PGA exopolysaccharide in biofilm formation and pathogenicity of Aggregatibacter a

描述(申请人提供)：Aggregatibacter放线菌(AA)和几种病原菌附着在非生物表面并产生胞外多糖，使这些定植表面的细菌细胞固定化。AA产生的胞外多糖是由N-乙酰-D-氨基葡萄糖(GlcNAc)单元组成的均聚物。AA利用与大肠杆菌的PGA、鼠疫耶尔森菌的HMS和表皮葡萄球菌的ICA同源的四个基因操纵子来产生这种胞外多糖(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，PGaB酶具有从PGA的GlcNAc中去除乙酰基的能力。更有趣的是，我们发现当添加到生长介质中时，PgaB可以分离预先形成的生物膜，并抑制AA、胸膜肺炎放线杆菌和表皮葡萄球菌的生物膜形成。我们以前已经证明，同样来自AA的一种酶分散体B(DspB)通过解聚PGA来防止几种革兰氏阴性和革兰氏阳性细菌的表面附着。与DspB不同，新发现的PGaB酶是PGA中GlcNAc残基的脱乙酰酶，通过脱乙酰基改变PGA的电荷状态(浓缩正电荷)。这一酶活性表明，脱附/抑制的机制可能是通过增加对PGA聚合物的排斥作用来实现的。重要的是，在这些生物中，尤其是在AA中，脱乙酰基在生物膜去除或形成中的作用还没有得到充分的研究。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致病中的作用，以及PGaB的脱乙酰酶活性是如何发挥这一作用的核心。我们将使用体外和体内模型来检验PGA在AA毒力中发挥关键作用的总体假设。具体地说，将研究以下目标：特定目标1.证明PgaB酶的PGA去乙酰化能力是其抗生物被膜活性的关键。具体目标2a。表明PGA的乙酰化状态和程度有助于AA生物膜的形成。具体目标2b。证明胞外多糖PGA参与了AA的致病作用。

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.