**3. Phosphoinositides as crucial regulators of vesicular trafficking**

Membrane compartments within eukaryotic cells are highly abundant, dynamic, and functionally distinct structures. Their movement must be tightly regulated to ensure that cargo carried by these structures is delivered to the proper destination. The cellular machinery recognizes and distinguishes these compartments based on the unique protein and lipid composition on the cytosolic leaflet of the membrane lipid bilayer [11]. Phosphoinositides are glycerophospholipids that amount to less than 15% of phospholipids within membranes but are essential for coordinating the spatiotemporal regulation of membrane trafficking events [11]. Phosphatidylinositol (PI), the precursor of phosphoinositides, can be reversibly phosphorylated at positions 3, 4, and 5 of its *myo*-inositol ring resulting in the

*Pathogenic Bacteria*

**immune cells**

membrane remodeling of the phagosomal compartment, employing a sizeable artillery of bacterial proteins that subvert multiple host cellular processes without compromising survival of the host cell throughout infection [2–4]. A specialized secretion system is responsible for translocating these proteins, known as effector proteins, from the bacterial milieu into the host cytosol [5–7]. Effector proteins do not share extensive homology with each other and are often composed of multiple domains that are functionally distinct [8, 9]. An emerging feature among effector proteins is their ability to recognize and bind host phosphoinositides (PIPs) [10], which are a series of phospholipids that play critical roles in coordinating cell signaling and membrane trafficking events in eukaryotic cells [11]. *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* [12]. Here we will discuss the *L. pneumophila* effector proteins that contribute to vacuole biogenesis and

maintenance through the exploitation of host phosphoinositides.

engulfed by a new host cell and tap into their nutrient resources.

**2.** *Legionella pneumophila* **replicates in protozoan and innate** 

*L. pneumophila* is ubiquitously found in aquatic environments forming close associations with protozoans and often found as an intracellular parasite of freeliving amoeba [13]. In the human lung, *L. pneumophila* infects resident alveolar macrophages leading to severe pneumonia, known as Legionnaires' disease, which can be fatal in immunocompromised individuals [14]. Outbreaks stem from

contaminated water systems such as those supplying water towers, cooling systems, and decorative fountains [15]. In 2017, a study by the Centers for Disease Control and Prevention (CDC) found that *L. pneumophila* was the leading bacterial agent responsible for public drinking water-associated outbreaks within the United States [14]. The number of reported Legionnaires' disease cases has been escalating since 2000, presumably due to an increase in urbanization, reliance on industrial water systems, as well as improved diagnostic methods [16]. *Legionella* spp. can exist within biofilms or amoebal hosts in freshwater systems, transitioning between a replicative and a transmissive/virulent phase life cycle [17, 18]. Nutrient deprivation within a biofilm or host triggers the upregulation of genes encoding virulence traits such as motility, osmotic stress resistance, pigmentation production, and multiple virulence factors [17]. This change in gene expression primes the bacterium to be

Inter-kingdom horizontal gene transfer events and circulating mobile genetic elements over long-term coevolution with multiple hosts have extensively reshaped the plasticity of the *Legionella* spp. genomes [19]. All *Legionella* spp. contain a highly conserved type IV secretion system (T4SS), yet there are differences in the combination of effectors present in each species. An analysis of 38 *Legionella* spp. genomes revealed that DNA exchange between species is rare and only seven core effectors are shared by all sequenced species [8]. *Legionella* effectors share more similarity with eukaryotic proteins than prokaryotic proteins, suggesting *Legionella* spp. have acquired their effector arrays from their hosts [20]. A striking number of effectors across the genus (>18,000) contain eukaryotic-derived domains [9]. This extensive combination of effectors likely stems from intimate coevolution between *Legionella* spp. with diverse protozoan hosts, such as *Acanthamoeba castellanii* [13], *Hartmannella vermiformis* [21], *Dictyostelium discoideum* [22, 23], *Tetrahymena* 

**38**

generation of seven PIP species [11]. Membrane compartments are characterized in part by the presence of distinct PIP species that essentially act as molecular anchors to facilitate protein recruitment and attachment to specific compartments [11]. PI is synthesized in the endoplasmic reticulum and delivered to membrane-bound compartments via vesicular transport or cytosolic PI transfer proteins [11]. The Golgi and plasma membrane are highly enriched with PI(4)P, while lower levels of PI(4)P are also found within membranes of the ER and late endosomes [11, 36, 37]. PI(3)P is mainly found on phagosomes, early endosomes, late endosomes, and multivesicular bodies (MVBs). MVBs and late endosomes also contain PI(3,5)P2, which is the dominant PIP on lysosomes. Phagocytosis and phagosome maturation are entirely dependent on phosphoinositide dynamics [38]. PI(4,5)P2 and PI(3,4,5) P3 are present on the plasma membrane and are critical for recruiting the cellular machinery for initiating phagocytosis. Once phagosomes have been formed, PI(3)P is the predominant PIP on the organelle [29]. PI(3)P then triggers the recruitment of proteins to the phagosome, such as EEA1 and its subsequent effector Rab5, to facilitate docking and fusion with early endosomes and progression down the phagolysosomal maturation pathway [39]. Blocking the formation of these PIP species results in robust inhibition of phagocytosis [40]. Given the crucial importance of PIPs for particle uptake and degradation, it is not surprising that intracellular bacteria have evolved molecular mechanisms to take command of these eukaryotic lipids.
