**4. Phosphoinositide dynamics on the LCV**

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 host membranes.

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

**41**

*expanding vacuole.*

**Figure 1.**

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

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

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

*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* 

gradual accumulation of PI(4)P-compartments around the LCV [42]. Together, these observations support the notion that *L. pneumophila* adopts a combined strategy to convert the LCV from a PI(3)P- to PI(4)P-enriched compartment, employing both direct modification of PIPs on the LCV membrane and selective association with

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

host vesicles.

**the LCV membrane**

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

gradual accumulation of PI(4)P-compartments around the LCV [42]. Together, these observations support the notion that *L. pneumophila* adopts a combined strategy to convert the LCV from a PI(3)P- to PI(4)P-enriched compartment, employing both direct modification of PIPs on the LCV membrane and selective association with host vesicles.
