Collinson Lucy M, Rangarajan Minnie, Curtis Michael A
MRC Molecular Pathogenesis Group, Department of Oral Microbiology, St Bartholomew's and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, University of London,32 Newark Street, London E1 2AA,UK.
Microbiology (Reading). 1998 Sep;144 ( Pt 9):2487-2496. doi: 10.1099/00221287-144-9-2487.
Proteases of Porphyromonas gingivalis are considered to be important factors in the virulence of this organism. A non-pigmenting mutant of P. gingivalis W50 (W50/BE1) has been shown to be less virulent in animal models and to produce significantly less Arg-specific protease activity than the parent strain. Three proteases are present in the culture supernatant of P. gingivalis W50: RI, RIA and RIB. All three proteases are derived from prpR1, which encodes a polypeptide of 1706 amino acids that is organized into distinct domains (pro, alpha, beta and gamma). The aim of the present investigation was to purify and characterize the Arg-specific proteases produced by the avirulent W50/BE1 strain. Significant differences were observed between the proteases of P. gingivalis W50 and W50/BE1. The levels of RI present in the culture supernatant of W50/BE1 were lower than those present in W50, and RIA and RIB were absent. RI from W50/BE1 was composed of three polypeptide chains, unlike the enzyme from W50, which is a heterodimer. The remainder of the Arg-specific protease activity in W50/BE1 was derived from a second gene, prR2, and was present in two fractions, RIIAs/BE (soluble) and RIIAv/BE (vesicle-bound). This activity contained two peptide chains: a approximately 54 kDa chain corresponding to the protease domain and a approximately 26 kDa chain, derived from the propeptide domain of the PrRII precursor. No enzyme with large glycan additions, equivalent to RIB in the vesicle fraction of the wild-type W50, was present. These data indicate that the reduced level of extracellular protease activity in W50/BE1 reflects reduced synthesis and/or export of prpR1 enzymes, which is only partially compensated by synthesis of prR2-derived enzymes, and that all of these proteases undergo altered post-translational modification compared to the parent strain.
牙龈卟啉单胞菌的蛋白酶被认为是该菌毒力的重要因素。牙龈卟啉单胞菌W50的一个无色素突变体（W50/BE1）已被证明在动物模型中毒力较低，且与亲本菌株相比，其精氨酸特异性蛋白酶活性显著降低。牙龈卟啉单胞菌W50的培养上清液中有三种蛋白酶：RI、RIA和RIB。这三种蛋白酶均来源于prpR1，该基因编码一个由1706个氨基酸组成的多肽，该多肽被组织成不同的结构域（前肽、α、β和γ）。本研究的目的是纯化和鉴定无毒力的W50/BE1菌株产生的精氨酸特异性蛋白酶。观察到牙龈卟啉单胞菌W50和W50/BE1的蛋白酶之间存在显著差异。W50/BE1培养上清液中RI的水平低于W50中的水平，且不存在RIA和RIB。与W50的酶（一种异二聚体）不同，W50/BE1的RI由三条多肽链组成。W50/BE1中精氨酸特异性蛋白酶活性的其余部分来自第二个基因prR2，并以两种组分存在，即RIIAs/BE（可溶性）和RIIAv/BE（囊泡结合型）。这种活性包含两条肽链：一条约54 kDa的链对应于蛋白酶结构域，另一条约26 kDa的链，来源于PrRII前体的前肽结构域。不存在与野生型W50囊泡部分中的RIB相当的、带有大量聚糖添加物的酶。这些数据表明，W50/BE1细胞外蛋白酶活性水平的降低反映了prpR1酶合成和/或分泌的减少，而prR2衍生酶的合成仅部分补偿了这一减少，并且与亲本菌株相比，所有这些蛋白酶都经历了翻译后修饰的改变。
