PGA exopolysaccharide in biofilm formation and pathogenicity of Aggregatibacter a

描述（由申请方提供）：伴放线聚集杆菌（Aa）和几种病原菌附着于非生物表面，并产生外泌多糖，这些外泌多糖覆盖这些定殖表面上的细菌细胞。由Aa产生的胞外多糖是一种<$-1，6-连接的N-乙酰基-D-葡糖胺（GlcNAc）单元的均聚物。Aa利用与大肠杆菌的pga、鼠疫耶尔森氏菌的hms和表皮葡萄球菌的伊卡同源的四基因操纵子产生这种外泌多糖（PGA）。在Aa中，操纵子pgaABCD携带由pgaB编码的脱乙酰酶（EC 3.5.1.41）。我们的初步数据表明，酶PgaB具有从PGA的GlcNAc去除乙酰基的能力。更有趣的是，我们发现PgaB分离预先形成的生物膜，并抑制Aa、胸膜肺炎放线杆菌和S.当加入到生长培养基中时，我们先前已经表明，也来自Aa的酶分散素B（Dsp B）通过PGA的解聚阻止几种革兰氏阴性和革兰氏阳性细菌物种的表面附着。与DspB不同，新发现的酶PgaB是PGA中GlcNAc残基的脱乙酰酶，并通过脱乙酰化改变PGA的电荷状态（正电荷的富集）。这种酶活性表明，分离/抑制的机制可能是通过增加PGA聚合物上的排斥相互作用。重要的是，脱乙酰化在生物膜去除或形成中的作用在这些生物体中，特别是在Aa中研究不足。我们将使用Aa的PgaB作为模型系统来理解这些过程。我们的长期目标是了解PGA在Aa致病性中的作用，以及PgaB的脱乙酰酶活性如何对这一作用起关键作用。我们将使用体外和体内模型来泰特PGA在Aa毒力中起关键作用的总体假设。具体而言，将研究以下目标：具体目标1。证明酶PgaB的PGA脱乙酰化能力对其膜活性至关重要。具体目标2a。证明PGA的乙酰化状态和程度有助于Aa中生物膜的形成。具体目标2b。证明胞外多糖PGA有助于Aa致病性。

DESCRIPTION (provided by applicant): Aggregatibacter actinomycetemcomitans (Aa) and several pathogenic bacteria attach to abiotic surfaces and produce exopolysaccharide that immobilize the bacterial cells on these colonized surfaces. The exopolysaccharide produced by Aa is a homopolymer of ¿-1,6-linked N-acetyl-D-glucosamine (GlcNAc) units. Aa produces this exopolysaccharide (PGA) utilizing a four-gene operon homologous to the pga of Escherichia coli, hms of Yersinia pestis, and ica of Staphylococcus epidermidis. In Aa, the operon pgaABCD carries a deacetylase enzyme (EC 3.5.1.41) encoded by pgaB. Our Preliminary Data show that the enzyme PgaB has the ability to remove acetyl groups from GlcNAc of PGA. More intriguingly, we discovered that PgaB detaches preformed biofilms and inhibits biofilm formation of Aa, Actinobacillus pleuropneumoniae and S. epidermidis when added to the growth medium. We have previously shown that an enzyme dispersin B (DspB) also from Aa prevents the surface attachment of several Gram negative as well as Gram-positive bacterial species through depolymerization of PGA. Unlike DspB, the newly discovered enzyme PgaB is a deacetylase of GlcNAc residues in PGA and through deacetylation alters the charge state of PGA (enrichment of positive charges). This enzyme activity suggests that the mechanism of detachment/inhibition might be through the increased repulsive interactions on the PGA polymer. Importantly, the role of deacetylation in biofilm removal or formation is understudied in these organisms, especially in Aa. We will use PgaB from Aa as a model system to understand these processes. Our long-range goal is to understand the role of PGA in Aa pathogenicity and how the deacetylase activity of PgaB is central to this role. We will use both in vitro as well as in vivo models to tet the overall hypothesis that PGA plays a critical role in Aa virulence. Specifically, the following aims will be studied: Specific Aim 1. Demonstrate that PGA deacetylating ability of the enzyme PgaB is critical for its antibiofilm activity. Specific Aim 2a. Demonstrate that the status and degree of acetylation of PGA contributes to the biofilm formation in Aa. Specific Aim 2b. Demonstrate that the exopolysaccharide PGA contributes to Aa pathogenicity.