Date on Master's Thesis/Doctoral Dissertation
Microbiology and Immunology
Microbiology and Immunology, PhD
Abu Kwaik, Yousef
Committee Co-Chair (if applicable)
Legionella pneumophila; effector protein; LCV biogenesis; MavE, ACBD3; vesicular trafficking
Legionella pneumophila is a Gram-negative facultative intracellular bacterium found in freshwater environments that has co-evolved to survive and proliferate in various amoeba and protozoan species, which serve as the natural host for the bacterium. Humans are an accidental host of L. pneumophila, where infection occurs upon inhalation of aerosolized water droplets that contain the bacteria. Intracellular proliferation of L. pneumophila in alveolar macrophages is essential for manifestation of pneumonia, designated as Legionnaires’ Disease. Biogenesis of the legionella containing vacuole (LCV) occurs via interception of ER-Golgi vesicle trafficking and avoids the default endosomal/lysosomal degradation pathway. Intracellular proliferation of L. pneumophila within protozoa and macrophages is dependent on the Dot/Icm type IV secretion system (T4SS) apparatus, which is comprised of 27 proteins and is responsible for translocating over 350 different effector proteins into the host cell. Many of these effector proteins contain eukaryotic-like domains and motifs, which have been acquired through interkingdom horizontal gene transfer from various aquatic eukaryotic hosts. While L. pneumophila contains the largest repertoire of effector proteins, known for an intracellular pathogen, most of which are not required for survival and proliferation in mammalian macrophages. It is more likely that the large repertoire of effector proteins constitutes a toolbox utilized by L. pneumophila to survive and replicate within various protozoan species. The diversion of the L. pneumophila-containing vacuole (LCV) from the host endosomal-lysosomal degradation pathway is one of the main virulence features essential for disease manifestation. Many of the ~350 Dot/Icm-injected effectors identified in L. pneumophila have been shown to interfere with various host pathways and processes; but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. While most effector mutants of L. pneumophila do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of L. pneumophilia in human monocyte-derived macrophages (hMDMs), amoebae and for intrapulmonary proliferation in mice. This is shown by both single cell analysis during confocal microscopy and by quantifying colony forming units (CFUs). We have shown the mavE null mutant fails to remodel the LCV with ER-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin-killed bacteria. Importantly, during infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE (39-172) was resolved to 1.8 Å, revealing a eukaryotic NPxY motif that binds with phosphotyrosine-binding domains present on signaling and adaptor eukaryotic proteins. We show that point mutations within the NPxY motif results in attenuation of L. pneumophila in both hMDMs and amoeba, and the substitution defects of P78 and D64 results in fusion of the LCV to the lysosomes, with no remodeling by the ER, leading to bacterial degradation. Following ectopic expression of MavE, a proximity-dependent biotin identification (BioID) strategy was used to screen for MavE-interacting proteins in mammalian cells. These data show that MavE interacts with a host protein, acyl-CoA binding domain containing 3 (ACBD3), which co-localizes with the LCV. ACBD3 plays an essential role in the sorting and modification of proteins exported from the endoplasmic reticulum through its interaction with the integral membrane protein giantin. We have shown the mavE null mutant-containing LCV fails to colocalize with ACBD3, similar to the Dot/Icm translocation-defective mutant. There are areas of homology of ACBD3 with proteins found in Amoebozoa, indicative of a possible conserved binding motif. We conclude that the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion by interacting with the host protein ACBD3, which is involved in ER-Golgi vesicle trafficking and is likely conserved throughout evolution.
Vaughn, Bethany, "Lysosomal evasion of legionella pneumophilia the effector." (2022). Electronic Theses and Dissertations. Paper 3833.