Pneumococcal capsule and host innate immunity

描述（由申请人提供）：肺炎链球菌是一种革兰氏阳性细菌，可表达90多种不同的荚膜类型（血清型），通常作为鼻咽（NPX）中的荚膜携带，但也可通过侵入更深的组织引起严重疾病。流行病学研究表明，不同血清型肺炎球菌的携带率和侵袭效率差异很大（>100倍）。随着肺炎球菌结合疫苗的使用，最常携带的血清型发生了变化（称为“血清型转变”），血清型11 A已成为其中之一。然而，血清型11 A具有非常低的侵袭性，并且11 A的低侵袭性和高携带率的分子基础尚不清楚。 通过灭活血清型11 A的wcjE，从而使其失去其多糖的乙酰基，我们发现，在个体患者的侵袭性肺炎球菌感染期间，11 A经常变成11 E。我们还发现11 A比11 E对溶菌酶的抗性更强，并且L-纤维胶凝蛋白与11 A结合而不与11 E结合。L-纤维胶凝蛋白是最近在血液中发现的先天调理素，其在结构上类似于甘露糖结合凝集素（MBL），靶向乙酰基，并通过激活MBL相关丝氨酸蛋白酶-2（MASP-2）来启动凝集素依赖性补体级联。基于这些观察结果，我们提出了总体假设，即血清型11 A保留WcjE以抵抗溶菌酶，从而在NPX中存活，并且其失去WcjE功能以变成11 E，从而避免L-纤维胶凝蛋白并在血液中存活。 为了研究该假设，我们将确定1）通过测试L-纤维胶凝蛋白调理血清型11 A而非11 E的吞噬作用的能力，通过测量患有血清型11 A肺炎球菌脓毒症的个体中L-纤维胶凝蛋白和MASP-2的血清水平，并通过测试另一种具有L-纤维胶凝蛋白反应性的常见wcjE+血清型以寻找未发现的无L-纤维胶凝蛋白反应性的wcjE-亚型; 2）血清型11 A的WcjE是否使肺炎球菌对Murami-通过比较血清型11 A和11 E分离物对正常和突变溶菌酶的体外抗性以及在有或没有溶菌酶的小鼠中的鼻咽携带，测定溶菌酶的活性;以及3）WcjE是否通过帮助Adr（药物抗性衰减剂）修饰肽聚糖而增强溶菌酶抗性。 我们的工作是广泛相关的，因为wcjE是在许多不同的肺炎球菌血清型中发现的，因为很少有人知道L-纤维胶凝蛋白在细菌感染中的作用。我们的工作也与肺炎球菌疫苗的开发直接相关，因为11 A已成为最常见的鼻咽血清型之一，因为其他新出现的血清型可能与11 A相似：它们可能与先天调理素相互作用，因此即使它们变得更常见，也不会对健康构成威胁。我们的工作还将扩大“血清型”的概念，包括血清型特异性与先天调理素的相互作用，以及抗体，这个新的概念可能会带来进一步的创新，针对许多病原体的疫苗设计。

DESCRIPTION (provided by applicant): Streptococcus pneumoniae is a Gram-positive bacterium that can express more than 90 different capsule types (serotypes) and is often carried as a commensal in the human nasopharynx (NPX), but can also cause serious diseases by invading deeper tissues. Epidemiologic studies show that the prevalence of carriage and the efficiency of invasion are highly variable (>100-fold) among different pneumococcal serotypes. With the use of pneumococcal conjugate vaccines, the most commonly carried serotypes have changed (called "serotype shift"), with serotype 11A having become one. However, serotype 11A has a very low invasiveness, and the molecular basis for 11A's low invasiveness and high rate of carriage is not known. By inactivating serotype 11A's wcjE and thus causing it to lose an acetyl group from its polysaccharide, we discovered that 11A often becomes 11E during the invasive pneumococcal infection of individual patients. We also found that 11A is more resistant to lysozyme than 11E is and that L-ficolin binds to 11A but not to 11E. L-ficolin is a recently discovered innate opsonin in the blood that resembles mannose binding lectin (MBL) in structure, targets acetyl groups, and initiates the lectin-dependent complement cascade by activating MBL- associated serine protease-2 (MASP-2). Based on these observations, we propose the overarching hypothesis that serotype 11A retains WcjE to resist lysozyme and thus survive in the NPX and that it loses WcjE function to become 11E and thus avoid L-ficolin and survive in the blood. To investigate this hypothesis, we will determine 1) whether L-ficolin is protective against serotype 11A but not against 11E isolates by testing L-ficolin's ability to opsonize serotype 11A but not 11E for phagocytosis, by measuring the serum levels of L-ficolin and MASP-2 in individuals with serotype 11A pneumococcal sepsis, and by testing another common wcjE+ serotype with L-ficolin reactivity for an undiscovered wcjE- subtype without L-ficolin reactivity; 2) whether the WcjE of serotype 11A makes pneumococci resistant to the murami- dase activity of lysozyme by comparing serotypes 11A and 11E isolates for their in vitro resistance to normal and mutant lysozymes and for their nasopharyngeal carriage in mice with or without lysozyme; and 3) whether WcjE enhances lysozyme resistance by helping Adr (attenuator of drug resistance) modify peptidoglycan. Our work is broadly relevant because wcjE is found in many different pneumococcal serotypes and because very little is known about L-ficolin's role in bacterial infections. Our work is also directly relevant to pneumococcal vaccin development because 11A has become one of the most common nasopharyngeal serotypes and because other newly emerging serotypes might be like 11A: they may interact with innate opsonins and thus be less of a health threat even if they become more common. Our work would also expand the concept of "serotype" to include serotype specific interactions with innate opsonins, as well as antibodies, and this new concept may bring further innovations in vaccine designs against many pathogens.