Role of phase-variable DNA methylase in virulence of Haemophilus influenzae

描述（由申请人提供）：流感嗜血杆菌是定植在人鼻咽部的正常菌群的一员。它也是上呼吸道感染的一个原因，如中耳炎和支气管炎，以及全身感染，如菌血症和脑膜炎。理想情况下，流感嗜血杆菌疾病的预防或治疗只会影响与疾病相关的细菌，而不会扰乱正常的菌群。这项工作的长期目标是了解从无症状定植到粘膜或全身感染的转变。我们假设鼻咽细菌适应新环境（如中耳或血液）的机制之一是DNA甲基化酶基因的相位变化，mod.表面结构（如外膜蛋白和脂多糖）的相位变化已经在流感嗜血杆菌中研究了几年。mod基因包含一个串联重复区域，类似于在流感嗜血杆菌中发现的其他已被充分表征的相位变量基因，但直到最近才知道mod相位变化的生理作用。我们的合作者通过微阵列分析比较了mod缺失突变体与mod+亲本，并报道mod影响至少15个基因的转录（Srikhanta et al. 2005 PNAS 102:5547-51），显然是通过启动子的甲基化。并非所有这些基因都有已知的功能，但通常被mod表达上调的基因与营养获取有关，而一些被mod表达下调的基因在热休克组中。有人提出，mod的相位变化是其他几个基因随机协调开关的机制。这些研究使用了实验室适应的无毒菌株Rd KW20。在流感嗜血杆菌的临床分离株和其他粘膜生物（如脑膜炎奈瑟菌）中也发现了相位可变mod基因。本探索性项目将研究强毒流感嗜血杆菌基因调控的新机制。

DESCRIPTION (provided by applicant): Haemophilus influenzae is a member of the normal bacterial flora colonizing the human nasopharynx. It is also a cause of infections of the upper respiratory tract, such as otitis media and bronchitis, and of systemic infections, such as bacteremia and meningitis. Ideally, prevention or treatment of H. influenzae disease would affect only disease-associated bacteria without perturbing normal flora. The long-term goal of this work is to understand the transition from asymptomatic colonization to mucosal or systemic infection. We hypothesize that one of the mechanisms by which nasopharyngeal bacteria adapt to a new environment such as the middle ear or the bloodstream is phase variation of a DNA methylase gene, mod. Phase variation of surface structures such as outer membrane proteins and lipopolysaccharide has been studied in H. influenzae for several years. The mod gene contains a tandem repeat region similar to those identified in other, well-characterized phase-variable genes in H. influenzae, but a physiologic role for mod phase variation was unknown until recently. Our collaborators compared a mod deletion mutant with a mod+ parent by microarray analysis and reported that mod affects transcription of at least 15 genes (Srikhanta et al. 2005 PNAS 102:5547-51) apparently via methylation of promoters. Not all of these genes have known functions, but generally genes upregulated by mod expression are involved in nutrient acquisition, while several of those downregulated by mod expression are in the heat-shock group. It is proposed that phase variation of mod is a mechanism for random, coordinated on/off switching of several other genes. These studies used the laboratory-adapted, avirulent strain Rd KW20. Phase-variable mod genes have also been identified in clinical isolates of H. influenzae and in other mucosal organisms such as Neisseria meningitidis. This exploratory project will study this novel mechanism of gene regulation in virulent H. influenzae, (i) using microarrays to identify mod-related genes in clinical isolates and (ii) examining the effect of mod phase in experimental infections. Finding that mod regulation is common in clinical isolates and affects pathogenesis will provide the basis for future studies on the physiological adaptation of bacteria to different anatomic niches within the human host. Haemophilus influenzae is a bacterium that usually grows harmlessly in the human throat, but is also able to cause disease. If we can understand how these bacteria change as they move from the throat to sites of infection (such as the ear, lungs, or blood), we may someday be able to prevent disease.