Opsonophagocytosis-Inhibiting Mac Protein of Group A Streptococcus: Identification and Characteristics of Two Genetic Complexes

Recently, it was reported that a streptococcal Mac protein (designated Mac(5005)) made by serotype M1 group A Streptococcus (GAS) is a homologue of human CD11b that inhibits opsonophagocytosis and killing of GAS by human polymorphonuclear leukocytes (PMNs) (B. Lei, F. R. DeLeo, N. P. Hoe, M. R. Graham, S. M. Mackie, R. L. Cole, M. Liu, H. R. Hill, D. E. Low, M. J. Federle, J. R. Scott, and J. M. Musser, Nat. Med. 7:1298-1305, 2001). To study mac variation and expression of the Mac protein, the gene in 67 GAS strains representing 36 distinct M protein serotypes was sequenced. Two distinct genetic complexes were identified, and they were designated complex I and complex II. Mac variants in each of the two complexes were closely related, but complex I and complex II variants differed on average at 50.66 ± 5.8 amino acid residues, most of which were located in the middle one-third of the protein. Complex I Mac variants have greater homology with CD11b than complex II variants. GAS strains belonging to serotypes M1 and M3, the most abundant M protein serotypes responsible for human infections in many case series, have complex I Mac variants. The mac gene was cloned from representative strains assigned to complexes I and II, and the Mac proteins were purified to apparent homogeneity. Both Mac variants had immunoglobulin G (IgG)-endopeptidase activity. In contrast to Mac(5005) (complex I), Mac(8345) (complex II) underwent autooxidation of its cysteine residues, resulting in the loss of IgG-endopeptidase activity. A Mac(5005) Cys94Ala site-specific mutant protein was unable to cleave IgG but retained the ability to inhibit IgG-mediated phagocytosis by human PMNs. Thus, the IgG-endopeptidase activity was not essential for the key biological function of Mac(5005). Although Mac(5005) and Mac(8345) each have an Arg-Gly-Asp (RGD) motif, the proteins differed in their interactions with human integrins α(v)β(3) and α(IIb)β(3). Binding of Mac(5005) to integrins α(v)β(3) and α(IIb)β(3) was mediated primarily by the RGD motif in Mac(5005), whereas binding of Mac(8345) involved the RGD motif and a region in the middle one-third of the molecule whose sequence is different in Mac(8345) and Mac(5005). Taken together, the data add to the emerging theme in GAS pathogenesis that allelic variation in virulence genes contributes to fundamental differences in host-pathogen interactions among strains.

近日有研究报道，M1群A链球菌（group A Streptococcus, GAS）血清型菌株分泌的链球菌Mac蛋白（命名为Mac(5005)）是人类CD11b的同源蛋白，可抑制人类多形核白细胞（polymorphonuclear leukocytes, PMNs）对GAS的调理吞噬作用与杀伤活性（B. Lei、F. R. DeLeo、N. P. Hoe等，Nat. Med. 7:1298-1305, 2001）。为研究Mac蛋白的基因变异与表达情况，研究人员对代表36种不同M蛋白血清型的67株GAS菌株中的mac基因进行了测序。研究共鉴定出两类独立的遗传复合体，分别命名为复合体I与复合体II。两类复合体各自的Mac变异体序列相似度较高，但复合体I与复合体II的变异体平均在50.66±5.8个氨基酸残基位点存在差异，其中多数差异位点位于蛋白的中间三分之一区域。相较于复合体II的Mac变异体，复合体I的Mac变异体与CD11b的同源性更高。在诸多临床病例系列中，M1和M3血清型是引发人类感染的最常见M蛋白血清型，这两类血清型对应的GAS菌株均携带复合体I型Mac变异体。研究人员从分别隶属于复合体I和II的代表性菌株中克隆了mac基因，并将表达的Mac蛋白纯化至表观均一状态。两类Mac变异体均具备免疫球蛋白G（immunoglobulin G, IgG）内肽酶活性。与复合体I型的Mac(5005)不同，复合体II型的Mac(8345)会发生半胱氨酸残基的自身氧化，进而丧失IgG内肽酶活性。Mac(5005)的Cys94Ala位点特异性突变蛋白无法切割IgG，但仍能抑制人类PMNs介导的IgG依赖性吞噬作用。由此可见，IgG内肽酶活性并非Mac(5005)核心生物学功能所必需的特性。尽管Mac(5005)与Mac(8345)均含有精氨酸-甘氨酸-天冬氨酸（Arg-Gly-Asp, RGD）基序，但二者与人类整合素α(v)β(3)和α(IIb)β(3)的相互作用模式存在差异。Mac(5005)与整合素α(v)β(3)和α(IIb)β(3)的结合主要由其自身的RGD基序介导，而Mac(8345)的结合则同时依赖于RGD基序，以及该蛋白中间三分之一区域的一段序列——该区域在Mac(8345)与Mac(5005)中的序列存在显著差异。综上，本研究数据进一步丰富了GAS致病机制的新兴研究范式：毒力基因的等位基因变异是导致不同菌株与宿主间相互作用存在本质差异的关键因素。