Identification and characterization of phosphoinositide-binding Legionella effector proteins
Date
2019
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Publisher
University of Delaware
Abstract
Bacterial pathogens have evolved diverse and effective strategies to promote their survival in human cells. Some bacteria can circumvent the innate immune response, managing to replicate within macrophages, which are the first line of defense against microbial pathogens and genetically programmed to eradicate foreign particles. Mechanisms that bacteria employ to survive in macrophages include (i) acclimating to the acidic environment within the host lysosome, (ii) escaping the phagosome to persist inside the host cell cytoplasm, or (iii) eluding the endolysosomal pathway by establishing a replication permissive vacuole within the host (Di Russo Case & Samuel, 2016). The Gram-negative facultative intracellular bacterium, Legionella pneumophila (hereafter Legionella), has adopted a survival strategy that relies on the establishment of a protective vacuole that avoids encounters with the endolysosomal pathway. Legionella is an important cause of hospital- and community-acquired pneumonia. Those with weakened immunity are particularly at risk, and in these cases the mortality rate can exceed 30% despite antibiotic treatment. The CDC reported Legionella as the leading bacterial cause of outbreaks associated with public drinking water, and was identified as the causative agent in all deaths related to public drinking water outbreaks reported in 2013-2014. Together these findings underscore the importance of understanding the molecular mechanisms behind Legionella pneumophila pathogenesis. ☐ In the human lung, macrophages internalize L. pneumophila into a membrane-bound compartment termed the phagosome. Once engulfed, L. pneumophila directs membrane remodeling of the phagosomal compartment, employing a sizeable artillery of bacterial proteins that subvert multiple host cellular processes without compromising the integrity of the host cell throughout infection (Hubber & Roy, 2010; Robinson & Roy, 2006; Swanson & Isberg, 1995). A specialized secretion system is responsible for translocating these proteins, known as effector proteins, from the bacterial milieu into the host cytosol (Berger & Isberg, 1993; Segal & Shuman, 1998; Vogel, Andrews, Wong, & Isberg, 1998). Effector proteins do not share extensive homology amongst each other and are often composed of multiple domains that are functionally distinct (Burstein et al., 2016; Gomez-Valero et al., 2019). An emerging feature among effector proteins is their ability to recognize and bind host phosphoinositides (PIPs) (Steiner, Weber, & Hilbi, 2018), which are a series of phospholipids that play critical roles in coordinating cell signaling and membrane trafficking events in eukaryotic cells (Di Paolo & De Camilli, 2006). L. pneumophila effector proteins exploit the spatiotemporal regulation of host PIPs to facilitate the formation of the Legionella-containing vacuole (LCV) and to avoid the endolysosomal pathway. Disruption of the PIP distribution on the LCV membranes leads to bacterial degradation, illustrating that controlling PIP dynamics on and around the LCV is crucial for intracellular survival of L. pneumophila (S. Weber, Wagner, & Hilbi, 2014). ☐ In chapter 2, we identified novel PIP-binding Legionella effector proteins by implementing a biochemical systematic approach combined with validation in mammalian cells and in the context of macrophage infection. From this analysis, we detected 66 effectors that are predicted to have similar secondary structures to eukaryotic PIP-binding proteins. Of these 66 candidates, we identified seventeen effectors that interact with PIPs in vitro. This newly identified cohort of effectors will provide the groundwork needed for studying the functions of novel PIP-binding effectors, which will fill important knowledge gaps regarding how L. pneumophila effectors selectively recognize host membranes and subvert host membrane trafficking. ☐ In chapters 3 and 4, we pursued the detailed characterization of two novel PI(3)P-binding effectors identified in our screen, RavD and Lpg2411. We found that RavD is present on the LCV membrane and is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways. Following the publication of chapter 3, RavD was reported to be a deubiquitinase (DUB) that cleaves linear ubiquitin present on the LCV. Establishing the link between RavD’s DUB activity and function in preventing phagosomal maturation of the LCV will certainly provide valuable insight into how this pathogen can circumvent endolysosomal degradation. In chapter 4, we report Lpg2411 as an effector involved in late stage infection that also associates with ubiquitinated-proteins. Cellular events that occur during late infection are not well established, thus further studies on Lpg2411 will be a significant contribution towards elucidating the host-pathogen dynamics during late infection. Overall, our data support the hypothesis that PIPs are a primary target of L. pneumophila effectors, and that PIP-binding effectors have diverse functions that warrant further investigation for revealing molecular mechanisms of intracellular bacterial pathogenesis.
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Keywords
Bacteria, Effector proteins, Host-pathogen interactions, Legionella, Pathogenesis