Staphylococcus aureus and Pseudomonas aeruginosa interactions in wound pathogenesis

项目摘要 慢性伤口感染的患者有丰富的微生物群落，几种细菌已经被 被确定为持续性疾病的主要驱动因素。最主要的两种伤口病原体是 金黄色葡萄球菌(包括耐甲氧西林金黄色葡萄球菌(MRSA))和铜绿假单胞菌。这个 哨兵抗菌素监测项目确认S。 三 11%， 铜绿假单胞菌和肠球菌。作为 美国引起皮肤软组织感染(SSTI)的主要病原体占44%， 占1998年和2004年所有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，复发率很高。S.aureus和P. 铜绿假单胞菌是最常见的致病菌，通常为耐药生物膜。 众所周知，生物被膜很难根除，因为它们对抗生素具有抵抗力，而且能够逃避清除。 通过免疫系统。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整个社区对抗生素的耐受性。具体地说，在体外 与金黄色葡萄球菌共培养研究增加了喹诺酮类假单胞菌基因的表达，调节 几种依赖群体感应的铜绿假单胞菌毒力因子和铁获取系统的表达。 随着耐甲氧西林金黄色葡萄球菌在美国的流行程度的增加，需要新的策略来治疗慢性和复发性 感染。因此，长期目标是为慢性创面患者开发替代治疗方案。 感染金黄色葡萄球菌和铜绿假单胞菌。这项建议的目标是确定生物 铜绿假单胞菌脓球蛋白MRSA修饰的后果。这项建议背后的理由是我们的 初步发现金黄色葡萄球菌和铜绿假单胞菌具有协同作用，这可能在一定程度上解释了为什么 感染这两种病原体会导致更糟糕的结果。在初步的成像质谱学研究中， 我们确定了在MRSA和P相互作用过程中产生了一种新的甲基化褐蛋白衍生物。 铜绿假单胞菌。我们确定了与MRSA有关的基因，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，绿脓素起源于铜绿假单胞菌，然后是 被MRSA酶转化为一种新的甲基化形式。我们假设金黄色葡萄球菌甲基化 在多菌环境中竞争的绿球蛋白。为了检验这一假设，我们将(I)刻画 耐甲氧西林金黄色葡萄球菌通过生化、遗传和感染模拟方法产生甲基化的绿球蛋白； (2)确定甲基化的绿脓菌素对铜绿假单胞菌生理和功能的影响 发病机制。这项工作的预期结果将增加我们对生物合成和 驱动种间相互作用以更好地预测感染的特定代谢物的生物活性 结果，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.