Microbial Response to Neutrophil Phagocytosis

描述(申请人提供)：中性粒细胞是宿主抵抗细菌和真菌病原体的关键组成部分。当中性粒细胞在数量或功能上严重减少时，宿主就会变得非常容易受到严重感染。中性粒细胞通常会在很短的时间内吞噬并杀死细菌。为了更好地了解细菌对吞噬作用的反应，将使用基因组阵列来评估中性粒细胞吞噬的大肠杆菌细胞的mRNA丰度的变化。我们希望了解摄取的细菌是如何优先考虑吞噬细胞中产生的压力的反应，以及这些反应是否提高了微生物的生存能力。由吞噬细胞NADPH氧化酶(CGD，慢性肉芽肿性疾病)或髓过氧化物酶介导的抗微生物系统中缺乏中性粒细胞的作用将与正常中性粒细胞进行比较。初步研究表明，正常的中性粒细胞，而不是CGD的中性粒细胞，会引起细菌转录因子OxyR介导的抗氧化反应。OxR基因的破坏产生了一种细菌菌株，它对正常中性粒细胞的杀伤敏感性增加了10倍。在上行性肾盂肾炎的小鼠模型中，oxR株的毒力似乎也明显减弱。这项建议试图更全面地表征Ox R的作用，并探索选定的其他主要转录反应对中性粒细胞吞噬和单独的抗菌模型系统的影响。它还寻求实施方法学，强调检测存在于致病性大肠杆菌菌株中但不存在于实验室适应菌株中的吞噬诱导的mRNA转录本的变化。后一个目标的驱动假说是：病原体特定的转录本对致病表型有很大贡献。吞噬细菌的表达谱似乎提供了有关吞噬细胞空泡中的条件以及响应这种恶劣环境的重要细菌防御系统的有用信息。

DESCRIPTION (provided by applicant): Neutrophils are a key component of host defense against bacterial and fungal pathogens. When neutrophils are severely diminished in number or function, the host becomes highly susceptible to serious infection. Neutrophils typically ingest and kill bacteria within a short space of time. In order to gain a better understanding of the bacterial response to phagocytosis, genomic arrays will be used to assess changes in mRNA abundance for Escherichia coli cells ingested by neutrophils. We expect to learn how ingested bacteria prioritize responses to stresses generated in the phagocyte and whether these responses enhance microbial survival. The effects of neutrophils deficient in antimicrobial systems mediated by the phagocyte NADPH oxidase (CGD, chronic granulomatous disease) or the enzyme myeloperoxidase will be compared with normal neutrophils. Initial studies indicate that normal neutrophils, but not CGD neutrophils, evoke an anti-oxidant response mediated by the bacterial transcription factor, OxyR. Disruption of the oxyR gene generated a bacterial strain that was 10-fold more susceptible to killing by normal neutrophils. The oxyR strain also appears to be markedly attenuated for virulence in a mouse model of ascending pyelonephritis. This proposal seeks to characterize oxyR effects more fully, and to explore the effects of selected other major transcriptional responses both to neutrophil phagocytosis and to isolated antimicrobial model systems. It also seeks to implement methodology that will emphasize detection of phagocytosis-induced changes for mRNA transcripts present in a pathogenic E. coli strain but not in a laboratory-adapted strain. The driving hypothesis for the latter goal being that: pathogen-specific transcripts contribute substantially to the pathogenic phenotype. Expression profiling of phagocytosed bacteria appears to provide useful information about conditions in the phagocytic vacuole and about important bacterial defenses mounted in response to this hostile environment.