Investigation of the role of the Dot/Icm Type Four Secretion System substrate SidL during Legionella pneumophila intracellular growth

Legionella pneumophila is a facultative intracellular pathogen capable of infecting protozoan hosts in aquatic environments. When aerosols contaminated with L. pneumophila are inhaled by humans, a similar infection is initiated in lung alveolar macrophages, resulting in a pneumonia called Legionnaire\u27s disease. After phagocytosis of the bacterium by a eukaryotic host cell, the Legionella containing vacuole (LCV) undergoes a unique route of endocytic transport in which the LCV does not fuse with lysosomal compartments and eventually acquires characteristics of rough endoplasmic reticulum. This modulation of host vesicle trafficking is dependent on the L. pneumophila Dot/Icm Type Four Secretion System (TFSS), since Dot/Icm mutants do not replicate and are trafficked to the lysosome. The Dot/Icm TFSS has been shown to translocate over 250 bacterial proteins into the host cell cytosol. Several of these translocated substrates have been shown to directly modulate host cell processes, such as vesicle trafficking and programmed cell death, to the benefit of bacterial replication. Some Dot/Icm effectors disrupt pathways essential to eukaryotic host cells, and therefore produce an inhibitory or toxic effect when ectopically expressed in yeast. We performed a screen to identify novel L. pneumophila Dot/Icm effectors that are toxic or inhibitory to the growth of yeast. Out of 80 L. pneumophila hypothetical proteins tested, eight were identified as novel substrates of the Dot/Icm TFSS, and two were found that inhibited yeast growth. One substrate of Dot/Icm that was found to be toxic when expressed in both yeast and mammalian cells, SidL, was subjected to further investigation. SidL has previously been identified as an inhibitor of eukaryotic protein synthesis, but the host proteins or pathway being manipulated by SidL to produce this effect are as of yet unknown. We identified two substitution mutants of SidL, SidL G234V and SidL T623N, which were no longer toxic when expressed in yeast. Additionally, we screened a library of yeast genomic DNA fragments to identify yeast proteins that, when co-expressed with SidL, could alleviate its toxic effects. We found that co-expression of the G-actin binding protein profilin with SidL could suppress the toxicity of SidL. This suppression was dependent on the ability of profilin to interact with actin, as co-expression of SidL with a mutant profilin deficient in actin binding could not rescue yeast growth. These results indicate that SidL may be manipulating a conserved process related to the actin cytoskeleton during L. pneumophila infection; however, the precise biochemical mechanism of this subversion, and the benefit that it confers to bacterial replication are still being determined