# Patent

## Title
Bacterial protein phosphoinositide probes and effectors

## Abstract

Methods for detecting and quantifying phosphoinositide lipids in a sample are provided, together with novel SidC- and SdcA-derived polypeptide fragments, or fusion proteins comprising such fragments that may be effectively employed as PI(4)P probes in biochemical and cell biological assays. Applications of the disclosed probes include: (i) detection and quantification of PI(4)P in vitro; and (ii) staining of intracellular compartments in live or fixed eukaryotic cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. Provisional Application No. 60/719,934 filed on Sep. 23, 2005.


FIELD OF THE INVENTION
The present invention relates to methods for determining the presence or absence of phosphoinositide lipids in a sample. More specifically the method of the present invention allows to determine the presence or absence of phosphatidylinositol(3) phosphate (PI(3)P), phosphatidylinositol(4) phosphate (PI(4)P) or phosphatidyl-inositol(5) phosphate (PI(5)P) in a sample. Additionally, the present invention relates to isolated proteins capable of binding phosphoinositides, protein-phosphatidylinositol constructs, methods for preparing phosphatidylinositol-binding polypeptides and methods for treating disorders.
BACKGROUND OF THE INVENTION
In the environment, the Gram-negative bacterium Legionella pneumophila colonizes biofilms and multiplies within various protozoa [1]. Upon transmission to the human lung, the bacteria replicate within alveolar macrophages, and may cause the severe pneumonia Legionnaires' disease [2, 3]. To establish its replicative niche, L. pneumophila prevents the fusion of its phagosome with lysosomes [4], and recruits early secretory vesicles at endoplasmic reticulum (ER) exit sites [5]. The resulting Legionella-containing vacuole (LCV) is characterized by the ER marker calnexin, the v-SNARE Sec22b, and the small GTPases Arf1 and Rab1 [6, 7]. Moreover, the LCV undergoes a transition from a tight to a spacious vacuole [8, 9] and eventually matures into an acidic vacuole [10], wherein the bacteria multiply independently of the bacterial Icm/Dot type IV secretion system (T4SS) [11]. The Icm/Dot T4SS, a conjugation machinery encoded by 25 different genes, is required for formation of the LCV [12, 13] as well as for modulation of phagocytosis [14, 15].
To date, more than 30 different Icm/Dot-secreted proteins have been identified as putative effectors, many of which form families of 2-6 paralogues [16-22]. The precise function of most of these proteins is not known, owing at least in part to the fact that L. pneumophila strains lacking even multiple family members do not show a phenotype with regard to intracellular replication [18, 20, 22]. However, the inability of Icm/dot mutants to direct phagocytosis and establish a LCV, suggests that at least some Icm/Dot-secreted proteins interfere with host cell phagocytosis or vesicle trafficking.
Indeed, the recently identified effectors LepA and LepB share homology with SNAREs and seem to promote the non-lytic release of vesicles containing L. pneumophila from amoebae [19]. The first Icm/Dot substrate to be functionally characterized, RalF, recruits the GTPase Arf1 to the LCV and acts as a guanine nucleotide exchange factor for the Arf family of small GTPases [16]. The Icm/Dot-secreted proteins SidC and its paralogue SdcA have no orthologues in the database, their function is unknown, and a sidC/sdcA double mutant shows no phenotype [18]. SdcA was recently identified in a screen using growth inhibition of yeast to select for putative L. pneumophila effector proteins [22]. To subvert host cell trafficking, the large number of Icm/Dot-secreted proteins is likely organized in a complex spatial and temporal manner.
In eukaryotic cells, the metabolism of phosphoinositide lipids is pivotal for the regulation of membrane dynamics during phagocytosis, endocytosis and exocytosis [23, 24]. Depending on phosphorylation at position 3, 4 and/or 5 of the D-myo-inositol ring, phosphoinositides recruit specific effectors to distinct membranes in a time- and organelle-dependent manner, thus coordinating intracellular membrane trafficking and actin remodelling, as well as receptor-mediated signal transduction. The central role of phosphoinositide second messengers is exploited by a number of intracellular bacterial pathogens [25], for example Shigella flexneri [26] and Salmonella enterica [27, 28] employ type III-secreted phosphoinositide phosphatases to modulate phosphoinositide metabolism during bacterial entry and intracellular replication.
Phosphoinositide metabolism is well characterized in the social amoeba Dictyostelium discoideum [29, 30], which supports Icm/Dot-dependent intracellular replication of L. pneumophila [31-33]. Here, we use a Dictyostelium strain lacking the class I PI(3) kinase (PI3K)-1 and -2 (ΔPI3K1/2; [34]) to demonstrate a role for phosphoinositide metabolism in phagocytosis, trafficking and intracellular replication of L. pneumophila. Furthermore, we identify Icm/Dot-secreted proteins, which specifically bind to PI(4)P, thus providing a mechanistic link between phosphoinositide metabolism and the subversion of host cell trafficking by L. pneumophila. 
SUMMARY OF THE INVENTION
The inventors have unexpectedly found that bacterial proteins, or functional equivalents thereof, directly and specifically bind to certain phosphoinositide lipids in vitro and on the Legionella-containing vacuole (LCV) in infected host cells, thus linking Icm/Dot-dependent secretion and bacterial replication to host cell phosphoinositide metabolism. The present invention provides a method for detecting and quantifying phosphoinositide lipids in a sample.
It was found, for example, that L. pneumophila SidC and SdcA directly and specifically bind to PI(4)P in vitro and on LCVs in infected host cells. It was also found that L. pneumophila LepB, LidA and Lpg2311 directly bind to mono-phosphorylated phosphoinositides in vitro.
Moreover the inventors have also surprisingly found that, in the context of liposomes, SidC also strongly binds PI(4)P, but not PI(3)P or PI(4,5)P2. SidC and SdcA are not predicted to contain a PH domain or other phosphoinositide-binding domains, and therefore, harbor a novel PI(4)P-binding domain or novel PI(4)P-binding domains.
In one aspect, the present invention comprises novel SidC- and SdcA-derived fragments, or fusion proteins based upon those fragments, as PI(4)P probes in biochemical and cell biological assays. Applications of the probes include: (i) detection and quantification of PI(4)P in vitro, (ii) staining of intracellular compartments in live or fixed eukaryotic cells, (iii) use in lipid-protein overlay experiments, (iv) enrichment of PI(4)P-containing cellular compartments by pull down assays, and (v) ectopical expression, e.g. as a GFP fusion protein.
According to a further aspect of the present invention, full length SidC, SdcA and further Legionella-derived proteins, and fragments thereof, are stable and robustly bind PI(4)P even after cycles of shock freezing and thawing.
The inventors have found that SidC specifically binds to PI(4)P but not to other phosphoinositides or other lipids. It was established that GST-SidC can be readily overexpressed in E. coli (e.g. strain BL21(DE3)) in a predominantly soluble form and purified by glutathione affinity chromatography. The yield is up to 25 mg/l LB medium. GST-SidC is stable and withstands repeated cycles of freezing/thawing without loss of PI(4)P-binding activity. Thus, SidC is a robust probe. Moreover, the PI(4)P-binding domain is significantly smaller than, for example, a PI(4)P-binding antibody.
The present invention is also directed towards pharmaceutical compositions and methods of preparing PI(4)P-binding polypeptides.
In a further aspect, a method of treating a disorder or disease in which abnormal levels of PI(4)P are implicated in a patient is provided. The treatment comprises administering to the patient a PI(4)P-binding polypeptide or a fusion protein comprising such a polypeptide.
Furthermore, the present invention provides a rationale for the observed toxicity of SdcA expressed in yeast. SidC and SdcA have a deleterious effect on essential PI(4)P- and/or PI(3)P-mediated vesicle trafficking, either by titrating PI(4)P membrane anchors for host cell effectors or by functioning as effectors that interfere with vesicle trafficking directly. Given that SidC and SdcA modulate vesicle trafficking, the proteins or fragments thereof can be used as therapeutic agents in human diseases caused by defects in phosphoinositide signaling, including, but not limited to, Lowe syndrome [55].
The Icm/Dot T4SS is well established as a pivotal virulence determinant of L. pneumophila, which governs phagocytosis as well as intracellular trafficking of the bacteria. Contrarily, the activities and host cell targets of most of the Icm/Dot-secreted effector proteins remain obscure. Here, we analyzed the role of host cell PI3Ks during phagocytosis and intracellular replication of L. pneumophila and identify Icm/Dot-secreted proteins which directly engage PI(4)P.
Wild-type L. pneumophila upregulates phagocytosis by Dictyostelium (FIG. 1, 2), as shown previously using macrophages or Acanthamoeba castellanii as host cells [14]. PI3Ks were found to be dispensable for uptake of wild-type L. pneumophila by Dictyostelium but involved in phagocytosis and degradation of an L. pneumophila ΔicmT mutant. Therefore, L. pneumophila apparently employs a specific phagocytic pathway, which bypasses a requirement for PI3Ks. This pathway is distinct from PI3K-dependent phagocytosis of non-invasive or other pathogenic bacteria, including Listeria monocytogenes or uropathogenic E. coli [25].
We provided genetic and pharmacological evidence that class I PI3Ks are involved in intracellular replication and trafficking of wild-type L. pneumophila (FIG. 2, 4). In the absence of PI3Ks, L. pneumophila replicated more efficiently and, at the same time, the transition from tight to spacious vacuoles was inhibited. In contrast, in a Dictyostelium rtoA mutant, the defective transition of LCVs from tight to spacious vacuoles coincided with a decreased efficiency of intracellular replication of L. pneumophila [8]. Our results suggest that the “maturation” of tight to spacious vacuoles is not required for formation of a replication-permissive vacuole. PI3Ks have been implicated in homotypic phagosome fusion and formation of spacious phagosomes [38]. Accordingly, PI3K-dependent formation of spacious phagosomes may represent a host cell process which does not support (or even counteracts) the formation of a replication-permissive LCV.
Formation of the LCV takes place at ER exit sites and requires a functional early secretory pathway [5-7]. Since class I PI3Ks play a role in degradation of ΔicmT (FIG. 3) and the modulation of the LCV (FIG. 4A, B), absence of PI3Ks might contribute to a more efficient intracellular replication of L. pneumophila in two synergistic ways: (i) by rendering the degradative endocytic pathway less efficient and (ii) by promoting interactions of the LCV with the secretory pathway. The PI3K products PI(3,4,5)P3 and PI(3)P have been shown to promote phagocytosis, endocytosis, and bacterial degradation [23]. Absence of these phosphoinositides might therefore account for the observed defects in degradation of ΔicmT and render evasion of the degradative pathway by wild-type L. pneumophila more efficient. On the other hand, the effect of PI3Ks on trafficking of LCVs along the secretory pathway perhaps involves PI(4)P, which in absence of PI3Ks is more abundant in Dictyostelium [36] and thus might accumulate locally on LCVs. The discovery that the Icm/Dot-secreted proteins SidC and SdcA bind PI(4)P in vitro (FIG. 5) and anchor to the LCV in infected Dictyostelium preferentially in the absence of PI3Ks (FIG. 6) supports this hypothesis. To account for their effect on the secretory pathway, PI3Ks may be recruited. More likely, however, the absence of PI3Ks affects the modulation of LCVs in trans by increasing the cellular concentration of PI(4)P.
The identification of Icm/Dot-secreted L. pneumophila proteins specifically binding to PI(4)P identifies this phosphoinositide as a lipid marker of the LCV. PI(4)P selectively accumulates in the trans Golgi complex and regulates exocytosis by a poorly defined mechanism [24]. PI(4)P is the first lipid marker and the first Golgi marker identified on the LCV. SidC secreted by L. pneumophila selectively bound to the LCV, but not to other cellular vesicles, even if overexpressed as an M45-tagged protein (FIG. 4C, 6). To account for this specificity, SidC perhaps engages a co-receptor on the LCV, which increases the affinity to PI(4)P of the purified protein alone observed in vitro (FIG. 5).
By anchoring to PI(4)P, SidC and SdcA either (i) directly engage in vesicle trafficking and formation of a replicative vacuole via (an) effector domains, or (ii) serve as adaptors for other L. pneumophila effectors involved in formation of the replicative vacuole or exit of the bacteria from host cells. SidC and its upstream paralogue SdcA have no orthologues in the database yet are found in the genomes of all three L. pneumophila strains (Philadelphia, Paris, Lens) sequenced to date [39, 40]. The proteins are not predicted to contain a PH or other phosphoinositide-binding domains and, therefore, likely harbor a novel PI(4)P-binding domain which, due to its exquisite specificity, may effectively be employed as a selective PI(4)P probe.
In one aspect, the present invention provides methods for determining the presence or absence of a phosphoinositide lipid in a sample, such as a biological sample. Such methods comprise contacting the sample with a polypeptide and determining whether the compound binds to the phosphoinositide lipid, wherein the polypeptide comprises a sequence selected from the group consisting of: a) sequences having at least 60% identity to a sequence of SEQ ID NO: 1 or 2; b) sequences having at least 80% identity to a sequence of SEQ ID NO: 1 or 2; c) sequences having at least 90% identity to a sequence of SEQ ID NO: 1 or 2; d) sequences having at least 95% identity to a sequence of SEQ ID NO: 1 or 2; d) naturally occurring allelic variants of SEQ ID NO: 1 or 2; e) a fragment of SEQ ID NO: 1 or 2; f) SEQ ID NO: 1 or 2; g) Legionella pneumophila SidC; h) Legionella pneumophila SdcA; i) Legionella pneumophila LidA; j) Legionella pneumophila LepA; k) Legionella pneumophila LepB; l) Legionella pneumophila Lpg2311; and m) C-terminal fragments of SidC: SidC—3C (36 kDa), SidC—3-4 (20 kDa). Diagnostic kits comprising such polypeptides are also provided.
In one embodiment, the phosphoinositide lipid is phosphatidylinositol(4) phosphate and the polypeptide comprises a sequence selected from the group consisting of: a) sequences having at least 60% identity to a sequence of SEQ ID NO: 1 or 2; b) sequences having at least 80% identity to a sequence of SEQ ID NO: 1 or 2; c) sequences having at least 90% identity to a sequence of SEQ ID NO: 1 or 2; d) sequences having at least 95% identity to a sequence of SEQ ID NO: 1 or 2; d) naturally occurring allelic variants of SEQ ID NO: 1 or 2; e) a fragment of SEQ ID NO: 1 or 2; f) SEQ ID NO: 1 or 2; g) Legionella pneumophila SidC; h) Legionella pneumophila SdcA; i) Legionella pneumophila LidA; j) Legionella pneumophila LepB; k) Legionella pneumophila Lpg2311; and 1) C-terminal fragments of SidC: SidC—3C (36 kDa), SidC—3-4 (20 kDa).
In a further embodiment, the phosphoinositide lipid is phosphatidylinositol(3) phosphate and the polypeptide comprises a sequence selected from the group consisting of: a) Legionella pneumophila LepA; b) Legionella pneumophila LepB; c) Legionella pneumophila Lpg2311; and d) Legionella pneumophila LidA. In yet another embodiment, the phosphoinositide lipid is phosphatidylinositol(5) phosphate and the polypeptide comprises a sequence selected from the group consisting of: a) Legionella pneumophila Lpg2311; and b) Legionella pneumophila LidA.
The polypeptides employed in such methods may be fused with or conjugated to a tag. For example, the tags and detection methods may include: i) GST, M45, Myc, HA, His and antibody (against tag), ii) fluorescent proteins and fluorescence, iii) fluorophores and fluorescence, iv) biotin and avidin-peroxidase or avidin-fluorophor, and v) colloidal gold and electron microscopy.
In another aspect, the present invention provides isolated polypeptides comprising an amino acid sequence of SEQ ID NO: 1 or 2, together with variants and functional portions, or fragments, thereof. In certain embodiments, such variants are capable of binding a phosphoinositide, such as phosphatidylinositol(4) phosphate.
Fusion proteins comprising such polypeptide are also provided. In certain embodiments, the polypeptide is operatively fused or conjugated to a tag selected from the group consisting of: i) GST, M45, Myc, HA, His, ii) fluorescent proteins, iii) fluorophores, iv) biotin, and v) colloidal gold.
In a further aspect, polypeptide-phosphatidylinositol constructs comprising a polypeptide bound to phosphatidylinositol(4) phosphate (PI(4)P) are provided, wherein the polypeptide comprises a sequence selected from the group consisting of: a) sequences having at least 60%, 80%, 90% or 95% identity to SEQ ID NO: 1 or 2; b) naturally occurring allelic variants of SEQ ID NO: 1 or 2; c) functional portions or fragments of SEQ ID NO: 1 or 2; d) SEQ ID NO: 1 or 2; e) Legionella pneumophila SidC; f) Legionella pneumophila SdcA; g) Legionella pneumophila LidA; h) Legionella pneumophila LepB; and k) Legionella pneumophila Lpg2311.
Pharmaceutical compositions comprising the polypeptides and/or fusion proteins disclosed herein together with a pharmaceutically acceptable carrier are also provided, together with methods of treating a disorder characterized by the presence of abnormal levels of PI(4)P by administering to a patient a therapeutically effective amount of the pharmaceutical composition.
In yet a further aspect, the present invention provides methods for preparing a PI(4)P binding polypeptide, a functional equivalent thereof or a fusion protein comprising such a polypeptide. In certain embodiments such methods comprise: (a) introducing a vector comprising a nucleic acid molecule coding for the polypeptide, functional equivalent or fusion protein into a host cell; (b) culturing the host cell under conditions wherein the PI(4)P binding polypeptide, functional equivalent or fusion protein is expressed; and (c) recovering the PI(4)P binding polypeptide, functional equivalent or fusion protein.



BRIEF DESCRIPTION OF THE FIGURES
The invention described herein will be more fully understood from the detailed description given below and the accompanying figures, which should not be considered limiting to the invention described in the appended claims.
FIG. 1 Phagocytosis of wild-type L. pneumophila or ΔicmT by Dictyostelium. Phagocytosis of gfp-expressing wild-type L. pneumophila or ΔicmT by Dictyostelium infected at (A, B) an MOI of 100, or (C) at the MOI indicated was analyzed by flow cytometry. The increase in GFP fluorescence (FL 1, x-axis) indicates that, at MOIs ranging from 1-100, the number of wild-type L. pneumophila phagocytosed is about one order of magnitude higher than the number of ΔicmT. Phagocytosis is blocked by latrunculin B or incubation at 4° C. The data shown (B, C) are the means and standard deviations of duplicates and representative of at least three independent experiments.
FIG. 2 A role for PI3Ks in intracellular replication but not phagocytosis of wild-type L. pneumophila. (A) Phagocytosis by Dictyostelium wild-type Ax3 or ΔPI3K1/2 (untreated or treated with 5 μM wortmannin (WM)) of GFP-labeled L. pneumophila wild-type (black bars) or a ΔicmT mutant strain (grey bars) was determined by flow cytometry. (B) Release of L. pneumophila wild-type (squares) or ΔicmT (circles) into the supernatant of Dictyostelium wild-type (closed symbols) or ΔPI3K1/2 (open symbols) was quantified by CFUs. (C) Release of wild-type L. pneumophila (CFUs) from Dictyostelium wild-type (untreated, ▪; 10 μM LY294002 (LY), ▴) or ΔPI3K1/2 (untreated, □; LY, Δ). (D) Quantification by flow cytometry of intracellular growth of GFP-labeled wild-type L. pneumophila within wild-type Dictyostelium or ΔPI3K1/2 in presence or absence of 20 μM LY. The data shown are means and standard deviations of duplicates (A) or triplicates (B, C) and representative of at least three independent experiments (A-D).
FIG. 3 A role for PI3Ks in degradation of L. pneumophila ΔicmT. Quantification by flow cytometry of intracellular degradation of GFP-labeled L. pneumophila ΔicmT (MOI 100, MOI 500) within wild-type Dictyostelium or ΔPI3K1/2 in presence or absence of 20 μM LY. In the absence of PI3Ks, ΔicmT is less efficiently phagocytosed and degraded.
FIG. 4 Trafficking of L. pneumophila within Dictyostelium lacking functional PI3Ks. (A) Confocal laser scanning micrographs of calnexin-GFP-labeled (green) Dictyostelium wild-type Ax3 (untreated or treated with 20 μM LY) or ΔPI3K1/2 infected with DsRed-labeled wild-type L. pneumophila (red) for 1 h or 2.5 h, respectively. DNA was stained with DAPI (blue). (B) Quantification over time of spacious Legionella-containing vacuoles (LCV) in calnexin-GFP-labeled Dictyostelium wild-type (untreated, ▪; or treated with LY, ▴) or ΔPI3K1/2 (□) infected with DsRed-labeled wild-type L. pneumophila. Data represent percentage of spacious vacuoles from 50-200 total vacuoles per time point scored in 4 independent experiments. (C) Confocal laser scanning micrographs of Dictyostelium wild-type or ΔPI3K1/2, infected for 75 min with wild-type L. pneumophila expressing M45-tagged SidC. Infected amoebae were stained with rhodamine-conjugated anti L. pneumophila antibody (red), FITC-conjugated anti M45-tag antibody (green), and DAPI (blue), respectively. Bars, 2 μm.
FIG. 5A Binding of L. pneumophila Icm/Dot-secreted proteins to phosphoinositides in vitro. Binding of affinity-purified GST fusion proteins of SidC, SdcA, SidD or SdhB (160 pmol) to different lipids (100 pmol; left panels) or twofold serial dilutions of phosphoinositides (100-1.56 pmol; right panels) immobilized on nitrocellulose membranes was analyzed by a protein-lipid overlay assay using an anti GST antibody and enhanced chemiluminescence. Phosphoinositides (PI), lysophosphatidic acid (LPA); lysophosphocholine (LPC), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingosine-1-phosphate (SP), phosphatidic acid (PA), phosphatidylserine (PS). The experiment was reproduced at least three times with similar results.
FIG. 5B Binding of L. pneumophila Icm/Dot-secreted proteins to phosphoinositides in vitro. Phospholipid (PL) vesicles (20 μl, 1 mM lipid) composed of PC (65%), PE (30%), and 5% (1 nmol) either PI(4)P, PI(3)P or PI(4,5)P2 were incubated with affinity-purified GST-SidC or GST-SidD (40 pmol), centrifuged and washed. Binding of GST fusion proteins to PL vesicles was assayed by Western blot with an anti GST antibody. Similar results were obtained in three separate experiments.
FIG. 5C Binding of L. pneumophila proteins LepA, LepB and Lpg2311 to phosphoinositides in vitro. Binding of GST fusion proteins to different synthetic di-hexadecanoyl-phosphoinositides (100 pmol) immobilized on nitrocellulose membranes was analyzed by a protein-lipid overlay assay using an anti GST antibody. The fusion proteins specifically bind mono-phosphorylated phosphoinositides.
FIG. 5D Binding of the L. pneumophila proteins SidC, LepB and LidA to phosphoinositides in vitro. Binding of GST effector fusion proteins to different synthetic di-hexadecanoyl-phosphoinositides (100 pmol) immobilized on nitrocellulose membranes was analyzed by a protein-lipid overlay assay using an anti GST antibody. While SidC specifically binds to PI(4)P, LepB binds PI(3)P and LidA binds mono-phosphorylated phosphoinositides. Abbreviation: phosphatidylinositol (PtdIns).
FIG. 5E Domain analysis of SidC (CC: coiled coils) and fragments tested (vertical lines).
FIG. 5F Binding of affinity-purified GST fusion proteins of SidC fragments to different phosphoinositides spotted in 2-fold serial dilutions (100-1.56 pmol) onto nitrocellulose membranes. Binding was determined by a protein-lipid overlay assay using an anti-GST antibody.
FIG. 6 PI3Ks affect the amount of SidC bound to Legionella-containing vacuoles in Dictyostelium. (A) Confocal laser scanning micrographs of calnexin-GFP-labeled Dictyostelium wild-type strain Ax3 (green), infected with DsRed-labeled wild-type L. pneumophila (red) for 1 h (left panel), and immuno-labeled for SidC (blue) with an affinity purified primary and Cy5-conjugated secondary antibody (middle panel). To quantify fluorescence intensity (right panel), the averaged fluorescence intensity within background areas (“B1-3”) was subtracted from the intensity of the sample area (“S”). Bar, 2 μm. (B) Dot plot of SidC fluorescence (average and variance) on LCVs within Dictyostelium wild-type (untreated, n=135; treated with 20 PM LY, n=94) or ΔPI3K1/2 (n=86). The data shown are combined from 6 independent experiments, each normalized to 100% (average SidC fluorescence on LCVs in wild-type Dictyostelium).
FIG. 7 PI(4)P is a lipid marker of LCVs harboring Icm/Dot-proficient L. pneumophila (A, B, and D). Confocal micrographs of LCVs in lysates of (A) calnexin-GFP-labeled Dictyostelium, (B) VatM-GFP-labeled Dictyostelium, or (D) RAW264.7 macrophages infected with DsRed-Express-labeled L. pneumophila are shown. The lysates were prepared with a ball homogenizer, and PI(4)P was visualized on the LCVs using as probes either the PH domain of the PI(4)P-binding protein FAPP1 fused to GST, an antibody against PI(4)P, or GST-SidC. Using GST alone or omission of the anti-PI(4)P antibody did not label the LCVs. Bar denotes 2 μm (magnification of all images is identical). (C) Quantification of PI(4)P-positive calnexin-GFP-labeled (n=300) or VatM-GFP-labeled (n=100) LCVs in Dictyostelium wild-type strain Ax3.
FIG. 8 Uptake and intracellular replication of L. pneumophila in Dictyostelium PI(5)P phosphatase mutants. Uptake of L. pneumophila was analyzed by FACS using GFP-expressing bacteria. (A) Deletion of Dictyostelium PI(5)P phosphatases barely affected uptake of L. pneumophila wild-type (black bars) or ΔicmT (grey bars). (B) L. pneumophila grew more efficiently in Dictyostelium lacking PI(5)P phosphatase-4 (open squares) but not other PI(5)P phosphatases.
FIG. 9 Polynucleotide sequence for Legionella pneumophila SidC (SEQ ID NO: 3).
FIG. 10 Polynucleotide sequence for Legionella pneumophila SdcA (SEQ ID NO: 4).
FIG. 11 Amino acid sequence for Legionella pneumophila LepA (SEQ ID NO: 5).
FIG. 12 Amino acid sequence for Legionella pneumophila LepB (SEQ ID NO: 6).
FIG. 13 Amino acid sequence for Legionella pneumophila LidA (SEQ ID NO: 7).
FIG. 14. Amino acid sequence for Legionella pneumophila Lpg2311 (SEQ ID NO: 8).



DETAILED DESCRIPTION
In certain embodiments, the present invention provides isolated polypeptides comprising a sequence selected from the group consisting of SEQ ID NO: 1 and 2 and variants thereof. As used herein, the term “variant” comprehends nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 60%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably yet at least 90%, and most preferably at least 95% or 98% identity to a specific sequence disclosed herein. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.
Polynucleotide and polypeptide sequences having a specified percentage identity to a polynucleotide or polypeptide disclosed herein share a high degree of similarity in their primary structure. In addition to a specified percentage identity to a polynucleotide or polypeptide of the present invention, variant polynucleotides and polypeptides preferably have additional structural and/or functional features in common with a polynucleotide or polypeptide of the present invention. Polynucleotides having a specified degree of identity to, or capable of hybridizing to, a polynucleotide disclosed herein preferably additionally have at least one of the following features: (1) they contain an open reading frame, or partial open reading frame, encoding a polypeptide, or a functional portion of a polypeptide, having substantially the same functional properties as the polypeptide, or functional portion thereof, encoded by a polynucleotide in a recited SEQ ID NO; or (2) they contain identifiable domains in common.
Polynucleotide or polypeptide sequences may be aligned, and percentages of identical nucleotides or amino acids in a specified region may be determined against another polynucleotide or polypeptide, using computer algorithms that are publicly available. The BLASTN and FASTA algorithms, set to the default parameters described in the documentation and distributed with the algorithm, may be used for aligning and identifying the similarity of polynucleotide sequences. The alignment and similarity of polypeptide sequences may be examined using the BLASTP algorithm. BLASTX and FASTX algorithms compare nucleotide query sequences translated in all reading frames against polypeptide sequences. The FASTA and FASTX algorithms are described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and in Pearson, Methods in Enzymol. 183:63-98, 1990. The FASTA software package is available from the University of Virginia by contacting the Assistant Provost for Research, University of Virginia, PO Box 9025, Charlottesville, Va. 22906-9025. The BLASTN software is available from the National Centre for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894. The BLASTN algorithm Version 2.0.11 [Jan-20-2000] set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of polynucleotide variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, BLASTP and BLASTX, is described in the publication of Altschul et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25:3389-3402, 1997.
The “hits” to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, FASTA, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
As noted above, the percentage identity of a polynucleotide or polypeptide sequence is determined by aligning polynucleotide and polypeptide sequences using appropriate algorithms, such as BLASTN or BLASTP, respectively, set to default parameters; identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide of the present invention; and then multiplying by 100 to determine the percentage identity. By way of example, a queried polynucleotide having 220 nucleic acids has a hit to a polynucleotide sequence in the EMBL database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the default parameters. The 23-nucleotide hit includes 21 identical nucleotides, one gap and one different nucleotide. The percentage identity of the queried polynucleotide to the hit in the EMBL database is thus 21/220 times 100, or 9.5%. The percentage identity of polypeptide sequences may be determined in a similar fashion.
Polypeptides comprising sequences that differ from the specific polypeptide sequences disclosed herein as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence length are contemplated by and encompassed within the present invention, provided the variant polypeptide has functional properties which are substantially the same as, or substantially similar to, those of the specific polypeptide disclosed herein.
All of the polynucleotides and polypeptides described herein are isolated and purified, as those terms are commonly used in the art. Preferably, the polypeptides and polynucleotides are at least about 80% pure, more preferably at least about 90% pure, and most preferably at least about 99% pure.
As used herein, the term “polypeptide” encompasses amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. The term “polypeptide encoded by a polynucleotide” as used herein, includes polypeptides encoded by a polynucleotide that comprises a partial isolated polynucleotide sequence provided herein. Polypeptides of the present invention may be naturally purified products, or may be produced partially or wholly using recombinant techniques.
Polypeptides of the present invention may be produced recombinantly by inserting a polynucleotide sequence of the present invention encoding the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a polynucleotide molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells. Preferably, the host cells employed are plant, E. coli, insect, yeast, or a mammalian cell line such as COS or CHO.
In a related aspect, polypeptides are disclosed that comprise at least a functional portion, or fragment, of a polypeptide having a specific SEQ ID NO: disclosed herein. As used herein, the “functional portion” of a polypeptide is that portion which contains the active site essential for affecting the function of the polypeptide, for example, the portion of the molecule that is capable of binding a phosphoinositide lipid. Such functional portions generally comprise at least about 5 amino acid residues, more preferably at least about 10, and most preferably at least about 20 amino acid residues. Functional portions of a polypeptide may be identified by first preparing fragments of the polypeptide, by either chemical or enzymatic digestion of the polypeptide or mutation analysis of the polynucleotide that encodes for the polypeptide, and subsequently expressing the resultant mutant polypeptides. The polypeptide fragments or mutant polypeptides are then tested to determine which portions retain the biological activity of the full-length polypeptide.
Portions and other variants of polypeptides may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain (Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963). Equipment for automated synthesis of polypeptides is available from suppliers such as Perkin Elmer/Applied BioSystems, Inc. (Foster City, Calif.), and may be operated according to the manufacturer's instructions. Variants of a native polypeptide may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see, for example, Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492, 1985). Sections of DNA sequence may also be removed using standard techniques to permit preparation of truncated polypeptides.
Fusion proteins comprising a first polypeptide disclosed herein and a second, known, polypeptide, together with variants of such fusion proteins, are also provided. Fusion proteins may include a linker peptide between the first and second polypeptides.
A polynucleotide encoding a fusion protein is constructed using known recombinant DNA techniques to assemble separate polynucleotides encoding the first and second polypeptides into an appropriate expression vector. The 3′ end of a polynucleotide encoding the first polypeptide is ligated, with or without a peptide linker, to the 5′ end of a DNA sequence polynucleotide encoding the second polypeptide so that the reading frames of the sequences are in phase to permit mRNA translation of the two polynucleotides into a single fusion protein that retains the biological activity of both the first and the second polypeptides.
A peptide linker sequence may be employed to separate the first and the second polypeptides by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. The linker sequence may be from 1 to about 50 amino acids in length. Peptide linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. The ligated polynucleotides encoding the fusion proteins are cloned into suitable expression systems using techniques known to those of ordinary skill in the art.
Methods are provided for using one or more of the disclosed polypeptides, functional portions thereof and fusion proteins to treat a disorder in a patient, such as a disorder characterized by an unwanted and/or deleterious level of phosphatidylinositol(4) phosphate. As used herein, a “patient” refers to any warm-blooded animal, preferably a human.
In this aspect, the polypeptide, functional portion thereof or fusion protein (referred to as the “active component”) is generally present within a composition, such as a pharmaceutical or immunogenic composition. Such compositions may comprise one or more active components and a physiologically acceptable carrier. Immunogenic compositions may comprise one or more of the active components and an immunostimulant, such as an adjuvant or a liposome.
Routes and frequency of administration, as well as dosage, vary from individual to individual. In general, the compositions may be administered by injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. In general, the amount of binding agent present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg per kg of host, and preferably from about 100 pg to about 1 μg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 ml to about 2 ml.
While any suitable carrier known to those of ordinary skill in the art may be employed in the compositions, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a lipid, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
Any of a variety of adjuvants may be employed in the compositions to non-specifically enhance the immune response. Most adjuvants contain a substance designed to protect against rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis or M. tuberculosis. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, Mich.), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A.
As noted above, the disclosed polypeptides, functional portions thereof and fusion proteins disclosed herein may be used to determine the presence or absence of a phosphoinositide lipid in a biological sample. Examples of biological samples that may be used in such methods include, but are not limited to, blood, sera, saliva, urine, cerebrospinal fluid, synovial fluid, mucus, and/or tissue biopsies.
There are a variety of assay formats known to those of ordinary skill in the art for use in such methods. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of a phosphoinositide lipid may be determined by (a) contacting a biological sample obtained from a patient with a polypeptide disclosed herein; (b) detecting in the sample a level of phosphoinositide lipid that binds to the polypeptide; and (c) comparing the level of phosphoinositide lipid with a predetermined cut-off value.
In one embodiment, the assay involves the use of polypeptide immobilized on a solid support to bind to and remove the phosphoinositide lipid from the sample. The bound phosphoinositide lipid may then be detected using a detection reagent that contains a reporter group and specifically binds to the phosphoinositide lipid/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide.
The solid support may be any material known to those of ordinary skill in the art to which the polypeptide may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The polypeptide may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. As used herein, the term “immobilization” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane may be preferred. In such cases, adsorption may be achieved by contacting the polypeptide, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of polypeptide ranging from about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is sufficient to immobilize an adequate amount of polypeptide.
Covalent attachment of the polypeptide to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the polypeptide.
The polypeptide may be fused with, or conjugated to, a tag that can be readily detected. The method employed for detecting the tag depends upon the nature of the tag. For example, scintillation counting or autoradiographic methods are generally appropriate for radioactive groups. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a reporter group (commonly a radioactive or fluorescent group or an enzyme).
To determine the presence or absence of a disorder characterized by an unwanted amount of phosphoinositide lipid, the signal detected in the assay is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value is the average mean signal obtained when the polypeptide is incubated with samples from patients without the disorder. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the disorder. Alternatively, the cut-off value may be determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a disorder.
Those of skill in the art will appreciate that numerous other assay protocols exist that are suitable for use with the polypeptides disclosed herein. The above descriptions are intended to be exemplary only.
EXAMPLES
Experimental Protocol
1. Bacteria, Dictyostelium, Media and Growth Conditions
The L. pneumophila strains used in this study were wild-type strain JR32 (a salt-sensitive derivative of a streptomycin-resistant strain Philadelphia-1), the isogenic ΔicmT deletion mutant GS3011, which lacks a functional Icm/Dot T4SS, and corresponding strains constitutively producing the green fluorescent protein GFP or the red fluorescent protein DsRed-Express (Table 1). L. pneumophila was routinely grown for 3 days on CYE agar plates containing charcoal yeast extract, buffered with N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) [41]. Liquid cultures were inoculated in AYE medium supplemented with BSA (0.5%) [42] at an OD600 of 0.1 and grown for 21 h at 37° C. (post exponential growth phase). To maintain plasmids, chloramphenicol (cam) was added at 5 μg/ml. As “input” controls, 20 μl of a 105/ml bacterial solution was plated and counted after 3 d incubation in all phagocytosis and intracellular growth experiments.
Dictyostelium discoideum wild-type strain Ax3 and the PI3K-1/2 double mutant (ΔPI3K1/2) were a gift from R. Firtel (University of California, San Diego, USA). The ΔPI3K1/2 mutant is lacking two PI3Ks which are related to the mammalian p110 catalytic subunit of class I PI3Ks. The mutant strain shows morphological, developmental and chemotactic phenotypes and is defective for vegetative growth in axenic medium and on bacterial lawns [34, 36, 38, 43]. Specifically, ΔPI3K1/2 is smaller than the isogenic wild-type strain and is impaired for (i) phagocytosis of live or autoclaved bacteria, (ii) pinocytosis of fluid markers, (iii) maturation of phagosomes to “spacious” phagosomes via homotypic fusion, and (iv) possibly exocytosis. A biochemical analysis of the phosphoinositide profile of ΔPI3K1/2 compared to the complemented strain revealed that the levels of PI(3,4)P2 and PI(3,4,5)P3 were reduced, while the level of PI(4)P was elevated, and PI(3)P as well as PI(4,5)P2 remained unchanged [36].
Dictyostelium amoebae were grown axenically at 23° C. in 75 cm2 tissue culture flasks in HL5 liquid medium (10 g glucose, 5 g yeast extract, 5 g proteose peptone, 5 g thiotone E peptone, 0.35 g Na2HPO4, 0.34 g KH2PO4 in 1 l H2O, pH 6.5) supplemented with 10 μg/ml G418 or blasticidin-S when necessary. The amoebae were split once or twice a week and fed with fresh HL5 medium 24 h before use. For viability assays, Dictyostelium was plated together with Klebsiella pneumoniae on SM/5 agar plates, and plaque forming units (PFU) were counted after 3-4 d incubation at 23° C. [44].
2. Plasmid Construction, Protein Purification and Antibody Preparation
Translational gst fusions of sidC, sdcA, sidD and sdhB, were constructed by PCR amplification of the putative ORFs using the primers listed in Table 2. For sidD the ATG at position 120 downstream of a TTG in the ORF was used as a start codon. The PCR fragments were cut with BamHI and SalI and ligated into plasmid pGEX-4T-1 yielding pCR2, pCR16, pCR10, and pCR8, respectively (Table 1). All constructs were sequenced. Production of the fusion proteins in E. coli BL21(DE3) was induced at a cell density of OD600 0.6 with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 3 h at 30° C. in LB medium. In all cases, this protocol resulted in a significant portion of soluble fusion protein of the expected size (132 kDa, 132 kDa, 79 kDa, 239 kDa, respectively). The fusion proteins were purified from lysates prepared by sonication using glutathione-sepharose beads in a batch procedure according to the manufacturer's recommendations (Amersham). Purity of the protein preparations was analyzed by SDS polyacrylamide gel electrophoresis.
According to a preferred embodiment GST-SidC was purified according to the following protocol:

    
    
        1) Freshly transform E. coli BL21(DE3) with the GST-SidC fusion construct.
    
    


Streak on LB agar plates containing ampicillin (150 μg/ml) and incubate over night (o/n) at 37° C.

    
    
        2) Wash the transformants with LB medium from the agar plate and inoculate 20 ml LB medium containing ampicillin (100 μg/ml). Incubate o/n (37° C.).
        3) Inoculate 1 liter LB medium with 20 ml o/n culture (approximate OD600 0.05). Incubate at 37° C. (180 rpm) until OD600 reaches approximately 0.5-0.8, preferably 0.6.
        4) Chill liquid culture on ice (5 min), induce production of GST fusion protein with 0.5-1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 3 h at 30° C. (180 rpm).

Under these conditions a significant portion of soluble fusion protein GST-SidC of the expected size of 132 kDa is produced.

        5) Pellet bacteria, freeze pellet preferably in liquid nitrogen.
        6) Cell lysis: Resuspend bacterial pellet in 30 ml cold PBS (80 g NaCl, 2 g KCl, 26.8 g Na2HPO4×7 H2O, 2.4 g KH2PO4 per liter, adjust pH to 7.4 with HCl). Optional: add approximately 1 mg lysozyme (15 min). Add 50 μl 1M MgCl2 and 1 mg DNAse. Pass the suspension 3×though a French Press (55 MPa). Centrifuge cell debris (2×15 min, 15′000 rpm, rotor SS34, 4° C.).
        7) Incubate supernatant with glutathione-sepharose beads (Amersham Biosciences; 1.2 ml slurry, washed with 10 ml PBS, resuspended in 1 ml PBS) for 1-1.5 h on a wheel (20 rpm) at 4° C.
        8) Wash 3× with 10 ml PBS (spin at approximately 550×g, 4° C.).
        9) Elute with 1 ml elution buffer (0.462 g reduced glutathione, 700 μl 1 M Tris, pH 8.0, per 10 ml H2O; final pH: 7.4-7.6).
        10) Shock freeze aliquots.

The yield is approximately 15 mg purified protein per liter liquid culture grown in LB medium.

    
    


A translational his-sidC fusion was constructed by moving the sidC ORF (cut with BamHI and SalI) from pCR2 into pET28a(+), yielding pCR1. Production of His6-SidC by E. coli BL21(DE3) was induced with 1 mM IPTG for 3-6 h at 30° C., resulting in a predominantly soluble protein of the expected size (109 kDa). The His6-SidC fusion protein was purified by Ni2+ affinity chromatography, and polyclonal antibodies against the purified protein were raised in rabbits (NeoMPS). The antibodies were affinity-purified from rabbit serum by using an Aekta liquid chromatography system (Amersham) and GST-SidC covalently linked to Affigel-10 beads (BioRad) [45]. Typical yield of purified anti SidC antibody was 2.2-3.2 mg/ml serum.
An L. pneumophila expression vector for M45-tagged SidC (pCR34) was constructed using pMMB207C [19] as a backbone. A ribosomal binding site (RBS) was introduced by moving an EcoRI/BamHI fragment from pUA26 [46] into pMMB207C, yielding pMMB207C-RBS-lcsC. To insert the DNA encoding the M45 tag, the oligos oCR-P1 and oCR-P2 harboring a mutation in the internal BamHI site of the M45 sequence (G to T in oligo oCR-P1) were used. The oligos were annealed (1 nmol each in 100 μl; heated to 94° C., followed by slow cooling to 4° C.) and ligated into pMMB207C-RBS-lcsC cut with NdeI and BamHI, yielding vector pMMB207C-RBS-M45. Finally, the fragment encoding SidC was moved from plasmid pCR2 into pMMB207C-RBS-M45 by using the BamHI and SalI restriction sites, yielding pCR34.
3. Analysis of Phagocytosis by Flow Cytometry
Phagocytosis of L. pneumophila by Dictyostelium was analyzed by flow cytometry using GFP-labeled bacteria. Exponentially growing Dictyostelium were seeded onto a 24-well plate (5×105 cells/ml HL5 medium per well) and allowed to adhere for 1-2 h. L. pneumophila grown for 21 h in AYE liquid culture was centrifuged, resuspended in HL5 medium and used to infect the amoebae at a multiplicity (MOI) of 100 or at the MOI indicated (OD600 of 0.3=2×109 bacteria/ml). The infection was synchronized by centrifugation (10 min at 880×g), infected cells were incubated at 25° C., and 30 min post-infection, extracellular bacteria were removed by washing three to five times with SorC (2 mM Na2HPO4, 15 mM KH2PO4, 50 μM CaCl2, pH 6.0). Infected Dictyostelium were detached by vigorously pipetting, and 2×104 amoebae per sample were analyzed using a FACSCalibur flow cytometer (Becton Dickinson). The GFP fluorescence intensity falling into a Dictyostelium scatter gate was quantified using the FlowJo software (Treestar).
To confirm that the fluorescence observed arises from internalized and not from adherent L. pneumophila, phagocytosis was inhibited in parallel experiments. One h prior infection the medium was exchanged, and Dictyostelium was incubated in HL5 medium containing either latrunculin B (1-50 μM), cytochalasin A (1-50 μM) or, as a solvent control, DMSO (0.5%). Alternatively, the infection was performed on ice, and the infected cells were incubated at 4° C. While latrunculin B or cytochalasin A blocked phagocytosis in a dose-dependent manner, cytochalasin D (up to 50 μM), which effectively blocks macrophage phagocytosis, did not prevent uptake of L. pneumophila by Dictyostelium (data not shown).
In experiments addressing the role of PI3Ks, the amoebae were incubated in HL5 medium containing the PI3K inhibitors wortmannin (WM; 0.1-10 μM) or LY294002 (LY; 5-25 μM) for 1 h prior infection, which was then performed in presence of the inhibitors. At the concentrations indicated, the pharmacological inhibitors did not affect viability of L. pneumophila or Dictyostelium, as determined by measuring colony forming units (CFU) or PFU (data not shown).
4. Intracellular Growth of L. pneumophila within Dictyostelium 
Release of L. pneumophila from Dictyostelium due to intracellular replication was quantified by determining CFUs in the supernatant as described [31, 33]. Briefly, exponentially growing Dictyostelium amoebae were washed with SorC and resuspended in MB medium (7 g yeast extract, 14 g thiotone E peptone, 20 mM MES in 1 l H2O, pH 6.9). 105 Dictyostelium cells per well were seeded onto a 96-well plate, let adhere for 1-2 h, and were infected at an MOI of 1 with L. pneumophila grown on CYE plates for 3-4 days and resuspended in MB medium. Occasionally, L. pneumophila grown in AYE medium for about 21 h was used as an inoculum. The infection was synchronized by centrifugation, and the infected amoebae were incubated at 25° C. At the time points indicated, the number of bacteria released into the supernatant was quantified by plating aliquots (10-20 μl) of appropriate dilutions on CYE plates. L. pneumophila did not grow in MB medium. Rather, the CFUs decreased 2-3 orders of magnitude within 3-6 days under these conditions (data not shown).
Intracellular bacterial growth before host cell lysis was quantified by counting CFU after selectively lysing infected Dictyostelium with saponin (“single round replication”). At the time points indicated, the MB medium was replaced by 100 μl 0.8% saponin and incubated for 15 min. The cells were lysed by pipetting, and aliquots were plated. Intracellular replication of GFP-labeled wild-type L. pneumophila or killing of GFP-labeled ΔicmT was also directly determined by flow cytometry. Here, the fluorescence intensity falling into a Dictyostelium scatter gate was quantified. Alternatively, the number of GFP-labeled L. pneumophila released into 120 μl Dictyostelium supernatant was quantified by flow cytometry using a scatter gate adjusted for bacteria.
To determine the effect of PI3K inhibitors on intracellular growth of L. pneumophila, Dictyostelium was incubated for 1 h in MB medium containing 5 μM wortmannin or 10-20 μM LY, respectively. The medium was not exchanged prior to an infection with L. pneumophila, leaving the inhibitors throughout the experiment. Since wortmannin is unstable in buffered aqueous solutions [47], LY was used preferentially. In some experiments, the inhibitors were added freshly to the medium every second day of the incubation period, yet, this protocol did not alter the results of the experiments. The PI3K inhibitors did not have an effect on L. pneumophila in MB medium (data not shown). Dictyostelium Ax3 wild-type cells treated with 5 μM wortmannin or 10 μM LY were as viable as untreated wild-type or ΔPI3K1/2 for up to 5 or 6 days in MB medium (data not shown). At later time points, cells treated with LY showed a reduced viability as determined by PFU on lawns of K. pneumoniae, and therefore, intracellular growth of L. pneumophila in presence of PI3K inhibitors was analyzed only up to 6 d.
5. Intracellular Trafficking of L. pneumophila 
For immuno-fluorescence, Dictyostelium was split and fed two days prior to an experiment, seeded on sterile coverslips in 24-well plates at 2.5×105 per well in 0.5 ml HL5 medium and let grow over night. The medium was exchanged about 1 h before the infection and contained 20 μM LY where indicated. The L. pneumophila strains used for the infections were grown for 21 h (OD600 of the inoculum: 0.1) in 3 ml AYE/BSA containing 5 μg/ml cam and 0.5 mM IPTG. Bacterial cultures were diluted in HL5 medium to a concentration of 5×108/ml, and 100 μl of the suspension were added to the amoebae (MOI=100). The infection was synchronized by centrifugation, and the cells were washed twice with HL5 medium.
At the time points indicated, the infected amoebae were washed 3 times with cold SorC buffer and fixed with 4% paraformaldehyde for 30 min at 4° C. The fixed cells were washed 3 times, permeabilized (0.1% Triton X-100, 10 min) and blocked with 2% normal human AB serum in SorC for 30 min. The coverslips were incubated for 1 h at room temperature on parafilm with 30 μl of primary antibodies diluted in blocking buffer (rhodamine-conjugated rabbit anti L. pneumophila Philadelphia-1 serogroup 1, 1:100 (m-Tech, Monoclonal Technologies); mouse anti M45 hybridoma supernatant, 1:4 [48]; affinity purified rabbit anti SidC (see above), 1:1000) and washed 3 times with blocking buffer after each antibody. Secondary antibodies were from Jackson Immuno Research Laboratories (FITC-conjugated goat anti mouse IgG, F(ab′)2-specific; Cy5-conjugated goat anti rabbit Fab fragment) and incubated at a 1:200 dilution in blocking buffer for 1 h at room temperature. Finally, DNA was stained with DAPI (1 μg/ml) in SorC for 5 min, the coverslips were washed twice and mounted using Vectashield (Vector Laboratories).
The samples were viewed with an inverted confocal microscope (Axiovert 200M; Zeiss), equipped with a 100×oil phase contrast objective (Plan Neofluar; Zeiss), an “Ultraview” confocal head (Perkin Elmer) and a krypton/argon laser (643-RYB-A01; Melles Griot). Data processing and three-dimensional reconstruction was performed with the “Volocity” 2.6.1 software (Improvision).
The amount of SidC on LCVs harboring DsRed-labeled L. pneumophila in calnexin-GFP-labeled wild-type Dictyostelium (untreated or treated with 20 μM LY) or in ΔPI3K1/2 was quantified by immuno-fluorescence using affinity purified anti SidC and CyS-conjugated secondary antibodies. The fluorescence intensity of an area identical for all samples and covering the LCV was quantified using the “Quantity One” software (BioRad) after background correction (averaged intensity of 3 areas within the infected amoeba). To standardize the procedure, all images were acquired with the same exposure time, only LCVs containing rod-shaped and non-permeabilized bacteria were considered, and “equatorial” sections along the z-axis through the bacteria were chosen.
6. Binding of Icm/Dot-Secreted L. pneumophila Proteins to Phosphoinositides and Other Lipids in vitro
Direct binding of L. pneumophila Icm/Dot-secreted putative effector proteins to phosphoinositides and other lipids was tested in a protein-lipid overlay assay [49]. The lipid compounds bound to nitrocellulose membranes were incubated with GST-effector fusion proteins, which were constructed and purified as described above. Preliminary binding experiments were performed using synthetic di-hexadecanoyl-phosphoinositides (Echelon) or purified authentic di-acyl- (preferentially 1-stearoyl-2-arachidonoyl) phosphoinositides (Sigma; Matreya LLC). 3 μl of diluted stock solutions in CHCl3:MeOH:H2O=1:2:0.8 (synthetic phosphoinositides) or MeOH (authentic phosphoinositides) were spotted onto nitrocellulose membranes yielding 6-200 pmol per spot. The membranes were blocked with 4% fat free milk powder in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween-20 (v/v), pH 8.0) for 1 h at room temperature and incubated with the fusion proteins (ca. 120 pmol/ml blocking buffer) over night at 4° C. Binding of the GST-effector fusion proteins to lipids was visualized by ECL (Amersham) using a monoclonal anti GST antibody (Sigma) and a secondary goat anti mouse peroxidase-labeled antibody (Sigma). The final experiments were done with commercially available PIP-stripsTM and PIP-arraysTM (Echelon), using GST-tagged PH domains of PLCδ1 (PIP2 GripTM) and LL5α (MultiPIP GriPTM) as control reagents for the presence of PI(4,5)P2 or all phosphoinositides on the nitrocellulose membranes.
To test whether SidC binds to phosphoinositides incorporated into phospholipid (PL) vesicles, we used affinity purified GST-SidC or GST-SidD and biotinylated PL vesicles (1 mM lipid) composed of 65% phosphatidylcholine (PC), 29% phophatidylethanolamine (PE), 1% biotinylated PE and 5% either PI(4)P, PI(3)P or PI(4,5)P2 (PolyPIPosomesTM; Echelon). The PL vesicles (20 μl, 1 nmol PI) were incubated for 20 min at 4° C. with GST-SidC or GST-SidD fusion proteins (40 pmol) in a total of 1 ml binding buffer (50 mM Tris, 150 mM NaCl, 0.05% Nonidet P40, pH 7.6). The liposomes were subsequently centrifuged (10 min, 20′800×g) and washed 5 times with 1 ml binding buffer. Finally, the pellet was resuspended in 25 μl SDS PAGE loading buffer, boiled and loaded onto an 8% SDS gel. GST fusion proteins were visualized by Western blot with a monoclonal anti GST antibody (Sigma).
7. Accession Numbers
The GenBank accession numbers for the proteins mentioned in this application are Dictyostelium calnexin, AF073837; Dictyostelium PI3K1 and PI3K2, U23476 and U23477, respectively; human FAPP1, AF286162; L. pneumophila Icm/Dot T4SS conjugation apparatus, Y15044; SidC, AY504673 (DNA sequence provided in FIG. 9; SEQ ID NO: 3) SidC paralog SdcA, AY504674 (DNA sequence provided in FIG. 10; SEQ ID NO: 4); LepA, AAP20592 (FIG. 11; SEQ ID NO: 5); LepB, AAP20593 (FIG. 12; SEQ ID NO: 6); LidA, AA061471 (FIG. 13; SEQ ID NO: 7); LPG 2311, AAU28373 (FIG. 14; SEQ ID NO: 8).
8. Strains, Plasmids, Oligonucleotides







TABLE 1







Strains and plasmids.









Name
Relevant characteristics
References





Strains




L. pneumophila


JR32
Salt-sensitive derivative of virulent
[50]



L. pneumophila Philadelphia-1 serogroup 1


GS3011
JR32 icmT3011::KanR
[51]


E. coli


TOP10

Invitrogen


BL21(DE3)

Novagen


Dictyostelium


Ax3
Wild-type
[34]


ΔPI3K1/2
Ax3 ΔPI3K1/2
[34]


Ax2
Wild-type
[52]


HG1769
Ax2 Δcalreticulin, blasticidin-SR (BlsR)
[52]


HG1770
Ax2 Δcalnexin, BlsR
[52]


HG1773
Ax2 Δcalnexin/calreticulin, BlsR, G418R
[52]


Plasmids


pCR1
sidC-his in pET28a(+)
This study


pCR2
sidC-gst in pGEX-4T-1
This study


pCR8
sdhB-gst in pGEX-4T-1
This study


pCR10
sidD-gst (120ATG) in pGEX-4T-1
This study


pCR16
sdcA-gst in pGEX-4T-1
This study


pCR33
pMMB207C-RBS-M45,
This study



from pMMB207C-RBS-lcsC


pCR34
pMMB207C-RBS-M45-SidC
This study


pET28A(+)
expression of N-terminal his fusions; PT7; KanR
Novagen


pGEX-4T-1
expression of N-terminal gst fusions; Ptac; AmpR
Amersham


pMMB207C
Legionella expression vector, ΔmobA, no RBS
[19]


pMMB207-Km14-gfpc
pMMB207-Km14, ΔlacIq, constitutive gfp
[53]


pMMB207-RBS-lcsC
expression vector for lcsC, RBS (= pUA26)
[46]


pMMB207C-RBS-lcsC
expression vector for lcsC, RBS, ΔmobA
This study


pMMB207C-RBS-M45
expression vector, ΔmobA, RBS, M45-(Gly)5
This study


pSW001
pMMB207C, ΔlacIq, constitutive dsred
[53]


pCalnexin-GFP
act15/calnexinA-RSSSKLK-gfp(S65T), G418R
[52]
















TABLE 2







Oligonucleotides.










Oligo-
Relevant characteristics




nucleotides
or sequencea
Comments





oCR1
AAAAACGCGGATCCATGGTGATAAACATGGT
5′ sidC;




TGACG (SEQ ID NO:9)
BamHI


 


oCR2
AAAAACGCGTCGACCTATTTCTTTATAATTC
3′ sidC;



CCGTGTAC (SEQ ID NO:10)
SalI


 


oCR4
AAAAACGCGTCGACTTAAATAGTAAGACTCG
3′ sidD;



AGTTAG (SEQ ID NO:11)
SalI


 


oCR7
AAAAACGCGTCGACTCATGCTACTATTAAGC
5′ sdhB;



ATAGAGG (SEQ ID NO:12)
SalI


 


oCR8
AAAAGAATGCGGCCGCTTACAATTTGGTAAA
3′ sdhB;



TTCGATTTCAC (SEQ ID NO:13)
NotI


 


oCR13
AAAAACGCGGATCCATGCGTTCGATTATTAC
5′ sidD,



ACAAATC (SEQ ID NO:14)
BamHI


 


oCR29
AAAAACGCGTCGACTCATGAACATGGTTGAC
5′ sdcA;



AAAATAAAATTC (SEQ ID NO:15)
SalI


 


oCR30
AAAAGAATGCGGCCGCCTATATTGTATTCCT
3′ sdcA;



AACAGTTTCTC (SEQ ID NO:16)
NotI


 


oCR-P1
TATGGCCATGGATCGGAGTAGGGATCGCCTA
‘NdeI,



CCTCCTTTTGAGACAGAGACGCGTATCCTCG
BamHI’



GTGGTGGTGGTGGTG



(SEQ ID NO:17)


 


oCR-P2
GATCCACCACCACCACCACCGAGGATACGCG
‘BamHI,



TCTCTGTCTCAAAAGGAGGTAGGCGATCCCT
NdeI’



ACTCCGATCCATGGCCA



(SEQ ID NO:18)





aRestriction sites are underlined.






Results

1. PI3Ks are Dispensable for Phagocytosis of Wild-Type L. pneumophila 

Phagocytosis of L. pneumophila by Dictyostelium was quantified by flow cytometry using bacteria constitutively expressing gfp. About 10 times more amoebae showed increased fluorescence, if infected with wild-type L. pneumophila compared to an icmT mutant strain (ΔicmT), which lacks a functional Icm/Dot T4SS (FIG. 1). This assay indicates that at least 10 times more wild-type L. pneumophila were phagocytosed compared to ΔicmT. Icm/Dot-dependent phagocytosis was observed at a multiplicity of infection (MOI) ranging from 1 to 100 and blocked by inhibitors of actin polymerization (latrunculin B, 20 μM; cytochalasin A, 10 μM), or by performing the infection at 4° C.
Wild-type L. pneumophila was only slightly less efficiently phagocytosed by Dictyostelium ΔPI3K1/2 (−4%), or wild-type Dictyostelium treated with the PI3K inhibitors wortmannin (WM; −19%) or LY294002 (LY; −33%), respectively (FIG. 2A, data not shown). Thus, genetic and pharmacological data indicate that phagocytosis of L. pneumophila by Dictyostelium does not require PI3Ks. This result is in agreement with the finding that uptake of L. pneumophila by macrophage-like cells occurs via a wortmannin-insensitive pathway [35]. Contrarily, phagocytosis of ΔicmT was reduced by 77-88% upon deletion or inhibition of PI3Ks, corresponding to reports that Dictyostelium PI3K1/2 is involved in phagocytosis of E. coli [36]. The addition of PI3K inhibitors to ΔPI3K1/2 did not further diminish phagocytosis of ΔicmT, indicating that other Dictyostelium class I PI3Ks present in the genome [37] are not involved in uptake.
2. PI3Ks are Involved in Intracellular Replication of Wild-Type L. pneumophila and Degradation of ΔicmT
The effect of PI3Ks on intracellular replication of L. pneumophila was quantified by determining colony forming units (CFUs) in the supernatant of infected Dictyostelium cultures. Compared to wild-type Dictyostelium, about a factor of 100 more wild-type L. pneumophila were released within 6-8 days from ΔPI3K1/2 (FIG. 2B) or amoebae treated with LY (FIG. 2C), indicating that functional PI3Ks restrict intracellular replication of L. pneumophila. To test intracellular growth of L. pneumophila more directly, we analyzed Dictyostelium infected with GFP-labeled L. pneumophila by flow cytometry (FIG. 2D). In this assay, GFP-labeled L. pneumophila grew earlier and more efficiently within ΔPI3K1/2 or wild-type Dictyostelium treated with LY compared to untreated wild-type amoebae. Treatment of ΔPI3K1/2 with LY did not enhance intracellular replication further, suggesting that no other class I PI3Ks are involved. Quantification by flow cytometry of GFP-labeled wild-type L. pneumophila released from Dictyostelium showed that L. pneumophila emerged earlier from Dictyostelium lacking PI3Ks, yet apparently grew with similar rates (data not shown). In a “single round” growth assay, where the amoebae were selectively lysed with saponin, L. pneumophila started to grow already after 1 d in absence of PI3Ks, while in wild-type Dictyostelium the numbers of wild-type L. pneumophila initially decreased (data not shown). Finally, while ΔicmT did not replicate within Dictyostelium in presence or absence of PI3Ks (FIG. 2B), the mutant bacteria were killed about 2 times slower within ΔPI3K1/2 (FIG. 3, data not shown). These results are in agreement with a requirement of class I PI3Ks for the endocytic degradative pathway [23].
3. Trafficking of L. pneumophila is Altered in Absence of Functional PI3Ks
The finding that L. pneumophila replicates more efficiently in absence of PI3Ks suggests that vesicle trafficking and formation of the LCV is altered. As a marker for LCVs, we used the ER membrane protein calnexin-GFP, which within 2 h co-localizes with about 65% LCVs harboring wild-type L. pneumophila but not at all with ΔicmT-containing LCVs (data not shown; [6, 9]). Calnexin does not profoundly affect trafficking of L. pneumophila, since intracellular replication within wild-type Dictyostelium was similar to replication in Dictyostelium mutants lacking calnexin, calreticulin, calnexin/calreticulin, or in a calnexin-GFP-expressing strain (data not shown).
In Dictyostelium wild-type and in strains lacking PI3Ks, the LCVs acquired calnexin-GFP with similar kinetics (data not shown), suggesting that initial docking and fusion of ER-derived vesicles with the Legionella phagosome is not affected by PI3Ks. However, the morphological dynamic of the LCV was altered, as the transition from tight to spacious vacuoles was severely impaired in Dictyostelium lacking functional PI3Ks (FIG. 4A). In wild-type Dictyostelium, 25% of the LCVs appeared spacious as early as 15 min post infection, and within 2 h, 40% spacious vacuoles were scored (FIG. 4B). In contrast, in Dictyostelium lacking PI3Ks, the portion of spacious LCVs was less than 5% at 15 min post infection, reached within 2 h only 10% (ΔPI3K1/2) or 20% (LY-treated wild-type Dictyostelium), and remained below the level observed in wild-type Dictyostelium throughout the 6 h observation period. At later time points, scoring of the vacuoles became difficult, since infected Dictyostelium easily detached from the substratum, and LCVs harboring replicating bacteria appeared spacious in presence or absence of PI3Ks. In summary, these results indicate that class I PI3Ks play a role in the modulation of the LCV and the formation of a replication-permissive vacuole.
4. The Icm/Dot-Secreted L. pneumophila Protein SidC Localizes to Tight and Spacious Vacuoles
To correlate the morphology of the LCV with the presence of a putative L. pneumophila effector protein, we stained for the Icm/Dot-secreted protein SidC (“Substrate of Icm/Dot transporter”, [18]). The function of SidC is unknown. However, the protein localizes to LCVs in Legionella-infected macrophages and is exposed to the cytoplasmic side of the vacuolar membrane. Immuno-staining of M45-tagged SidC within Legionella-infected Dictyostelium amoebae revealed its presence on tight as well as spacious LCVs (FIG. 4C). Similar to LCVs labeled with calnexin-GFP, the majority of M45-SidC-labeled LCVs formed in wild-type Dictyostelium after 75 min appeared spacious, while at the same time the LCVs in ΔPI3K1/2 were all tight-fitting (data not shown). Some punctate background staining was also visible in uninfected Dictyostelium and thus is not due to association of SidC with cellular organelles. As even upon overexpression, M45-tagged SidC localized exclusively to the LCV, but not to other cellular vesicles, SidC anchors with high affinity and specificity to the LCV membrane.
5. SidC and SdcA Directly and Specifically Bind to PI(4) Phosphate in vitro
Intracellular replication of L. pneumophila depends on phosphoinositide metabolism as well as on the Icm/Dot T4SS. A direct link between these host cell and pathogen factors would exist, if secreted L. pneumophila proteins bind to phosphoinositides on the LCV. SidC is an attractive candidate to test this hypothesis, since the protein binds to the LCV membrane, yet no transmembrane helices are predicted from its primary sequence. To determine whether SidC interacts with phosphoinositides in vitro, we assayed binding of an N-terminal GST-SidC fusion protein to phosphoinositides and other lipids immobilized on nitrocellulose membranes. Under these conditions, SidC directly and almost exclusively bound to PI(4)P and to a much weaker extent to PI(3)P but not to other phosphoinositides or lipids (FIG. 5A). Estimated from binding of SidC to phosphoinositides arrayed in twofold serial dilutions, the affinity of SidC for PI(4)P was a factor of 50-100 higher than for PI(3)P. The SidC paralogue SdcA (72% identity on an amino acid level) also specifically bound to PI(4)P and PI(3)P, yet compared to SidC with apparently lower affinity to PI(4)P and higher affinity to PI(3)P.
We also tested GST fusion proteins of SidD and SdhB for binding to phosphoinositides (FIG. 5A). SdhB is a paralogue of SidH and predicted with low stringency by the “scansite” algorithm (available from the Massachusetts Institute of Technology) to contain an ANTH domain putatively binding PI(4,5)P2. While the GST-SidD fusion protein did not bind to any of the lipids tested in vitro, the GST-SdhB fusion protein bound very weakly only to PI(3)P but not to other lipids.
To investigate the binding specificity of SidC to phosphoinositides incorporated into phospholipid (PL) vesicles, we incubated GST-SidC with PL vesicles composed of phosphatidylcholine (65%), phosphatidylethanolamine (30%), and 5% either PI(4)P, PI(3)P or PI(4,5)P2. GST-SidD was used as a putative negative control. The PL vesicles were incubated with GST-SidC or GST-SidD, centrifuged and washed several times, prior to analyzing binding of the proteins by Western blot with an anti GST antibody (FIG. 5B). Under the conditions used, SidC almost exclusively bound to PI(4)P, while binding to PI(3)P was negligible. Binding of SidC to PI(4,5)P2 was in the range observed for SidD and thus considered unspecific. Estimated by densitometry, about 200 times more SidC bound to PL vesicles harboring PI(4)P compared to vesicles containing PI(3)P or PI(4,5)P2. Taken together, using two different biochemical assays SidC was found to specifically bind to PI(4)P in vitro.
6. SidC Preferentially Binds to LCVs in Absence of Functional PI3Ks
To address the question of whether SidC binds to PI(4)P on LCVs in infected Dictyostelium amoebae, we determined whether altering the ratio of cellular phosphoinositides affects the amount of SidC bound to LCVs. In Dictyostelium ΔPI3K1/2 the level of the PI3K products PI(3,4)P2 and PI(3,4,5)P3 is decreased, while the level of the PI3K substrate PI(4)P is increased compared to the complemented strain [36]. Accordingly, if the level of phosphoinositides on LCVs mirrors the cellular phosphoinositide levels, SidC is predicted to preferentially bind to LCVs in absence of PI3Ks. To test whether PI3Ks affect the amount of SidC on LCVs, SidC bound to LCVs was quantified by immuno-fluorescence using an affinity purified antibody (FIG. 6A). SidC and calnexin-GFP always and strictly co-localized on LCVs regardless of whether PI3Ks were present or not. We found that with high statistical significance (p<10−9) about a factor of 1.5 more SidC localized to LCV membranes in wild-type Dictyostelium treated with LY or ΔPI3K1/2, compared to LCVs formed in wild-type Dictyostelium (FIG. 6B). This result is in agreement with the notion that in absence of functional PI3Ks the amount of cellular and vacuolar PI(4)P is increased, allowing more SidC to bind to the LCV in L. pneumophila-infected host cells.
7. PI(4)P is a Lipid Marker of LCVs Harboring Icm/Dot-Proficient L. pneumophila 
SidC specifically binds to PI(4)P in vitro and to the membrane of the LCV, suggesting that PI(4)P is a constituent of the LCV. To test whether PI(4)P is indeed a lipid marker of the LCV, we used as probes a PI(4)P-specific antibody or the PH domain of FAPP1 (phosphatidylinositol(4) phosphate adaptor protein-1) fused to GST. FAPP1 is required for transport from the trans Golgi network to the plasma membrane and has been shown to specifically bind PI(4)P [49,54]. The PI(4)P-specific antibody, as well as the GST FAPP1-PH probe, labeled calnexin-GFP-positive LCVs in homogenates of Dictyostelium infected with L. pneumophila (FIG. 7A). Similarly, GST-SidC stained the LCV in homogenates of L. pneumophila-infected Dictyostelium. Using the PI(4)P-specific antibody, we found that 80% of calnexin-GFP-positive, wild-type L. pneumophila-containing vacuoles stain positive for PI(4)P. Omission of the anti-PI(4)P antibody or using GST alone did not label the LCV. These results establish PI(4)P as a lipid marker of the LCV in L. pneumophila infected Dictyostelium. In intact calnexin-GFP-labeled Dictyostelium infected with L. pneumophila, the PI(4)P probes produced a punctate staining on the cytoplasmic membrane and in the cytoplasm, rendering it difficult to detect PI(4)P on the LCVs (unpublished data).
To address the question of whether the presence of PI(4)P on LCVs is dependent on the Icm/Dot T4SS, we used the Dictyostelium wild-type strain Ax3 expressing VatM-GFP. VatM is the 100-kDa transmembrane subunit of the vacuolar H+-translocating adenosine triphosphatase (V-ATPase), which is excluded from LCVs harboring wild-type L. pneumophila but is delivered to LCVs containing ΔicmT by fusion with endolysosomes [9,19]. One hour post-infection, only 15% of wildtype but 41% of ΔicmT mutant L. pneumophila resided in vacuoles staining positive for VatM-GFP. Interestingly, however, 42% of the VatM-GFP-positive LCVs harboring wildtype L. pneumophila stained positive for PI(4)P, compared to only 6% of VatM-positive LCVs containing ΔicmT (FIGS. 7B and 7C). These results indicate that the presence of PI(4)P on LCVs is Icm/Dot-dependent.
The mechanism of intracellular replication of L. pneumophila within amoebae and macrophages appears to be very similar. To test whether LCVs formed in macrophages also contain PI(4)P, we used RAW264.7 cells. L. pneumophila grows within these macrophages [1,46] and therefore the corresponding LCVs represent replication-permissive compartments. In lysates of RAW264.7 macrophages infected with L. pneumophila, the LCVs were labeled by an anti-PI(4)P antibody as well as by an anti-SidC antibody (FIG. 7D). As expected, upon omission of the anti-PI(4)P antibody, only SidC was detected on the LCV. These results demonstrate that PI(4)P is also a lipid component of the LCV in macrophages, and the results further underscore the structural similarity of LCVs within amoebae and macrophages. Similar to Dictyostelium, in intact L. pneumophila-infected macrophages, the PI(4)P probes led to a punctate staining pattern on the cytoplasmic membrane and in the cytoplasm.
8. Fragments of SidC Directly and Specifically Bind to PI(4) Phosphate
The L. pneumophila SidC protein (105 kDa) as shown in FIG. 5E specifically binds PI(4)P. Smaller, C-terminal fragments of SidC (SidC—3C: 36 kDa, SidC—3-4: 20 kDa; FIG. 5E) bind PI(4)P with the same specificity (FIG. 5F).
According to preferred embodiments of the present invention, the segments comprise the following amino acid sequences:








SidC-3-4 (169 AA) (SEQ ID NO: 1):



SKYSSKPLLDVELNKIAEGLELTAKIYNEKRGREWWFKGSRNEARKTQCE


 


ELQRVSKEINTLLQSESLTKSQVLEKVLNSIETLDKIDRDISAESNWFQS


 


TLQKEVRLFRDQLKDICQLDKYAFKSTKLDEIISLEMEEQFQKIQDPAVQ


 


QIVRDLPSHCHNDEAIEFF


 


SdcA-3-4 (168 AA) (SEQ ID NO: 2):


SKYSSKPLLDVELNKIAEGLDLTAKIYNEKRKSEWFKGSRNEARKTQCEE


 


LQRVSQEINALLQSESLTKSQVLEKVLNSIEALDKIDRDISAEYNLFKST


 


LQKEVQSFRDQLKDICQLDNYAFKSTKLDEIISLEMEEQFQMIKDPAVQQ


 


IVRDLPSHCHNNEVIEFF






The SidC-3-4 and SdcA-3-4 sequences are 90.5% identical (ClustalW algorithm).

Similar to full length SidC, the fragments are stable and robustly bind PI(4)P even after cycles of shock freezing and thawing. The proteins lose their PI(4)P-binding activity upon freezing at −20° C. 25-100 μg of the affinity purified GST fusion protein is routinely used per protein-lipid overlay assay. These features allow the use of SidC- or SdcA-fragments as specific probes for PI(4)P in cell biological and biochemical assays.
To produce the SidC-derived PI(4)P-binding proteins, translational GST fusions were constructed by PCR amplification. All constructs were sequenced. The following preferred constructs are provided to produce recombinant SidC-derived PI(4)P-binding GST fusion proteins:

    
    
        pGEX-4T-1-SidC
        pGEX-4T-1-SidC—3C
        pGEX-4T-1-SidC—3-4

9. Applications of the PI(4)P-Binding Proteins SidC and SdcA and Fragments Thereof

    
    


SidC, SdcA and peptide fragments thereof may be employed as probes in biochemical assays as well as in cell biological assays to detect and quantify PI(4)P. The probes are either recombinant tagged proteins, which are purified and can be used in a soluble form or immobilized on surfaces, or ectopically expressed fusion proteins (Table 3).
The information disclosed above allows the person skilled in the art to perform, for example, the following diagnostic and analytic applications:

    
    
        Diagnostic assays to detect and quantify PI(4)P in biological samples.
        Analytic assays to stain and quantify PI(4)P in sub-cellular compartments of cells.
        Assays for phosphoinositide kinases or phosphatases which turn over or produce radio- or fluorescence-labeled PI(4)P. Screens for enzyme inhibitors or agonists.
        Lipid-protein overlay or pull-down assays using phosphoinositides immobilized on membranes or beads.
        Pull down assays to enrich PI(4)P-containing cellular compartments and lipid vesicles.
        Fusion assays using PI(4)P-containing liposomes.
    
    













Tag
Detection







None
Antibody (against SidC)


GST, M45, Myc, HA, His, etc
Antibody (against tag)


Fluorescent proteins (GFP, etc)
Fluorescence


Fluorophores (FITC, etc)
Fluorescence


Biotin
Avidin-peroxidase, Avidin-fluorophor


Colloidal gold
Electron microscopy









Table 3. Examples of detectable tags fused or conjugated to SidC/SdcA or their PI(4)P-binding domains.
SidC, SdcA and PI(4)P-binding peptide fragments thereof interfere with phosphoinositide-dependent vesicle trafficking and are advantageously used to modulate such trafficking processes, which are impaired under certain pathological conditions.
The therapeutic applications include:

    
    
        Ectopical or retroviral expression of SidC/SdcA or fragments derived thereof.
        Delivery of purified recombinant PI(4)P-binding SidC/SdcA-derived peptides.

10. Other L. pneumophila Effector Proteins Binding to Phosphoinositides

    
    


In our attempts to systematically screen secreted L. pneumophila proteins for binding to phosphoinositides, we identified the Icm/Dot-secreted effectors LepA and LepB [19], LidA [17], as well as the putative Icm/Dot substrate Lpg2311 [39] as candidate proteins binding to mono-phosphorylated phosphoinositides (FIG. 5C, 5D). Given the large number of Icm/Dot-secreted proteins, we expect further phosphoinositide-binding L. pneumophila proteins to be identified.
11. Other L. pneumophila Effectors Interfering with Phosophoinositide Metabolism
Several lines of evidence show that in addition to the effectors identified above, other L. pneumophila proteins also interfere with host cell phosphoinositide metabolism:
(i) The modulation of phosphoinositide metabolism has profound effects on intracellular replication of L. pneumophila, yet a sidC-sdcA double mutant is not impaired for intracellular replication. Therefore, other phosphoinositide-binding effector proteins might have redundant functions.
(ii) While PI3Ks are required for phagocytosis of an L. pneumophila ΔicmT mutant strain lacking a functional Icm/Dot T4SS, wild-type L. pneumophila is phagocytosed one order of magnitude more efficiently than ΔicmT and independently of PI3Ks. These findings suggest that wild-type L. pneumophila bypasses a requirement for PI3Ks by actively modulating phosphoinositide metabolism of the host cell.
(iii) Experiments performed with Dictyostelium PI(5)P phosphatase mutant strains revealed that L. pneumophila grows 2-3 orders of magnitude more efficiently in a strain lacking PI(5)P phosphatase-4 (PI5P-4), compared to wild-type Dictyostelium or other PI(5)P phosphatase mutants (FIG. 8). Dictyostelium PI5P-4 contains a functionally important RhoGAP domain and is the homologue of human PI(5)P phosphatase OCRL-1, which is implicated in the severe disease oculocerebrorenal syndrome of Lowe [55]. OCRL-1 localizes to the trans Golgi network (TGN) and likely regulates bidirectional endosome to TGN trafficking as well as actin dynamics. In absence of PI5P-4, the amount of its substrates PI(4,5)P2 and PI(3,4,5)P3 is increased, indicating that L. pneumophila effectors also interact with poly-phosphorylated phosphoinositides.
Although the present invention has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will become apparent to those skilled in the art. It is preferred, therefore, that the present invention be limited not by the specific disclosure herein, but only by the appended claims.
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## Claims

1. A method for determining the presence or absence of a phosphoinositide lipid in a sample, the method comprising:
i) contacting the sample with a polypeptide comprising a sequence selected from the group consisting of:
a) SEQ ID NO: 1 and
b) Legionella pneumophila SidC encoded by SEQ ID NO: 3; and

ii) determining whether the compound binds to the phosphoinositide lipid, wherein the phosphoinositide lipid is phosphatidylinositol (4) phosphate.

2. The method of claim 1, wherein the polypeptide is fused with or conjugated to a tag.

3. The method of claim 2, wherein the tag is selected from the group consisting of: GST, M45, Myc, HA, His, fluorescent proteins, fluorophores, biotin, avidin-peroxidase, avidin-fluorophor and colloidal gold.

4. The method of claim 1, wherein binding of the polypeptide to the phosphoinositide lipid is detected by antibody, fluorescence or electron microscopy.

5. A method for quantifying the amount of a phosphoinositide lipid in a sample, the method comprising:
i) contacting the sample with a polypeptide comprising a sequence selected from the group consisting of:
a) SEQ ID NO: 1 and
b) Legionella pneumophila SidC encoded by SEQ ID NO: 3; and

ii) determining whether the compounds binds to the phosphoinositide lipid; and
iii) quantifying the amount of bound phosphoinositide lipid,
wherein the phosphoinositide lipid is phosphatidylinositol 94) phosphate.

6. The method of claim 5, wherein the polypeptide is fused with or conjugated to a tag.

7. The method of claim 6, wherein the tag is selected from the group consisting of: GST, M45, Myc, HA, His, fluorescent proteins, fluorophores, biotin, avidin-peroxidase, avidin-fluorophor and colloidal gold.

8. The method of claim 5, wherein binding of the polypeptide to the phosphoinositide lipid is detected by antibody, fluorescence or electron microscopy.