MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China; National Research Center of Veterinary Biologicals engineering and Technology, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China; Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou, China.
Vet Microbiol. 2019 Jun;233:28-38. doi: 10.1016/j.vetmic.2019.04.023. Epub 2019 Apr 17.
The anti-phagocytic abilities of bacteria often affect bacterial pathogenicity. Here, random mutant library of Streptococcus equi subsp. zooepidemicus (SEZ) was constructed using transposon mutagenesis. After careful screening, 30 transposon mutants with different transposon insertion sites were identified by conducting quantitative phagocytosis and insertion-site confirmation assays, whose anti-phagocytic abilities were significantly reduced relative to the wild-type strain. Insertion sites of 19 strains were monocistronic, including genes coding membrane proteins, transporters, and enzymes with unknown pathological function, such as sadM, adhP, purD, guaA, alpha-galactosidase coding gene, ABC transporter permease coding gene, metallo-beta-lactamase coding gene, and three secreted enzyme coding genes spuZ, slaB, and endoS, as well as known virulence factor coding genes, such as hasA and szM. The insertion sites of another 11 strains were polycistronic. We focused on four monocistronic-mutant strains: MhtpZ, MspuZ, MslaB, and MendoS. The anti-phagocytic abilities of not only the mutants that were precoincubated with the recombinant proteins, but also the complement strains were significantly more pronounced than those of all four corresponding mutants. The polyclonal antiserum against SlaB or EndoS also significantly decreased the anti-phagocytic capacity of wild-type SEZ. All four mutants exhibited significantly decreased viability in whole blood and reduced lethality in mice relative to the wild-type strain. Thus, we identified a variety of new anti-phagocytic factors, particularly multiple SEZ secreted enzymes. These factors are instrumental in the phagocytic resistance of SEZ in the absence of opsonin. Our results provide a framework for further studies of SEZ pathogenesis and relevant vaccine development for novel potential targets.
细菌的抗吞噬能力通常会影响其致病性。在这里，我们使用转座子突变技术构建了马链球菌兽疫亚种（SEZ）的随机突变文库。经过仔细筛选，通过定量吞噬和插入位点确认实验，鉴定出 30 个具有不同转座子插入位点的转座子突变体，它们的抗吞噬能力与野生型菌株相比显著降低。19 株的插入位点为单顺反子，包括编码膜蛋白、转运体和具有未知病理功能的酶的基因，例如 sadM、adhP、purD、guaA、编码α-半乳糖苷酶的基因、ABC 转运体通透酶编码基因、金属β-内酰胺酶编码基因和三个分泌酶编码基因 spuZ、slaB 和 endoS，以及已知的毒力因子编码基因，如 hasA 和 szM。另外 11 株的插入位点为多顺反子。我们重点研究了四个单顺反子突变株：MhtpZ、MspuZ、MslaB 和 MendoS。不仅与重组蛋白预先孵育的突变株，而且与互补菌株的抗吞噬能力都明显更为显著。针对 SlaB 或 EndoS 的多克隆抗血清也显著降低了野生型 SEZ 的抗吞噬能力。与野生型菌株相比，所有四个突变株在全血中的存活率明显降低，在小鼠中的致死率降低。因此，我们鉴定了多种新的抗吞噬因子，特别是多种 SEZ 分泌酶。这些因子对于 SEZ 在缺乏调理素的情况下的吞噬抵抗至关重要。我们的研究结果为进一步研究 SEZ 发病机制和相关疫苗开发提供了新的潜在靶点。
