Defining the mitochondrial targeting mechanisms of a Legionella pneumophila effector
Date
2024
Authors
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Publisher
University of Delaware
Abstract
Diseases caused by bacterial pathogens are an increasingly worrisome threat to public health, both in the United States and globally. This is due to the ever-looming possibilities for pathogens to develop resistance towards commonly used antibiotics for treatment of bacterial infections used in growth of livestock for human consumption and those that are leached into the environment (Mancuso et al., 2021). This has been extensively documented for several bacterial pathogens, including those that can replicate and proliferate intracellularly (Aslam et al., 2018), highlighting the need to both understand and target mechanisms that contribute to their pathogenesis. Although documented antibiotic resistance cases are far and few in between for the facultative, intracellular pathogen Legionella pneumophila (Mondino et al., 2020), this may not be the case in the near future. L. pneumophila is an aerobic, Gram-negative bacterium whose replication and proliferation inside human alveolar macrophages ultimately manifests in Legionnaires’ disease in immunocompromised individuals, characterized by non-specific symptoms of pneumonia and often leads to additional symptom manifestation involving other organs (Cunha, 2010). L. pneumophila evolved its mechanisms for intracellular survival in macrophages through replication in its native hosts, unicellular eukaryotic protists such as amoeba and can be phagocytosed through a similarly-conserved pathway between macrophages and amoeba (Best & Abu Kwaik, 2018). ☐ L. pneumophila quickly commandeers the host cell upon entry through remodeling both the nascent phagosome and pathways involved in cellular homeostasis by the actions of its type 4 secretion system (T4SS)-secreted effector proteins (herein effectors) that are injected into the host cytosol at either early or delayed time points. Individual or coordinated efforts of some effectors establish the Legionella-containing vacuole and divert it from the phagolysosomal maturation pathway. Numerous other effectors are redirected to host organelles and compartments to exert spatiotemporal control to promote intracellular survival. Signaling peptides that direct localization to organelles (Rolando et al., 2013; Schator et al., 2023)(Dolezal et al., 2012) and lipid modifications (Price et al., 2010) are both documented methods for effector recruitment. However, an increasingly apparent motif for intracellular targeting is through binding eukaryotic phosphoinositides, lipids that differentially decorate host organelles and subcellular compartments. For example, different pools of phosphoinositides can be found adjacent to or on mitochondria, a target for an increasing number of effectors in manipulating its many internal and external functions. Therefore, understanding the preferences for phosphoinositide binding, as well as their timing for intracellular accumulation, for these effectors can shape our understanding of their predicted localization and functions in promoting L. pneumophila pathogenesis. ☐ In this work, we functionally characterize one of the previously-validated T4SS-secreted effectors, Ceg29/Lpg2409, which was demonstrated by members of our lab to bind the phosphoinositide PI(3)P for an unknown purpose. Its optimized expression and purification workflow, as well as its structural characterization, is outlined in Chapter 2. Utilizing a site-directed mutagenesis strategy, we demonstrated that C-terminal residues of Ceg29 are likely involved in binding to PI(3)P, and that both N and C-terminal truncation constructs of Ceg29 can localize with the PI(3)P marker EGFP-2x-FYVE when co-transfected in mammalian cells, indicating a potentially low affinity for binding. However, we were able to successfully characterize the tight binding to the soluble head group of PI(3)P by effector RavD/Lpg0160 using a fluorescence polarization-based approach. These findings indicate potentially different and physiologically-relevant binding strategies for effectors that target PI(3)P, and may shed light on the duration that they remain on host membranes. ☐ In Chapter 3, we identified that N-terminal residues of Ceg29 demonstrate increased targeting to mammalian mitochondria through fluorescence-based and biochemical fractionation assays employing N and C-terminally tagged Ceg29, and that this targeting is retained through cellular fractionation of infected macrophages. This likely directs its import into the inner mitochondrial membrane, intermembrane space, and matrix as confirmed by extraction and Proteinase K protection assays. We determined the putative host protein interactome of Ceg29 using a combined immunoprecipitation/MS approach in HEK293T cells, and in doing so demonstrated significant enrichment of hits in ubiquitin-dependent pathways, as well as membrane-bound proteins. Among the top candidates was a protein crucial for preserving mitochondrial cristae junctions, Mic60, and we demonstrated through site-directed mutagenesis that both N and C-terminal residues of Ceg29 may be involved in binding. Co-immunoprecipitation with PINK1, as well as increased ubiquitination of Mic60 in mammalian cells upon overexpression of Ceg29 and its N-terminal residues, pointed to a potential link for mediation of mitophagy by Ceg29. This work marks the first documented case of a L. pneumophila effector targeting any protein involved in mitochondrial cristae structure, and is additionally the first instance of a direct means of mitophagy regulation by the pathogen. ☐ Lastly, to circumvent the possibility for inaccurate localization through ectopic overexpression of Ceg29, we validated antibodies for the use of detecting endogenous Ceg29 during infection, as well as overexpressed Ceg29 by L. pneumophila. In tandem, we performed preliminary experiments for labeling and tracking HaloTagged-Ceg29 during late infection using commercially-available HaloTag ligands, and validated that HaloTag-Ceg29 can still retain localization to the mitochondria of infected macrophages. Although future optimization is still required to determine the true subcellular localization of Ceg29, we were able to successfully apply the HaloTag labeling system to a L. pneumophila effector, a strategy that will be invaluable for live-cell tracking of Ceg29 and its influence on host mitochondrial dynamics. Taken together with the findings from Chapter 2, we surmise that Ceg29 localizes to mitochondrial-ER contact sites enriched in PI(3)P, where it targets mitochondria and is imported via N-terminal residues. Once inside, Ceg29 binds to Mic60 and triggers its ubiquitination by an unknown mechanism, potentially stimulating mitophagy. This work highlights an entirely new method for mitochondrial access by a bacterial pathogen and suggests that L. pneumophila engages in manipulating mitochondrial structure and stress responses at later stages in its intracellular life cycle than what has previously been documented.
Description
Keywords
Host-pathogen interactions, Legionella pneumophila, Mitochondria, Protein-lipid interactions, Organelles