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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=543851
• 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.

溶血性曼氏菌是一种革兰氏阴性菌，是牛鼻咽通道中的一种肠道微生物。当动物受到压力或同时受到病毒感染时，它们的免疫系统受到损害，无法清除吸入肺部的细菌。该细菌能够粘附/定殖于肺，逃避免疫清除，并由于肺组织的破坏而引起肺炎。该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。白细胞毒素溶解牛巨噬细胞以防止它们攻击入侵的细菌。巨噬细胞的裂解释放细胞裂解组分，其导致肺组织的破坏和肺功能的丧失。白细胞毒素是在许多细菌病原体中发现的溶细胞素的RTX家族的成员。尽管编码白细胞毒素的基因已被鉴定超过25年，但对它们的表达调控知之甚少。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溶血突变体的分离来完成。该遗传筛选利用白细胞毒素的溶血活性，其在羊血琼脂平板上的细菌菌落周围产生清除区。非溶血性突变体可能在实际的白细胞毒素基因中有缺陷，或者在白细胞毒素基因表达所需的另一个“因子”中有缺陷。在将突变体分选到上述两组中之后，将通过与基因组文库互补来进一步表征那些具有不在白细胞毒素基因中的突变的突变体，以筛选溶血表型的恢复。任何能够恢复溶血表型的重组质粒都应该含有缺失或突变的功能。这将确定白细胞毒素基因表达所需的任何新因子。本研究的结果可以扩展到其他细菌病原体中RTX基因表达的调控。