Department of Animal Science, The University of Tennessee, 2506 River Drive, Knoxville, TN 37996, USA.
Microb Pathog. 2018 Feb;115:304-311. doi: 10.1016/j.micpath.2017.12.046. Epub 2017 Dec 16.
In spite of the increasing prevalence of Streptococcus uberis mastitis, its pathogenesis and associated virulence factors are not clearly defined. The aim of this study was to identify virulence associated genes and their products that can be used to develop effective vaccine to control bovine S. uberis mastitis. S. uberis was co-cultured with primary bovine mammary epithelial cells (PBMEC) or infused into mammary gland of dairy cows. The messenger RNA (mRNA) from S. uberis associated with PBMEC after 2 h or 4 h of co-culture was purified and sequenced. Results showed that virulence-associated genes such as surface lipoprotein (slp), infection induced histidine kinase (iihK), infection induced response regulator (iirR) and extracellular sugar binding protein 1 and 2 (exsbP1 and exsbP2) were among the top-up-regulated genes. To verify this observation in vivo, quantitative real time PCR (qRT - PCR) was conducted on mRNA of S. uberis recovered from milk of infected mammary glands 24 h post infection. Results revealed that in vitro up-regulated virulence-associated genes were also significantly up regulated under in vivo conditions. The iihK and iirR are flanked by exsbP1 and exsbP2 genes and this entire operon seems to be involved in adaptation to glands micro-environment, survival and colonization of the bovine mammary glands. Based on immunogenic epitope prediction of proteins encoded by these up-regulated genes during early stages of host-bacterial interactions slp, exsbP1 and exsbP2 genes were selected, cloned and expressed in E. coli. The purified recombinant proteins (rSlP, rExsbP1 & rExsbP2) reacted strongly with convalescent serum from cows experimentally infected with S. uberis confirming that they are immunogenic. These proteins may serve as potential targets to develop an effective vaccine against S. uberis mastitis.
尽管无乳链球菌乳腺炎的患病率不断增加，但它的发病机制和相关的毒力因子尚不清楚。本研究旨在鉴定与毒力相关的基因及其产物，以便开发有效的疫苗来控制牛无乳链球菌乳腺炎。将无乳链球菌与原代奶牛乳腺上皮细胞（PBMEC）共培养或注入奶牛乳腺。在共培养 2 小时或 4 小时后，从与 PBMEC 相关的无乳链球菌中提取信使 RNA（mRNA）并进行纯化和测序。结果表明，表面脂蛋白（slp）、感染诱导组氨酸激酶（iihK）、感染诱导反应调节剂（iirR）和细胞外糖结合蛋白 1 和 2（exsbP1 和 exsbP2）等毒力相关基因是上调表达基因之一。为了在体内验证这一观察结果，对感染后 24 小时从感染乳腺中回收的无乳链球菌 mRNA 进行了定量实时 PCR（qRT-PCR）。结果表明，在体外上调的毒力相关基因在体内条件下也显著上调。iihK 和 iirR 位于 exsbP1 和 exsbP2 基因的两侧，整个操纵子似乎参与了对乳腺微环境的适应、牛乳腺的生存和定植。基于宿主-细菌相互作用早期这些上调基因编码蛋白的免疫表位预测，选择、克隆并在大肠杆菌中表达了 slp、exsbP1 和 exsbP2 基因。纯化的重组蛋白（rSlP、rExsbP1 和 rExsbP2）与牛无乳链球菌感染后康复血清强烈反应，证实它们具有免疫原性。这些蛋白可能成为开发无乳链球菌乳腺炎有效疫苗的潜在靶标。
