Interactions between Pseudomonas aeruginosa and Streptococcus salivarius and effects on the host immune response

项目概要 铜绿假单胞菌是一种具有多重耐药性的病原体，可引起慢性肺部感染，是一种 囊性纤维化（CF）患者死亡的主要原因。铜绿假单胞菌产生生物膜基质，保护 抗菌剂中的细菌并有助于宿主免疫逃避。该生物膜基质的主要成分 是三种胞外多糖——Pel、Psl 和藻酸盐。此外，铜绿假单胞菌会导致炎症和 通过激活宿主细胞中的促炎性 NF-κB 通路，导致产生肺损伤 炎症细胞因子和炎症免疫细胞募集到肺部，导致随后的组织 损坏。 CF 患者肺部的 NF-κB 激活自然升高，而 P. 铜绿假单胞菌感染。尽管铜绿假单胞菌通常被研究为一种孤立的感染，但其他物种也可以定殖 CF 患者的肺部，可能会调节铜绿假单胞菌的毒力。口腔链球菌定植 最近发现，肺部的炎症与 CF 肺稳定性有关。这些口腔链球菌中，链球菌 唾液链球菌被发现是 CF 患者肺部最常见的链球菌。唾液链球菌 已被证明可以抑制多种呼吸道病原体的生长，并抑制 NF-κB 的激活 人支气管上皮细胞中的途径。尽管唾液链球菌与稳定的肺功能相关 在 CF 患者中，没有研究检验唾液链球菌和铜绿假单胞菌之间的相互作用及其影响 关于主机响应。为了描述两个物种之间的相互作用，我们首先测量了 共培养时两个物种生物膜的形成。我们的初步结果表明，S. salivarius 存在胞外多糖 Psl 时，生物膜形成显着增加，胞外多糖 Psl 是由 非粘液铜绿假单胞菌菌株 PAO1。此外，唾液链球菌麦芽糖结合蛋白 MalE 在铜绿假单胞菌存在下过度表达。目标 1 将检验 MalE 与 Psl 相互作用的假设 促进唾液链球菌生物膜的形成。全细胞 ELISA 和等温滴定量热法将用于 表征两个目标的结合亲和力。我们的初步数据还表明，果蝇 在唾液链球菌存在的情况下，黑腹果蝇免受铜绿假单胞菌介导的杀死，强调 需要进一步研究唾液链球菌对铜绿假单胞菌发病机制的影响。目标 2 将测试 假设 CF 肺中存在唾液链球菌，通过下调亲 响应铜绿假单胞菌感染而产生的 NF-κB 下游的炎性细胞因子。我们将开发 CF大鼠混合感染模型，我们测量特定的促炎细胞因子、中性粒细胞募集、 以及在存在或不存在唾液链球菌的情况下铜绿假单胞菌感染期间的肺组织病理学。数据 该提案产生的结果将促进我们对 S. salivarius 如何融入 P. 铜绿假单胞菌生物膜并调节宿主对铜绿假单胞菌感染的反应。

PROJECT SUMMARY Pseudomonas aeruginosa is a multi-drug resistant pathogen which causes chronic lung infections and is a leading cause of mortality in cystic fibrosis (CF) patients. P. aeruginosa produces a biofilm matrix which protects the bacterium from antimicrobials and aids in host immune evasion. The main component of this biofilm matrix are three exopolysaccharides- Pel, Psl, and alginate. Additionally, P. aeruginosa contributes to inflammation and lung damage by activating the pro-inflammatory NF-κB pathway in host cells, leading to the production of inflammatory cytokines and recruitment of inflammatory immune cells to the lungs, causing subsequent tissue damage. NF-κB activation is naturally elevated in the lungs of CF patients, which is further exacerbated by P. aeruginosa infection. Although P. aeruginosa is generally studied as an isolated infection, other species colonize the lungs of CF patients and could potentially modulate P. aeruginosa virulence. Colonization of oral streptococci in the lungs has recently been associated with CF lung stability. Of these oral streptococci, Streptococcus salivarius, was found to be the most prevalent streptococcal species in the lungs of CF patients. S. salivarius has been shown to inhibit growth of multiple respiratory pathogens, as well as inhibit activation of the NF-κB pathway in human bronchial epithelial cells. Although S. salivarius has been correlated with stable lung function in CF patients, no studies have examined interactions between S. salivarius and P. aeruginosa and their impact on the host response. To characterize the interactions between the two species, we first measured changes in biofilm formation of the two species when co-cultured. Our preliminary results demonstrated that S. salivarius biofilm formation is significantly increased in the presence of the exopolysaccharide Psl, which is produced by the non-mucoid P. aeruginosa strain PAO1. Additionally, the S. salivarius maltose-binding protein MalE was overexpressed in the presence of P. aeruginosa. Aim 1 will test the hypothesis that MalE interacts with Psl to promote biofilm formation of S. salivarius. Whole-cell ELISA and isothermal titration calorimetry will be used to characterize the binding affinity of the two targets. Our preliminary data also demonstrated that Drosophila melanogaster were protected from P. aeruginosa-mediated killing in the presence of S. salivarius, highlighting the need to further examine the impact of S. salivarius on P. aeruginosa pathogenesis. Aim 2 will test the hypothesis that the presence of S. salivarius in the CF lung improves lung function by downregulating pro- inflammatory cytokines downstream of NF-κB produced in response to P. aeruginosa infection. We will develop a CF rat model of co-infection in which we measure specific pro-inflammatory cytokines, neutrophil recruitment, and lung histopathology during P. aeruginosa infection in the presence or absence of S. salivarius. Data generated from this proposal will promote our understanding of how S. salivarius incorporates into the P. aeruginosa biofilm and modulates the host response to a P. aeruginosa infection.