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

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

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* 

**39**

*Exploitation of Phosphoinositides by the Intracellular Pathogen,* Legionella pneumophila

*pyriformis* [24], and *Naegleria fowleri* [25]. Only 20 of the 65 known species have been associated with human disease, suggesting that perhaps *Legionella* species are better adapted for infection within their amoebal hosts [9]. A clear set of effectors that render *Legionella* better suited for human infection is not apparent, although conservation of ankyrin motifs, F-box, or Set18 domains was predominantly found

The prevailing thought is that the mechanisms that enable *L. pneumophila* to infect and proliferate within protozoa have equipped this bacterium with the ability to survive within innate immune cells. This ability could be due to the high conservation of the pathways involved in uptake and microbial degradation between protozoa and human macrophages. In the lung, resident macrophages and neutrophils engulf *L. pneumophila* by phagocytosis but are often unable to degrade it through phagosome maturation [26–28], a process that entails sequential fusion of the phagosome with endocytic compartments and ultimately the lysosome [29]. *L. pneumophila* is initially encased within a phagosome after macrophage engulfment, but within minutes, the membrane of this phagosome is drastically remodeled into a compartment resembling the endoplasmic reticulum (ER) [2, 4]. Tubular ER and secretory vesicles are rapidly routed toward the phagosome where some eventually fuse with the phagosomal membrane, allowing the phagosome to adopt the identity of the recruited host membrane [30]. While promoting LCV membrane fusion with the ER and Golgi-derived vesicles, *L. pneumophila* prevents fusion with endosomal compartments. Studies have found that *L. pneumophila* effector proteins can target specific host membrane compartments, including early endosomes, recycling endosomes, and autophagosomes. Collectively, these effectors help *L. pneumophila* evade the macrophage's pre-programmed lysosomal degradation pathway [10], although

precisely how these events are choreographed is not well understood.

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

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

Membrane compartments within eukaryotic cells are highly abundant, dynamic,

The extensive remodeling of the vacuolar membrane is entirely dependent on a specialized Dot/Icm T4SS that delivers a staggering number of bacterial effector proteins (over 350) [8] into the host cytosol, many of which target membrane transport pathways [31, 32]. Disruption of the T4SS results in lysosomal degradation of the bacterium, indicating that the actions of effector proteins are paramount to bacterial survival [33]. However, it is often a challenge to identify an observable phenotype that can be attributed to a single effector because of functional redundancy among bacterial effectors [34]. Many advances have been made to dissect the molecular contribution of individual effectors toward bacterial infection (reviewed in [35]). A number of these effectors have been reported to hijack host vesicular trafficking pathways. An emerging feature among some of the effectors that target membrane trafficking is the ability to bind key host regulatory lipids,

*DOI: http://dx.doi.org/10.5772/intechopen.89158*

in more virulent strains [9].

phosphoinositides (PIPs).

#### *Exploitation of Phosphoinositides by the Intracellular Pathogen,* Legionella pneumophila *DOI: http://dx.doi.org/10.5772/intechopen.89158*

*pyriformis* [24], and *Naegleria fowleri* [25]. Only 20 of the 65 known species have been associated with human disease, suggesting that perhaps *Legionella* species are better adapted for infection within their amoebal hosts [9]. A clear set of effectors that render *Legionella* better suited for human infection is not apparent, although conservation of ankyrin motifs, F-box, or Set18 domains was predominantly found in more virulent strains [9].

The prevailing thought is that the mechanisms that enable *L. pneumophila* to infect and proliferate within protozoa have equipped this bacterium with the ability to survive within innate immune cells. This ability could be due to the high conservation of the pathways involved in uptake and microbial degradation between protozoa and human macrophages. In the lung, resident macrophages and neutrophils engulf *L. pneumophila* by phagocytosis but are often unable to degrade it through phagosome maturation [26–28], a process that entails sequential fusion of the phagosome with endocytic compartments and ultimately the lysosome [29]. *L. pneumophila* is initially encased within a phagosome after macrophage engulfment, but within minutes, the membrane of this phagosome is drastically remodeled into a compartment resembling the endoplasmic reticulum (ER) [2, 4]. Tubular ER and secretory vesicles are rapidly routed toward the phagosome where some eventually fuse with the phagosomal membrane, allowing the phagosome to adopt the identity of the recruited host membrane [30]. While promoting LCV membrane fusion with the ER and Golgi-derived vesicles, *L. pneumophila* prevents fusion with endosomal compartments. Studies have found that *L. pneumophila* effector proteins can target specific host membrane compartments, including early endosomes, recycling endosomes, and autophagosomes. Collectively, these effectors help *L. pneumophila* evade the macrophage's pre-programmed lysosomal degradation pathway [10], although precisely how these events are choreographed is not well understood.

The extensive remodeling of the vacuolar membrane is entirely dependent on a specialized Dot/Icm T4SS that delivers a staggering number of bacterial effector proteins (over 350) [8] into the host cytosol, many of which target membrane transport pathways [31, 32]. Disruption of the T4SS results in lysosomal degradation of the bacterium, indicating that the actions of effector proteins are paramount to bacterial survival [33]. However, it is often a challenge to identify an observable phenotype that can be attributed to a single effector because of functional redundancy among bacterial effectors [34]. Many advances have been made to dissect the molecular contribution of individual effectors toward bacterial infection (reviewed in [35]). A number of these effectors have been reported to hijack host vesicular trafficking pathways. An emerging feature among some of the effectors that target membrane trafficking is the ability to bind key host regulatory lipids, phosphoinositides (PIPs).
