**6.2** *L. pneumophila* **T4SS effectors that bind PI(3)P**

Multiple PI(3)P-binding effectors have been identified, and several were shown to be involved in preventing the LCV from entering the phagosomal maturation pathway. AnkX binds both PI(3)P and PI(4)P *in vitro*, and in macrophages infected with a mutant strain lacking AnkX, the lysosomal marker, LAMP1, accumulates around the LCV indicating it is being routed for endolysosomal degradation [70]. AnkX's N-terminal FIC domain harbors phosphocholine transferase activity catalyzing, the covalent attachment of a phosphocholine moiety onto a serine or threonine residue of Rab1 and Rab35 [83, 84]. It is unknown whether AnkX localizes to the LCV, and despite its ability to covalently modify Rab1, it does not enhance retention of Rab1 on the LCV as observed for SidM-catalyzed adenylylation of Rab1. Phosphocholination locks Rab35 in an inactive conformation by preventing interaction with its cognate GEF, connecdenn; however, phosphocholinated Rab1 was still able to interact with SidM, which also acts as a GEF [85]. AnkX disrupts endocytic recycling in infected macrophages in a phosphocholination-dependent manner, suggesting that phosphocholination of Rab35, a key regulator of endocytic recycling, may be responsible for this phenotype [86].

The PI(3)P-binding effector, RavD, also contributes to preventing encounters between lysosomes and the LCV. Transmission electron microscopy and structuredillumination microscopy revealed RavD is present on the LCV membrane and vesicles adjacent to the LCV; however the identity of these vesicles has not yet been revealed. RavD binds PI(3)P via a C-terminal region [67]. A recent study reported that RavD's N-terminal region harbors deubiquitinase activity (DUB) that specifically cleaves linear ubiquitin chains from the LCV using a Cys-His-Ser triad [87]. Deletion of *ravD*

**47**

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

causes the LCV to become decorated with linear ubiquitin and triggers subsequent activation of the NF-κB pathway [87]. Since *Legionella* species have not coevolved with macrophages, it is possible that RavD's DUB activity would be functional in both macrophages and protozoan hosts. It would be interesting to determine RavD's substrates in the context of a macrophage versus amoebae infection. Understanding the functional link between RavD's DUB activity and its contribution to the prevention of LCV-endolysosomal fusion could provide novel insight into why pathogens exploit

*L. pneumophila*'s cohort of effectors includes multiple deubiquitinases that have

evolved to act on different ubiquitin chains. Effector LotA localizes to the LCV through interaction with PI(3)P and harbors dual DUB activity to remove ubiquitin from the LCV [88]. LotA uses a C13 residue that acts against K6 linkages and a C303 residue that acts against K48 and K63 linkages, although C303 has a more considerable contribution to removing ubiquitin from the LCV. A *Legionella* strain lacking LotA and the ubiquitin-associated SidE family of effectors resulted in impaired bacterial growth within murine bone marrow-derived macrophages, indicating LotA has coordinated activity with other *L. pneumophila* ubiquitin-modifying enzymes [88]. While it has not been reported whether the SidE effector family interacts with PIPs, it cannot be ruled out that these ubiquitin-modifying enzymes may also rely on PIPs to correctly direct them to the sites where their enzymatic

The effector RidL binds PI(3)P and inhibits retrograde transport through molecular mimicry. Retrograde trafficking serves as a conduit that connects endosomes, the trans-Golgi network, and the ER [89]. Cargo that is cycled from endosomes to the Golgi is recognized and sorted by a retromer complex. Ectopically expressed RidL blocks retrograde trafficking at endosome exit sites through interactions with the retromer complex protein, Vps29 [68]. RidL is present on the LCV membrane and endosomes but does not localize to endosomes through interactions with PI(3)P. Instead, RidL inserts itself into the endosomal retromer complex through interactions with Vps29, displacing Vps29 from binding to the Rab7 GAP, TBC1D5. RidL interacts with Vps29 using a hairpin loop that mimics the same manner in which TBC1D5 interacts with Vps29 [90]. This displacement blocks the movement of retrograde vesicles through an unknown mechanism. In the absence of *ridL*, LCVs accumulate lysosomal markers and retrograde cargo such as CI-MPR, which delivers acidic hydrolases to endocytic compartments [90]. This suggests the LCV may accept cargo or membranes from a subset of endosomal pathways and

PI(3)P is also present on autophagosomes [91], and studies found that indeed *L. pneumophila* effectors also interfere with the dynamics of these compartments [69]. Autophagy is a conserved process across eukaryotic species that is triggered by cellular stress and serves as an additional defense mechanism against intracellular pathogens. Autophagy progression relies on a series of membrane reconstruction events, starting with phagophore membrane nucleation, to phagophore elongation and fusion to form the PI(3)P-rich autophagosome and ultimately fusion with lysosomes to degrade the internal cargo [91]. Early phagophore formation events are dependent on the presence of PI(3)P, which stimulates the recruitment of PI(3)P-binding proteins on ER-derived omegasomes [91]. Phagophore closure is completed through conjugation of LC3 to phosphoethanolamine (PE) on the phagophore membrane [92, 93]. Effector RavZ inhibits autophagy by extracting lipidated LC3 from autophagosome membranes and generating a modified LC3 product that lacks the essential C-terminal glycine required for reconjugation back

that RidL could intercept these incoming vesicles.

onto autophagosome membranes.

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

ubiquitin during infection.

activity is required.

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

causes the LCV to become decorated with linear ubiquitin and triggers subsequent activation of the NF-κB pathway [87]. Since *Legionella* species have not coevolved with macrophages, it is possible that RavD's DUB activity would be functional in both macrophages and protozoan hosts. It would be interesting to determine RavD's substrates in the context of a macrophage versus amoebae infection. Understanding the functional link between RavD's DUB activity and its contribution to the prevention of LCV-endolysosomal fusion could provide novel insight into why pathogens exploit ubiquitin during infection.

*L. pneumophila*'s cohort of effectors includes multiple deubiquitinases that have evolved to act on different ubiquitin chains. Effector LotA localizes to the LCV through interaction with PI(3)P and harbors dual DUB activity to remove ubiquitin from the LCV [88]. LotA uses a C13 residue that acts against K6 linkages and a C303 residue that acts against K48 and K63 linkages, although C303 has a more considerable contribution to removing ubiquitin from the LCV. A *Legionella* strain lacking LotA and the ubiquitin-associated SidE family of effectors resulted in impaired bacterial growth within murine bone marrow-derived macrophages, indicating LotA has coordinated activity with other *L. pneumophila* ubiquitin-modifying enzymes [88]. While it has not been reported whether the SidE effector family interacts with PIPs, it cannot be ruled out that these ubiquitin-modifying enzymes may also rely on PIPs to correctly direct them to the sites where their enzymatic activity is required.

The effector RidL binds PI(3)P and inhibits retrograde transport through molecular mimicry. Retrograde trafficking serves as a conduit that connects endosomes, the trans-Golgi network, and the ER [89]. Cargo that is cycled from endosomes to the Golgi is recognized and sorted by a retromer complex. Ectopically expressed RidL blocks retrograde trafficking at endosome exit sites through interactions with the retromer complex protein, Vps29 [68]. RidL is present on the LCV membrane and endosomes but does not localize to endosomes through interactions with PI(3)P. Instead, RidL inserts itself into the endosomal retromer complex through interactions with Vps29, displacing Vps29 from binding to the Rab7 GAP, TBC1D5. RidL interacts with Vps29 using a hairpin loop that mimics the same manner in which TBC1D5 interacts with Vps29 [90]. This displacement blocks the movement of retrograde vesicles through an unknown mechanism. In the absence of *ridL*, LCVs accumulate lysosomal markers and retrograde cargo such as CI-MPR, which delivers acidic hydrolases to endocytic compartments [90]. This suggests the LCV may accept cargo or membranes from a subset of endosomal pathways and that RidL could intercept these incoming vesicles.

PI(3)P is also present on autophagosomes [91], and studies found that indeed *L. pneumophila* effectors also interfere with the dynamics of these compartments [69]. Autophagy is a conserved process across eukaryotic species that is triggered by cellular stress and serves as an additional defense mechanism against intracellular pathogens. Autophagy progression relies on a series of membrane reconstruction events, starting with phagophore membrane nucleation, to phagophore elongation and fusion to form the PI(3)P-rich autophagosome and ultimately fusion with lysosomes to degrade the internal cargo [91]. Early phagophore formation events are dependent on the presence of PI(3)P, which stimulates the recruitment of PI(3)P-binding proteins on ER-derived omegasomes [91]. Phagophore closure is completed through conjugation of LC3 to phosphoethanolamine (PE) on the phagophore membrane [92, 93]. Effector RavZ inhibits autophagy by extracting lipidated LC3 from autophagosome membranes and generating a modified LC3 product that lacks the essential C-terminal glycine required for reconjugation back onto autophagosome membranes.

*Pathogenic Bacteria*

membrane [82].

infection has yet to be determined.

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

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.

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

Multiple PI(3)P-binding effectors have been identified, and several were shown to be involved in preventing the LCV from entering the phagosomal maturation pathway. AnkX binds both PI(3)P and PI(4)P *in vitro*, and in macrophages infected with a mutant strain lacking AnkX, the lysosomal marker, LAMP1, accumulates around the LCV indicating it is being routed for endolysosomal degradation [70]. AnkX's N-terminal FIC domain harbors phosphocholine transferase activity catalyzing, the covalent attachment of a phosphocholine moiety onto a serine or threonine residue of Rab1 and Rab35 [83, 84]. It is unknown whether AnkX localizes to the LCV, and despite its ability to covalently modify Rab1, it does not enhance retention of Rab1 on the LCV as observed for SidM-catalyzed adenylylation of Rab1. Phosphocholination locks Rab35 in an inactive conformation by preventing interaction with its cognate GEF, connecdenn; however, phosphocholinated Rab1 was still able to interact with SidM, which also acts as a GEF [85]. AnkX disrupts endocytic recycling in infected macrophages in a phosphocholination-dependent manner, suggesting that phosphocholination of Rab35, a key regulator of endocytic recycling, may be responsible for this pheno-

The PI(3)P-binding effector, RavD, also contributes to preventing encounters between lysosomes and the LCV. Transmission electron microscopy and structuredillumination microscopy revealed RavD is present on the LCV membrane and vesicles adjacent to the LCV; however the identity of these vesicles has not yet been revealed. RavD binds PI(3)P via a C-terminal region [67]. A recent study reported that RavD's N-terminal region harbors deubiquitinase activity (DUB) that specifically cleaves linear ubiquitin chains from the LCV using a Cys-His-Ser triad [87]. Deletion of *ravD*

It is unknown whether the PIP interaction contributes to LidA's function.

**6.2** *L. pneumophila* **T4SS effectors that bind PI(3)P**

**46**

type [86].

RavZ localizes to autophagosome membranes through a C-terminal domain that recognizes PI(3)P. RavZ1–331 contains catalytic activity yet displays reduced LC3-PE extraction, indicating proper localization to phagosomes is needed to inhibit autophagy [94]. This high-affinity PI(3)P-binding domain, termed LED027, contains two conserved tyrosine and lysine residues that are key for PI(3)P binding. LED027 is found in two other effectors, Lpg1121 (Ceg19) and Lpg1961, although Lpg1961 did not display lipid-binding activity when tested *in vitro* [66]. It would be interesting to determine if these LED027-containing effectors also preferentially localize to PI(3)P on autophagosomes, possibly unveiling a novel conserved domain that confers autophagy-related activity in bacterial effectors.

While effectors rely on PIPs for proper localization, binding to PIPs can also induce the enzymatic activity of some effectors. Effector protein SetA possesses an N-terminal region with glucosyltransferase activity and a C-terminal PI(3) P-binding region responsible for LCV localization [95]. Notably, PI(3)P binding enhances SetA's glucosyltransferase activity [96]. *In vitro* SetA has multiple substrates including actin, vimentin, and the chaperonin CCT5 [96], although it is unclear if these substrates are modified during infection.

The cohort of T4SS substrates is not conserved across all *L. pneumophila* strains. Strains harbor variations in their combinations of effectors that have been presumably acquired during the course of coevolution with a variety of protozoan hosts [97]. Despite these variations, PIP binding is emerging as a common feature among effectors of *L. pneumophila* strains. The *L. pneumophila* Paris strain encodes the glucosyltransferase LtpM that resembles the Philadelphia strain effector, SetA, in domain structure and the ability to cause a growth defect in yeast [96, 98]. Unlike SetA which uses a typical DxD motif for catalysis, LtpM harbors a noncanonical DxN motif. The glucosyltransferase activity LtpM is also stimulated by PI(3)P, indicating multiple effectors have evolved to exploit PI(3)P for purposes other than directing proper localization.
