Abstract：
Bacteria possess the ability to develop diverse and ingenious strategies to outwit the host immune system, and proteases are one of the many weapons employed by bacteria. This study sought to identify S. agalactiae additional serine protease and determine its role in virulence. The S. agalactiae THN0901 genome features one S8 family serine peptidase B (SfpB), acting as a secreted and externally exposed entity. A S8 family serine peptidase mutant strain (ΔsfpB) and complement strain (CΔsfpB) were generated through homologous recombination. Compared to the wild-type strain THN0901, the absorption of EtBr dyes was significantly reduced (P < 0.01) in ΔsfpB, implying an altered cell membrane permeability. In addition, the ΔsfpB strain had a significantly lower survival rate in macrophages (P < 0.01) and a 61.85 % lower adhesion ability to the EPC cells (P < 0.01) compared to THN0901. In the in vivo colonization experiment using tilapia as a model, 210 fish were selected and injected with different bacterial strains at a concentration of 3 × 106 CFU/tail. At 6, 12, 24, 48, 72 and 96 h post-injection, three fish were randomly selected from each group and their brain, liver, spleen, and kidney tissues were isolated. Subsequently, it was demonstrated that the ΔsfpB strain exhibited a markedly diminished capacity for colonization in tilapia. Additionally, the cumulative mortality of ΔsfpB in fish after intraperitoneal injection was reduced by 19.92-23.85 %. In conclusion, the findings in this study have demonstrated that the SfpB plays a significant role in S. agalactiae cell membrane stability and immune evasion. The immune evasion is fundamental for the development and transmission of invasive diseases, the serine protease SfpB may be a promising candidate for the development of antimicrobial agents to reduce the transmission of S. agalactiae.
摘要：
细菌具有开发多样化和巧妙的策略来胜过宿主免疫系统的能力，蛋白酶是细菌使用的众多武器之一。这项研究试图鉴定无乳链球菌的其他丝氨酸蛋白酶并确定其在毒力中的作用。无乳链球菌THN0901基因组具有一个S8家族丝氨酸肽酶B（SfpB），充当分泌和外部暴露的实体。通过同源重组产生S8家族丝氨酸肽酶突变株(ΔsfpB)和补体株(CΔsfpB)。与野生型菌株THN0901相比，EtBr染料在ΔsfpB中的吸收显着降低（P<0.01），暗示细胞膜通透性改变。此外,与THN0901相比，ΔsfpB菌株在巨噬细胞中的存活率显着降低（P<0.01），对EPC细胞的粘附能力降低了61.85％（P<0.01）。在以罗非鱼为模型的体内定植实验中，选择210条鱼并以3×106CFU/尾的浓度注射不同的细菌菌株。注射后6、12、24、48、72和96小时，每组随机选择三条鱼，它们的大脑，肝脏,脾,脾分离肾组织。随后，已证明，ΔsfpB菌株在罗非鱼中的定殖能力显着降低。此外,腹腔注射后，鱼中ΔsfpB的累积死亡率降低了19.92-23.85％。总之,这项研究的结果表明，SfpB在无乳链球菌细胞膜稳定性和免疫逃避中起着重要作用。免疫逃避是侵袭性疾病发展和传播的基础,丝氨酸蛋白酶SfpB可能是开发抗微生物剂以减少无乳链球菌传播的有希望的候选物。
