Bacterial strains, media, and growth conditions
This study used H. pylori strain ATCC 700392 as a reference strain for in vitro bacteriostasis experiments. The H. pylori were cultured in solid and liquid media under microaerobic conditions at 37℃ for about 48–72 h. The solid medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., which contained 4 g/l bovine brain extract powder, 4 g/l bovine heart extract powder, 5 g/l peptone, 16 g/l casein peptone, 2 g/l glucose, 5 g/l sodium chloride, 2.5 g/l disodium hydrogen phosphate, and 13.5 g/l agar. The liquid culture medium was also purchased from Qingdao Haibo Biotechnology Co., Ltd., which contained 10 g/l bovine brain extract powder, 9 g/l bovine heart extract powder, 10 g/l peptone, 2 g/l glucose, 5 g/l sodium chloride, and 2.5 g/l disodium hydrogen phosphate. After high-pressure steam sterilization at 121 °C for 15 min of the above two media, 7% sterile defibrinated sheep blood and H. pylori bacteriostatic agent (containing 1 mg nalidixic acid, 0.5 mg trimethoprim, 0.3 mg vancomycin, and 0.2 mg amphotericin B) were added. The above-mentioned sterile defibrinated sheep blood and H. pylori bacteriostatic agent were provided by the manufacturer when the mediums were purchased. E. coli strain BL21(DE3) was cultured in LB medium (15 g/l yeast extract powder, 1 g/l glucose, and 10 g/l sodium chloride) at 37 ℃. When necessary, an additional 40 µg/ml kanamycin was added to LB medium.
Design of artilysin genes
Bioinformatics prediction and analysis were performed using the published complete genome sequence of H. pylori phage KHP30 (NC_019928.1) and 1961P (NC_019512.1) (Lehours et al. 2011; Luo et al. 2012; Uchiyama et al. 2012, 2013; Abdel-Haliem and Askora 2013; Takeuchi et al. 2018). Preliminary annotations and reports of H. pylori phage endolysin and holin were used in combination with the NCBI BLAST program (e-values ≤ 0.01 were considered credible); the smallest item in the credible range was subjected to the next round of BLAST. This was repeated until no obvious homologues appeared, and possible enzymatic functions were identified using the annotated H. pylori phage endolysin. The (ExPASy) Protparam software was used to analyze basic biochemical properties (e.g., amino acid length, molecular weight, isoelectric point, charge number, and hydrophilicity). Signal P3.0 software was used to analyze N-terminal amino acid sequences to determine whether they contained signal peptides. The SWISS-MODEL automatic matching method was used to predict and compare tertiary structures, using all default parameters. The above analysis and comparison were expected to identify multiple H. pylori bacteriophage endolysin and holin sequences, including Holin A, Holin B, Endolysin A and Endolysin B (see Additional file 1: Table S1). In addition, during the early stages of this study, extensive literature review and data analysis revealed several transmembrane peptides with different physical and chemical properties. Their sequences or optimized sequences (see Additional file 1: Table S2) were connected to H. pylori phage endolysin and holin using a linker (GAGA), thereby producing artilysin genes (see Additional file 1: File S1).
Early experiments showed that endolysins formed inclusion bodies during prokaryotic expression in vitro, which reduced their cleavage activity. Therefore, the pSUMO soluble E. coli expression vector purchased from Miao Ling Biological Technology Co., Ltd. was used, which had His-Tag and could follow by nickel column affinity chromatography to capture the target protein. An enzyme digestion–enzyme ligation method was used to construct recombinant pSUMO-artilysin plasmids. For the artilysin genes and pSUMO, Xho I and Sac I were used for digestion. The optimized target genes did not contain Xho I or Sac I linearization sites.
Construction and expression of engineered bacteria
For the E. coli expression system, competent E. coli BL21 cells were prepared using the calcium chloride method and then heat shocked for plasmid transformation. Kanamycin-containing agar was used to obtain clones expressing the plasmid. Sequences were verified to confirm the construction of BL21-pSUMO-artilysin bacteria.
Protein expression conditions (e.g., induction temperature, induction timing, induction time, inducing agent, medium composition, and nutrients) were adjusted as necessary to optimize expression in the engineered bacteria. Two recombinant engineered bacterias (BL21-pSUMO-artilysin) were cultured in LB liquid medium on a shaker at 37 ℃ and 250 rpm until the OD600nm was between 0.6 and 1. Then add IPTG with a final concentration of 0.5 mM, and incubate on a shaker at 25 ℃ and 250 rpm for 16–20 h. SDS-PAGE was used to verify artilysin expression.
In the E. coli expression system, the induced bacteria were centrifuged, and the supernatant were discarded. Bacterial cells were then crushed using a high pressure homogenizer at the condition of 850 bar. After further centrifugation at the condition of 8500 rpm and 30 min, the precipitate were discarded, and the supernatant were separated by nickel-chelating affinity chromatography to obtain purer target proteins (Ding et al. 2020). The Tris–HCl system was selected as the mobile phase, and 200 mM imidazole was added to the eluent, and the elution peak was collected by gradient elution. Because proteins expressed using the pSUMO vector was fused to the SUMO protein, they were digested with the SUMO enzyme (500 U/g) purchased from Solarbi Co., Ltd. to obtain the target proteins. The molecular weight and the pI of artilysin 1 were 30.69 kDa and 9.69, while the molecular weight and the pI of artilysin 2 were 22.02 kDa and 9.01. According to the protein content of the elution peak, add SUMO enzyme according to the enzyme activity of 500 u/g, and digest the collected elution peak for 16 h. SDS-PAGE was used for analysis. After the above purification, preliminary artilysins were obtained. A 3K hollow fiber column was used to concentrate the artilysins to 103 μg/ml and replace the artilysins with 20 mM phosphate buffer.
The Kirby–Bauer test was used as an in vitro antibacterial assay. H. pylori was spread on solid medium, and sterile filter paper was attached to the plates after inoculation of the H. pylori. Subsequently, 20–30 µl artilysins with a concentration of 103 μg/ml were added to the filter paper. Antibiotics and lysozyme were used as positive controls, while 20 mM phosphate buffer and the supernatant of BL21 broken bacteria with 500 u/g SUMO enzyme were used as negative controls. The antibiotic mixture was composed of gentamicin, levofloxacin and amoxicillin, and three antibiotics were formulated into a mixed solution with a final concentration of 103 μg/ml in equal proportions. All three antibiotics were purchased from Shanghai Macleans Biochemical Technology Co., Ltd. Lysozyme was purchased from Solebold Technology Co., Ltd., and was prepared at 103 μg/ml with purified water. Bacteria were incubated at 37℃ under microaerobic conditions for 48 h, and inhibition zones were examined at 48 h. The size of the inhibition zone was compared between artilysin and positive controls.
In addition, artilysins were added directly to H. pylori following centrifugation. After the bacteria had been incubated for 2 h on a shaker, they were observed under an electron microscope at 20 min, 1 h, and 2 h. The bacteria were collected by centrifugation, fixed with 2.5% glutaraldehyde and 1% osmium acid. After rinsing with phosphate buffer, dehydrating with ethanol, drying the sample with LEICA CPD-300 automatic critical point dryer, and then using ion sputtering to make the sample conductive with LEICA ACE-600 high vacuum coating machine, and finally can be observed by HITACHI SU8200 scanning electron microscope. The liquid method was also used to verify whether the bacterial concentration decreased after the addition of artilysins. Lysozyme was used as a positive control, and artilysin buffer was used as a negative control. Artilysins were added to robust H. pylori cultures at a final concentration of 100 or 500 μg/ml. The bacteria were cultivated for 30 h at 37 ℃ under microaerobic conditions with shaking at 150 rpm. At 0, 6, 24, and 30 h, samples of the bacteria were collected to measure the absorbance at 600 nm (Aiba et al. 2019; Knaack et al. 2019).