**6.** *L. pneumophila* **effector proteins specifically bind phosphoinositides**

Central to the ability of *L. pneumophila* to grow within both mammalian and protozoan cells is the remodeling of the phagosomal membrane through the manipulation of host secretory and endosomal trafficking. The loss of PI(3)P and the acquisition of PI(4)P on the phagosome membrane are achieved through a concerted mechanism carried out by the actions of multiple effector proteins. The acquisition of PI(4)P on the phagosome membrane is imperative for the subsequent recruitment of membranes to promote vacuole expansion [12]. PI(4)P on the LCV can serve as a docking site for effector attachment to ensure effectors are directed to the correct compartment within the cell [65]. Many effectors that bind to PI(4)P on the LCV are involved in the recruitment and fusion of secretory vesicles and ER. In addition to directly producing PI(4)P on the LCV via effector-driven phosphorylation and dephosphorylation of PIPs, it was recently reported that the phagosome also derives PI(4)P from the membrane material of secretory vesicles, demonstrating *L. pneumophila* employs multiple tactics to acquire PI(4)P. A number of PI(3)P-binding effectors have also been identified [66]. The few whose functions have been characterized interfere with phagosomal maturation, retrograde trafficking, and autophagy [67–70]. An overview of *L. pneumophila* effectors that target PIPs is in **Table 1**.

#### **6.1** *L. pneumophila* **T4SS effectors that bind PI(4)P**

Bacterial effectors translocated early during infection have been shown to facilitate the recruitment and fusion of ER/secretory vesicles with the LCV. SidM

**45**

**Table 1.**

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

(DrrA), an effector protein translocated immediately upon infection, localizes to the LCV and plays a crucial role in ER recruitment by exploiting the activity of Rab1, a small GTPase responsible for the transport of vesicles between the ER and Golgi [71–74]. SidM is a modular protein consisting of an N-terminal adenylyltransferase domain, a C-terminal PI(4)P-binding domain, and a central guanine nucleotide exchange factor (GEF) domain that activates the small GTPase Rab1 by facilitating the exchange of GDP with GTP [73]. SidM's adenylyltransferase activity covalently adds an adenosine monophosphate moiety onto Tyr 77 of Rab1, locking this small GTPase in its active conformation. Activated Rab1 is required for the recruitment of secretory vesicles to the LCV [73, 74]. SidM then promotes the tethering and fusion of these compartments with the phagosome membrane by interacting with an exocyst complex comprised of Sec5 and Sec15 [75].

Legionella pneumophila *effectors targeting PI(3)P and PI(4)P.*

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

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



#### **Table 1.**

*Pathogenic Bacteria*

detection methods.

growth in a mouse model [60].

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

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

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].

**6.** *L. pneumophila* **effector proteins specifically bind phosphoinositides**

Central to the ability of *L. pneumophila* to grow within both mammalian and

Bacterial effectors translocated early during infection have been shown to facilitate the recruitment and fusion of ER/secretory vesicles with the LCV. SidM

protozoan cells is the remodeling of the phagosomal membrane through the manipulation of host secretory and endosomal trafficking. The loss of PI(3)P and the acquisition of PI(4)P on the phagosome membrane are achieved through a concerted mechanism carried out by the actions of multiple effector proteins. The acquisition of PI(4)P on the phagosome membrane is imperative for the subsequent recruitment of membranes to promote vacuole expansion [12]. PI(4)P on the LCV can serve as a docking site for effector attachment to ensure effectors are directed to the correct compartment within the cell [65]. Many effectors that bind to PI(4)P on the LCV are involved in the recruitment and fusion of secretory vesicles and ER. In addition to directly producing PI(4)P on the LCV via effector-driven phosphorylation and dephosphorylation of PIPs, it was recently reported that the phagosome also derives PI(4)P from the membrane material of secretory vesicles, demonstrating *L. pneumophila* employs multiple tactics to acquire PI(4)P. A number of PI(3)P-binding effectors have also been identified [66]. The few whose functions have been characterized interfere with phagosomal maturation, retrograde trafficking, and autophagy [67–70]. An overview of *L. pneumophila* effectors that target PIPs is in **Table 1**.

**6.1** *L. pneumophila* **T4SS effectors that bind PI(4)P**

**44**

Legionella pneumophila *effectors targeting PI(3)P and PI(4)P.*

(DrrA), an effector protein translocated immediately upon infection, localizes to the LCV and plays a crucial role in ER recruitment by exploiting the activity of Rab1, a small GTPase responsible for the transport of vesicles between the ER and Golgi [71–74]. SidM is a modular protein consisting of an N-terminal adenylyltransferase domain, a C-terminal PI(4)P-binding domain, and a central guanine nucleotide exchange factor (GEF) domain that activates the small GTPase Rab1 by facilitating the exchange of GDP with GTP [73]. SidM's adenylyltransferase activity covalently adds an adenosine monophosphate moiety onto Tyr 77 of Rab1, locking this small GTPase in its active conformation. Activated Rab1 is required for the recruitment of secretory vesicles to the LCV [73, 74]. SidM then promotes the tethering and fusion of these compartments with the phagosome membrane by interacting with an exocyst complex comprised of Sec5 and Sec15 [75].

A high-resolution crystal structure of SidM revealed a novel fold within the protein structure, termed P4M, that was responsible for binding PI(4)P with an unprecedented high affinity in the nanomolar range [76]. Two additional PI(4)P-binding effectors, Lem4 and Lem28, contain C-terminal domains similar to the P4M domain [77]. While Lem4 and Lem28 localize to the LCV through their PI(4)P-binding domains, they do not act on Rab1. Lem4 was recently demonstrated to be a phosphotyrosine phosphatase [78], although how this enzymatic function contributes to infection has yet to be determined.

Multiple effectors manipulate Rab1 to exploit secretory trafficking [44, 79]. While SidM is required for activating this small GTPase on the LCV, the PI(3)P and PI(4)P binder, LidA, protects Rab1 from being inactivated [73, 74, 80]. LidA also localizes to the early LCV as well as other uncharacterized membrane compartments [73, 74, 80]. Unlike P4M-containing effectors, LidA interacts with PIPs through a central coiled-coil region. LidA interacts with AMPylated Rab1 through the same coiled-coil domain, preventing GAPs from accessing Rab1 to deactivate it. It is unknown whether the PIP interaction contributes to LidA's function.

In addition to SidM, the PI(4)P binders SidC and its paralogue, SdcA, are also required for the recruitment of ER proteins to the LCV. In the absence of *sidC*, only 20% of LCVs acquire the ER marker calnexin, indicating that the interaction of LCVs with the ER is severely impaired upon deletion of this gene [81]. SidC and SdcA interact with PI(4)P using a 20 kDa C-terminal-binding domain (P4C) that does not share similarities with P4M or other eukaryotic PIP-binding motifs. Mutations that abolish P4C-PI(4)P interactions reduced ER recruitment to the LCV, indicating that SidC's PI(4)P-binding activity is critical for remodeling the LCV membrane [82].
