Structural Identification of Legionella pneumophila Effector Protein LEM23 as a Fic Protein and its Functional Implications

Abstract

Legionella pneumophila (L. pneumophila) is an opportunistic human pathogen known to cause severe pneumonia in those infected. It primarily infects human alveolar macrophage cells, altering vesicular trafficking while evading innate immune response. Once internalized through phagocytosis by the host macrophage cell, L. pneumophila hijacks the host cell’s signaling and vesicular trafficking pathways. To accomplish this, L. pneumophila has evolved a type IVB secretion system that is capable of transporting effector proteins through its inner and outer membranes and through the vacuole into the host cytoplasm. Once in the cytoplasm, the effector proteins perform post-translational modifications (PTMs) to the host proteins, altering their functions. The aim of this thesis was to characterize an effector protein that was identified using the process of “machine learning”, aptly named Lem23 (Legionella effector identified by machine learning). Using protein crystallography, poorly diffracting Lem23 crystals were obtained and, subsequently optimized using reductive lysine methylation. The optimized crystals led to a structure with a resolution of 1.55 Å; further analysis revealed that Lem23 is a Fic protein. Lem23 contains the required 6 α-helices that constitute a Fic core, a slightly modified catalytic loop, and a large insertion that may be an interaction domain for its target proteins. Using sequence analysis and structural knowledge the possibility of Lem23 AMPylating its protein target was assessed. AMPylation is a post- translational modification common to Fic proteins. Finally a HEK293 expression and co- immunoprecipitation procedure was used to pull-down Lem23’s target proteins during host cell infection. Preliminary mass spectrometry results of co-immunoprecipitated proteins revealed Lem23 interacts with two proteins involved with the cellular apoptotic pathway. This study provides further information about one effector protein, Lem23, and its potential importance in the infection process to inhibit the host cells innate immune response.