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Four ST-7 N. meningitidis isolates were cultured on Columbia agar containing 5% sheep blood (BAP) (Thermo Fisher Oxoid, Beijing, China) and incubated at 37 °C in 5% CO2 for 24 h. Information related to the ST-7 isolates used in this study is provided in Table 1 and the Supplementary Table S1 (available in www.besjournal.com). All experiments were performed in a safety cabinet.
Table 1. N. meningitidis isolates used in the present study
Isolate No. Serogroup Isolate source Year of isolation Sequence type Clonal complex 510612 A Patient 2006 7 5 441212 B Patient 2012 7 5 630601 C Patient 2006 7 5 331401 X Patient 2014 7 5 Table S1. Detail information of strains used in this study
Bacteria No. Serogroup Isolates source Specimen type Year of isolated Isolate district Sequence type Clonal complex PorA FetA NadA_ variant NHBA_ peptide FHbp 510612 A Patient Throat swab 2006 Sichuan 7 5 P1.20,9 F3-1 NadA-2/3 126 1.5 441212 B Patient CSF 2012 Guangdong 7 5 P1.20,9 F3-1 NadA-2/3 126 1.5 630601 C Patient CSF 2006 Qinghai 7 5 P1.20,9 F3-1 NadA-2/3 126 1.5 331401 X Patient CSF 2014 Zhejiang 7 5 P1.20,9 F3-1 NadA-2/3 126 1.5 -
A549 cells (#TCHu-150) were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). A549 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco by Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (#FS201-02; Transgene Biotech, Beijing, China) and 1% penicillin-streptomycin solution (final concentration: penicillin, 100 units/mL; streptomycin, 100 μg/mL) (#10378016; Thermo Fisher Scientific, Shanghai, China). The cells were maintained in a humid atmosphere with 5% CO2 at 37 °C.
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A549 cells were seeded in 12-well cell culture plates at a density of 1 × 105 cells/well. The day before infection, the cells were starved by culturing them in DMEM without supplements. On the next day, a suspension of the overnight bacterial culture in DMEM was adjusted to an optical density of 0.200 at 600 nm. After 1 × 105 times dilution, 100 μL of the bacterial suspension was streaked on BAP and incubated at 37 °C in 5% CO2 for 24 h to calculate the initial number of bacteria infecting the host cells. Then, 1 mL of the bacterial suspension was added into each of the eleven wells and further incubated at 37 °C with 5% CO2. One well containing non-infected cells was used in each experiment as a control (CTR).
After 4 h of infection, unbound bacteria were removed by three rounds of washing with sterile phosphate-buffered saline (PBS). In each well, A549 cells with adherent bacteria were lysed with 1 mL of sterile double-distilled water (DDW) and scratched off the well bottom with a cell scraper. After 1 × 103 times dilution, 100 μL of the cell suspension was streaked on BAP and incubated at 37 °C in 5% CO2 for 24 h. Adhesion of N. meningitidis to the host cells was determined by counting the number of colony-forming units (CFUs). The adhesion rate was calculated as the number of adherent bacteria divided by the initial number of bacteria.
For the invasion experiment, after 4 h of infection, the cells were further incubated for two h after the addition of 100 μg/mL gentamicin in PBS to each well (L1312; Solarbio Life Sciences, Shanghai, China) to kill any remaining extracellular bacteria. The bacterial killing was confirmed by streaking 100 μL of the supernatants on BAP and incubation at 37 °C in 5% CO2 for 24 h. Cells were washed with sterile PBS to eliminate the remaining gentamicin, and then they were lysed with sterile DDW and removed with a cell scraper, as described earlier. After 1 × 102 times dilution, 100 μL of the cell suspension was streaked on BAP and incubated at 37 °C in 5% CO2 for 24 h. The number of intracellular cc4821 isolates was determined based on the number of CFUs. The invasion rate was calculated as the number of invasive bacteria divided by the number of adherent bacteria.
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An essential factor that determines pathogenicity is the inflammatory state of the host after N. meningitidis infection[11,12]. Therefore, after four h of infection, A549 cells were further cultured for 12, 24, 36, and 48 h, and the pro-inflammatory cytokine levels were detected by enzyme-linked immunosorbent assay (ELISA). The interleukin-6 (IL-6) level was detected with the human IL-6 ELISA set (#555220; BD Bioscience, San Diego, USA); the interleukin-8 (IL-8) level, with the human IL-8 ELISA Kit II (#550999; BD Bioscience, San Diego, USA); and the interleukin-1β (IL-1β) level, with the human IL-1β ELISA Set II (#557953; BD Bioscience, San Diego, USA). The tumor necrosis factor-α (TNF-α) level was detected with the human TNF ELISA Kit II (#550610; BD Bioscience, San Diego, USA), and the release of interferon (IFN) was detected with the human IFN ELISA set (#555142; BD Bioscience, San Diego, USA). The cytokines mentioned above were assayed according to the manufacturer’s instructions. The cytokine levels for non-infected A549 cells were considered as the CTR values.
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A549 cells were transferred into 96-well plates after 4 h of infection. The cells were allowed to grow for 24 and 48 h. At each of the indicated time points, the number of non-viable cells was assessed by Trypan blue staining (C0040; Solarbio Life Sciences, China). The cells were collected and centrifuged at 1,000–2,000 rpm for 1 min, and the supernatant was discarded. Following this, a single-cell suspension was prepared from the pellet and diluted appropriately. The cell suspension was mixed with 0.4% Trypan blue solution at a ratio of 9:1 (final concentration: 0.04%) and stained for 3 min. A small amount of the stained cell suspension was taken for cell counting with the blood cell counting plate. The percentage of non-viable cells was calculated by dividing the number of non-viable cells by the total number of cells. The number of non-viable non-infected cells was considered as the CTR.
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After 4 h of infection, A549 cells were further cultured for 24 h and 48 h before apoptosis analysis with the FITC Annexin V Apoptosis Detection Kit I according to the manufacturer’s protocol (#556547; BD Biosciences, United States). The cells were collected and washed twice with cold PBS, and they were then resuspended in 1× binding buffer at a concentration of 1 × 105 cells/mL. Following this, 100 µL of the solution (1 × 105 cells) was transferred to a 5-mL culture tube, and 5 µL of FITC Annexin V and 5 µL PI were added to the solution. The cells were gently vortexed and incubated for 15 min at room temperature (25 °C) in the dark. Next, 400 µL of 1× binding buffer was added to each tube and flow cytometric (FCM) analysis was performed within 1 h. Cells that stained positive for FITC Annexin V and negative for PI were considered to be undergoing apoptosis. Cells that stained positive for both FITC Annexin V and PI were considered to be in the end stage of apoptosis, undergoing necrosis, or already non-viable. Cells that were negative for both FITC Annexin V and PI were considered alive and not undergoing measurable apoptosis. Cells that were not infected with bacteria were used as CTRs.
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All analyses were conducted using SPSS version 20.0 (IBM, SPSS, Chicago, IL, USA) and GraphPad Prism 5 (GraphPad, CA, USA). Differences between means were analyzed by a two-way ANOVA. A two-sided P value of < 0.05 was considered to indicate statistical significance.
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The detailed results for the adhesion and invasion rates of the four N. meningitidis isolates are presented in Table 2 and Figure 1. Serogroup A isolate 510612 had a significantly higher adhesion rate than the other serogroup ST-7 isolates (P < 0.05 vs. the other three isolates). Serogroup X isolate 331401 had the lowest adhesion capability (P < 0.05 vs. serogroup A isolate 510612, P < 0.01 vs. serogroup B isolate 441212, P < 0.001 vs. serogroup C isolate 630601) (Figure 1A). Serogroup A isolate 510612 also had the highest invasion rate (P < 0.01 vs. the other two isolates). In addition, Serogroup X isolate 331401 also had the weakest invasion capability (P < 0.01 vs. serogroup A isolate 510612 and serogroup C isolate 630601, P < 0.05 vs. serogroup B isolate 441212) (Figure 1B, Table 2).
Figure 1. Adhesion and invasion rates of the four N. meningitidis ST-7 isolates examined in this study. The adhesion rate was calculated as the percentage of the number of adherent bacteria divided by the number of total bacteria (A), and the invasive rate was calculated as the percentage of the number of intracellular bacteria divided by the number of total associated bacteria (B). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the indicated groups.
Table 2. Adhesion and invasion rates of the ST-7 isolates in A549 cells (Mean ± SD)
Isolate No. Adhesion rate (‰) Invasion rate (%) 510612 11.17 ± 2.18 11.95 ± 2.32 441212 7.35 ± 1.10 8.36 ± 0.95 630601 8.04 ± 0.36 9.99 ± 0.87 331401 6.18 ± 0.62 5.62 ± 0.18 -
A549 cells were infected with the isolates as described in the previous section, and the release of cytokines (IL-6, IL-1β, IFN, IL-8, and TNF-α) was examined for each serogroup ST-7 isolate. Cytokine analysis was performed using ELISA at 12, 24, 36, and 48 h after infection. Challenge with the four N. meningitidis isolates induced time-dependent secretion of IL-6, IL-1β, and IFN. Cytokine release by uninfected cells was analyzed to determine the CTR values.
The ELISA data shown in Table 3 illustrate that in accordance with the adhesion and invasion rates, serogroup A isolate 510612 had the strongest ability to promote the release of IL-6 in host cells (P < 0.05) and serogroup X isolate 331401 triggered significantly less IL-6 release than the other ST-7 isolates did (P < 0.05).
Table 3. IL-6 release in response to the challenge with ST-7 isolates (pg/mL) (Mean ± SD)
Isolate No. Infection time 12 h 24 h 36 h 48 h CTR 3.93 ± 0.14 4.11 ± 0.05 4.40 ± 0.06 4.77 ± 0.10 510612 19.83 ± 0.60 42.42 ± 0.59b 61.06 ± 4.05b 67.05 ± 1.69b 441212 19.37 ± 0.07 38.77 ± 2.69a 58.55 ± 1.06b 62.65 ± 0.77a 630601 20.15 ± 1.24 44.14 ± 0.58b 59.61 ± 0.97b 64.89 ± 0.71b 331401 20.69 ± 0.42 38.12 ± 1.52a 53.25 ± 1.10a 60.99 ± 1.47a Note. aP < 0.05 compared with serogroup A isolate 510612, bP < 0.05 compared with serogroup X isolate 331401. The findings for IL-1β release were similar to those for IL-6 release. As shown in Table 4, infection with serogroup A isolate 510612 resulted in the highest levels of IL-1β release (P < 0.05), while infection with serogroup X isolate 331401 resulted in the lowest levels of IL-1β secretion in host cells (P < 0.05).
Table 4. IL-1β release in response to the challenge with ST-7 isolates (pg/mL) (Mean ± SD)
Isolate No. Infection time 12 h 24 h 36 h 48 h CTR 1.59 ± 0.04 1.63 ± 0.03 1.61 ± 0.07 1.75 ± 0.03 510612 1.66 ± 0.14 2.45 ± 0.06b 2.72 ± 0.06b 4.50 ± 0.12b 441212 1.51 ± 0.04 2.17 ± 0.11ab 2.68 ± 0.06b 4.27 ± 0.11ab 630601 1.48 ± 0.09 2.21 ± 0.15ab 2.54 ± 0.06b 4.26 ± 0.05ab 331401 1.50 ± 0.07 1.90 ± 0.05a 2.20 ± 0.20a 3.45 ± 0.04a Note. aP < 0.05 compared with serogroup A isolate 510612, bP < 0.05 compared with serogroup X isolate 331401. The variations in IFN release also showed similar patterns to IL-6 and IL-1β release: that is, serogroup A isolate 510612 exhibited the strongest capability to induce the release of IFN (P < 0.05), while serogroup X isolate 331401 exhibited the weakest capability for IFN secretion (P < 0.05) (Table 5).
Table 5. IFN release in response to the challenge with ST-7 isolates (pg/mL) (Mean ± SD)
Isolate No. Infection time 12 h 24 h 36 h 48 h CTR 1.17 ± 0.09 1.42 ± 0.05 1.44 ± 0.02 1.55 ± 0.03 510612 1.48 ± 0.01bc 1.98 ± 0.04bc 2.74 ± 0.02bc 2.96 ± 0.06bc 441212 1.23 ± 0.01a 1.56 ± 0.02ac 1.97 ± 0.02abc 2.32 ± 0.01abc 630601 1.30 ± 0.07a 1.66 ± 0.05a 2.22 ± 0.08ac 2.55 ± 0.16ac 331401 1.28 ± 0.03a 1.70 ± 0.05a 1.83 ± 0.05ab 2.07 ± 0.02ab Note. aP < 0.05 compared with serogroup A isolate 510612, bP < 0.05 compared with serogroup C isolate 630601, cP < 0.05 compared with serogroup X isolate 331401. No significant difference in TNF-α and IL-8 release was found between the four ST-7 N. meningitidis isolates (Tables 6 & 7).
Table 6. TNF-α release in response to the challenge with ST-7 isolates (pg/mL) (Mean ± SD)
Isolate No. Infection time 12 h 24 h 36 h 48 h CTR 0.24 ± 0.02 0.23 ± 0.01 0.25 ± 0.02 0.29 ± 0.02 510612 0.26 ± 0.02 0.32 ± 0.01 0.25 ± 0.02 0.29 ± 0.03 441212 0.23 ± 0.01 0.25 ± 0.02 0.25 ± 0.03 0.30 ± 0.03 630601 0.23 ± 0.02 0.25 ± 0.02 0.26 ± 0.01 0.29 ± 0.02 Table 7. IL-8 release in response to the challenge with ST-7 isolates (pg/mL) (Mean ± SD)
Isolate No. Infection time 12 h 24 h 36 h 48 h CTR 0.91 ± 0.04 0.93 ± 0.04 0.92 ± 0.07 1.08 ± 0.05 510612 1.11 ± 0.08 0.93 ± 0.05 1.09 ± 0.06 0.92 ± 0.09 441212 0.93 ± 0.03 0.96 ± 0.04 1.07 ± 0.02 1.02 ± 0.04 630601 1.08 ± 0.05 1.05 ± 0.03 1.04 ± 0.03 1.01 ± 0.11 331401 0.97 ± 0.12 1.02 ± 0.06 1.05 ± 0.03 0.99 ± 0.02 -
The effect of infection with the different serogroups on host cells was further clarified with an analysis of cell death performed by Trypan blue staining and apoptosis analysis performed with FCM. As reported for the other experiments, non-infected cells were used as CTR.
As shown in Figure 2A and Table 8, the ST-7 isolates induced significant cell death from 24 h to 48 h after infection, as compared with the cell death rate from 0 h to 24 h after infection. Further, at 24 h and 48 h after infection, the serogroup X isolate 331401 was associated with the lowest cell death rate among the ST-7 isolates (P < 0.001 vs. the other isolates at both time points; P < 0.01 vs. the serogroup C isolate 630601 at 48 h). This finding is in accordance with data for adhesion and invasion rates and cytokine release. However, unlike the findings for IFN release, the host cell death rate was higher in response to the challenge with the serogroup B isolate 441212 than with the serogroup C isolate 630601 (P < 0.05) (Figure 2A & Table 8).
Figure 2. Cell death and apoptosis induced by the examined N. meningitidis isolates. (A) A549 cells were infected with four ST-7 isolates belonging to four serogroups (A, B, C, and X) or left as uninfected controls (CTR). Non-viable cells were stained with Trypan blue. Cell death induced by different serogroup ST-7 isolates at different time points was calculated and compared. (B) A549 cells infected with the same four ST-7 isolates were analyzed for apoptosis using FITC Annexin V by FCM. Q2-1 represents non-viable non-apoptotic cells (NVN); Q2-2, non-viable apoptotic cells (NVA); Q2-3, viable non-apoptotic cells (VN); Q2-4, viable apoptotic cells (VA). The apoptosis rate was calculated as the percentage of the number of Q2-2 cells plus the number of Q2-4 cells. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the indicated groups.
Table 8. Cell death in response to the challenge with ST-7 isolates (%) (Mean ± SD)
Isolate No. Infection time 24 h 48 h CTR 5.82 ± 0.95 6.86 ± 0.25 510612 16.33 ± 1.77 44.41 ± 0.16 441212 19.46 ± 0.35 44.75 ± 3.59 630601 16.10 ± 0.78 41.44 ± 0.85 331401 8.62 ± 0.19 36.22 ± 2.06 The results of Trypan blue straining were verified by host cell apoptosis analysis with FCM. As shown in Table 9 and Figure 2B, the host cell apoptosis rate was higher from 24 h to 48 h after infection than from 0 h to 24 h. Among the ST-7 isolates, the serogroup A isolate 510612 had the most powerful apoptotic effect, and the serogroup X isolate 331401 had the weakest apoptotic effect. This is also in agreement with the other findings.
Table 9. Apoptosis rate in response to the challenge with ST-7 isolates (%)
Isolate No. Infection time Percentage of cells Q2-1 Q2-2 Q2-3 Q2-4 CTR 24 h 1.19 1.06 95.06 2.70 48 h 1.56 1.88 93.97 2.59 510612 24 h 3.45 6.15 84.78 5.62 48 h 1.18 17.01 60.51 21.31 441212 24 h 1.61 7.65 80.96 9.77 48 h 8.48 21.01 59.68 10.84 630601 24 h 0.78 4.22 84.74 10.26 48 h 7.91 21.34 59.82 10.93 331401 24 h 1.39 6.48 91.22 0.91 48 h 12.70 19.91 63.60 3.79 Note. Q2-1, non-viable non-apoptotic cells (NVN); Q2-2, non-viable apoptotic cells (NVA); Q2-3, viable non-apoptotic cells (VN); Q2-4, viable apoptotic cells (VA). The percentage of apoptosis was calculated by dividing the number of cells in Q2-2 by the number of cells in Q2-4. The in vitro data confirm that the pathogenicity of the four serogroups belonging to the same ST differ, with serogroup X having the lowest pathogenicity. Therefore, capsule structure affects the interaction and adaptation between N. meningitidis and host cells. The overall effects of capsule structure on N. meningitidis pathogenicity are presented in Table 10.
Table 10. Overall effects of capsule structure on N. meningitidis pathogenicity of the four serogroups
Isolate No. Serogroup Sequence type Adhesion Invasion Cytokine release (48 h) Apoptosis (48 h) IL-6 IL-1β IFN 510612 A 7 ++++ ++++ ++++ ++++ ++++ ++++ 441212 B 7 ++/+++ ++/+++ ++ +++/++ ++ ++/+++ 630601 C 7 +++/++ +++/++ +++/++ ++/+++ +++ +++/++ 331401 X 7 + + + + + +
doi: 10.3967/bes2020.015
Comparison of the Pathogenicity of Neisseria meningitidis Isolates of Hyperinvasive Sequence Type 7 Belonging to Serogroups A, B, C, and X
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Abstract:
Objective To compare the pathogenicity of isolates of sequence type 7 (ST-7) Neisseria meningitidis (N. meningitidis) belonging to four different serogroups (A, B, C, and X). Methods Four ST-7 N. meningitidis isolates serogrouped as A, B, C, and X and characterized by different capsule structures, were examined for their adhesion and invasion properties, and their ability to induce cytokine release and apoptosis in the host cell (the A549 cell line). Results Among the four ST-7 N. meningitidis isolates, the serogroup A isolate possessed the strongest adhesion and invasion ability. This isolate also induced the release of the highest levels of the pro-inflammatory mediators interleukin-6, interleukin-1β, and interferon, and the highest apoptosis rate in the host cells. However, there was no significant difference in interleukin-8 and tumor necrosis factor-α secretion between the four isolates. Based on the findings, the serogroup X N. meningitidis isolate had the weakest pathogenicity, whereas there was almost no difference in the pathogenicity of the isolates from serogroups B and C. Conclusions The differences in the capsular structure of the four isolates of ST-7 N. meningitidis affected their pathogenic capacities. The findings also imply that the hyperinvasive ST-7 N. meningitidis lineage may include hypoinvasive isolates. -
Key words:
- ST-7 N. meningitidis /
- Capsule /
- Pathogenicity /
- Adaptation
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Figure 1. Adhesion and invasion rates of the four N. meningitidis ST-7 isolates examined in this study. The adhesion rate was calculated as the percentage of the number of adherent bacteria divided by the number of total bacteria (A), and the invasive rate was calculated as the percentage of the number of intracellular bacteria divided by the number of total associated bacteria (B). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the indicated groups.
Figure 2. Cell death and apoptosis induced by the examined N. meningitidis isolates. (A) A549 cells were infected with four ST-7 isolates belonging to four serogroups (A, B, C, and X) or left as uninfected controls (CTR). Non-viable cells were stained with Trypan blue. Cell death induced by different serogroup ST-7 isolates at different time points was calculated and compared. (B) A549 cells infected with the same four ST-7 isolates were analyzed for apoptosis using FITC Annexin V by FCM. Q2-1 represents non-viable non-apoptotic cells (NVN); Q2-2, non-viable apoptotic cells (NVA); Q2-3, viable non-apoptotic cells (VN); Q2-4, viable apoptotic cells (VA). The apoptosis rate was calculated as the percentage of the number of Q2-2 cells plus the number of Q2-4 cells. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the indicated groups.
Table 1. N. meningitidis isolates used in the present study
Isolate No. Serogroup Isolate source Year of isolation Sequence type Clonal complex 510612 A Patient 2006 7 5 441212 B Patient 2012 7 5 630601 C Patient 2006 7 5 331401 X Patient 2014 7 5 S1. Detail information of strains used in this study
Bacteria No. Serogroup Isolates source Specimen type Year of isolated Isolate district Sequence type Clonal complex PorA FetA NadA_ variant NHBA_ peptide FHbp 510612 A Patient Throat swab 2006 Sichuan 7 5 P1.20,9 F3-1 NadA-2/3 126 1.5 441212 B Patient CSF 2012 Guangdong 7 5 P1.20,9 F3-1 NadA-2/3 126 1.5 630601 C Patient CSF 2006 Qinghai 7 5 P1.20,9 F3-1 NadA-2/3 126 1.5 331401 X Patient CSF 2014 Zhejiang 7 5 P1.20,9 F3-1 NadA-2/3 126 1.5 Table 2. Adhesion and invasion rates of the ST-7 isolates in A549 cells (Mean ± SD)
Isolate No. Adhesion rate (‰) Invasion rate (%) 510612 11.17 ± 2.18 11.95 ± 2.32 441212 7.35 ± 1.10 8.36 ± 0.95 630601 8.04 ± 0.36 9.99 ± 0.87 331401 6.18 ± 0.62 5.62 ± 0.18 Table 3. IL-6 release in response to the challenge with ST-7 isolates (pg/mL) (Mean ± SD)
Isolate No. Infection time 12 h 24 h 36 h 48 h CTR 3.93 ± 0.14 4.11 ± 0.05 4.40 ± 0.06 4.77 ± 0.10 510612 19.83 ± 0.60 42.42 ± 0.59b 61.06 ± 4.05b 67.05 ± 1.69b 441212 19.37 ± 0.07 38.77 ± 2.69a 58.55 ± 1.06b 62.65 ± 0.77a 630601 20.15 ± 1.24 44.14 ± 0.58b 59.61 ± 0.97b 64.89 ± 0.71b 331401 20.69 ± 0.42 38.12 ± 1.52a 53.25 ± 1.10a 60.99 ± 1.47a Note. aP < 0.05 compared with serogroup A isolate 510612, bP < 0.05 compared with serogroup X isolate 331401. Table 4. IL-1β release in response to the challenge with ST-7 isolates (pg/mL) (Mean ± SD)
Isolate No. Infection time 12 h 24 h 36 h 48 h CTR 1.59 ± 0.04 1.63 ± 0.03 1.61 ± 0.07 1.75 ± 0.03 510612 1.66 ± 0.14 2.45 ± 0.06b 2.72 ± 0.06b 4.50 ± 0.12b 441212 1.51 ± 0.04 2.17 ± 0.11ab 2.68 ± 0.06b 4.27 ± 0.11ab 630601 1.48 ± 0.09 2.21 ± 0.15ab 2.54 ± 0.06b 4.26 ± 0.05ab 331401 1.50 ± 0.07 1.90 ± 0.05a 2.20 ± 0.20a 3.45 ± 0.04a Note. aP < 0.05 compared with serogroup A isolate 510612, bP < 0.05 compared with serogroup X isolate 331401. Table 5. IFN release in response to the challenge with ST-7 isolates (pg/mL) (Mean ± SD)
Isolate No. Infection time 12 h 24 h 36 h 48 h CTR 1.17 ± 0.09 1.42 ± 0.05 1.44 ± 0.02 1.55 ± 0.03 510612 1.48 ± 0.01bc 1.98 ± 0.04bc 2.74 ± 0.02bc 2.96 ± 0.06bc 441212 1.23 ± 0.01a 1.56 ± 0.02ac 1.97 ± 0.02abc 2.32 ± 0.01abc 630601 1.30 ± 0.07a 1.66 ± 0.05a 2.22 ± 0.08ac 2.55 ± 0.16ac 331401 1.28 ± 0.03a 1.70 ± 0.05a 1.83 ± 0.05ab 2.07 ± 0.02ab Note. aP < 0.05 compared with serogroup A isolate 510612, bP < 0.05 compared with serogroup C isolate 630601, cP < 0.05 compared with serogroup X isolate 331401. Table 6. TNF-α release in response to the challenge with ST-7 isolates (pg/mL) (Mean ± SD)
Isolate No. Infection time 12 h 24 h 36 h 48 h CTR 0.24 ± 0.02 0.23 ± 0.01 0.25 ± 0.02 0.29 ± 0.02 510612 0.26 ± 0.02 0.32 ± 0.01 0.25 ± 0.02 0.29 ± 0.03 441212 0.23 ± 0.01 0.25 ± 0.02 0.25 ± 0.03 0.30 ± 0.03 630601 0.23 ± 0.02 0.25 ± 0.02 0.26 ± 0.01 0.29 ± 0.02 Table 7. IL-8 release in response to the challenge with ST-7 isolates (pg/mL) (Mean ± SD)
Isolate No. Infection time 12 h 24 h 36 h 48 h CTR 0.91 ± 0.04 0.93 ± 0.04 0.92 ± 0.07 1.08 ± 0.05 510612 1.11 ± 0.08 0.93 ± 0.05 1.09 ± 0.06 0.92 ± 0.09 441212 0.93 ± 0.03 0.96 ± 0.04 1.07 ± 0.02 1.02 ± 0.04 630601 1.08 ± 0.05 1.05 ± 0.03 1.04 ± 0.03 1.01 ± 0.11 331401 0.97 ± 0.12 1.02 ± 0.06 1.05 ± 0.03 0.99 ± 0.02 Table 8. Cell death in response to the challenge with ST-7 isolates (%) (Mean ± SD)
Isolate No. Infection time 24 h 48 h CTR 5.82 ± 0.95 6.86 ± 0.25 510612 16.33 ± 1.77 44.41 ± 0.16 441212 19.46 ± 0.35 44.75 ± 3.59 630601 16.10 ± 0.78 41.44 ± 0.85 331401 8.62 ± 0.19 36.22 ± 2.06 Table 9. Apoptosis rate in response to the challenge with ST-7 isolates (%)
Isolate No. Infection time Percentage of cells Q2-1 Q2-2 Q2-3 Q2-4 CTR 24 h 1.19 1.06 95.06 2.70 48 h 1.56 1.88 93.97 2.59 510612 24 h 3.45 6.15 84.78 5.62 48 h 1.18 17.01 60.51 21.31 441212 24 h 1.61 7.65 80.96 9.77 48 h 8.48 21.01 59.68 10.84 630601 24 h 0.78 4.22 84.74 10.26 48 h 7.91 21.34 59.82 10.93 331401 24 h 1.39 6.48 91.22 0.91 48 h 12.70 19.91 63.60 3.79 Note. Q2-1, non-viable non-apoptotic cells (NVN); Q2-2, non-viable apoptotic cells (NVA); Q2-3, viable non-apoptotic cells (VN); Q2-4, viable apoptotic cells (VA). The percentage of apoptosis was calculated by dividing the number of cells in Q2-2 by the number of cells in Q2-4. Table 10. Overall effects of capsule structure on N. meningitidis pathogenicity of the four serogroups
Isolate No. Serogroup Sequence type Adhesion Invasion Cytokine release (48 h) Apoptosis (48 h) IL-6 IL-1β IFN 510612 A 7 ++++ ++++ ++++ ++++ ++++ ++++ 441212 B 7 ++/+++ ++/+++ ++ +++/++ ++ ++/+++ 630601 C 7 +++/++ +++/++ +++/++ ++/+++ +++ +++/++ 331401 X 7 + + + + + + -
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