In the pulmonary form of anthrax caused by the pathogen Bacillus anthracis (Ba), spores in the lung are ingested by macrophages. Once phagocytosed, the spores are transported to regional lymph nodes and germinate inside macrophages. In order for the disease to progress, Ba must resist killing by the macrophage and further disseminate into the blood for vegetative growth. The exact means by which Ba survives and escapes the macrophage are unknown. Recent work has shown that newly vegetative bacilli escape from the phagocytic vesicles of macrophages and replicate in the cytosol, a process that was first described and best studied in another human pathogen Listeria monocytogenes (Lm). Lm escapes from the primary phagocytic vacuole of a macrophage using listeriolysin O (LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC). Genes orthologous to LLO and PI-PLC have recently been discovered in Ba by the genome sequencing project. In order to investigate the functions of the Ba orthologs of LLO and PI-PLC, Lm will be used as a heterologous host to express these proteins and to analyze their role in mediating bacterial escape from phagocytic vesicles and release from the host cell. For safety reasons a strain of Lm that has been developed as a potential vaccine vector and is unable to grow in cells without a D-alanine supplement will be used.
The first aim i s to determine if the LLO ortholog permits escape of Lm from the macrophage phagosome. Its potential for signaling through a recently discovered protein kinase C signaling pathway, thought to be needed for escape from the phagosome, will also be explored. Since the Ba PI-PLC ortholog is 94% identical in amino acid sequence to the PI-PLC from Bacillus cereus, it almost certainly has that enzymatic activity, but there are potential structural differences between the Bacillus enzymes and the Lm enzyme that would affect their activity both outside and inside the host. The effects of these structural differences will be examined in the second aim, in which the role of Ba PI-PLC in escape from the phagosome and host cell signaling is explored. Lastly, studies on inhibitors of PI-PLC will test their efficacy in blocking the biological role of this enzyme and may lead to eventual high throughput screening for drugs to combat anthrax infections. The results of this study will provide evidence on the potential role of the LLO and PI-PLC orthologs of Ba in mediating its survival, growth and escape from the macrophage, essential elements in its ability to cause a devastating disease with high mortality in humans.
|Wei, Zhengyu; Schnupf, Pamela; Poussin, Mathilde A et al. (2005) Characterization of Listeria monocytogenes expressing anthrolysin O and phosphatidylinositol-specific phospholipase C from Bacillus anthracis. Infect Immun 73:6639-46|
|Zenewicz, Lauren A; Wei, Zhengyu; Goldfine, Howard et al. (2005) Phosphatidylinositol-specific phospholipase C of Bacillus anthracis down-modulates the immune response. J Immunol 174:8011-6|