Ethics statement
All study participants provided written informed consent allowing use of human blood samples in adherence with guidelines established by the Committee for Regulation of Human Specimens and Volunteers at the National Taiwan University Hospital (NTUH, 202301127RINB).
Bacterial strains and plasmids
Wild-type S. gordonii strains were grown in brain heart infusion broth (BHI, Difco Laboratories Inc., Detroit, MI, USA). For essC (ΔessC) and esxA-deficient (ΔesxA) strains, the BHI contained 500 µg/mL kanamycin. To generate the complementation strain (comΔesxA), bacteria were transformed with the shuttle plasmid pDL278 containing the promoter region and the esxA gene, and grown in BHI supplemented with 500 µg/mL spectinomycin, as previously described52,55. The vector without the promoter and esxA gene regions was also transformed into the esxA-deficient mutant and served as the vector control (ΔesxA-pDL278) to exclude any effect of pDL278. GFP-labeled bacteria were generated by transformed with pPDGFPuv56, which carries the GFPuv sequence and a chloramphenicol resistance cassette. To maintain the GFP-labeled strains, BHI broth was supplemented with 10 µg/mL chloramphenicol to ensure retention of the plasmids during bacterial growth.
Construction of essC and esxA deletion mutants and esxA complementation strains
All methods used in this study were carried out in accordance with relevant guidelines and regulations, and all experimental protocols were approved by Taipei Medical University and National Taiwan University.
Strains having deletion of essC or esxA were generated using a ligation-PCR mutagenesis strategy as described previously that involved insertion of promoter-less kanamycin cassettes to disrupt the corresponding genes in the S. gordonii strain DL152,55. Briefly, gene sequences upstream and downstream of essC and esxA as well as the promoter-less kanamycin resistance gene were amplified from S. gordonii DL1 chromosomal DNA and the pALH124 plasmid, respectively, using primers listed in Table 1. The primers were designed according to the S. gordonii DL1 genomic database (https://www.ncbi.nlm.nih.gov/genome/). The resulting PCR products were digested with EcoRI and ligated with T4 ligase. The ligation products were transformed into wild-type S. gordonii DL1 and mutant strains were verified by PCR amplification and western blotting analysis. To construct the comΔessC and comΔesxA strains, essC and esxA were amplified using the primers listed in Table 1. The PCR products were digested with EcoRI and ligated into the pDL278 plasmid carrying a spectinomycin resistance cassette. The resulting plasmids were then used to transform ΔessC and ΔesxA to generate the comΔessC and comΔesxA strains, respectively. Expression of esxA was further confirmed by western blotting analysis.
Rat model of S. gordonii-induced IE and in vivo bacteria clearance assay
A rat model of streptococcal endocarditis was performed as previously described with the following modifications5. All experiments involving animals were performed according to protocols approved by the National Taiwan University Institutional Animal Care and Use Committee (Taipei, Taiwan; IACUC 20220439). The study was carried out in accordance with the relevant guidelines and regulation with the ARRIVE guidelines 2.0 (https://arriveguidelines.org/arrive-guidelines). All animals were handled in full compliance with the National Institutes of Health guidelines for the care and use of laboratory animals, ensuring ethical and humane treatment throughout the research process. Thirty adult rats (8–10 weeks old, weighing 500–600 g) were obtained from BioLASCO Taiwan Co., Ltd. All animals were housed under standardized laboratory conditions, with ad libitum access to water and food, in an environment maintained at a temperature of 20–24 °C and a relative humidity of 40–75%. For anesthetization, each rat was initially anesthetized with 4% isoflurane for induction and maintained on 2% isoflurane. Additionally, Zoletil (20–40 mg/kg) and xylazine (5–10 mg/kg) were administered intraperitoneally. No repeated anesthesia was performed on the same animal. Briefly, the ventral neck surgical site was shaved and disinfected using an aqueous iodine solution. The aortic valve was injured using a polyethylene tube containing a stainless-steel wire inserted into the left carotid artery. At 24 h post-catheterization, rats were intravenously infected with 109 colony forming units (CFUs) of wild-type or isogenic mutant T7SSb strains. The rats were sacrificed 24 h after infection using 50% CO2 for 10 min, whereupon all cardiac vegetations were harvested and weighed. The vegetations were then homogenized in saline, serially diluted, and plated onto BHI agar to determine bacterial counts, which were normalized according to the vegetation weight. To observe NETs, the vegetations were fixed with 2% paraformaldehyde and stained with an anti-rat myeloperoxidase (MPO) antibody (diluted 1:50; Santa Cruz Biotechnology, California, United States), followed by rhodamine red-conjugated anti-rabbit immunoglobulin G (IgG) antibody (diluted 1:500; Jackson ImmunoResearch Labs, West Grove, Pennsylvania, United States). DNA was stained with Hoechst 33,258 (Sigma-Aldrich, St. Louis, Missouri, United States) and NET structures were observed using a Leica SP8 confocal microscope. For biofilm observation, S. gordonii DL1 wild-type and mutant strains were transformed with pDGFPuv56, and the biofilm structures were monitored using ImarisViewer10.2 Image Analysis Software (https://imaris.oxinst.com/). The bacterial clearance assay was performed as we previously described56. Briefly, 109 CFUs of bacteria were intravenously into groups of 5 Wistar rats for each strain. Each rat represented an independent experiment with distinct bacterial cultures.
Recombinant EsxA purification and anti-EsxA antibody preparation
Purification of DL1 EsxA was performed as previously described with some modifications55. The esxA gene of DL1 was cloned into the BamHI site of the pQE30 expression vector that contains an N-terminal in-frame 6XHis-tag encoding sequence. The resultant EsxA expression plasmid was transformed into M15 Escherichia coli. EsxA expression was induced in M15 E. coli during the exponential growth phase growth by addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After 4 h of induction, the bacterial cells were collected, resuspended in binding buffer (30µM imidazole, 0.5µM, 20µM Tris-HCl, pH 7.9) and then disrupted by sonication on ice. The expressed proteins were purified using a nickel chelating affinity chromatography system, dialyzed into 10mM NaPB buffer (1 M NaH2PO4, 1 M Na2HPO4, pH 6.0) and checked by SDS-PAGE. The rat anti-EsxA polyclonal antibody was isolated as previously described56. Briefly, purified rEsxA was mixed and intracutaneously injected into Wistar rats on the back. A second booster injection with 1:1 Freund’s complete adjuvant and rEsxA was given two weeks later and serum was collected two days after the second boost. Anti-EsxA sera titers were analyzed using enzyme-linked immunosorbent assay (ELISA), and the anti-EsxA rat serum specificity was measured by western blotting using recombinant EsxA protein, total protein extracts from wild-type S. gordonii and isogenic mutant T7SSb strains.
Detection of cell wall-associated and secreted S. gordonii EsxA
To detect secreted EsxA in S. gordonii culture supernatants, wild-type and isogenic mutant T7SSb and complementation strains were grown in BHI broth for 16 h at 37 °C. The bacteria were centrifuged at 5000xg for 10 min at 4 °C. EDTA-free protease inhibitor cocktail (1:200 dilution; MCE, catalog: HY-K0010) was added to the supernatants and the flow-through was precipitated with trichloroacetic acid (TCA; final concentration 20%) for 24 h at 4 °C. The precipitated proteins were gently washed with acetone, dried and resuspended in sample buffer (10% SDS, 4% β-mercaptoethanol, 0.5 M Tris, pH 6.8). Total bacterial pellets were gently washed with phosphate buffered saline (PBS) and resuspended in sample buffer. Precipitates and supernatants were boiled for 10 min before SDS-PAGE and western blotting. For western blots, protein samples were transferred to PVDF membranes that were blocked in fast blocking buffer (BioMAN; catalog: FBB01-500) for 30 min at room temperature. After blocking, the membranes were hybridized with an anti-EsxA rat polyclonal antibody in blocking buffer overnight at 4 °C. The membranes were then washed in 1X Tris buffered saline, 0.1% Tween 20 (TBST) and incubated with goat-anti-rat secondary antibodies (1:10,000; Abcam; ab97057) for 2 h at room temperature. After washing three times with TBST, the western blots were visualized using a Muti-function Gel Image system.
Preparation of neutrophils and in vitro induction of NET formation
As mentioned above, this research received approval from the Committee for Regulation of Human Specimens and Volunteers at National Taiwan University Hospital and written informed consent was obtained from all study participants. Volunteers were healthy individuals older than 18 years, regardless of sex, and without prior use of antibacterial or anti-inflammatory drugs. Neutrophils were isolated from heparinized venous blood samples using Ficoll-Histopaque (Histopaque 1119 and Histopaque 1083, Sigma-Aldrich). Neutrophils were collected and seeded onto plasma-coated coverslips (100 µL of the suspension in Roswell Park Memorial Institute (RPMI) medium contains 106 cells/mL), which were placed into the wells of a 24-well plate and incubated in 5% CO2 at 37 °C for 1 h. After removing non-adherent neutrophils, the remaining neutrophils on the coverslip were subsequently stimulated with recombinant S. gordonii EsxA (0.25, 0.5, 1, and 10 µg/mL) in RPMI medium for 4 h. A transwell assay was performed in a 24-well plate using polycarbonate transwell inserts with 8.75 mm diameter membranes having 0.4 µM pores (SPL life science; catalog:35324). The neutrophils were seeded in the lower compartment of the 24-well plate, and wild-type and isogenic S. gordonii strains were added to the transwell bucket at an MOI of 100. The plate was then incubated for 8 h in 5% CO2 at 37 °C. To observe NETs, samples were stained with two primary antibodies: a mouse anti-human MPO antibody (dilution 1:200; Santa Cruz Biotechnology, sc-52707) and a rabbit anti-human citrullinated histone H3 (citH3) antibody (dilution 1:100; Abcam, ab5103, Cambridge, MA). MPO was detected using a FITC- or Texas Red-labeled anti-mouse IgG secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA, USA), while citH3 was followed with an anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA, USA). DNA was stained with Hoechst 33,258 (Sigma-Aldrich, St. Louis, Missouri, United States). NET formation was observed by confocal microscopy (TCS Leica SP5 or SP8) and analyzed using the Volocity program (PerkinElmer; https://www.volocity4d.com/).
Cell cytotoxicity assay
For neutrophil cytotoxicity assays, bacterial cells were inoculated with neutrophils at a multiplicity of infection (MOI) of 100 and incubated for 8 h. LDH release was then quantified using an LDH assay kit (Abcam ab65393) according to the manufacturer’s instructions. Transwell cytotoxicity assays were performed in 24-well plates with cells seeded in the bottom compartment. Wild-type and isogenic S. gordonii strains were added to the transwell bucket at an MOI of 100. The plate was then incubated for 8 h in 5% CO2 at 37 °C. To achieve maximum LDH release, cell lysis solution was added to each well, followed by addition of 50 µl of the reconstituted substrate mix. The reaction was then stopped, and the absorbance was recorded at 450 nm using an ELISA plate reader. Cytotoxicity was quantified as a percentage relative to an uninfected control that was treated with 1% Triton X-100.
Statistical analysis
Analyses of the statistical significance of differences between two datasets were conducted using an unpaired, two-tailed Student’s t-test. For comparison of more than two datasets, a one-way ANOVA followed by a Bonferroni post-hoc test was utilized. For data with a non-normal distribution, Mann-Whitney U and Kruskal-Wallis tests followed by a Dunn’s post-test were employed. These analyses were performed using GraphPad Prism version 8 (GraphPad Software). A p-value < 0.05 indicated statistical significance.