File(s) under embargo
Reason: Wish to publish Chapter 3
until file(s) become available
REGULATION OF HOST CELL VESICLE TRAFFICKING AND PROTEIN TRANSLATION BY LEGIONELLA PNEUMOPHILA EFFECTORS
thesisposted on 15.11.2019, 16:17 by Alix McCloskey
The intracellular bacterial pathogen Legionella pneumophila is the etiological agent of Legionnaires’ disease, a severe pneumonia; it has also served as a valuable tool in studying host-pathogen interactions. The study of L. pneumophila pathogenesis has led to the discovery of novel biochemical and enzymatic mechanisms and a better understanding of host cell immune responses and signaling. L. pneumophila replicates within eukaryotic cells through the use of a type IV secretion system and over 330 effector proteins injected into the host cell. Only approximately 10% of these effectors have been characterized, but regardless of the small fraction, the complexity of L. pneumophila infection is clear. A good demonstration of this complexity is the large number of effector activities the bacteria uses to manipulate the small GTPase involved in ER to Golgi trafficking, Rab1. Six different effectors with eight separate activities modulate the activity of Rab1 to aid in the replication of the bacteria. We recently discovered that the protein SetA is yet another effector targeting Rab1. SetA glucosylates Rab1 using a canonical DxD motif and the glucose moiety interferes with both GTP hydrolysis and guanosine nucleotide dissociation inhibitor (GDI) binding. Based on our findings, the role of SetA is likely to aid in maintaining a pool of free Rab1, increasing availability for use by other L. pneumophila effectors. Another example of the complexity of L. pneumophila pathogenesis is the use of metaeffectors. Metaeffectors are effectors that regulate other effectors, both being produced by L. pneumophila. Three mechanisms of metaeffector regulation have been identified: 1) removal of a modification on host proteins placed by the cognate effector, 2) direct modification of the cognate effector or 3) direct binding to the cognate effector. Through the use of Size Exclusion Chromatography (SEC), binding assays with purified proteins and bacterial two-hybrid analysis, we found the mechanism of regulation for the SidI metaeffector Lpg2505 to be inactivation through direct binding. Atypical of previously identified effector characteristics, the binding of SidI by Lpg2505 occurs within the bacterial cell prior to translocation. The expression pattern of both effectors in L. pneumophila in addition to the other findings suggest a temporal role for Lpg2505 activity in which inactivation of SidI occurs after sufficient bacterial replication has occurred.