Functional Analysis Of The Type Ⅳ Effector Protein Ceg14from Legionella Pneumophila

Legionella pneumophila is the causative agent of a pneumonia known as Legionnaires’ disease. These bacteria are ubiquitous in aquatic environments and the protozoan amebae are their natural host. Inhalation of aerosol contaminated by L. pneumophila introduces the bacterium to alveolar macrophage, within which it replicates by mechanisms similar to those observed in its natural host. After phagocytosis, Legionella pneumophila can form an ER-like compartment, also known as legionella containing vacuole (LCV). The LCV can escape from endocytic pathway and avoid the fusion with lysosome where the bacteria will be degraded. Intracellular bacterial replication entirely depends upon a Type IVB secretion system termed Dot/Icm, which delivers a lager cohort of protein substrates into host cells. These proteins, also called effectors function to modify fundamental cellular process, such as cell death, membrane trafficking, stress response, and cytoskeleton to create an environment permissive for bacterial survival and multiplication. Although functional dissection of some of these effectors has revealed exiciting insights into the intricate manipulation of host activity by L. pneumophila, the activity of most of these genes reamains unknown.The yeast saccharomyces cerevisiae is a powerful tool in functional study of bacterial effectors. Many effectors of L. pneumophila have been identified by their ability to inhibit yeast growth. In our lab, one of the focuses is to dissect the function of effectors exhibiting strong toxicity to yeast. Here we found Cegl4, an effector of L. pneumophila, completely inhibits yeast growth. By random chemical mutagenesis, we found that residues Gly234or Thr623is critical for yeast toxicity of Ceg14. Further, we found that the yeast profilin gene strongly suppresses the toxicity of Ceg14. Consistent with the suppressor phenotype, we found that wild type Ceg14but not the two mutants, disrupted yeast cytoskeletonal structure.Direct analysis of the effects of Ceg14on actin polymerization by biochemical approaches revealed that this protein interferes with actin polymerization, causing the formation of shorter actin filaments. Again, mutations in Gly234or Thr623abolished the effects of Ceg14on actin assembly. These activities are opposite from those observed with VipA, suggesting that the bacterium targets host cytoskeleton in a balanced manner. We also examined the effects of Ceg14on the intracellular growth of L. pneumophila. Similar to most Dot/Icm effectors, deletion of ceg14did not cause detectable defect in intracellular bacterial growth in amoeba or primary mouse macrophages. Mutants lacking both ceg14and vipA also did not display detectable defect in intracellular bacterial growth, suggesting the existence of more effectors involved in targeting the host cytoskeleton. The identification and functional study of such effectors likely will reveal the role of host cytoskeleton in the formation of vacuole supportive for L. pneumophila replication.