Sanders J D, Cope L D, Jarosik G P, Maciver I, Latimer J L, Toews G B, Hansen E J
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Infect Immun. 1993 Sep;61(9):3966-75. doi: 10.1128/iai.61.9.3966-3975.1993.
The major outer membrane protein (OmpP2) of nontypeable Haemophilus influenzae (NTHI) has been shown to vary markedly with respect to both size and the presence of specific surface-exposed epitopes among strains of this unencapsulated pathogen. In contrast, the OmpP2 proteins of H. influenzae type b (Hib) strains are well conserved at the level of primary protein structure and have in common several surface-exposed antigenic determinants that have not been detected in NTHI strains. The availability of an isogenic, avirulent Hib ompP2 mutant made it possible to investigate whether an NTHI OmpP2 protein could function properly in the Hib outer membrane. A plasmid shuttle vector (pGJB103) was used to clone the ompP2 gene from NTHI TN106 into a recombination-deficient H. influenzae strain in which expression of the NTHI OmpP2 protein was detected by means of an NTHI TN106 OmpP2-specific monoclonal antibody. The amino acid sequence of this NTHI OmpP2 protein, as deduced from the nucleotide sequence of the NTHI TN106 ompP2 gene, was determined to be 83% identical to that of the Hib OmpP2 protein. Transformation of this cloned NTHI ompP2 gene into the Hib ompP2 mutant yielded a Hib transformant strain that expressed the NTHI OmpP2 protein. Expression of this NTHI OmpP2 protein allowed the Hib ompP2 mutant, which normally grows poorly in vitro, to grow in a manner indistinguishable from that of the wild-type Hib strain. More importantly, the introduction of this functional NTHI ompP2 gene into the avirulent Hib ompP2 mutant restored the virulence of this strain to wild-type levels. These results indicate that an NTHI OmpP2 protein can be expressed and function properly in the Hib outer membrane.
不可分型流感嗜血杆菌（NTHI）的主要外膜蛋白（OmpP2）在大小和特定表面暴露表位的存在方面，在这种无荚膜病原体的不同菌株间有显著差异。相比之下，b型流感嗜血杆菌（Hib）菌株的OmpP2蛋白在一级蛋白质结构水平上高度保守，并且具有一些在NTHI菌株中未检测到的共同表面暴露抗原决定簇。一个同基因的、无毒力的Hib ompP2突变体的可得性使得研究NTHI OmpP2蛋白是否能在Hib外膜中正常发挥功能成为可能。使用一个质粒穿梭载体（pGJB103）将来自NTHI TN106的ompP2基因克隆到一个重组缺陷型流感嗜血杆菌菌株中，通过一种NTHI TN106 OmpP2特异性单克隆抗体检测NTHI OmpP2蛋白的表达。根据NTHI TN106 ompP2基因的核苷酸序列推导的这种NTHI OmpP2蛋白的氨基酸序列，被确定与Hib OmpP2蛋白的氨基酸序列有83%的同一性。将这个克隆的NTHI ompP2基因转化到Hib ompP2突变体中产生了一个表达NTHI OmpP2蛋白的Hib转化菌株。这种NTHI OmpP2蛋白的表达使得通常在体外生长不良的Hib ompP2突变体能够以与野生型Hib菌株难以区分的方式生长。更重要的是，将这个功能性的NTHI ompP2基因导入无毒力的Hib ompP2突变体中，使该菌株的毒力恢复到野生型水平。这些结果表明，一种NTHI OmpP2蛋白可以在Hib外膜中表达并正常发挥功能。
