Benešík Martin, Nováček Jiří, Janda Lubomír, Dopitová Radka, Pernisová Markéta, Melková Kateřina, Tišáková Lenka, Doškař Jiří, Žídek Lukáš, Hejátko Jan, Pantůček Roman
Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, Brno, 611 37, Czech Republic.
CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, 625 00, Czech Republic.
Virus Genes. 2018 Feb;54(1):130-139. doi: 10.1007/s11262-017-1507-2. Epub 2017 Aug 29.
The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.
自发宿主范围突变体812F1和K1/420源自多价噬菌体812，该噬菌体与噬菌体K几乎相同，属于肌尾噬菌体科凯病毒属。噬菌体K1/420用于葡萄球菌感染的噬菌体疗法。这些突变体的溶菌酶LysF1由N端半胱氨酸-组氨酸依赖性氨基水解酶/肽酶（CHAP）结构域和C端SH3b细胞壁结合结构域组成，与野生型溶菌酶相比，中间的酰胺酶结构域缺失。在这项工作中，LysF1及其两个结构域均作为重组蛋白制备并分析了其功能。LysF1对43种测试的葡萄球菌中的31种具有抗菌作用。SH3b结构域影响LysF1的抗菌活性，因为仅包含CHAP结构域的截短变体的裂解活性降低。SH3b结构域的共沉降分析结果表明，它能够结合肽桥不同的三种纯化葡萄球菌肽聚糖11.2、11.3和11.8，也能结合乳房链球菌的肽聚糖11.5型，并且使用与绿色荧光蛋白的融合蛋白和荧光显微镜在体内验证了这种能力。使用包括核磁共振在内的几种不同方法，我们尚未证实先前提出的SH3b结构域与细菌细胞壁中五肽甘氨酸桥的相互作用。新的天然产生的缺失突变体溶菌酶LysF1比LysK小，具有广泛的裂解谱，因此是一种适合实际应用的酶。SH3b结构域覆盖所有已知葡萄球菌肽聚糖类型的结合谱是通过将其与更有效的催化结构域结合来创建新的嵌合溶菌酶的一个有前景的特征。
