Pathogen Synergy Through Cross-Species Induction of Outer Membrane Vesicle Biogenesis

摘要 在整个进化过程中，细菌一直在合作和竞争。然而，直到最近， 在传染病机制的背景下，开始认真考虑这一点。扩展能力 研究复杂财团中的细菌（即它们通常如何生活）引发了人们对细菌的重新认识。 真实的细菌种群的复杂性和它们如何相互作用的检查。共感染研究表明， 病原体群落的毒性更强，这与流行病学报告中将多个物种联系在一起的观点一致 感染导致患者预后更差。一个常见的假设是，这种病原体协同作用是因为 在感染点的多个物种之间的竞争和交流。我们假设外部 膜囊泡（OMV）作为这些相互作用的主要介质，因为它们已知促进 细菌之间的竞争和交流（除了对宿主细胞的直接毒力）。 我们在铜绿假单胞菌中提出了一个模型，其中OMV的生物合成是由自分泌的 产生的小分子进入外膜的外小叶。我们最近发现这种分子 在低浓度时，可以诱导相关物种过量产生OMV， 种产生自己的OMV交叉诱导因子。这提出了跨物种诱导的可能性， OMV的生物发生可以作为一种机制，多物种社区协同增加 毒性在强有力的初步数据的指导下，我们将追求两个具体目标，以描述多重 感染部位的病原体可能通过OMV生物发生的跨种诱导相互作用， 这将对宿主细胞产生：（1）我们将开始通过使用 已知的铜绿假单胞菌OMV诱导化合物PQS针对一组重要的人类病原体， 在感染点相遇我们的既定方法已经确定了几个这样的 γ-Proteobacteria之间的相互作用。然后，我们将测试是否真的在一起种植物种（相比之下， 使用具有如上所述的外源化合物的单一培养物）将导致增加的OMV产量， 社区初步结果表明，这对于我们的铜绿假单胞菌+ E.杆菌(2)的omv 将测试来自单一培养物与共培养物的抗巨噬细胞和肺上皮细胞的细胞毒性潜力 以及它们降解或隔离抗生素的能力（OMV的另一种疾病相关功能）。初步 铜绿假单胞菌+ E.大肠杆菌共培养OMV表明，细胞毒性和诱导凋亡都是 增加了单一栽培OMV，证明了该方法的可行性，并为 假设跨物种OMV诱导可能有助于病原体协同作用。 了解病原体在感染部位的相互作用对于理解为什么多物种感染至关重要 导致更严重的疾病。考虑到OMV生产在革兰氏阴性生物中的普遍性， 从这项研究中获得的见解有望广泛影响我们对多物种发病机制的理解。

Abstract Bacteria have been cooperating and competing for all of evolutionary time. Only recently, however, have we seriously begun to take this into account in the context of infectious disease mechanisms. The expanding ability to study bacteria in complex consortia (i.e. how they normally live) has sparked a renewed appreciation for the complexity of real bacterial populations and an examination of how they interact. Co-infection studies show that pathogen communities are more virulent, and this aligns with epidemiological reports that connect multispecies infections to worse patient outcomes. A common hypothesis is that this pathogen synergy comes about because of competition and communication between multiple species at an infection site. We hypothesize that Outer Membrane Vesicles (OMVs) serve as a major mediator of these interactions because they are known to facilitate both competition and communication between bacteria (in addition to direct virulence against host cells). We proposed a model in P. aeruginosa where OMV biogenesis is driven by the secretion and insertion of a self- produced small molecule into the outer leaflet of the outer membrane. We recently showed that this molecule could induce related species to overproduce OMVs when given at low concentration, and that those recipient species produced their own OMV cross-inducing factors. This raised the possibility that cross-species induction of OMV biogenesis could serve as a mechanism for multispecies communities to synergize for increased virulence. Guided by strong preliminary data, we will pursue two specific aims to characterize how multiple pathogens at an infection site might interact through cross-species induction of OMV biogenesis and what effects this would have on host cells: (1) We will begin by testing for small molecule-induced OMV formation using the known P. aeruginosa OMV-inducing compound PQS against a broad panel of important human pathogens likely to encounter each other at an infection site. Our established methods have already identified several such interactions among the γ-proteobacteria. We will then test whether actually growing species together (in contrast to using monocultures with exogenous compounds as above) will result in increased OMV production for the community. Preliminary results show that this is true for our pilot pairing of P. aeruginosa + E. coli. (2) OMVs from mono- vs. co-culture will be tested for their cytotoxic potential against macrophage and lung epithelial cells as well as their ability to degrade or sequester antibiotics (another disease-related function of OMVs). Preliminary results with P. aeruginosa + E. coli co-culture OMVs show that cytotoxicity and induction of apoptosis are both increased over monoculture OMVs, demonstrating feasibility of the approach and providing support for the hypothesis that cross-species OMV induction may contribute to pathogen synergy. Understanding pathogen interactions at infection sites is critical to understanding why multispecies infections lead to more severe disease. Given the ubiquity of OMV production among Gram-negative organisms, the insights gained from this study promise to broadly impact our understanding of multi-species pathogenesis.