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: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=568105
• 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.

溶血性甘露氏菌是一种革兰氏阴性细菌，是牛鼻咽中的共生微生物。当动物受到压力或同时感染病毒感染时，它们的免疫系统就会受到损害，无法清除吸入肺部的细菌。细菌能够粘附/定居肺，逃避免疫清除率并导致由于肺组织破坏而导致肺炎。细菌产生的主要毒力因子之一是靶向牛白细胞的白细胞毒素。白细胞毒素裂解牛巨噬细胞，以防止它们攻击入侵细菌。巨噬细胞的裂解释放了导致肺组织破坏和肺功能丧失的细胞裂解成分。白细胞毒素是在许多细菌病原体中发现的RTX家族的成员。即使将白细胞毒素编码的基因表征了25年以上，但对它们的表达调节知之甚少。为了更好地了解细菌如何以及何时决定产生白细胞毒素，我建议研究调节白细胞毒素基因表达的遗传机制。这将通过诱变实验和非血液突变体的分离来实现。该遗传筛查利用白细胞毒素的溶质化活性，该溶质毒素周围在绵羊血琼脂平板上产生清除区域。非血压突变体在实际白细胞毒素基因中可能有缺陷，或者在表达白细胞毒素基因所需的另一个“因子”中。将突变体分解为上述两组之后，没有在白细胞毒素基因中的突变的人将进一步以与基因组文库的互补为特征，以筛选恢复溶血性表型。任何可以恢复溶血表型的重组质粒都应包含缺失或突变功能。这应该确定表达白细胞毒素基因所需的新因素。这项研究的结果可以扩展到其他细菌病原体中RTX基因表达的调节。