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

项目总结 铜绿假单胞菌是一种对多种药物耐药的病原体，可导致慢性肺部感染，是一种 囊性纤维化(CF)患者死亡的主要原因。铜绿假单胞菌产生一种生物膜基质，可以保护 细菌来自抗菌剂和帮助宿主免疫逃避。该生物膜基质的主要成分 有三种胞外多糖--Pel、PSL和藻酸盐。此外，铜绿假单胞菌还会引起炎症和 肺损伤通过激活宿主细胞中促炎症的NF-κB途径，导致产生 炎性细胞因子和炎性免疫细胞向肺部募集，导致随后的组织 损坏。在CF患者的肺中，NF-κB的活性自然升高，而P。 铜绿假单胞菌感染。虽然铜绿假单胞菌通常被研究为一种孤立的感染，但其他物种也会定居。 可能对铜绿假单胞菌的毒力有潜在的调节作用。口腔链球菌的定植 最近发现，肺中的炎症与肺纤维化的稳定性有关。在这些口腔链球菌中，链球菌 唾液链球菌是CF患者肺部最常见的链球菌。唾液链霉菌 已被证明可以抑制多种呼吸道病原体的生长，以及抑制核因子-κB的激活 人支气管上皮细胞中的通路。尽管唾液葡萄球菌与稳定的肺功能相关 在慢性阻塞性肺疾病患者中，还没有研究检查唾液葡萄球菌和铜绿假单胞菌之间的相互作用及其影响。 在主机响应上。为了描述这两个物种之间的相互作用，我们首先测量了 两种生物共培养时生物膜的形成。我们的初步结果表明，唾液链球菌 在胞外多糖PSL的存在下，生物被膜的形成显著增加，PSL是由 非粘液型铜绿假单胞菌PAO1。此外，唾液链霉菌麦芽糖结合蛋白雄性 在铜绿假单胞菌存在时过表达。目标1将检验男性与PSL相互作用的假设 促进唾液葡萄球菌生物被膜的形成。将采用全细胞酶联免疫吸附试验和等温滴定量热法测定 表征两个靶标的结合亲和力。我们的初步数据还表明，果蝇 在唾液葡萄球菌存在的情况下，黑素胃鼠受到保护，不受铜绿假单胞菌介导的杀灭，强调 需要进一步研究唾液葡萄球菌对铜绿假单胞菌发病的影响。目标2将测试 假设唾液链霉菌存在于CF肺中，通过下调Pro-2来改善肺功能。 铜绿假单胞菌感染后，NF-κB下游产生炎性细胞因子。我们将发展 我们建立了一种混合感染的CF大鼠模型，在该模型中，我们测量了特定的促炎细胞因子、中性粒细胞募集、 在存在或不存在唾液葡萄球菌的情况下，铜绿假单胞菌感染时的肺组织病理学改变。数据 这一建议将促进我们对唾液链霉菌如何整合到P. 铜绿假单胞菌生物被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.