PGA exopolysaccharide in biofilm formation and pathogenicity of Aggregatibacter a

描述（由申请人提供）：聚集菌放线菌（放线菌）和几种致病菌附着在非生物表面并产生胞外多糖，将细菌细胞固定在这些定植表面上。Aa产生的胞外多糖由？-1,6链n -乙酰- d -氨基葡萄糖（GlcNAc）单元。Aa利用一个与大肠杆菌pga、鼠疫杆菌hms和表皮葡萄球菌同源的四基因操纵子产生这种胞外多糖。在Aa中，操纵子pgaABCD携带一个由pagB编码的去乙酰化酶（EC 3.5.1.41）。我们的初步数据表明，PgaB酶具有从PGA的GlcNAc的n -乙酰基上去除乙酰基的能力。更有趣的是，我们发现PgaB在Aa以及表皮葡萄球菌和胸膜肺炎放线杆菌中分离生物膜并抑制其形成。我们之前已经证明，一种同样来自Aa的分散酶B （DspB）可以通过解聚外多糖（PGA）阻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酶改变了PGA的电荷状态，这表明分离/抑制的机制可能是由于去乙酰化导致聚合物上正电荷的增加。重要的是，去乙酰化在生物膜去除和

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 made up of ?-1,6-linked N-acetyl-D-glucosamine (GlcNAc) units. Aa produces this exopolysaccharide utilizing a four-gene operon homologous to E. coli pga, Y. pestis hms, and S. epidermidis ica. In Aa, the operon pgaABCD carries a deacetylase enzyme (EC 3.5.1.41) encoded by pagB. Our Preliminary Data show that the enzyme PgaB has the ability to remove acetyl groups from the N-acetyl groups of GlcNAc of PGA. More intriguingly, we discovered that PgaB detaches biofilms and inhibits their formation in Aa as well as S. epidermidis and Actinobacillus pleuropneumoniae. We have previously shown that an enzyme dispersin B (DspB) also from Aa prevents surface attachment of several Gram negative as well as Gram-positive bacterial species through depolymerization of the exopolysaccharide (PGA). Unlike DspB, the newly discovered enzyme PgaB alters the charge state of PGA suggesting that the mechanism of detachment/inhibition might be through the increase of positive charges on the polymer as a result of deacetylation. Importantly, the role of deacetylation in biofilm removal and formation is understudied in these organisms, especially 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. We will use both in vitro as well as in vivo models to test 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 enzyme PgaB is critical for Aa biofilm formation. Specific Aim 2a. Demonstrate that the state of acetylation of PGA contributes to the biofilm formation in Aa. Specific Aim 2b. Establish that PGA contributes to Aa pathogenicity.