Role of Burkholderia Cenocepacia Adhesin, AdhA, in Cystic Fibrosis Infections

描述（由申请人提供）：囊性纤维化（CF）是一种常染色体隐性遗传疾病，影响约1：2700活产婴儿。在美国，大约有30，000人患有CF。该病表现为多器官功能障碍，最常见的死亡原因是慢性肺部感染引起的呼吸衰竭。铜绿假单胞菌是大多数肺部感染的原因，影响>80%的CF患者;然而，属于洋葱伯克霍尔德菌复合体（Bcc）的细菌已经成为威胁生命的呼吸道病原体。虽然感染不到10%的CF患者，但值得注意的是，Bcc与发病率和死亡率的增加密切相关，能够在患者间传播，以及急性菌血症和肺炎综合征，称为“洋葱综合征”。在Bcc的物种中，新洋葱伯克霍尔德氏菌“流行”菌株是造成不成比例的CF肺部感染的原因，并且更可能引起严重感染、患者间传播和更高的死亡率。B。属于ET 12流行谱系的新洋葱病在CF中尤其成问题。这一谱系是独特的电缆菌毛和相关的22 kDa粘附素（AdhA）的表达。我们以前已经表明，AdhA是本地化沿着轴的电缆毛和介导的绑定和入侵的宿主细胞。缺乏缆毛或AdhA的突变体不能结合、损伤或穿过鳞状上皮细胞，表明这些结构在发病机制中的重要性。电缆皮利组装和分泌通过典型的伴侣-引导途径。相比之下，22-kDa AdhA粘附素包含在开放阅读框内，其编码具有至少12个大重复序列的>278 kDa蛋白。该蛋白质与革兰氏阴性菌和革兰氏阳性菌中推定的粘附素蛋白质具有同源性。AdhA在这个蛋白质家族中似乎是独特的，因为它被加工成小得多的形式，并与细胞表面附属物相关联。adhA基因的下游是编码与I型分泌装置同源的蛋白质的基因。我们推测AdhA粘附素在B的发病机制中起重要作用。新洋葱肺部感染。因此，在这项为期两年的探索性资助的第一个具体目标中，我们将通过比较野生型ET 12谱系菌株与我们先前在补充小鼠模型中表征的同基因AdhA突变体来确定AdhA对毒力的贡献，该模型突出了Bcc呼吸道感染的不同阶段。在第二个具体目标中，我们将确定AdhA是否由adhA基因下游的基因编码的I型分泌装置分泌。这些实验的结果将确定这种粘附素分子在毒力中的作用以及AdhA分泌的步骤。这些研究代表了开发抑制这些相互作用的试剂的合理的第一步，从而抑制CF患者Bcc感染的发病机制。公共卫生相关性：这项资助的重点是新洋葱伯克霍尔德氏菌的粘附素分子AdhA，这是囊性纤维化患者中一种新出现的肺部病原体。特别是，我们将（1）比较我们已经构建和表征的粘附素突变体与野生型菌株，在小鼠感染模型中评估发病机制的不同方面，和（2）确定这种不寻常的蛋白质粘附素是如何从B分泌的。cenocepacia。这些实验是制定预防这种细菌引起的危及生命的感染的策略的第一步。

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is an autosomal recessive disease that affects approximately 1:2700 live births. In the United States there are about 30,000 individuals living with CF. The disease manifests as multi-organ dysfunction with the most common cause of death is respiratory failure due to chronic lung infections. Pseudomonas aeruginosa is responsible for most lung infections, affecting >80% of CF patients; however, bacteria belonging to the Burkholderia cepacia complex (Bcc) have emerged as life-threatening respiratory pathogens. While infecting less than 10% of CF patients, Bcc is noteworthy for being strongly associated with increased rates of morbidity and mortality, capable of patient-to-patient spread, and a syndrome of acute bacteremia and pneumonia, referred to as "cepacia syndrome". Among the species of the Bcc, Burkholderia cenocepacia "epidemic" strains are responsible for a disproportionate degree of CF lung infections and are more likely to cause severe infections, inter-patient spread, and greater rates of mortality. B. cenocepacia belonging to the ET12 epidemic lineage are especially problematic in CF. This lineage is distinctive in the expression of both a cable pilus and an associated 22-kDa adhesin (AdhA). We have previously shown that AdhA is localized along the shaft of the cable pilus and mediates binding to and invasion of host cells. Mutants that lack either the cable pilus or AdhA fail to bind, damage, or transmigrate across squamous epithelial cells, indicating the importance of these structures in pathogenesis. Cable pili are assembled and secreted via the typical chaperone-usher pathway. In contrast, the 22-kDa AdhA adhesin is contained within an open reading frame that encodes a >278 kDa protein that has at least 12 large repeated sequences. This protein shows homology to putative adhesin proteins in both Gram-negative and Gram-positive bacteria. AdhA appears to be unique among this family of proteins in that it is processed to a much smaller form and is associated with a cell-surface appendage. Downstream of the adhA gene are genes encoding proteins homologous to a type I secretion apparatus. We hypothesize the AdhA adhesin plays an important role in the pathogenesis of B. cenocepacia lung infections. Therefore, in the first specific aim of this two-year exploratory grant, we will determine the contribution of AdhA to virulence by comparing a wild type ET12 lineage strain to an isogenic AdhA mutant, which we have previously characterized, in complementary murine models that highlight different stages of Bcc respiratory infection. In the second specific aim, we will determine whether AdhA is secreted by a type I secretion apparatus, encoded by the genes downstream of the adhA gene. Findings from these experiments will define the role of this adhesin molecule in virulence as well as the steps in the secretion of AdhA. These studies represent a logical first step in the development of reagents to inhibit these interactions and thereby the pathogenesis of Bcc infections in patients with CF. PUBLIC HEALTH RELEVANCE: This grant focuses on the adhesin molecule, AdhA, of the bacteria Burkholderia cenocepacia, which is an emerging lung pathogen in cystic fibrosis patients. In particular, we will (1) compare adhesin mutants, which we have already constructed and characterized, to the wild type strain, in mice models of infection that assess different aspects of pathogenesis, and (2) determine how this unusual protein adhesin is secreted from B. cenocepacia. These experiments are the first steps in developing strategies to prevent the life threatening infections caused by this bacterium.