**7. Eukaryotic and bacterial phosphoinositide-binding domains**

In eukaryotes, proteins bind PIPs via domains that are highly conserved. Protein-lipid binding typically occurs through electrostatic interactions between positively charged amino acid residues and the negative phosphate(s) on the *myo*-inositol ring. These protein domains vary in their binding affinity and specificity for the seven PIP species [99]. The well-characterized pleckstrin homology (PH) domain is the eleventh most common domain in humans, found in 275 proteins [100]. Proteins harboring the PH domain are recruited to membranes through interactions with either PI(3,4)P2, PI(4,5)P2, or PI(3,4,5)P3. The FYVE domain confers high specificity for PI(3)P and is present in many proteins that localize to endosomes [101, 102]. The phox domain (PX) is commonly found in sorting nexins and preferentially binds PI(3)P and in some cases PI(3,4)P2 [103]. Intriguingly, bacterial proteins that specifically bind host PIPs do not use eukaryotic-like domains.

Bacteria can acquire protein domains by horizontal gene transfer from the hosts they infect [97]. A number of *L. pneumophila* effectors harbor eukaryoticlike domains such as ankyrin repeats, U-Box, F-box, and Sel1 repeats [9]. Interestingly, prokaryotic PIP-binding domains were not derived from their eukaryotic hosts. Global bioinformatic analysis of 38 *Legionella* genomes revealed a conserved PI(4)P-binding domain found in 36 putative effectors, while a

**49**

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

domain termed LED006 is found in 136 effectors from 30 species [8]. The PI(4) P-binding domain was experimentally validated to be functional in SidM,

A recent study identified three conserved PI(3)P-binding domains present in 14 *Legionella* effectors across 41 *Legionella* species: LED006, LED027, and LED025 [66]. All three domains rely on positively charged or aromatic residues confined to the C-terminus and are accompanied by an adjacent enzymatic or proteinbinding domain. LED006 displayed the weakest affinity for PI(3)P yet is the most conserved, found in eight *L. pneumophila* effectors: CegC2, LegK1, Ceg22, LegC5, Lem9, LegC6, LepB, and Lpg2327. Only LegK1, LegC5, and LepB have been studied and shown to possess catalytic activity. While the C-terminal region of these proteins is conserved, the catalytic activity harbored by their N-terminal region varies. LegK1 is a serine/threonine kinase that targets the NF-κB pathway, LegC5 is a glucosyltransferase that modifies eEF1A, and LepB has dual PI 4-kinase activity and a Rab GAP domain. LED027 binds PI(3)P with high affinity and is found in RavZ, Lpg1121 (Ceg19), and Lpg1961, although Lpg1961 did not display lipid-binding activity when tested in vitro. LED035 is present in RavB, Lem21, and MavH,

Biochemical analysis of a *Vibrio parahaemolyticus* effector revealed a conserved type III secreted bacterial phosphoinositide-binding domain (BPD) domain that mediates membrane localization in eukaryotic cells. The BPD domain is the first instance of a domain found in both plant and animal pathogens yet shares no homology to eukaryotes suggesting this domain is the result of convergent evolution [104]. Despite the recent discoveries of novel PIP-binding domains, the PI(3) P-binding regions in effectors SetA, RavD, LotA, and AnkX have not been linked to any conserved domains. We could speculate that perhaps this is because phosphoinositide binding is mediated by small, variable motifs or that lipid-binding domains may be quite diverse, as is the case for eukaryotic proteins. A clear perspective on this issue requires further identification, domain mapping, and computational analysis of known and novel phosphoinositide-binding effectors. Therefore, there is much to be learned about the molecular details underlying interactions

What enables *L. pneumophila* effectors to target multiple membrane trafficking pathways stems in part from their modular structures consisting of various combinations of protein domains. Many of the PIP binding effectors are characterized by the presence of a C-terminal PIP-binding region and an N-terminal region that

The presence of PI(3)P on phagosomal membranes serves as a signpost for the recruitment of endocytic proteins that promote fusion with subsequent endocytic compartments and ultimately the lysosome. PI(3)P is therefore an attractive target for intracellular pathogens to eliminate entry into the phagosomal maturation pathway. It is well-established that after phagocytosis, PI(3)P on the nascent phagosome is rapidly depleted in conjunction with PI(4)P acquisition [12, 42]. Multiple studies have supported that this lipid rearrangement is accomplished through the actions of PIP-modifying effectors and effectors that promote the recruitment and fusion of PI(4)P-rich compartments with the LCV (reviewed in [105]). The recent evidence demonstrated that this lipid can also be removed from on or around the LCV in the form of PI(3)P-positive vesicles that are shed from

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

although none have been functionally characterized.

between bacterial proteins and host phosphoinositides.

harbors enzymatic activity or interacts with host proteins.

**8. Conclusions and perspectives**

Lpg1101, and Lpg2603 [73, 77].

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

domain termed LED006 is found in 136 effectors from 30 species [8]. The PI(4) P-binding domain was experimentally validated to be functional in SidM, Lpg1101, and Lpg2603 [73, 77].

A recent study identified three conserved PI(3)P-binding domains present in 14 *Legionella* effectors across 41 *Legionella* species: LED006, LED027, and LED025 [66]. All three domains rely on positively charged or aromatic residues confined to the C-terminus and are accompanied by an adjacent enzymatic or proteinbinding domain. LED006 displayed the weakest affinity for PI(3)P yet is the most conserved, found in eight *L. pneumophila* effectors: CegC2, LegK1, Ceg22, LegC5, Lem9, LegC6, LepB, and Lpg2327. Only LegK1, LegC5, and LepB have been studied and shown to possess catalytic activity. While the C-terminal region of these proteins is conserved, the catalytic activity harbored by their N-terminal region varies. LegK1 is a serine/threonine kinase that targets the NF-κB pathway, LegC5 is a glucosyltransferase that modifies eEF1A, and LepB has dual PI 4-kinase activity and a Rab GAP domain. LED027 binds PI(3)P with high affinity and is found in RavZ, Lpg1121 (Ceg19), and Lpg1961, although Lpg1961 did not display lipid-binding activity when tested in vitro. LED035 is present in RavB, Lem21, and MavH, although none have been functionally characterized.

Biochemical analysis of a *Vibrio parahaemolyticus* effector revealed a conserved type III secreted bacterial phosphoinositide-binding domain (BPD) domain that mediates membrane localization in eukaryotic cells. The BPD domain is the first instance of a domain found in both plant and animal pathogens yet shares no homology to eukaryotes suggesting this domain is the result of convergent evolution [104]. Despite the recent discoveries of novel PIP-binding domains, the PI(3) P-binding regions in effectors SetA, RavD, LotA, and AnkX have not been linked to any conserved domains. We could speculate that perhaps this is because phosphoinositide binding is mediated by small, variable motifs or that lipid-binding domains may be quite diverse, as is the case for eukaryotic proteins. A clear perspective on this issue requires further identification, domain mapping, and computational analysis of known and novel phosphoinositide-binding effectors. Therefore, there is much to be learned about the molecular details underlying interactions between bacterial proteins and host phosphoinositides.

## **8. Conclusions and perspectives**

What enables *L. pneumophila* effectors to target multiple membrane trafficking pathways stems in part from their modular structures consisting of various combinations of protein domains. Many of the PIP binding effectors are characterized by the presence of a C-terminal PIP-binding region and an N-terminal region that harbors enzymatic activity or interacts with host proteins.

The presence of PI(3)P on phagosomal membranes serves as a signpost for the recruitment of endocytic proteins that promote fusion with subsequent endocytic compartments and ultimately the lysosome. PI(3)P is therefore an attractive target for intracellular pathogens to eliminate entry into the phagosomal maturation pathway. It is well-established that after phagocytosis, PI(3)P on the nascent phagosome is rapidly depleted in conjunction with PI(4)P acquisition [12, 42]. Multiple studies have supported that this lipid rearrangement is accomplished through the actions of PIP-modifying effectors and effectors that promote the recruitment and fusion of PI(4)P-rich compartments with the LCV (reviewed in [105]). The recent evidence demonstrated that this lipid can also be removed from on or around the LCV in the form of PI(3)P-positive vesicles that are shed from

*Pathogenic Bacteria*

bacterial effectors.

directing proper localization.

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

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

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

**7. Eukaryotic and bacterial phosphoinositide-binding domains**

In eukaryotes, proteins bind PIPs via domains that are highly conserved. Protein-lipid binding typically occurs through electrostatic interactions between positively charged amino acid residues and the negative phosphate(s) on the *myo*-inositol ring. These protein domains vary in their binding affinity and specificity for the seven PIP species [99]. The well-characterized pleckstrin homology (PH) domain is the eleventh most common domain in humans, found in 275 proteins [100]. Proteins harboring the PH domain are recruited to membranes through interactions with either PI(3,4)P2, PI(4,5)P2, or PI(3,4,5)P3. The FYVE domain confers high specificity for PI(3)P and is present in many proteins that localize to endosomes [101, 102]. The phox domain (PX) is commonly found in sorting nexins and preferentially binds PI(3)P and in some cases PI(3,4)P2 [103]. Intriguingly, bacterial proteins that specifically bind host PIPs do not use eukary-

Bacteria can acquire protein domains by horizontal gene transfer from the hosts they infect [97]. A number of *L. pneumophila* effectors harbor eukaryoticlike domains such as ankyrin repeats, U-Box, F-box, and Sel1 repeats [9]. Interestingly, prokaryotic PIP-binding domains were not derived from their eukaryotic hosts. Global bioinformatic analysis of 38 *Legionella* genomes revealed a conserved PI(4)P-binding domain found in 36 putative effectors, while a

unclear if these substrates are modified during infection.

**48**

otic-like domains.

the LCV. This would indicate that somehow microdomains of PI(3)P within membranes are being recognized, sequestered, and sorted into vesicles for removal or that perhaps PI(3)P-positive vesicles do not stably interact with the LCV. How the LCV can distinguish the simultaneous shedding of PI(3)P-compartments with the fusion of PI(4)P-compartments has yet to be determined. We can speculate that *L. pneumophila* has evolved cohorts of effectors that can independently regulate the acceptance of PI(4)P-rich membrane or the egress of PI(3)P-rich membrane from the LCV.

PI(3)P is completely lost from the LCV membrane after 2 hours; however, it is unclear why there is a strong presence of PI(3)P-binding effectors that are on the LCV membrane after this time point (LpnE, SetA, LotA, RidL, LtpM, LtpD, RavD). At later stages of infection, an accumulation of stagnant PI(3)P-positive vesicles can be seen surrounding the LCV. It is possible that effectors anchored to the LCV could be interacting with these vesicles by recognizing multiple membrane compartments. Most LCV localization studies are assessed using light microscopy, in which the resolution may not be high enough to visualize smaller distinct structures around the LCV. Light microscopy showed RavD is present on the LCV membrane; however higher-resolution imaging techniques like structured illumination and transmission electron microscopy revealed RavD is also present on a subset of unidentified vesicles adjacent to the LCV. It is most likely these vesicles are PI(3) P-rich, as RavD does not localize to PI(4)P-positive compartments. Moreover, RavD does not rely on PI(3)P binding to anchor to the LCV, supporting that effectors may exhibit dual localization patterns and that RavD may interact with the LCV and vesicles through different domains.

*L. pneumophila* has developed intricate strategies to facilitate intracellular growth by circumventing essential host cellular processes. The arsenal of effectors secreted by the type IV secretion system has evolved to target specific eukaryotic components such as proteins and lipids. Localization to the correct compartments within this host cell is imperative for protein function. A number of *Legionella* effectors rely on phosphoinositides to confer this directionality during infection. Not only are phosphoinositides needed to govern organelle identity, but they also dictate the path the phagosome embarks on once engulfed into the host cell. Thus, some effectors are ingeniously equipped to directly modify the lipid content on the phagosome membrane to avoid being routed toward degradation. Only a small percentage of effectors have been reported to interact with or modify phosphoinositides. Future studies that continue to expand on the repertoire of PIP-binding effectors will undoubtedly enhance our understanding of how intracellular pathogens survive within membrane-bound compartments within eukaryotic hosts.

**51**

**Author details**

Colleen M. Pike, Rebecca R. Noll and M. Ramona Neunuebel\*

\*Address all correspondence to: neunr@udel.edu

provided the original work is properly cited.

Department of Biological Sciences, University of Delaware, Newark, DE, USA

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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

*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*
