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

项目摘要 铜绿假单胞菌是一种多药耐药病原体，其引起慢性肺部感染，并且是一种耐药病原体。 囊性纤维化（CF）患者死亡的主要原因。铜绿假单胞菌产生一种生物膜基质，可以保护 细菌从抗菌剂和艾滋病在宿主免疫逃避。这种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，铜绿假单胞菌有助于炎症， 通过激活宿主细胞中的促炎性NF-κB通路，导致产生 炎症细胞因子和炎症免疫细胞向肺部的募集，引起随后的组织 损害NF-κB活化在CF患者的肺中自然升高，P. 铜绿感染虽然铜绿假单胞菌通常作为一种孤立的感染进行研究，但其他物种也会定植 CF患者的肺部，并可能调节铜绿假单胞菌的毒力。口腔链球菌定植 最近与CF肺稳定性有关。在这些口腔链球菌中， 唾液链球菌是CF患者肺部最常见的链球菌。S.唾液 已显示抑制多种呼吸道病原体的生长，以及抑制NF-κB的活化 人支气管上皮细胞中的信号通路。虽然S.流涎与稳定的肺功能相关 在CF患者中，没有研究检查S.唾液腺杆菌和铜绿假单胞菌及其影响 主机响应。为了描述这两个物种之间的相互作用，我们首先测量了 当共培养时两个物种的生物膜形成。初步结果表明，S.唾液 生物膜的形成在胞外多糖Psl的存在下显著增加，所述胞外多糖Psl由 非粘液铜绿假单胞菌菌株PAO 1。此外，S.唾液麦芽糖结合蛋白MalE 在铜绿假单胞菌存在下过表达。目的1将检验MalE与Psl相互作用以 促进S.唾液将使用全细胞ELISA和等温滴定量热法， 表征两个靶标的结合亲和力。我们的初步数据还表明， 在S.唾液，突出显示 需要进一步研究S.对铜绿假单胞菌致病机制的影响。目标2将测试 假设S. CF肺中的唾液腺通过下调前体蛋白来改善肺功能， NF-κB下游的炎性细胞因子响应铜绿假单胞菌感染而产生。我们将开发 CF大鼠共感染模型，其中我们测量特异性促炎细胞因子，中性粒细胞募集， 和肺组织病理学在铜绿假单胞菌感染的存在或不存在的S。唾液数据 这一建议将促进我们对S. salivarius并入P. 铜绿假单胞菌感染可以通过调节铜绿假单胞菌生物膜来调节宿主对铜绿假单胞菌感染的应答。

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.