Staphylococcus aureus and Pseudomonas aeruginosa interactions in wound pathogenesis

项目摘要 患有慢性伤口感染的患者具有丰富的微生物群落，并且已经发现了几种细菌物种。 被确定为持续性疾病的主要驱动因素。两种最突出的伤口病原体是 金黄色葡萄球菌（包括耐甲氧西林S.金黄色葡萄球菌（MRSA））和铜绿假单胞菌。的 SENTRY抗菌素监测计划确定了S。 三 百分之十一 金黄色葡萄球菌、铜绿假单胞菌和肠球菌属（Enterococcus spp.）为 引起皮肤和软组织感染（SSTI）的主要病原体在美国占44%， 在1998年和2004年期间，占所有SSTI的9%。 , , 慢性腿部溃疡（CLU）影响1-2%的人 在世界范围内，是长期发病的主要原因，复发率高。S. aureus和P. 铜绿假单胞菌是从CLU中分离的最常见的病原体，通常作为对抗生素治疗具有抗性的生物膜。 众所周知，生物膜难以根除，因为它们对抗生素的耐受性和逃避清除的能力 免疫系统的影响。有证据表明，这两种病原体可以交换专门的代谢物， 有可能改变细胞行为并导致社区范围内的抗生素耐受性。具体来说，在体外 与S.金黄色葡萄球菌增加了调节细胞凋亡的假单胞菌喹诺酮基因表达， 几种群体感应依赖性铜绿假单胞菌毒力因子和铁获取系统的表达。 随着MRSA在美国的流行率增加，有必要制定新的策略来治疗慢性和复发性MRSA。 感染.因此，长期目标是为慢性伤口患者开发替代治疗方案 感染S.金黄色葡萄球菌和铜绿假单胞菌。本提案的目的是确定生物学 铜绿假单胞菌绿脓菌螯铁蛋白的MRSA修饰的后果。这一建议的基本原理是， 初步研究表明，S.金黄色葡萄球菌和铜绿假单胞菌协同作用，这可能部分解释了为什么共同 这两种病原体的感染导致更坏的结果。在初步的成像质谱研究中， 我们确定在MRSA和P. 铜绿。我们确定了负责的MRSA基因，我们可以在小鼠伤口中检测到这种新的代谢物， 感染模型我们的初步研究证实绿脓菌螯铁蛋白来源于铜绿假单胞菌， 被MRSA酶促转化为新的甲基化形式。我们假设MRSA甲基化 绿脓菌螯铁蛋白在多微生物环境中竞争。为了验证这一假设，我们将（i）描述 通过生物化学、遗传学和感染建模方法由MRSA产生的甲基化绿脓菌螯铁蛋白; (ii)确定甲基化绿脓菌螯铁蛋白对铜绿假单胞菌生理学的功能后果， 发病机制这项工作的预期结果将增加我们对生物合成的理解， 驱动种间相互作用以更好地预测感染的专门代谢物的生物活性 并开发新的治疗方法。

PROJECT ABSTRACT Patients with chronic wound infections have a rich microbial community and several bacterial species have been identified as the main drivers of persistent disease. Two of the most prominent wound pathogens are Staphylococcus aureus (including methicillin-resistant S. aureus (MRSA)), and Pseudomonas aeruginosa. The SENTRY antimicrobial surveillance program identified S. three 11%, aureus P. aeruginosa and Enterococcus spp. as the main pathogens causing skin and soft tissue infections (SSTI) in the United States accounting for 44%, and 9% of all SSTI during the years 1998 and 2004. , , Chronic leg ulcers (CLUs) affect 1-2% of people worldwide, are a major cause of prolonged morbidity and have a high recurrence rate. S. aureus and P. aeruginosa are the most common agents isolated from CLUs usually as biofilm resistant to antibiotic therapy. Biofilms are notoriously difficult to eradicate, due to their recalcitrance to antibiotics and ability to evade clearance by the immune system. There is evidence that these two pathogens can exchange specialized metabolites that have the potential to alter cellular behavior and lead to community-wide antibiotic tolerance. Specifically, in vitro co-cultivation studies with S. aureus increased Pseudomonas quinolone gene expression that regulates the expression of several quorum-sensing dependent P. aeruginosa virulence factors and iron acquisition systems. As the prevalence of MRSA increases in the US, new strategies are necessary to treat chronic and recurrent infections. Therefore, the long-term goal is to develop alternative treatment options for chronic wound patients infected by S. aureus and P. aeruginosa. The objective of this proposal is to determine the biological consequences of MRSA modification of P. aeruginosa pyochelin. The rationale underlying this proposal is our preliminary findings that S. aureus and P. aeruginosa act synergistically, which may in part explain why co- infections with these two pathogens lead to worse outcomes. In preliminary imaging mass spectrometry studies, we determined that a novel methylated pyochelin derivative is produced during the interaction of MRSA and P. aeruginosa. We identified the MRSA gene responsible and we can detect this new metabolite in a mouse wound infection model. Our initial studies confirm that the pyochelin originates from P. aeruginosa and then is enzymatically converted by MRSA into a new methylated form. We hypothesize that MRSA methylates pyochelin to compete in a polymicrobial environment. In order to test this hypothesis we will (i) characterize methylated pyochelin generated by MRSA through biochemical, genetic and infection modeling methods and; (ii) determine the functional consequences of methylated pyochelin on P. aeruginosa physiology and pathogenesis. The expected outcomes of this work will increase our understanding the biosynthesis and biological activities of specialized metabolites that drive inter-species interactions to better predict infection outcomes and to develop new treatment approaches.