an effective index for discriminating between potentially clinically significant, highly toxigenic (ILY high-producing) strains with lacR mutations and ILY low-producing strains. Interestingly, some exceptions were observed: both ILY high-producing strain UNS40 and ILY low-producing strain JICC32122 had wild-type LacR but showed higher M/N ratios. Strains F600, JICC33405, JICC674 and UNS42 exhibited ILY high-producing phenotypes but had wild-type LacR and lower M/N ratios (<2). These data suggest that ily expression in these strains is activated by a mutation other than in the LacR gene. Further elucidation of the mechanisms underlying ILY expression levels in S. intermedius will contribute to a greater understanding of the variation in virulence observed for strains of this human pathogen. It has been shown that S. intermedius is more virulent in patients immunocompromised due to aging, diabetes, cirrhosis and cancer than in immunocompetent individuals (Murray et al.1978; Jacobs et al.1995; Jerng et al.1997; Bert, Bariou-Lancelin and Lambert-Zechovsky 1998; Takayanagi et al.2010; Noguchi et al.2015). It is thought that this is especially so for ILY high-producing strains that would pose a higher risk to immunocompromised individuals (Tomoyasu et al.2017). Therefore, an increasing number of serious infections with ILY high-producing strains can be anticipated in populations with increasing proportions of elderly people. Our screening method adopting the ratio of two glycosidase activities can be carried out in virtually identical test formats making testing simpler and therefore less prone to experimental error. This screening method will be useful for detecting the existence of ILY high-producing, potentially pathogenic strains of this increasingly recognized clinically important species in the normal flora and at sites of focal infections. SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. Acknowledgements We thank Miyako Ishizu for experimental assistance. FUNDING This work was supported by KAKENHI (Grants-in-Aid for Scientific Research (C) 26460528) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of the Japanese Government. Conflict of interest. None declared. REFERENCES Bert F , Bariou-Lancelin M, Lambert-Zechovsky N. Clinical significance of bacteremia involving the “Streptococcus milleri” group: 51 cases and review. Clin Infect Dis  1998; 27: 385– 37. Google Scholar CrossRef Search ADS PubMed  Giddings KS , Zhao J, Sims PJet al.   Human CD59 is a receptor for the cholesterol-dependent cytolysin intermedilysin. 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FEMS Microbiology LettersOxford University Press

Published: Feb 1, 2018

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