Examination of the regulation of the leukotoxin operon in Mannheimia haemolytica.

检查溶血曼海姆氏菌中白细胞毒素操纵子的调节。

• 批准号: 246-2012
• 负责人: Lo, Reggie
• 金额: $ 1.89万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2012
• 资助国家: 加拿大
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=505671
• 关键词: Examination; regulation; leukotoxin; operon; Mannheimia

Mannheimia haemolytica is a Gram negative bacterium and a commensal microorganism in the nasal pharyngeal passage of cattle. When the animals are stressed or suffer from concurrent viral infections, their immune systems are compromised and can not clear the bacterium inhaled into the lungs. The bacterium is able to adhere/colonize the lungs, evade the immune clearance and cause pneumonia due to destruction of the lung tissue. One of the major virulence factor produced by the bacterium is a leukotoxin which targets the bovine leukocytes. The leukotoxin lyses the bovine macrophages to prevent them from attacking the invading bacterium. Lysis of the macrophages release cellular lytic components that leads to destruction of the lung tissue and the loss of lung function. The leukotoxin is a member of the RTX family of cytolysins found in many bacterial pathogens. Even though the genes that code for the leukotoxin have been characterized for over 25 years, very little is known about their regulation of expression. To gain a better understanding on how and when the bacterium decides to produce the leukotoxin, I proposed to investigate the genetic mechanism that regulate expression of the leukotoxin genes. This will be accomplished by mutagenesis experiments and the isolation of non-haemolytic mutants. This genetic screen utilized the haemolytic activity of the leukotoxin which produces a zone of clearance around the bacterial colony on sheep's blood agar plates. The non-haemolytic mutant could be defective in the actual leukotoxin genes, or in another 'factor' required for expression of the leukotoxin genes. After sorting out the mutant into the above two groups, those with mutations not in the leukotoxin genes will be further characterized by complementation with a genomic library to screen for restoration of the haemolytic phenotype. Any recombinant plasmid that can restore the haemolytic phenotype should contain the missing or mutated function. This should identify any novel factor required for expression of the leukotoxin genes. The results from this study could be extended to the regulation of expression of RTX genes in other bacterial pathogens.

溶血曼海氏菌是一种革兰氏阴性菌，是牛鼻咽通道的一种共生微生物。当动物感到压力或同时遭受病毒感染时，它们的免疫系统就会受损，无法清除吸入肺部的细菌。这种细菌能够附着/定植在肺部，逃避免疫清除，并由于肺部组织的破坏而引起肺炎。细菌产生的主要毒力因子之一是一种以牛白细胞为目标的白毒素。白毒素溶解牛巨噬细胞以阻止它们攻击入侵的细菌。巨噬细胞的裂解释放细胞裂解成分，导致肺组织的破坏和肺功能的丧失。白质毒素是在许多细菌病原体中发现的细胞溶解素RTX家族的一员。尽管编码白质毒素的基因已经被鉴定了25年以上，但对它们的表达调控却知之甚少。为了更好地了解细菌如何以及何时决定产生白质毒素，我建议研究调节白质毒素基因表达的遗传机制。这将通过诱变实验和非溶血突变体的分离来完成。这种遗传筛选利用了白细胞毒素的溶血活性，白细胞毒素在羊血琼脂板上的细菌菌落周围产生一个清除区。非溶血突变体可能在实际的白毒素基因中存在缺陷，或者在表达白毒素基因所需的另一个“因子”中存在缺陷。在将突变体分类为上述两组后，将非白毒素基因突变的突变体进一步与基因组文库进行互补表征，筛选溶血表型的恢复。任何可以恢复溶血表型的重组质粒都应该包含缺失或突变的功能。这应该确定任何新因子所需的白质毒素基因的表达。本研究结果可推广到其他病原菌中RTX基因的表达调控。