Inzana Thomas J, Glindemann Gretchen, Cox Andrew D, Wakarchuk Warren, Howard Michael D
Center for Molecular Medicine and Infectious Diseases, Virginia Polytechnic Institute & State University, Blacksburg, Virginia 24061-0342, USA.
Infect Immun. 2002 Sep;70(9):4870-9. doi: 10.1128/IAI.70.9.4870-4879.2002.
Haemophilus somnus isolates from cases of thrombotic meningoencephalitis, pneumonia, and other disease sites are capable of undergoing a high rate of phase variation in the oligosaccharide component of their lipooligosaccharides (LOS). In contrast, the LOS of commensal strains isolated from the normal reproductive tract phase vary little or not at all. In addition, the LOS of H. somnus shares conserved epitopes with LOS from Neisseria gonorrhoeae, Haemophilus influenzae, and other species that can incorporate sialic acid into their LOS. We now report that growth of disease isolates of H. somnus with CMP-N-acetylneuraminic acid (CMP-NeuAc) or NeuAc added to the medium resulted in incorporation of NeuAc into the LOS. However, NeuAc was not incorporated into the LOS of commensal isolates and one disease isolate following growth in medium containing CMP-NeuAc or NeuAc. Sialylated LOS was detected by an increase in the molecular size or an increase in the amount of the largest-molecular-size LOS electrophoretic bands, which disappeared following treatment with neuraminidase. Sialylated LOS could also be detected by reactivity with Limax flavus agglutinin lectin, which is specific for sialylated species, by dot blot assay; this reactivity was also reversed by neuraminidase treatment. H. somnus strain 2336 LOS was found to contain some sialic acid when grown in medium lacking CMP-NeuAc or NeuAc, although supplementation enhanced NeuAc incorporation. In contrast strain 738, an LOS phase variant of strain 2336, was less extensively sialylated when the growth medium was supplemented with CMP-NeuAc or NeuAc, as determined by electrophoretic profiles and electrospray mass spectrometry. The sialyltransferase of H. somnus strain 738 was confirmed to preferentially sialylate the Gal(beta)-(1-3)-GlcNAc component of the lacto-N-tetraose structure by capillary electrophoresis assay. Enhanced sialylation of the strain 2336 LOS inhibited the binding of monoclonal antibodies to LOS by enzyme immunoassay and Western blotting. Furthermore, sialylation of the LOS enhanced the resistance of H. somnus to the bactericidal action of antiserum to LOS. Sialylation and increased resistance to killing by normal serum also occurred in a deletion mutant that was deficient in the terminal Gal-GlcNAc disaccharide. LOS sialylation may therefore be an important virulence mechanism to protect H. somnus against the host immune system.
从血栓性脑膜脑炎、肺炎及其他疾病部位分离出的睡眠嗜血杆菌菌株，其脂寡糖（LOS）的寡糖成分能够进行高频相变。相比之下，从正常生殖道分离出的共生菌株的LOS很少发生相变或根本不变。此外，睡眠嗜血杆菌的LOS与淋病奈瑟菌、流感嗜血杆菌及其他能将唾液酸掺入其LOS的物种的LOS具有保守表位。我们现在报告，在培养基中添加CMP-N-乙酰神经氨酸（CMP-NeuAc）或NeuAc培养睡眠嗜血杆菌的疾病分离株时，NeuAc会掺入LOS。然而，在含有CMP-NeuAc或NeuAc的培养基中培养后，NeuAc并未掺入共生分离株及一种疾病分离株的LOS。通过分子大小增加或最大分子大小的LOS电泳条带数量增加来检测唾液酸化的LOS，用神经氨酸酶处理后这些条带消失。通过斑点印迹法用对唾液酸化物种特异的黄蛞蝓凝集素检测，也能检测到唾液酸化的LOS；这种反应性也会被神经氨酸酶处理逆转。发现睡眠嗜血杆菌菌株2336在缺乏CMP-NeuAc或NeuAc的培养基中生长时，其LOS含有一些唾液酸，尽管添加后会增强NeuAc的掺入。相比之下，菌株2336的LOS相变变体菌株738在添加CMP-NeuAc或NeuAc的生长培养基中唾液酸化程度较低，这通过电泳图谱和电喷雾质谱法确定。通过毛细管电泳分析证实，睡眠嗜血杆菌菌株738的唾液酸转移酶优先使乳糖-N-四糖结构的Gal(β)-(1-3)-GlcNAc成分唾液酸化。通过酶免疫测定和蛋白质印迹法，菌株2336的LOS唾液酸化增强抑制了单克隆抗体与LOS的结合。此外，LOS的唾液酸化增强了睡眠嗜血杆菌对LOS抗血清杀菌作用的抗性。在缺乏末端Gal-GlcNAc二糖的缺失突变体中也发生了唾液酸化和对正常血清杀伤抗性的增加。因此，LOS唾液酸化可能是保护睡眠嗜血杆菌抵御宿主免疫系统的一种重要毒力机制。
