Characterization of a bacteriophage lysin (Ply700) from Streptococcus uberis
Characterization of a bacteriophage lysin (Ply700) from Streptococcus uberis
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DOI：
10.1016/j.vetmic.2007.12.004
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发表时间：
2008-07-27
影响因子：
3.3
通讯作者：
Kerr, David E.
中科院分区：
文献类型：
--
作者：
Celia, Laura K.;Nelson, Daniel;Kerr, David E.
噬菌体裂解酶的抗菌特性可能在未来的乳腺炎控制计划中具有重要意义。从暴露于丝裂霉素C的链球菌菌株（ATCC 700407）中分离原噬菌体。噬菌体DNA的部分测序揭示了基于与其他链球菌噬菌体溶素的序列相似性的推定溶素。推定的溶素（Ply 700）在大肠杆菌中重组表达，并层析纯化。将纯化的Ply 700加入S.链球菌、酿脓链球菌和停乳链球菌引起快速的钙依赖性溶解，而对无乳链球菌、金黄色葡萄球菌或大肠杆菌几乎没有活性。杀死S。Ply 700（50 μ g/ml）对牛奶的抑制作用通过平板计数试验得到证实。活性与细菌的初始浓度相关，因为在接种物剂量为约4500 cfu/ml时观察到81%的杀灭（P < 0.05），而当接种物减少至约600 cfu/ml时观察到81%的杀灭（P < 0.01）。相反，在悬浮于测定缓冲液中的平行培养物中观察到完全灭菌，表明乳中的因子能够中和溶素。作为GFP融合蛋白的一个组成部分，C-末端结构域的功能表征揭示了其钙依赖性结合S.不稳定。C-末端结构域可能在靶向S中具有效用。但仍需确定溶素本身是否在牛奶中具有足够的效力以有效地用于控制S.乳房炎C）2007 Elsevier B. V.保留所有权利。
The antibacterial properties of bacteriophage lytic enzymes may be of importance in future mastitis control programs. A prophage was isolated from a strain of Streptococcus uberis (ATCC 700407) following exposure to mitomycin C. Partial sequencing of the phage DNA revealed a putative lysin based on sequence similarity to other streptococcal phage lysins. The putative lysin (Ply700) was recombinantly expressed in Escherichia coli, and chromatographically purified. Addition of the purified Ply700 to bacterial suspensions of S. uberis, Streptococcus pyogenes, and Streptococcus dysgalactiae caused a rapid, calcium-dependent lysis while there was little activity against Streptococcus agalactiae, Staphylococcus aureus, or E coli. Killing of S. uberis in milk by Ply700 (50 mu g/ml) was confirmed by plate count assay. Activity was related to the initial concentration of bacteria in that 81 % killing (P < 0.05) was observed with an inoculating dose of approximately 4500 cfu/ml, while 81% killing (P < 0.01) was observed when the inoculum was reduced to approximately 600 cfu/ml. In contrast, complete sterilization was observed in parallel cultures suspended in assay buffer indicating that factors in milk are able to neutralize the lysin. Functional characterization of the C-terminal domain, as a component of a GFP fusion protein, revealed its calciumdependent ability to bind to S. uberis. The C-terminal domain may have utility in targeting S. uberis while it remains to be determined if the lysin by itself has sufficient potency in milk for effective use in the control of S. uberis mastitis. C) 2007 Elsevier B.V. All rights reserved.