**5.** *L. pneumophila* **effector proteins alter the PIP composition of the LCV membrane**

To manipulate the PIP composition on the LCV, *L. pneumophila* uses both genetically encoded and host-derived PI kinases and phosphatases (**Figure 1**). Converting the PI(3)P-enriched phagosome to a predominantly PI(4)P-positive compartment requires a concerted effort between enzymes that add and remove

#### **Figure 1.**

*Pathogenic Bacteria*

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.

The PIP composition on the LCV membrane has profound effects on the fate of the bacteria-bearing vacuole. PI conversion that accompanies LCV maturation was deciphered by tracking the localization of fluorescent PI probes produced in the soil amoeba, *Dictyostelium discoideum*, which serves as a model organism for the study of host-pathogen interactions [41]. As *L. pneumophila* enters *D. discoideum*, the phagocytic cup is coated with PI(3,4,5)P3. On the membrane of the newly formed phagosome, PI(3,4,5)P3, PI(3,4)P2, and PI(4)P persist for less than 60 s on average. By 60 s, the phagosome begins to accumulate PI(3)P. Over the next 2 h, PI(4)P levels increase, the LCV lumen expands, and PI(3)P is slowly lost and excluded from the maturing LCV. The mature LCV maintains a discrete pool of PI(4)P separate from the surrounding ER, in which it acquires 30 to 60 min after uptake. As the bacterium continues to replicate, PI(4)P levels are steadily maintained on the LCV but are present in pools distinct from the surrounding ER network. The conversion from a PI(3)P to a PI(4)P-positive compartment is secretion system-dependent: a mutant strain lacking a functional T4SS accumulates PI(3)P on the LCV, PI(4) P is never acquired, and the LCV is destined for lysosomal degradation [12]. Thus, translocated effectors control the PIP composition of the LCV and potentially other

In a recent study, Weber and colleagues [42] pursued the source of the PI(4) P on the LCV membrane. Real-time high-resolution confocal laser scanning microscopy (CLSM) revealed that LCVs of infected *D. discoideum* capture PI(4)P from trans-Golgi-derived vesicles. PI(4)P-enriched vesicles accumulate close to the LCV, even in the absence of the T4SS, but retention of these vesicles relies on the T4SS. This observation indicates that while PI(4)P-positive compartments localize to phagosomes regardless of the internalized cargo, effector proteins are needed to prolong this interaction. The removal of PI(3)P from the phagosome membrane was thought to occur through the actions of PIP-modifying enzymes; however, CSLM imaging of infected *D. discoideum* revealed shedding of PI(3)P-positive vesicles from the LCV. Moreover, the timing of PI(3)P shedding coincided with the

**4. Phosphoinositide dynamics on the LCV**

**40**

host membranes.

*L. pneumophila converts the phagosome to a PI(4)P-rich vacuole. Within a minute of uptake into the host cell, the LCV acquires the endosomal phosphoinositide, PI(3)P. Within an hour of infection, the LCV starts to accumulate PI(4)P until the bacteria are completely encapsulated in a PI(4)P-rich membrane. To avoid progression down the phagosome maturation pathway, L. pneumophila translocates effectors that alter the phosphoinositide composition on the LCV membrane to a PI(4)P-positive compartment (inset). This process is a result of close association and fusion with host vesicles as well as the direct conversion of existing phosphoinositides by kinases and phosphatases. Golgi-derived PI(4)P-positive vesicles accumulate around the LCV and later fuse with the vacuolar membrane. In contrast, PI(3)P-containing vesicles traffic toward the LCV but do not fuse with it. Additionally, the Legionella effector LepB is a PI kinase that phosphorylates PI(3)P and generates PI(3,4)P2 on the LCV membrane. This PI is a substrate for SidF which dephosphorylates PI(3,4)P2 to PI(4)P. While the origin of PI(3)P that LepB utilizes as a substrate is undetermined, LegA5 is a PI 3-kinase produced by Legionella that phosphorylates PI and could lead to additional PI(3)P on the LCV for conversion to PI(4)P. In combination, LegA5, LepB, and SidF may provide a cascade of enzymatic events for converting the LCV into a PI(4)P-positive compartment. SidP, another direct modifier of phosphoinositides produced by Legionella, may also contribute to the avoidance of the endocytic pathway by removing the phosphate from PI(3)P to hinder vesicle fusion. VipD localizes to endosomes and hydrolyzes a lipid tail from PI(3)P to potentially limit their interaction with the LCV. During this phosphoinositide conversion, Legionella effectors associate with the LCV through phosphoinositide binding domains. Some effectors localize by binding PI(3)P (RavD, LidA, SetA, LpnE, RidL, LtpD, LtpM), and some can associate via PI(4)P-binding (LidA, Lem4, SidM, SidC, Lem28, SdcA). During the later stages of infection, PI(3)P is undetectable and PI(4)P has become enriched on the expanding vacuole.*

a phosphate group of the *myo*-inositol head group. The direct PI 4-kinase activity of the effector LepB could potentially initiate the conversion process to a PI(4) P-positive membrane by converting PI(3)P to PI(3,4)P2. LepB was initially identified as an effector that is involved in bacterial egress [43]. Since then, LepB was found to localize to the LCV, where it contributes to the dynamics of Rab1 by acting as a GTPase-activating protein (GAP) [44, 45]. Found between amino acids 313 and 618, the structure and mechanism of the GAP domain is now well understood [46–48]. The N-terminal domain consisting of the first 311 amino acids garnered interest as this domain alone could disrupt the structure and function of the Golgi. The crystal structure of LepB1–618 revealed homology to atypical kinases such as CtkA from *Helicobacter pylori* and actin-fragmin kinase from *Physarum polycephalum*. When mutating residues capable of performing phosphorylation, the yeast toxicity phenotype was found to be suppressed. While this suggested a kinase functionality, the pocket for a substrate was too small to accommodate proteins any larger than Rab GTPases. However, LepB did not phosphorylate any of the tested Rab GTPases. Instead, the LepB substrates were revealed to be phosphoinositides. Studies showed that LepB, but not the catalytically inactive mutant LepBH154A, caused the sensor for PI(3,4)P2 and PI(3,4,5)P3 to relocate from a cytosolic to punctate distribution, while the signal for PI(3)P diminished dramatically. Ultimately, an in vitro kinase assay validated that LepB is a PI 4-kinase with specificity for PI(3) P and a level of activity comparable to the host kinase PI4KIII. By phosphorylating PI(3)P on the LCV, LepB could be initiating the vacuole's phosphoinositide conversion to PI(4)P by providing the PI(3,4)P2 intermediate step [49].

As PI(3,4)P2 is generated on the LCV, it is thought that SidF can dephosphorylate this lipid to PI(4)P. SidF is a membrane protein containing a large N-terminal domain followed by two transmembrane domains triggering localization to the LCV. SidF was the first *L. pneumophila* effector found to directly modify phosphoinositides through a screen for the well-known CX5R phosphatase motif in the effector repertoire. SidF is a PI 3-phosphatase: this effector hydrolyzes the phosphate group at the third position of PI(3,4)P2 or PI(3,4,5)P3 to produce PI(4)P or PI(4,5) P2, respectively; however, it displays a preference for PI(3,4)P2. The mutation of the catalytic cysteine at residue 645 to a serine resulted in the abrogation of this phosphatase activity. As described in the following sections, PI(4)P on the LCV membrane can serve as a means for effectors, such as SidC, to anchor onto the LCV. Infection with a mutant lacking *sidF* shows significantly fewer vacuoles positive for SidC, suggesting that SidF contributes to the generation of PI(4)P on the LCV.

Ultimately, the functions of LepB and SidF suggest that PI(3)P can be converted to PI(4)P through the sequential efforts of these enzymes. The deletion of *lepB* and *sidF* individually shows a significant deficiency of SidC on the LCV membrane at similar levels [49, 50]. The deletion of both effectors simultaneously causes a decrease in SidC acquisition on the membrane no greater than the single-mutant strains, suggesting that these effectors are functioning in a linear pathway [49]. However, the complete loss of SidC was not seen in the infection with a *lepB sidF* double-deletion mutant. Additionally, both *lepB* and *sidF* are not always found in other *Legionella* species. Together, this suggests that there are other *Legionella* effectors or host proteins manipulating the LCV PIP landscape.

The screen that identified SidF as a PI phosphatase also yielded SidP as another direct modifier of phosphoinositides. SidP was identified as a candidate due to its CX5R motif. It was found to have PI 3-phosphatase activity, cleaving PI(3) P and PI(3,5)P2 in vitro. The *L. longbeachae* orthologue of SidP was only found to hydrolyze PI(3)P, suggesting this lipid may be the true target. SidP was also found to act as a PI 3-phosphatase *in vivo* when it suppressed yeast toxicity in a PI 3-phosphatase-deficient mutant but not a mutant lacking PI 4-phosphatases. This

**43**

alters the PI composition of the LCV.

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

activity was confirmed when the levels of PI(3)P, but not PI(4)P or PI(4,5)P2, were decreased in the presence of SidP [51]. Nonetheless, the purpose of SidP's phosphatase activity for successful infection has not yet been determined. We can speculate that SidP may work alongside LepB to quickly eliminate PI(3)P from the vacuole. As LepB converts PI(3)P to PI(3,4)P2, SidP may be dephosphorylating PI(3)P to PI to completely deplete the membrane of this phospholipid that would otherwise trigger

As part of an effort to determine the function of a *Francisella* effector, OpiA, LegA5 was found to possess PI 3-kinase activity. LegA5 contains two motifs, DXHXXN and IDH, separated by 14 amino acids that are characteristic of the catalytic and activation loops of PI 3-kinases (and PI 4-kinases) [52]. PI(3)P has been shown to accumulate on the LCV early during infection in a manner independent of effector protein translocation [12]. This lipid is speculated to be the substrate LepB that acts on to initiate the PI(3)P to PI(4)P conversion on the phagosome membrane. However, *Legionella* may encode an effector that also contributes to the PI(3) P pool. These proteins may be delivered to the LCV in a complex so that PI is efficiently converted to PI(4)P. Alternatively, or perhaps in addition, PI(3)P-positive vesicles that accumulate around the nascent *Legionella*-containing phagosome may

Aside from kinases and phosphatases that change the phosphorylation state of PIPs, *Legionella* also encodes 19 phospholipases. Phospholipases differ from PI phosphatases by cleaving the phospholipid backbone instead of hydrolyzing a phosphate on the *myo*-inositol head group. While these proteins can enter the host through different systems such as the Sec, Tat, T2SS, T4SS, and outer membrane vesicles (OMVs), only phospholipases translocated via T4SS will be discussed here [53]. The best characterized T4SS-secreted phospholipase is VipD. VipD has three paralogs, VpdA, VpdB, and VpdC, which are also T4SS substrates but have yet to be studied in detail [54]. The structure of VipD shows two distinct domains: the N-terminal domain has phospholipase activity A, indicating cleavage of the ester bond releasing a fatty acid chain, and the C-terminal domain causes localization to early endosomes and interacts directly with Rab5 and Rab22 [55]. The phospholipase activity is activated when VipD is bound to Rab5 due to a conformational change that exposes the active site [56, 57]. The activation of VipD causes cleavage of PI(3)P on endosomal membranes that prevents normal localization of membrane trafficking regulators, contributing to endosomal avoidance by *Legionella* [56]. While VipD has phospholipase A activity, *Legionella* also translocates two T4SS effectors with phospholipase C and D activity. A phospholipase C hydrolyzes the phosphorus-oxygen bond, releasing the phosphate of the phospholipid and the attached head group, and a phospholipase D solely cleaves off the attached head group. The phospholipase C effector protein, PlcC, is able to cleave phospholipids such as phosphatidylglycerol, phosphatidylcholine, and phosphatidylinositol [58]. Alone or in combination with two other phospholipase C effectors, PlcA and PlcB, translocated by the T2SS, these effectors were dispensable for growth in amoeba and macrophages. However, a triple mutant of these phospholipases displayed inefficient killing of larvae in the *G. mellonella* infection model compared to the wild type [58]. It is not yet known how this function may contribute to intracellular survival. We speculate that perhaps removing the head group on these phospholipids, specifically PI, would render them incapable of being modified by PI kinases and phosphatase and prevent the vacuole from being quickly converted to an endosomelike membrane. It would be interesting to determine if this phospholipase activity

Lastly, the phospholipase D effector, LpdA, was first identified due to its homology with known phospholipase D enzymes [59]. LpdA specifically cleaves the head

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

serve as a source for the initial wave of PI(3)P.

the phagocytic maturation.

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

activity was confirmed when the levels of PI(3)P, but not PI(4)P or PI(4,5)P2, were decreased in the presence of SidP [51]. Nonetheless, the purpose of SidP's phosphatase activity for successful infection has not yet been determined. We can speculate that SidP may work alongside LepB to quickly eliminate PI(3)P from the vacuole. As LepB converts PI(3)P to PI(3,4)P2, SidP may be dephosphorylating PI(3)P to PI to completely deplete the membrane of this phospholipid that would otherwise trigger the phagocytic maturation.

As part of an effort to determine the function of a *Francisella* effector, OpiA, LegA5 was found to possess PI 3-kinase activity. LegA5 contains two motifs, DXHXXN and IDH, separated by 14 amino acids that are characteristic of the catalytic and activation loops of PI 3-kinases (and PI 4-kinases) [52]. PI(3)P has been shown to accumulate on the LCV early during infection in a manner independent of effector protein translocation [12]. This lipid is speculated to be the substrate LepB that acts on to initiate the PI(3)P to PI(4)P conversion on the phagosome membrane. However, *Legionella* may encode an effector that also contributes to the PI(3) P pool. These proteins may be delivered to the LCV in a complex so that PI is efficiently converted to PI(4)P. Alternatively, or perhaps in addition, PI(3)P-positive vesicles that accumulate around the nascent *Legionella*-containing phagosome may serve as a source for the initial wave of PI(3)P.

Aside from kinases and phosphatases that change the phosphorylation state of PIPs, *Legionella* also encodes 19 phospholipases. Phospholipases differ from PI phosphatases by cleaving the phospholipid backbone instead of hydrolyzing a phosphate on the *myo*-inositol head group. While these proteins can enter the host through different systems such as the Sec, Tat, T2SS, T4SS, and outer membrane vesicles (OMVs), only phospholipases translocated via T4SS will be discussed here [53]. The best characterized T4SS-secreted phospholipase is VipD. VipD has three paralogs, VpdA, VpdB, and VpdC, which are also T4SS substrates but have yet to be studied in detail [54]. The structure of VipD shows two distinct domains: the N-terminal domain has phospholipase activity A, indicating cleavage of the ester bond releasing a fatty acid chain, and the C-terminal domain causes localization to early endosomes and interacts directly with Rab5 and Rab22 [55]. The phospholipase activity is activated when VipD is bound to Rab5 due to a conformational change that exposes the active site [56, 57]. The activation of VipD causes cleavage of PI(3)P on endosomal membranes that prevents normal localization of membrane trafficking regulators, contributing to endosomal avoidance by *Legionella* [56].

While VipD has phospholipase A activity, *Legionella* also translocates two T4SS effectors with phospholipase C and D activity. A phospholipase C hydrolyzes the phosphorus-oxygen bond, releasing the phosphate of the phospholipid and the attached head group, and a phospholipase D solely cleaves off the attached head group. The phospholipase C effector protein, PlcC, is able to cleave phospholipids such as phosphatidylglycerol, phosphatidylcholine, and phosphatidylinositol [58]. Alone or in combination with two other phospholipase C effectors, PlcA and PlcB, translocated by the T2SS, these effectors were dispensable for growth in amoeba and macrophages. However, a triple mutant of these phospholipases displayed inefficient killing of larvae in the *G. mellonella* infection model compared to the wild type [58]. It is not yet known how this function may contribute to intracellular survival. We speculate that perhaps removing the head group on these phospholipids, specifically PI, would render them incapable of being modified by PI kinases and phosphatase and prevent the vacuole from being quickly converted to an endosomelike membrane. It would be interesting to determine if this phospholipase activity alters the PI composition of the LCV.

Lastly, the phospholipase D effector, LpdA, was first identified due to its homology with known phospholipase D enzymes [59]. LpdA specifically cleaves the head

*Pathogenic Bacteria*

a phosphate group of the *myo*-inositol head group. The direct PI 4-kinase activity of the effector LepB could potentially initiate the conversion process to a PI(4) P-positive membrane by converting PI(3)P to PI(3,4)P2. LepB was initially identified as an effector that is involved in bacterial egress [43]. Since then, LepB was found to localize to the LCV, where it contributes to the dynamics of Rab1 by acting as a GTPase-activating protein (GAP) [44, 45]. Found between amino acids 313 and 618, the structure and mechanism of the GAP domain is now well understood [46–48]. The N-terminal domain consisting of the first 311 amino acids garnered interest as this domain alone could disrupt the structure and function of the Golgi. The crystal structure of LepB1–618 revealed homology to atypical kinases such as CtkA from *Helicobacter pylori* and actin-fragmin kinase from *Physarum polycephalum*. When mutating residues capable of performing phosphorylation, the yeast toxicity phenotype was found to be suppressed. While this suggested a kinase functionality, the pocket for a substrate was too small to accommodate proteins any larger than Rab GTPases. However, LepB did not phosphorylate any of the tested Rab GTPases. Instead, the LepB substrates were revealed to be phosphoinositides. Studies showed that LepB, but not the catalytically inactive mutant LepBH154A, caused the sensor for PI(3,4)P2 and PI(3,4,5)P3 to relocate from a cytosolic to punctate distribution, while the signal for PI(3)P diminished dramatically. Ultimately, an in vitro kinase assay validated that LepB is a PI 4-kinase with specificity for PI(3) P and a level of activity comparable to the host kinase PI4KIII. By phosphorylating

PI(3)P on the LCV, LepB could be initiating the vacuole's phosphoinositide conversion to PI(4)P by providing the PI(3,4)P2 intermediate step [49].

As PI(3,4)P2 is generated on the LCV, it is thought that SidF can dephosphorylate this lipid to PI(4)P. SidF is a membrane protein containing a large N-terminal domain followed by two transmembrane domains triggering localization to the LCV. SidF was the first *L. pneumophila* effector found to directly modify phosphoinositides through a screen for the well-known CX5R phosphatase motif in the effector repertoire. SidF is a PI 3-phosphatase: this effector hydrolyzes the phosphate group at the third position of PI(3,4)P2 or PI(3,4,5)P3 to produce PI(4)P or PI(4,5) P2, respectively; however, it displays a preference for PI(3,4)P2. The mutation of the catalytic cysteine at residue 645 to a serine resulted in the abrogation of this phosphatase activity. As described in the following sections, PI(4)P on the LCV membrane can serve as a means for effectors, such as SidC, to anchor onto the LCV. Infection with a mutant lacking *sidF* shows significantly fewer vacuoles positive for SidC, suggesting that SidF contributes to the generation of PI(4)P on the LCV. Ultimately, the functions of LepB and SidF suggest that PI(3)P can be converted

to PI(4)P through the sequential efforts of these enzymes. The deletion of *lepB* and *sidF* individually shows a significant deficiency of SidC on the LCV membrane at similar levels [49, 50]. The deletion of both effectors simultaneously causes a decrease in SidC acquisition on the membrane no greater than the single-mutant strains, suggesting that these effectors are functioning in a linear pathway [49]. However, the complete loss of SidC was not seen in the infection with a *lepB sidF* double-deletion mutant. Additionally, both *lepB* and *sidF* are not always found in other *Legionella* species. Together, this suggests that there are other *Legionella* effec-

The screen that identified SidF as a PI phosphatase also yielded SidP as another direct modifier of phosphoinositides. SidP was identified as a candidate due to its CX5R motif. It was found to have PI 3-phosphatase activity, cleaving PI(3) P and PI(3,5)P2 in vitro. The *L. longbeachae* orthologue of SidP was only found to hydrolyze PI(3)P, suggesting this lipid may be the true target. SidP was also found to act as a PI 3-phosphatase *in vivo* when it suppressed yeast toxicity in a PI 3-phosphatase-deficient mutant but not a mutant lacking PI 4-phosphatases. This

tors or host proteins manipulating the LCV PIP landscape.

**42**

group from PI, PI(3)P, PI(4)P, and phosphatidylglycerol *in vitro* [60]. While LpdA localizes to the LCV [59], it is not known if or how this effector contributes to phosphoinositide dynamics. Nonetheless, deleting this gene results in the attenuation of growth in a mouse model [60].

LppA is a phytase enzyme that dephosphorylates the compound *myo*-inositol hexakisphosphate, known as phytate. While LppA's phosphatase activity on phytate may play various roles during infection, of interest to this review are its effects on phosphoinositides. The inositol phosphate head group of PIPs is similar in structure to phytate. LppA was shown to dephosphorylate PI(3,4)P2 and PI(4,5)P2 as well as, but less efficiently, PI(3,4,5)P3 to PI(4)P in vitro. However, infection with an *lppA* deletion strain did not impact the presence of PI(4)P on the LCV [61]. It is possible that lack of LppA generates a more subtle phenotype that requires more sensitive detection methods.

In addition to directly manipulating the phosphoinositide composition of the vacuolar membrane, *Legionella* may change the PIP landscape by enlisting host enzymes. For instance, the host PI 5-phosphatase OCRL1 is recruited to the LCV in a T4SS-dependent manner. OCRL1 preferentially removes a phosphate from PI(4,5)P2 to generate PI(4)P [62]. The homolog of OCRL1 in *Dictyostelium*, Dd5P4, was found to localize to LCVs where it is catalytically active and therefore able to dephosphorylate PIPs [63]. How OCRL1 is recruited to the LCV is not yet clear, but it is thought that *Legionella* protein LpnE may contribute to this process. LpnE is a Sel1-like repeat protein translocated into host cells in a T4SS-independent manner, and it seems to be exported extracellularly through an unknown mechanism [64]. LpnE is important for entry into amoebae and macrophages as well as intracellular replication. *In vitro* LpnE binds PI(3)P and interacts with OCRL1, but it does not seem to be essential for recruitment of OCRL1 to the LCV. It may be that LpnE synergizes with other effectors to stably recruit OCRL1, but this idea remains to be tested [63].
