The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is responsible for a high number of acute and chronic hospital-acquired infections. As it develops more and more resistances against existing antibiotics, P. aeruginosa has been placed highest on the global priority list of antibiotic-resistant bacteria for which alternative treatments are urgently needed. Former studies have highlighted the crucial role of the bacterial lectin LecA and the host cell glycosphingolipid globotriaosylceramide (Gb3) for the cellular uptake of P. aeruginosa into epithelial cells via the lipid zipper mechanism. To further characterize the host cell plasma membrane domain for LecA-driven attachment and invasion, we analyzed the protein and lipid composition of pulled-down membrane domains for novel interaction partners of LecA by mass spectrometry. We unraveled a predilection of LecA for binding to saturated Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate clusters at the intracellular leaflet co-localizing with sites of LecA binding. Moreover, we identified the GPI-anchored protein CD59 and flotillins, known as cargo and eponymous component of flotillin-assisted endocytosis, as LecA interaction partners. Depletion of each of these host cell proteins resulted in more than 50% of reduction in invasiveness of the P. aeruginosa strain PAO1 highlighting the importance of this LecA-induced plasma membrane domain. Our strategy to reduce the complexity of host-pathogen interactions by first identifying interaction partners of a single virulence factor and subsequently transferring these findings to the bacterium has been proven to be a successful approach in elucidating the molecular mechanisms of bacterial infections.


Introduction
Pseudomonas aeruginosa (PA), a multi-drug resistant bacterium takes a spot on WHO's highest priority list [1]. This pathogen infects lungs, skin wounds and burns as well as the urinary and gastrointestinal tract of immuno-compromised individuals [2,3]. The very first contact between a pathogenic bacterium and its host cell can already decide the course of infection. Proteins aiding bacteria to successfully colonize epithelial cells are broadly classified into adhesins and invasins. The two lectins of PA, namely, LecA and LecB, were initially defined as exemplary adhesion proteins [4], however, several reports now suggest an additional role of LecA in the pathogenicity of PA [5][6][7][8]. The homotetrameric protein is localized to the outer membrane of the bacterium [9] and preferentially binds to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; also known as CD77 or the P k blood group antigen). Gb3 is well known to be the receptor for the B-subunit of AB 5 toxins produced by Shigella dysenteriae and enterohemorrhagic strains of Escherichia coli (Shiga toxin and Shiga-like toxin B-subunit, respectively, here referred to as StxB [10]). We previously demonstrated the importance of the interaction between Gb3 and LecA for PA uptake into lung epithelial cells [8]. Moreover, a novel divalent LecA ligand identified from a galactoside-conjugate array, bound to the lectin with high affinity and lowered the invasiveness of PA by up to 90% [11].
Pathogens hijack GSLs to induce plasma membrane bending and to transduce signals promoting endocytosis [12,13]. Both PAO1 and its lectin LecA trigger the activation of CrkII, an adaptor protein implicated in various cellular processes including cell adhesion and cytoskeletal reorganization [14,15], by phosphorylation of CrkII at Tyr 221 [16,17]. Interestingly, induction of this signaling pathway was dependent on binding of LecA to Gb3 located in the extracellular leaflet of the plasma membrane. Clearly, these observations raise the question of how the signal generated from receptor binding at the extracellular membrane leaflet, is transmitted to the intracellular site, activating proteins such as CrkII. One possibility could be the involvement of GSLs with long fatty acyl chains interdigitating into the inner leaflet of the membrane bilayer [18][19][20]. Additionally, the local lipid and protein environment of Gb3 may affect the induced signaling events and endocytic trafficking routes. The 7 involvement of CD59, flotillins, Src family kinases and phosphatidylinositol (3,4,5)trisphosphate (PIP 3 ) in the process of LecA binding to the plasma membrane. As major finding, we demonstrate that CD59 and flotillins promote PA invasion into lung epithelial cells. Deepening our understanding of the interplay between virulence factors and the host cell plasma membrane is crucial to develop novel treatment strategies and to minimize the rising risk of untreatable bacterial infections.

Cell culture and lectin stimulation
The human lung epithelial cell line H1299 (American Type Culture Collection, CRL-5803) was cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine at 37 °C and 5% CO 2 . LecA was expressed in E. coli BL21 (DE3), transformed with the plasmid pET25-pa1l encoding the lectin and purified as published in [7]. The B-subunit of Shiga toxin 1 (StxB) was bought from Sigma Aldrich. Lyophilized StxB was resuspended in ultrapure water, while LecA was diluted in calcium-and magnesiumfree Dulbecco's phosphate-buffered saline (DPBS-/-). Both lectins were filtered sterile and stored at 4 °C. Cells were seeded in appropriate amounts and stimulated at a confluency of 70-80% with a final concentration of 100 nM LecA or StxB for indicated time points. For cholesterol depletion, cells were pre-treated with 10 mM methyl-beta-cyclodextrin (MβCD) in RPMI for 30 min at 37 °C and washed once with DPBS-/-before lectin stimulation.

SDS-PAGE and immunoblot analysis
Stimulated H1299 cells were harvested in radio-immunoprecipitation assay (RIPA) buffer (20 mM Tris (pH 8.0), 0.5% (w/vol) sodiumdeoxycholate, 13.7 mM NaCl, 10% (vol/vol) Glycerol, 0.1% (w/vol) SDS, 2 mM EDTA, freshly supplemented with phosphatase and protease inhibitors (Sigma)) for 60 min at 4 °C. Protein concentrations were determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Cell lysates were denatured in SDS loading buffer and boiled at 95 °C for 7 min. Equal amounts of proteins were separated on 8 or 12% Tris-glycine gels and transferred on a nitrocellulose membrane by application of 0.12 A/gel. BSA (3% in TBS-T (50 mM Tris (pH 7.6), 154 mM NaCl, 0.5% (vol/vol) Tween 20)) was used to inhibit unspecific binding to the membrane before primary antibodies (1:1000) were added at 4 °C overnight. The corresponding horseradish peroxidase-conjugated secondary antibody (1:2000) was incubated with the membrane for another hour at room temperature (RT) the following day. Finally, ClarityTM Western ECL Chemiluminescent Substrate (Bio-Rad) was used to detect the luminescence signal by a Vilber Lourmat Fusion FX chemiluminescence imager. For the detection of LecA-biotin, an IRDye 800CW Streptavidin antibody (926-32230) was used and fluorescence signal was measured by the Odyssey CLx (LI-COR). Protein levels were analyzed by means of Fiji ImageJ 1.0 software and gel analysis tools.

Pull-down and immunoprecipitation
For pull-down studies, lectins were biotinylated by NHS-ester conjugation (Thermo Fisher Scientific) and dialyzed against DPBS-/-overnight. H1299 cells treated with 100 nM LecA-biotin or StxB-biotin for indicated time points were lysed by a buffer composition consisting of 25 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP40 and 5% glycerol for 60 min at 4 °C. After a short clarification centrifugation step, normalized protein lysates were incubated with magnetic streptavidin beads (Thermo Fisher Scientific) for 3 h at 4 °C. Subsequently, beads were washed three times with lysis buffer and in between rotated for 10 min at 4 °C. Pulled-down proteins were eluted by 2x SDS loading buffer and subjected to immunoblot analysis for further characterization. For co-immunoprecipitation, the Capturem Protein A technology from Takara was employed according to the manufacturer's protocol.
Briefly, H1299 cells were stimulated with LecA as described above and lysed by the provided lysis buffer freshly supplemented with protease inhibitor cocktail. Cell lysates were incubated with the target primary antibody for 60 min at 4 °C and endto-end rotation. The antibody/antigen complex was loaded on equilibrated spin columns, washed and eluted in elution buffer. Finally, 5x SDS loading buffer was added to the eluate and samples were boiled and analyzed by immunoblot analysis as described. For depletion of GSLs, cells were cultivated four days in the presence of 2.5 µM D-threo-l-phenyl-2-palmitoylarmino-3-morpholino-l-propanol (PPMP; Santa Cruz) to inhibit synthesis of glucosylceramide-based GSLs [41].

Lipid analysis by MS
Untreated or MβCD pre-incubated H1299 cells were stimulated with biotinylated lectins for 20 min at 37 °C. Pull-down was performed as described above with the exception that streptavidin beads were finally eluted in methanol/water (1:1) and stored at -80 °C until further usage. Lipid extraction by centrifugation with chloroform was performed as previously described [42]. Lipids were reconstituted in isopropanol:acetonitrile:water 2:1:1. The Gb3 composition and binding affinity of the cells were assessed by high-resolution mass spectrometry (Synapt G2Si, Waters Corporation) operated in positive MS E mode. Cholesterol and its influence on Gb3 binding were analyzed by targeted LC-QqQ-MS (6460, Agilent Technologies) operated in SRM/MRM mode. Chromatographic separation was achieved on a BEH C18 column (100 mm x 2.1 mm, 1.8 µm, Waters Corporation) as previously described [43]. For evaluation of the lectin preferences, pulled-down Gb3 species were normalized to their input values (i.e. the total cell lysates before incubation with streptavidin-coated beads). Additionally, to correct for different binding affinities, numbers were normalized to the species C16:0.

Protein analysis by MS
The pull-down of H1299 cells stimulated with LecA for 5 and 15 min was conducted as described above. After incubation of the cell lysates with magnetic streptavidin beads, beads were washed three times with lysis buffer. MS analysis was conducted on a LTQ Orbitrap XL mass-spectrometer (Thermo Scientific), which was coupled to the HPLC-system. The mass spectrometer was operated in the so-called "data-dependent" mode where after each global scan the five most intense peptide signals are chosen automatically for MS/MS-analysis.
The LC-ESI-MS/MS data were used for a database search with the software Mascot (Matrix Science) using the SwissProt database, species: human. Peptide mass tolerance was set to 50 ppm, fragment mass tolerance was set to 0.6 Da, a significance threshold p<0.05 was used. Carbamidomethylation at cystein was set as fixed modification; oxidation at methionine was set as variable modification. Peptides with up to 1 missed cleavage site were searched. Identified proteins are sorted by the Exponentially Modified Protein Abundance Index (emPAI), which offers approximate, label-free, relative quantitation of the proteins in a mixture based on protein coverage by the peptide matches in a database search result [44]. for 30 min at RT in the dark. Subsequently, the nucleus was counterstained with DAPI (1:1000) and the samples were mounted on cover slips using Mowiol (containing the anti-bleaching reagent DABCO). Samples were imaged by means of a laser scanning confocal microscope system from Nikon (Eclipse Ti-E, A1R), equipped with a 60x oil immersion objective and a numerical aperture of 1.49. Colocalization was calculated in Fiji ImageJ 1.0 software using the Coloc2 plugin. A minimum of three biological replicates and five to ten areas (one to five cells) per condition and replicate were analyzed.

G-LISA
Rac1 activation was measured by means of a G-LISA Activation Assay with luminescence read-out (Cytoskeleton, Inc.) according to the manufacturer's protocol.
Briefly, LecA-treated and snap frozen cell lysates were subjected to a 96 Rac-GTP affinity well plate and incubated for 30 min. Following a 2 min incubation with antigen presenting buffer, anti-Rac1 antibody was added and the plate was vigorously shaken for 45 min at RT. The procedure was repeated with a corresponding secondary antibody. Subsequently, the luminescence signal was detected using a HRP detection reagent and a microplate reader (Synergy H4, Biotek).

Live-cell imaging
H1299 cells were grown on glass cover slips (Thermo Fisher Scientific) until 80% confluency. On the day of the experiment, cells were pre-washed with and kept in Hanks' Balanced Salt Solution (HBSS, Thermo Fisher Scientific) while imaging. Live cell imaging was performed at 37 °C by using an incubator stage (OKOLab) mounted onto a confocal laser scanning microscope (Nikon Eclipse Ti-E, A1R).

Creation of ΔFLOT1 cell lines with CRISPR/Cas9 system
Knockout of Flotillin-1 was accomplished according to the protocol previously described [45]. Briefly, single guide RNAs (sgRNAs, designed using crispr.mit.edu, listed in Table S1) were cloned into pX458 vector (Addgene plasmid #48138) and transfected into H1299 cells. After 72 h, the transfected cells were trypsinized and resuspended in FACS buffer. GFP positive cells were sorted into 96-well plates.
After two weeks, single cell clones were expanded into bigger culture dishes. The initial screening for knockout clones was conducted by immunoblotting against flotillin-1 antibody. The genomic locus was amplified by PCR and analyzed by Sanger sequencing in order to confirm the knockout.

Quantitative RT-PCR
The single clones were subjected to TRIzol (Sigma-Aldrich) treatment in order to extract mRNA. Subsequently, the extracted mRNA was transcribed into cDNA using the Maxima First Strand cDNA Synthesis kit. SYBR Select Master Mix for CFX was used for qPCR according to the manufacturer's protocol. Samples were analyzed using the CFX384 qPCR system (Biorad, version 3.0). Relative flotillin mRNA levels were normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA levels. Primer sequences are listed in Table S1. Sequences of the siRNA are listed in Table S1.

Invasion assay
P. aeruginosa strain PAO1 was cultivated, GFP-tagged and deleted of LecA as described before [8]. For the invasion assay, overnight cultures of PAO1 wild-type (WT) and ΔLecA were centrifuged before resuspension of the pellet in RPMI

Antibodies and chemical reagents
The following antibodies were obtained from commercial sources: monoclonal Amikacin sulphate and Gentamicin were obtained from Sigma-Aldrich. StxB was purchased from Sigma-Aldrich (Cat. No. SML0562).

Statistical analysis
All data in graphs are presented as mean ± standard deviation (SD) and were calculated from the results of independent experiments. Statistical testing was performed with GraphPad Prism software using data of ≥ 3 biological replicates.
When appropriate, one-way analysis of variance (ANOVA), two-way ANOVA or twotailed unpaired t-test was conducted to determine the significance of the data. Tests with a p-value < 0.05 are considered statistically significant and marked by asterisks.

LecA preferentially binds to Gb3 species with saturated fatty acyl chains
The length and saturation level of Gb3 affects binding and subsequent trafficking of StxB [22,46], however, the influence on LecA binding is not known. First, we   S2e) and a strong decrease of up to 90% in the purified samples as compared to untreated H1299 cells (Fig. S2f). Subsequently, we addressed the question whether cholesterol depletion alters the Gb3 binding preference of LecA. Of note, the treatment significantly diminished binding to Gb3 overall (Fig. 1b). The strongest effect was observed for the species C16:0 and C24:1, whose binding was significantly reduced as compared to untreated cells. However, the same cannot be   To characterize the identified hits in more detail, we preformed confocal microscopy,

LecA activates Src kinases and Rac-1
We recently demonstrated that the adaptor protein CrkII is activated upon stimulation with LecA in H1299 cells [17]. Src family kinases were identified as a crucial upstream factor of LecA-induced CrkII activation. Since we detected the Src kinase Yes in the protein MS analysis, we further analyzed the role of Src family kinases in processes related to LecA binding. Using immunoblot analysis, we confirmed the LecA-mediated increase in phosphorylation of Src kinases at Tyr 416 in a timedependent manner (Figs. 4a and b, also compare with [17]). We also validated the presence of Src in the LecA plasma membrane domain by pull-down of LecA-biotin ( Fig. 4c). We performed Rac1 G-LISA to validate the role of Rac1, an important GTPase found in our MS hits (Fig. 4d). The small GTPase was activated upon LecA stimulation, however, great variability between the biological replicates was observed. Potentially, this was owed to the short lifetime of Rac1 activation.

PIP 3 clusters are induced upon LecA treatment
Phosphatidylinositols (PIPs) naturally bear a long saturated C18 acyl chain and, therefore, reach across bilayer enabling an interaction with long chains of lipids in the extracellular leaflet [52,53]. To investigate a potential coupling mechanism between the extracellular and the intracellular leaflet of the host cell plasma membrane, we studied the influence of LecA-treatment on phosphatidylinositol (4,5)bisphosphate (PIP 2 ) phosphorylation in H1299 cells. The PH domain of the PI3kinase downstream signaling molecule Protein kinase B (Akt) can sense PIP 3 and is commonly used as a GFP-fusion protein to study PIP 3 dynamics in living cells [54,55]. We transfected H1299 cells with PH-Akt-GFP and traced GFP signal for These observations encouraged us to study a potential link between the PI3-kinase and flotillins by co-transfection of H1299 cells with PH-Akt-GFP and flotillin-1-mCherry (Fig. 6). Strikingly, a clear co-localization of all three proteins (PH-Akt, flotillin-1 and LecA) was observed after 30 min of stimulation (Fig. 6a). As described before, flotillin-1 was recruited to the plasma membrane and accumulated at LecA nucleation domains. Addition of Wortmannin, a potent inhibitor of PI3-kinase activity [56], disrupted the co-localization of PH-Akt-GFP and LecA (Fig. 6b) and also fewer PH-Akt-GFP clusters were detected. Interestingly, this reduced the co-localization of flotillin-1-mCherry and LecA as well (Fig. 6c). Moreover, Wortmannin-treatment abrogated recruitment of flotillin-1 to the plasma membrane. Similar results were obtained for a co-transfection of PH-Akt-GFP and flotillin-2 (Fig. S5). Taken together, we propose that the LecA-induced recruitment of flotillins to the plasma membrane depends on PI3-kinase activity leading to phosphorylation of PIP 2 . The induced processes are important for efficient internalization of LecA.

H1299 cells
LecA is crucial for the pathogenicity of PA [5][6][7][8]. Here, we identified the proteins CD59, flotillin-1 and -2, the Src family kinases, Rac1 and PIP 3 as components of the plasma membrane domain for LecA-induced signaling and entry. Src kinases, small GTPases and PIPs are host cell factors with an established role in PA infection [16,57,58]. CD59 and flotillins, however, have not been studied in this context yet and we, therefore, aimed to understand the impact of these newly identified proteins for host cell invasion of PA. The invasion efficiency of PAO1 was analyzed with respect to the presence of flotillins and CD59 in H1299 cells (Fig. 7). To do so, we first created a CRISPR-Cas9 knockout model of FLOT1 (ΔFLOT1) in H1299 cells (Fig. S6). To negate the effects of flotillin-2, we additionally silenced FLOT2 expression by transfection of ΔFLOT1 cells with siRNA (Fig. 7a). Here, the interdependency of the two flotillins could be highlighted in an immunoblot of WT and

Discussion
Membrane processes related to the uptake of PA into host cells are only beginning to be elucidated, as this bacterium is mostly considered as an opportunistic extracellular pathogen [59]. However, at certain sites of colonization, for example in the lungs, invasive strains of PA cause severe and chronic infections. So far, PIPs [58], the Src family kinases [57,60], the actin cytoskeleton and the microtubule network [8,61,62] are known host factors necessary for the internalization of PA.
Here, we demonstrate that the flotillin family and the GPI-anchored protein CD59 (one of the cargo proteins of flotillin-assisted endocytosis) play a significant role for PAO1 invasion into lung epithelial cells. Flotillins are evolutionary conserved and ubiquitously expressed proteins with particularly high expression levels in heart, brain and lungs [63]. Interaction partners of LecA were identified by purification of proteins directly or indirectly bound by LecA-biotin using proteomic analysis. Strikingly, many detected proteins are known lipid raft components including the GPI-anchored protein CD59, the flotillin proteins and the Src family kinase Yes [75][76][77]. The complement regulation factor CD59 was already functionally linked to Src family kinases in studies demonstrating the recruitment and activation of trimeric G proteins and the Src kinase Lyn to CD59 clusters in the plasma membrane [75,78]. Additionally, CD59 is a known cargo of flotillin-assisted endocytosis [31] and highlighted in several studies as an interaction partner of flotillins [79,80].
Moreover, both flotillins can be tyrosine-phosphorylated by members of the Src family kinases and closely associate at the plasma membrane [77,81]. As we previously demonstrated [17] and tails has been shown to drastically affect lipid bilayer phases [83]. Therefore, it is unsurprising that several studies describe the critical impact of acyl chain length and degree of saturation on binding and trafficking of protein toxins [21,26,84,85]. The detected preference of LecA towards saturated Gb3 species in our MS analysis suggests preferential binding to rather tightly packed membrane domains, a characteristic feature of lipid rafts. Gb3 species with little abundance in the plasma membrane in our study, e.g. hydroxylated Gb3 species, were reported to play a crucial role in the scission of StxB-induced membrane tubules [86].
Several studies investigated the binding of StxB to its receptor Gb3 in synthetic model membranes with diverse results [22,84,85]. Interestingly, it was shown for StxB and simian virus 40 that successful trafficking to the endoplasmic reticulum (ER) and induction of toxicity requires binding to long saturated GSLs [87][88][89], while cholera toxin only sorted efficiently from the plasma membrane to the Golgi network and ER by interaction with unsaturated ceramide chains of its receptor, the GSL GM1 [69]. This provides further evidence that subtle changes in the lipid bilayer influence carbohydrate exposure and consequently allow, weaken or restrict lectin binding. Furthermore, the surrounding membrane environment is critical for Gb3 receptor function [90,91]. Depletion of cholesterol by MβCD significantly decreased levels of isolated Gb3 C16:0 and C24:1 species. The amount of measured Gb3 C24:0 varied between the three replicates but did not change significantly. The predilection of LecA-biotin towards the saturated Gb3 species was, however, maintained.
Of note, LecA and StxB both bind to Gb3, but partially localize to different membrane domains [23] and exhibit distinct trafficking routes [92]. In our analysis, no clear difference in binding behavior to Gb3 species of LecA in comparison to StxB was observed. Therefore, we suggest that the fates of the endocytosed lectins are rather determined by the set of cellular interaction partners responsible for downstream signal transduction and cargo sorting.
The binding of LecA to Gb3 generates a signal at the extracellular leaflet but so far no transmembrane-spanning protein linking Gb3 with the intracellular membrane leaflet is known. How is the signal then communicated to flotillins and Src kinases? A long-standing, popular hypothesis suggests a role for fatty acids in signal transduction [19,93]. Pinto and colleagues studied the influence of ceramide (i.e. the backbone of Gb3) structure and acyl chain length and demonstrated that long-chain ceramides (C24) induce strong alterations in the bilayer, suggesting the formation of interdigitating phases [94]. Further, GPI-anchored proteins with long saturated acyl chains can interdigitate and connect the membrane leaflets given that one of the two leaflets is immobilized [20]. A good example of transbilayer coupling through GPIanchored proteins might be the prion protein (PrP). PrP clustering in the extracellular leaflet was suggested to influence flotillins at the intracellular leaflet through interaction with flotillin myristoyl-and palmitoyl-residues [38]. Recently, PIPs were proposed to participate in transbilayer coupling [53]. Since PIPs naturally bear a long saturated C18 acyl chain, they reach across the bilayer and interact with long fatty acyl chains of lipids located in the extracellular leaflet. Here, we demonstrate an induction of PIP 3 clustering upon LecA stimulation of H1299 cells. Cluster formation was crucial for flotillin recruitment to the plasma membrane. Co-clustering of long, saturated Gb3 species and the GPI-anchored protein CD59 with PIPs might therefore enable the communication across the plasma membrane, inducing intracellular processes that culminate in endocytosis. Flotillins functionally scaffold and enhance signal transduction [32,48]. Since internalization of PA is known to require PI3-kinase and Src family kinase activity [57,58,60], the induction of these processes by LecA primes the cellular plasma membrane for PA uptake (Fig. S7, potential model).

Conclusion
In this study, we characterize the host cell plasma membrane domain to which the P. aeruginosa virulence factor LecA binds selectively. This membrane domain is composed of saturated Gb3 species along with the GPI-anchored protein CD59.
Upon LecA binding and clustering of Gb3, intracellular flotillins are recruited and scaffold active signaling platforms including Src family kinases. Flotillin recruitment is dependent on active PI3-kinases. The lectin-induced signal may therefore be transmitted from the extra-to the intracellular leaflet by transbilayer coupling between long fatty acyl chains of Gb3 or CD59 and PIP 3 . In addition, we unravel a crucial role of flotillins and CD59 in the invasion of PAO1 into lung epithelial cells.
We, therefore, propose a model in which LecA prepares and reorganizes the plasma membrane for the entry of P. aeruginosa.