A type II double gene deletion strain of Streptococcus suis and its application

Abstract

This invention discloses a *Streptococcus suis* type II double-gene deletion strain and its application. The strain preservation number of the *Streptococcus suis* type II double-gene deletion strain is: CCTCC NO: M 2023119, and its name is: Streptococcus suis II ZJJX081101‑ΔSodA‑ΔTrxB.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a type II double-gene deletion strain of Streptococcus suis and its applications. Background Technology

[0002] Streptococcus suis type 2 (SS2) is an important zoonotic pathogen. In pigs, infection manifests clinically as meningitis, arthritis, and septicemia. It can also infect humans, causing endocarditis, arthritis, septic shock, and in severe cases, death. While significant progress has been made in recent years regarding the pathogenic factors and mechanisms of Streptococcus suis type 2, further in-depth research is needed to understand the specific mechanisms. Pathogenesis of Streptococcus suis type 2 is closely related to bacterial adhesion, invasion of respiratory epithelial cells or vascular endothelial cells, anti-macrophage phagocytosis, and activation of immune cell inflammatory responses. Streptococcus suis surface proteins can bind to complement negative regulators in human serum to evade phagocytosis by human immune cells, thereby enhancing its survival in the bloodstream. Components of Streptococcus suis type 2 that resist macrophage phagocytosis include capsular polysaccharides, peptidoglycan deacetylase proteins, and superoxide dismutase. Superoxide dismutase (SMO) is a crucial virulence factor in Streptococcus suis type II, capable of scavenging superoxide ions and enhancing bacterial resistance to oxidative stress. Bacterial deficiency of SMO results in increased adhesion to epithelial and endothelial cells, but significantly decreased survival in macrophages. Regarding the mechanism by which SMO induces increased adhesion, the applicant's preliminary research showed elevated expression of thioredoxin 2 in Streptococcus suis. However, no reports have been submitted regarding the molecular mechanisms by which thioredoxin induces the antioxidant capacity and virulence of Streptococcus suis type II, requiring further investigation. Summary of the Invention

[0003] This invention aims to overcome the shortcomings of existing technologies and provide a type II double-gene deletion strain of Streptococcus suis and its applications. To achieve the above objective, the technical solution provided by this invention is as follows:

[0004] This invention amplifies the upstream and downstream homologous arms of the TrxB gene using designed primers, ligates them into the suicide plasmid pSET4S to construct a recombinant shuttle plasmid, and then electroporates the recombinant plasmid into a Streptococcus suis type II ΔSodA single-gene deletion strain. Screening is performed using temperature and resistance to obtain a Streptococcus suis type II ΔSodA-ΔTrxB double-gene deletion strain. This strain was deposited at the China Center for Type Culture Collection on February 14, 2023, with accession number CCTCC NO: M 2023119, and named: Streptococcus suisⅡZJJX081101-ΔSodA-ΔTrxB.

[0005] The experimental results of this invention show that *Streptococcus suis* type II strains lacking the SodA gene exhibit significantly increased adhesion to HEp-2 and bEnd.3 cells compared to both the parent and complement strains. Surface proteins of the parent and ΔSodA strains were extracted and analyzed by two-dimensional electrophoresis to determine protein expression differences. Mass spectrometry and quantitative fluorescence analysis revealed that the ΔSodA strain showed significantly increased expression and transcription of thioredoxin-A (TrxA) and thioredoxin-B (TrxB) compared to the parent strain. Prokaryotic expression proteins TrxA and TrxB, along with corresponding rabbit polyclonal antibodies, were prepared and validated by Western blotting. The results showed that the ΔSodA strain did not show a significant increase in TrxA expression, while TrxB protein expression was significantly increased.

[0006] This invention relates to a double-gene deletion strain of Streptococcus suis type II (ΔSodAΔTrxB) constructed based on a single-gene deletion strain of SodA. After the deletion of the TrxB gene, its virulence is significantly reduced, its safety level is high, its toxicity is irreversible, and its immunogenicity is high. It can protect immune pigs against attacks from highly virulent strains of Streptococcus suis type II. Moreover, the manufacturing process is simple and low-cost, providing effective assistance for controlling the occurrence and spread of streptococcal disease in pigs in my country. Attached Figure Description

[0007] Figure 1 Flowchart of the construction process of the thermosensitive suicide plasmid pSET4s-ΔTrxB;

[0008] Figure 2 Electrophoresis images of PCR products from the *Streptococcus suis* type II mutant strain with gene knockout. Lane 1 is the DL5000 marker; lane 2 is the PCR product of the *Streptococcus suis* mutant strain with both SodA and TrxB genes knocked out; lane 3 is the PCR product of the *Streptococcus suis* mutant strain with TrxB gene knockout; lane 4 is the PCR product of the *Streptococcus suis* mutant strain with Trx1 gene knockout; lane 5 is the PCR product of the *Streptococcus suis* mutant strain with SodA gene knockout; lane 6 is the wild-type positive control.

[0009] Figure 3 The enzyme digestion identification results of the suicide plasmid pSET4s and the primers SS2-TrxB-up-1F / R and SS2-TrxB-Down-1F / R were obtained. The results of amplification of the upstream and downstream fragments of TrxB were also presented. Lane 1 was the DL 5000 marker; lane 2 was before pSET4s digestion; lane 3 was after pSET4s digestion; lane 8 was the amplified upstream fragment of TrxB; and lane 9 was the amplified downstream fragment of TrxB.

[0010] Figure 4 Streptococcus suis type II SodA mediates cell adhesion and antiphagocytosis;

[0011] Figure 5 The effect of SodA gene deletion on the expression of virulence-related factors in Streptococcus suis type II;

[0012] Figure 6 Expression, purification and activity detection of type II SodA protein from Streptococcus suis (A); Prokaryotic expression and purification of thioredoxins TrxA and TrxB (C);

[0013] Figure 7 Antibody titers in rabbit serum immunized with Streptococcus suis type II thioredoxin TrxA and TrxB;

[0014] Figure 8 Immunoblotting was used to detect the difference in TrxB protein expression between the SodA-deficient strain and the parental strain. Detailed Implementation

[0015] 1. Design of strains, plasmids, and primers

[0016] In this invention, *Streptococcus suis* type II isolate 1105 was a highly virulent strain isolated from the lungs of diseased pigs in 2008 by the Zhejiang Provincial Key Laboratory of Animal Preventive Medicine (collected by Tang Yulong in November 2008). pSET4S is a temperature-sensitive suicide plasmid containing elements such as the spectinomycin resistance gene (Spc), a temperature-sensitive replicon, a ColE1 replicon, and a multiple cloning site. These elements enable the plasmid to replicate in *Escherichia coli* at 37°C under spectinomycin-containing conditions; however, it replicates in *Streptococcus suis* at 28°C under spectinomycin-containing conditions, but cannot replicate at 37°C. Based on the sequences of various sorting enzyme genes in GeBank, homologous arms were designed flanking Sod and TrxB for amplification. Primers designed using DNAStar software are shown in Table 1. All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0017] Table 1

[0018]

[0019] 2. Preparation of competent cells from Streptococcus suis type II isolate 1105

[0020] Preparation of competent Streptococcus suis cells: Overnight cultured Streptococcus suis culture was placed in 200 mL of fresh BHI medium (containing 40 mmol / L DL-threonine) and cultured at 37°C with shaking at 250 rpm / min until OD≈0.4. The culture was then incubated on ice for 30 min, centrifuged at 4°C for 4000 g × 15 min to collect the cells. The cells were washed twice with 20 mL of ice-cold sterile double-distilled water, then twice with 20 mL of ice-cold 0.3 mol / L sucrose solution, and finally once with 20 mL of ice-cold 0.3 mol / L sucrose + 15% glycerol solution. The cells were then resuspended in a specific volume of 0.3 mol / L sucrose / 15% glycerol solution to achieve a cell density of 1.0 × 10⁻⁶ cells / mL. 10 Approximately CFU / mL, dispensed into 100L EP tubes, and stored at -80℃ for later use.

[0021] 3. Construction of pSET4S-TrxB recombinant transfer plasmid ( Figure 1 )

[0022] Construction of recombinant plasmid pSET4S-ΔTrxB: Primers were designed based on the TrxB sequence (CP000407.1) of Streptococcus suis type II 05ZYH33 from NCBI (see Table 1 above). The upstream and downstream homologous arms of the TrxB gene, TrxB-up (821 bp) and TrxB-down (833 bp), were amplified from the SS2 genome using the following reaction program: 95℃ pre-denaturation for 3 min; 95℃ for 15 sec, 50℃ for 15 sec, 68℃ for 30 sec, 34 cycles, followed by a final extension at 68℃ for 1 min. The amplification results were detected by 0.8% agarose gel electrophoresis. The target fragment was purified and recovered. The amplified product of the purified upstream homologous arm of the TrxB gene was digested with HindIII and BamHI and directly ligated to the HindIII and BamHI sites of plasmid pSET4S (Invitrogen). The resulting recombinant plasmid pSET4S-SU was confirmed to be correctly constructed by HindIII and BamHI digestion. The purified downstream homologous arm amplification product of the TrxB gene was then digested with BamHI and EcoRI and ligated to the BamHI and EcoRI sites of the constructed plasmid pSET4S-SU. Double digestion with HindIII and EcoRI confirmed correct construction, and sequencing confirmed the absence of base mismatches. The obtained plasmid was named pSET4S-TrxB. Plasmid recombination, preparation, and restriction enzyme analysis were all performed according to standard methods.

[0023] 4. Screening and identification of Streptococcus suis type II ΔSodAΔTrxB double gene deletion strains

[0024] Following the method described in the paper (A Serine / threonine phoSphataSe 1 of Streptococcus suis type 2iS an important virulence factor. Fang L, Zhou J, Fan P, Yang Y, Shen H, and Fang WJ Vet Sci, 2017.18(4):p.439-447.), the obtained suicide recombinant transfer plasmid pSET4S-TrxB was electrotransformed into electrotransformed competent cells prepared from Streptococcus suis type II ΔSodA single gene deletion strain under electrotransformation parameters of 2.5 KV / cm, 200 Ω and 25 μF. After electrotransformation, the cells were plated on 100 μg / ml spectinomycin-resistant BHI plates and cultured overnight at 28°C. After single cells grew, they were cultured at 37°C for 48 h. In this process, the recombinant transfer plasmid pSET4S-TrxB undergoes a single homologous recombination with the genome of a Streptococcus suis type II ΔSodA single-gene deletion strain, resulting in a single crossover. After the single crossover is correctly identified, it is passaged to screen for mutant strains. A second homologous recombination occurs, yielding a double-gene deletion mutant. This second homologous recombination may produce two different strains: one that produces the desired Streptococcus suis type II ΔSodAΔTrxB double-gene deletion mutant; and another that produces the reverting parental strain.

[0025] Mass screening of Streptococcus suis type II ΔSodAΔTrxB double gene deletion strains was conducted using PCR. Single colonies from each generation of single-crossover culture after electroporation were selected and simultaneously inoculated into BHI plates containing both spectinomycin-free and spectinomycin-resistant bacteria. Single colonies that failed to grow under spectinomycin-resistant conditions but grew well on antibiotic-free plates were selected and cultured at 37°C with shaking as templates. PCR amplification was performed using primer pair SS2-double-T2 under the following conditions: 95°C pre-denaturation for 3 min; 34 cycles of 95°C for 30 s, 50°C for 15 s, and 68°C for 1 min, followed by a final extension at 10°C for 10 min. PCR products were detected by 0.8% agarose gel electrophoresis. For Streptococcus suis type II ΔSodAΔTrxB double gene deletion strains, primer pair SS2-double-T2 amplification should yield a 250 bp DNA fragment; for reverting to the parental strain, a 500 bp DNA fragment should be obtained. After screening for mutant strains via PCR amplification, the SS2-double-T2 primer pair was used for further amplification, and the amplified fragments were sequenced. The results confirmed the successful construction of the *Streptococcus suis* type II ΔSodA-ΔTrxB double-gene deletion mutant. This *Streptococcus suis* type II ΔSodA-ΔTrxB double-gene deletion mutant was deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2023119, and named *Streptococcus suisⅡZJJX081101-ΔSodA-ΔTrxB*.

[0026] 5. Biological characteristics analysis of a Streptococcus suis type II ΔSodA-ΔTrxB double gene deletion strain (Streptococcus suisⅡZJJX081101-ΔSodA-ΔTrxB)

[0027] (1) Comparison of biological function differences between gene-deleted strains and parental strains

[0028] Comparison of biological functions among deletion strains, and analysis of pathogenicity-related phenotypic differences among SS2-ΔSodA, SS2-ΔTrxB, SS2-ΔSodA / ΔTrxB, and their parental strains: including differences in growth or survival (BHI, whole blood), oxidative stress (H2O2, Paraquat), HEp-2 cell adhesion, bEnd3.0 cell adhesion, and survival differences in RAW264.7 cell line and primary porcine blood mononuclear macrophages. Antioxidant differential assay: Each bacterial strain was added at a 1:100 ratio to BHI medium containing different concentrations of H2O2 and Paraquat oxidative stress. Blank BHI medium served as a negative control. Bacterial OD620 was measured at time points from 1 to 8 hours, and the data were statistically analyzed. HEp-2 and bEnd.3 cells were inoculated at 2-4 × 10⁻⁴ cells / mL. 5The density of each well was determined by seeding 500 μL of 1640 cell culture medium per well into a 24-well plate and incubating overnight at 37°C. Bacterial infection of cells was performed, and the lysed cell solutions were serially diluted and spotted onto BHI plates at 10 μL per gradient. The plates were incubated overnight at 37°C, and bacterial counts were performed. Cell adhesion rates were calculated. In vitro animal infection experiments were conducted, and the pathogenicity differences in mice were analyzed. Different strains with the same infectious dose were observed for mortality curves, clinical symptoms, organ bacterial load, and histological changes, thus reflecting the role of the deleted gene in pathogenicity and the LD50. Results showed that the SodA gene-deleted strain had a significantly higher cell adhesion rate than the parental strain: the ΔSodA strain had an adhesion rate of 22.6‰ to vascular endothelial cells, while the parental strain had an adhesion rate of 0.3‰ (p<0.01); the ΔSodA strain had an adhesion rate of 21.3‰ to HEp-2 cells, while the parental strain had an adhesion rate of 0.4‰ (p<0.01). The phagocytosis rate of mouse macrophages RAW264.7 with SodA deletion was significantly higher than that of the parental macrophage (15.71% vs 0.67%, p<0.01).

[0029] (2) Protein prokaryotic expression, purification, polyclonal antibody preparation, and functional analysis

[0030] Previous research has completed the prokaryotic expression of SodA, TrxB, and Trx1 proteins and antibody preparation, demonstrating good specificity (see Basic Research). To facilitate protein isolation and purification, pET30 and pET32a vector systems were used for expression, followed by purification using a Ni-column. The purified proteins were used for differential expression detection of bacterial proteins via Western blotting. Rabbit polyclonal antibodies were prepared; if the polyclonal antibodies showed poor specificity, monoclonal antibodies were then prepared. In vitro redox function analysis of the proteins was performed using NBT (the substrate corresponding to SodA) and insulin (the substrates corresponding to Trx-1 and Trx-2). A 600 μL reaction system was infused with potassium phosphate buffer (100 mmol / L), oxidized glutathione (100 mmol / L), EDTA (2 mmol / L), bovine insulin (0.13 mmol / L), purified protein (5 mmol / L), and DTT (1 mmol / L) to initiate the reduction reaction. The OD650 absorbance was recorded every 1 minute using a spectrophotometer.

[0031] Differential expression analysis of surface proteins in whole WT and ΔSodA bacteria was performed using two-dimensional electrophoresis. Results showed that protein spots with elevated levels in WT were compared to those with elevated levels in ΔSodA. Differentially expressed protein spots were identified using biological software. Based on protein spots with significant differences in grayscale ratio, 20 spots were selected for mass spectrometry analysis, and 17 protein spots were successfully identified. Following the successful identification, six other relatively well-studied protein spots from other bacteria were selected, and quantitative real-time primers were designed to identify differences in transcriptional levels of these selected proteins.

[0032] (3) Preparation of whole bacterial protein and culture supernatant protein

[0033] Whole-cell proteins from the parental Streptococcus suis type II strain and its mutants SS2-ΔTrxB and SS2-ΔTrxB / SodA were prepared using a high-speed homogenization method with intermittent magnetic beads under low-temperature conditions to lyse the cell walls. The whole-cell proteins obtained using this method were well separated in 2D-PAGE (see “Research Basis”). Specific procedures were performed according to existing surface protein preparation methods. (ASerine / threoninephoSphataSe1ofStreptococcuSSuiStype2iSanimportantvirulencefactor.Fang L,Zhou J,Fan P,Yang Y,Shen H,andFang W.JVetSci,2017.18(4):p.439-447.)

[0034] (4) Detection of autophagy and apoptosis in RAW264.7 cells infected with deletion strains

[0035] Macrophages were infected with TrxB single deficiency and TrxB / SodA double deficiency. The difference in autophagy-related protein LC3-II / I was detected by immunoblotting. The aggregation of differentially expressed LC3-GFP in stable cell lines was observed by confocal microscopy. The changes in intracellular reactive oxygen species were detected by flow cytometry. Apoptosis and necrosis were detected by Annexin V-FITC / PI apoptosis detection kit, and the expression of apoptosis-related proteins Bcl-2 and CaSpaSe3 was detected by immunoblotting.

[0036] (5) Intracellular ROS detection

[0037] Dihydroethidium (DHE) was used for the specific detection of O2, and Dichlorodihydrofluore Sceindiacetate acetyle Ster (CM-DCFDA) was used for the detection of H2O2. DHE can be reacted with O2. - DHE, oxidized to red, and CM-DCFDA, oxidized to green dichlorofluore Scein by H2O2. In streptococcal-infected cells, after 1 and 2 hours, cell samples were digested with trypsin for 30 seconds in each well, washed with PBS, and 800 μL of each well was added to an EP tube with DHE (final concentration 3.2 mM) and CM-DCFDA (final concentration 5 mM) and mixed thoroughly. The mixture was incubated at 37°C in the dark for 15 min. The cell resuspended cells were then transferred to 5 mL flow cytometry tubes and analyzed by flow cytometry (FL1 channel). One sample was analyzed within 10 min, with a cell count of 1 × 10⁶ cells per sample.4 Cells were analyzed using a flow cytometer (Becton Dickinson, San Jose, CA, USA) (FL2 channel). The acquired data were analyzed using CellQueSt software.

[0038] The specific results of the above-mentioned experimental procedures 4 and 5 are as follows: Figures 2 to 8 As shown.

[0039] The nucleotide sequence of the deleted gene SodA in this invention is shown in SEQ ID NO: 11 (GenBank accession number: GeneID: 5099098); the nucleotide sequence of the deleted gene TrxB is shown in SEQ ID NO: 12 (GenBank accession number: GeneID: 5099385).

Claims

1. A bi- gene deletion strain of Streptococcus suis type II, characterized in that, The pig streptococcus type II double gene deletion strain has a strain preservation number of CCTCC NO: M 2023119, named as ZJJX081101-ΔSodA-ΔTrxB. Streptococcus suis II ZJJX081101-ΔSodA-ΔTrxB.

Images