PNAS-2014-Eierhoff-12895-900, Gb3 and Zipper, bacterial invasion

A lipid zipper triggers bacterial invasion

Thorsten Eierhoff a,b,1,Bj?rn Bastian c,Roland Thuenauer a,b,Josef Madl a,b,Aymeric Audfray d,Sahaja Aigal a,b,e,

Samuel Juillot a,b,f,Gustaf E.Rydell g,Stefan Müller a,b,Sophie de Bentzmann h,Anne Imberty d,Christian Fleck c,i,1,

and Winfried R?mer a,b,f,1

a Faculty of Biology,

b BIOSS Centre for Biological Signalling Studies,

c Centre for Biological Systems Analysis,an

d f Spemann Graduat

e School o

f Biology and Medicine,Albert Ludwigs University Freiburg,79104Freiburg,Germany;d Centre de Recherches sur les Macromolécules Végétales,UniversitéGrenoble Alpes and Centre National de la Recherche Scientifique,F38000Grenoble,France;e International Max Planck Research School for Molecular and Cellular Biology, Max Planck Institute of Immunobiology and Epigenetics,79108Freiburg,Germany;

g Department of Infectious Diseases,Section for Clinical Virology, University of Gothenburg,S-41346Gothenburg,Sweden;

h Laboratoire d’Ingénierie des Systèmes Macromoléculaires,UMR7255Centre National de la Recherche Scientifique and Aix Marseille University,Institut de Microbiologie de la Méditerranée,13402Marseille,France;and

i Laboratory for Systems and Synthetic Biology,Wageningen University,6703HB,Wageningen,The Netherlands

Edited by Kai Simons,Max Planck Institute of Molecular Cell Biology and Genetics,Dresden,Germany,and approved July23,2014(received for

review February11,2014)

Glycosphingolipids are important structural constituents of cellular membranes.They are involved in the formation of nanodomains (“lipid rafts”),which serve as important signaling platforms.Inva-sive bacterial pathogens exploit these signaling domains to trigger actin polymerization for the bending of the plasma membrane and the engulfment of the bacterium—a key process in bacterial up-take.However,it is unknown whether glycosphingolipids directly take part in the membrane invagination process.Here,we dem-onstrate that a“lipid zipper,”which is formed by the interaction between the bacterial surface lectin LecA and its cellular receptor, the glycosphingolipid Gb3,triggers plasma membrane bending during host cell invasion of the bacterium Pseudomonas aerugi-nosa.In vitro experiments with Gb3-containing giant unilamellar vesicles revealed that LecA/Gb3-mediated lipid zippering was suf-ficient to achieve complete membrane engulfment of the bacte-rium.In addition,theoretical modeling elucidated that the adhe-sion energy of the LecA–Gb3interaction is adequate to drive the engulfment process.In cellulo experiments demonstrated that inhi-bition of the LecA/Gb3lipid zipper by either lecA knockout,Gb3 depletion,or application of soluble sugars that interfere with LecA binding to Gb3significantly lowered P.aeruginosa uptake by host cells.Of note,membrane engulfment of P.aeruginosa occurred in-dependently of actin polymerization,thus corroborating that lipid zippering alone is sufficient for this crucial first step of bacterial host-cell entry.Our study sheds new light on the impact of glycosphin-golipids in the cellular invasion of bacterial pathogens and provides a mechanistic explication of the initial uptake processes. membrane curvature|glycolipid|infection

G lycosphingolipids(GSLs)are essential components of bio-

logical membranes that have significant impact on the or-ganization and the molecular architecture of the membrane(1,2). Preferential association of GSLs with cholesterol,various other lipid species,and proteins induces formation of micro-and nano-domains.Such domains,which are also termed“lipid rafts,”are involved in the signal transduction across the membrane(3).Sev-eral invasive bacterial pathogens hijack these membrane domains as signaling platforms for actin polymerization,which leads to the engulfment of the bacterium by the host plasma membrane(PM) (4–6).For example,Listeria monocytogenes exploits lipid raft-dependent E-cadherin and HGF-R/Met signaling in the host cell to trigger actin polymerization for bacterial uptake(7).The oppor-tunistic bacterium Pseudomonas aeruginosa can also invade and survive in diverse epithelial and endothelial cells(8–14),which significantly contributes to its pathogenicity(8,10,15).Numerous reports suggested various host cell factors,including cell surface receptors and signaling components,includingα5β1integrin,cystic fibrosis transmembrane conductance regulator,and Abelson tyro-sine-protein kinase1-dependent pathway(16–18),that are involved in the cellular uptake of P.aeruginosa.Host-cell GSLs have been identified as important molecules contributing to host specificity

and adhesion of P.aeruginosa(19,20).Recent observations suggest

that GSLs might be of critical importance for the internalization of

P.aeruginosa into nonphagocytic cells(9).The homotetrameric, galactophilic lectin LecA,which is localized to the outer bacterial membrane(21),belongs to the carbohydrate binding proteins expressed by P.aeruginosa that recognize GSLs(22,23)and rep-resents one of the virulence factors(24).The preferential binding

of LecA to the GSL globotriaosylceramide(also known as

Gb3/CD77or Pk histo-blood group antigen)(22,25)prompted

us to investigate the role of LecA–Gb3interaction in the cellular uptake of P.aeruginosa.

In our study,we demonstrated that GSLs have a major impact on

the bending of the host plasma membrane and engulfment of the pathogen.Binding of GSL by P.aeruginosa induced negative membrane curvature on synthetic,giant unilamellar vesicles (GUVs),solely driven by LecA–Gb3interaction.This interaction resulted in a bacterium tightly engulfed by the lipid bilayer of the GUV.Because the glycolipid component plays the major role in

this process,we termed this mechanism“lipid zipper”as a novel zipper-type mode of bacterial entry(26).Further experiments re-vealed that the lipid zipper has a significant impact on P.aeruginosa uptake in several epithelial cell lines.Interestingly,

actin

Author contributions:T.E.,B.B.,A.I.,C.F.,and W.R.designed research;T.E.,B.B.,J.M.,G.E.R.,

S.M.,C.F.,and W.R.performed research;R.T.,A.A.,S.A.,S.J.,S.d.B.,and A.I.contributed

new reagents/analytic tools;T.E.,B.B.,C.F.,and W.R.analyzed data;and T.E.,C.F.,and W.R.

wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence may be addressed.Email:thorsten.eierhoff@bioss.uni-freiburg.de, christian.fleck@wur.nl,or winfried.roemer@bioss.uni-freiburg.de.

This article contains supporting information online at https://www.360docs.net/doc/cd9607008.html,/lookup/suppl/doi:10.

1073/pnas.1402637111/-/DCSupplemental.

polymerization,which was previously identified as a major driving force for membrane deformation and engulfment,was not required for the lipid-triggered process.Moreover,ectopic expression of LecA in Escherichia coli was sufficient to greatly increase its invasion efficiency in Gb3-positive cells.Results

A LecA –Gb3Lipid Zipper Is Sufficient for Membrane Engulfment of P.aeruginosa in a Synthetic Lipid Bilayer System.We used Gb3-

containing GUVs as a membrane model system to directly ad-dress the impact of LecA –Gb3interactions on the curvature of a lipid bilayer without any potential interference of cellular fac-tors,particularly actin.We incubated GUVs with the invasive P.aeruginosa PAO1WT strain and a lecA deletion mutant (ΔlecA )that did not express LecA (Fig.S1,Inset ).Both strains were chromosomally GFP-tagged and did not differ in their growth rates (Fig.S1).PAO1WT not only bound to Gb3-con-taining GUVs but also induced a highly curved GUV membrane at the contact point,which finally led to a complete engulfment of the WT bacteria by the GUV membrane (Fig.1A and Fig.S2A –C ).Remarkably,we also observed membrane-engulfed bac-teria that were connected via tether-like structures to the sur-rounding GUV membrane (Fig.S2A ,arrowhead and B ).Inter-estingly,we observed clustering of lipid material at sites where bacteria attached to the GUVs and were engulfed (Fig.1A ).In total,82±6.5%of the analyzed GUVs were bound by PAO1WT and of these 45±6.4%showed bacteria,which were engulfed by

the GUV membrane (Fig.1B ).In contrast,the ΔlecA mutant essentially did not induce membrane invaginations (Fig.S2D –F ).Although still 22±7.7%of the GUVs were bound by the ΔlecA mutant (Fig.1B ),only 1of 102GUVs analyzed in total showed a membrane-engulfed bacterium.Lowering of the Gb3concen-tration in GUVs from 5to 0mol%led to a dose-dependent decrease of GUVs containing membrane-engulfed bacteria (Fig.1C ),clearly indicating a crucial role of Gb3in the process of bacterial membrane engulfment.Without Gb3,only 1of the 106GUVs analyzed showed a wrapped bacterium.In the next step we addressed the impact of the mechanical properties of the GUV membrane.When decreasing the surface tension by applying a hyperosmolar buffer solution from the outside (550mOsm ·L ?1outside vs.290mOsm ·L ?1inside the GUVs),GUVs containing 0.1mol%Gb3showed a number of membrane-wrapped bacteria comparable to that obtained with 5mol%Gb3GUVs at iso-osmolar conditions (Fig.S3and Fig.1C ).

Cholesterol plays a pivotal role in membrane physiology because it is implicated in the generation of lipid raft domains and can in-fluence membrane rigidity,which might affect the formation of the lipid zipper.Therefore,we compared the efficiency of lipid zipper formation on GUVs in the presence or absence of cholesterol.The efficiency was reduced by 65%in the absence of cholesterol (45±6.4%vs.15.7±4.8%),which,apparently,was not due to a decrease in binding of bacteria to GUVs,which was just 10%less (70±4.2%)compared with cholesterol-containing GUVs (81.7±5.4%)(Fig.1D ).In summary,these data provide clear evidence that binding of LecA to Gb3mediates a cholesterol-dependent,zipper-like engulfment of bacteria by the GUV membrane.

The LecA –Gb3Interaction Provides Enough Energy for Complete Engulfment of P.aeruginosa by a Lipid Membrane.We developed

a theoretical model to investigate whether the LecA –Gb3in-teraction is sufficient to mediate membrane engulfment of bac-teria.The GUV was described as an elastic surface consisting of mobile lipid constituents,and P.aeruginosa was modeled as a LecA-covered,rigid rod (Fig.2A and SI Materials and Methods ).The total energy of the system consists of three terms:(i )me-chanical Helfrich energy (27),which favors flat membrane con-figurations;(ii )curvature-promoting adhesion energy owing to LecA –Gb3interaction;and (iii )mixing entropy (28)of mobile lipids.The final configuration of the bacterium relative to the GUV membrane is the result of minimizing the total energy and depends solely on the physical properties of the system,that is,size of the bacterium,bending rigidity,and surface tension of the GUV membrane,as well as Gb3density.In Fig.2B we show the wrapping height h of a bacterium (L =2.5μm)in dependence of surface tension γand Gb3density σfor constant bending rigidity (SI Materials and Methods ).For realistic values of GUV Gb3densities (σ≈105μm ?2),the model predicts a complete mem-brane engulfment of the bacterium.Similar effects are predicted for the host-cell PMs,for which Gb3densities in the range of σ=1,400?7,400μm ?2(SI Materials and Methods )and surface ten-sions between 10μJ ·m ?2and 300μJ ·m ?2(29–31)are realistic.Furthermore,the model reveals that LecA-dependent clustering of mobile Gb3lipids is crucial.When Gb3is kept immobile,the global Gb3concentration required to achieve full engulfment increased by two orders of magnitude (Fig.2C ;compare curves in color and black for mobile and immobile Gb3,respectively).Full engulfment occurred at local Gb3densities well below critical values (i.e.,close packing of Gb3)(compare Fig.2C and Fig.S4C ).

To further explore the dependence of the wrapping on the global Gb3density and the vesicle size,we incubated GUVs prepared from lipid mixtures containing different Gb3concen-trations (Materials and Methods )with P.aeruginosa PAO1WT.By microscopic analysis we measured the equatorial diameter of GUVs in a random field of view.On the basis of our

model

Fig.1.P.aeruginosa induces a lipid zipper on giant unilamellar vesicles by binding of LecA to Gb3.(A )GUVs reconstituted with Texas Red-labeled 1,2-dihexadecanoyl-sn -glycero-3-phosphoethanolamine (DHPE-TR)and Gb3were inoculated with GFP-tagged P.aeruginosa PAO1WT (PAO1-GFP)for 15min at 37°C.Lipid clustering at the bacterial attachment site is visible (ar-rowhead).(Scale bar,5μm.)(B )Relative number of GUVs containing bound or membrane-wrapped P.aeruginosa PAO1WT or PAO1ΔlecA .Bound GUVs were normalized to the total number of GUVs analyzed and invaginated GUVs were normalized to GUVs bound by bacteria.Bars represent mean values ±SEM of n =4independent experiments with ≥100GUVs analyzed in total.(C )Relative number of GUVs with indicated Gb3concentrations con-taining membrane-engulfed P.aeruginosa PAO1WT.Bars represent mean values ±SEM of n ≥3independent experiments.For 0mol%Gb3,106GUVs were analyzed;for 0.01mol%Gb3,71GUVs;for 0.1mol%Gb3,105GUVs;for 1mol%,54GUVs;and for 5mol%Gb3,100GUVs.(D )Relative number of GUVs with bound or invaginated bacteria as normalized in B with lipid mixtures containing 5mol%Gb3with or without 30mol%cholesterol.Bars represent mean values ±SEM of n ≥3independent experiments with ≥100GUVs analyzed in total.

(see above),we calculated the minimal vesicle size for which the bacteria would be fully wrapped (h =2.5μm)for the global Gb3density σon the vesicle at the applied Gb3concentrations.Fig.2D compares the experimental results with the theoretical pre-diction for three different surface tensions (solid line,γ=800μJ ·m ?2).The shaded area in Fig.2D denotes the uncertainty range for the minimal vesicle size assuming that the bacterium is at least wrapped up to the spherical cap (h =2.25μm)and a 40%variation in the Gb3content (SI Materials and Methods ).Addi-tionally,we experimentally tested our model by variation of the surface tension of GUVs as a critical parameter for bacterial

wrapping (Materials and Methods ).According to the model the critical vesicle size should decrease with decreasing surface tension (compare in Fig.2D the solid line for γ=800μJ ·m ?2with the dashed line for γ=600μJ ·m ?2).This should lead to an in-creased fraction of bacteria-wrapped GUV in the low-diameter range.The experimental results for GUVs with a diameter of ≤20μm show that the fraction of bacteria-wrapped GUVs is increased by 25%at higher osmolarity compared with iso-osmolar con-ditions (compare in Fig.2E GUVs with 0.1mol%Gb3at 290vs.550mOsm ·L ?1).Taken together,our theoretical model shows that for typical Gb3densities and surface tensions of GUVs and cells the energy provided by LecA –Gb3binding and clustering is sufficient to induce total membrane engulfment of bacteria-sized objects.For engulfment of smaller particles such as viruses ad-ditional mechanisms were required (SI Materials and Methods ),which is in agreement with previous experimental and theoretical results (32,33).

LecA Promotes a Lipid Zipper on Epithelial Cells Independent of Actin Polymerization.In further experiments,we directly assessed whether

a lipid zipper mechanism is implicated in bacterial invasion of living host cells.We first verified that cell entry of P.aeruginosa into our cell model,the human lung epithelial cell line H1299,indeed depends on GSL expression,as previously reported (9).Quantification of the cellular invasion of P.aeruginosa showed an 80%reduction of invasion upon inhibition of GSL synthesis by D -threo-l-phenyl-2-palmitoylarmino-3-morpholino-l-propanol (PPMP)treatment,whereas bacterial attachment was not affected (Fig.S5A ).Disappearance of Gb3expression was verified by a loss of Shiga toxin B-subunit (StxB)binding to cells (Fig.S5B ).In the following we studied the entry process of P.aeruginosa into H1299cells by confocal fluorescence microscopy.

Shortly after binding to H1299cells,bacteria induced negative PM curvature at the cellular entry site (Fig.S6A )that progressed into the engulfment of the bacterium by PM (Fig.3A ,arrow-heads).For the ΔlecA mutant,the number of PM-surrounded bacteria was significantly lower (about 64.5%)at 1h postin-fection compared with the WT strain (Fig.3B ),suggesting that mainly LecA contributes to the zipper-like engulfment of bac-teria by PM.In line with the GUV data,extraction of cholesterol by methyl-β-cyclodextrin treatment lowered the occurrence of zipper-like,PM-engulfed bacteria by about 50%(Fig.S7)and thereby to the same extent as observed for GUVs,with and without cholesterol (Fig.1D ).Interestingly,actin polymerization does not play a role for the PM engulfment of the bacteria,because actin-related protein 2/3complex (Arp2)depletion (Fig.3D and Fig.S6B and D )(34)or latrunculin A treatment (Fig.S6E )(35)did not prevent initial envelopment of bacteria by PM.Nevertheless,accumulation of actin,as well as of the PM marker GPI-mCherry,was frequently seen close to invading bacteria even in Arp2-depleted cells (Fig.3D ).However,actin coverage seems not to be functionally linked to the formation of mem-brane invaginations,because we observed actin-covered,PM-enveloped bacteria under all perturbations (Fig.3D and Fig.S6E ,arrowheads).Most likely,actin coverage around invading bacteria represents persistent,cortical actin filaments.To further test whether actin polymerization is redundant for the zipper process,we studied the interactions of WT bacteria with plasma membrane spheres (PMS)induced in H1299cells that stably expressed the actin marker LifeAct-mCherry.In PMS,no endo-cytic machinery is present and (cortical)actin is excluded (36).Intriguingly,we observed bacteria that were tightly engulfed by the membrane of PMS,which demonstrates membrane wrapping even in the absence of actin (Fig.S8).These PM-enveloped bacteria closely resembled those wrapped by GUV membranes (Fig.S2B and C ).Therefore,we propose a mechanistic model in which actin polymerization is dispensable for membrane engulf-ment but required for completion of the invasion process

and

Fig.2.Gb3binding and clustering predicts complete membrane engulfment of P.aeruginosa .(A )Rotationally symmetric geometry of a modeled rod-shaped bacterium engulfed by GUV membrane.(B )Wrapping height in de-pendence of surface tension and Gb3surface density (GUV diameter d =16μm).(C )Invagination depth and local Gb3density as function of the global Gb3density and different GUV diameters for intermediate surface tension (γ=10?4J ·m ?2).Clustering of mobile Gb3reduces the global Gb3concen-tration necessary for full wrapping (compare colored curves for mobile vs.black curve for immobile Gb3).Shaded area corresponds to Gb3densities in the PM of host cells.(D )Critical GUV sizes.Circles represent observed vesicles of different size and with different Gb3content (they are scattered around x-values for improved visibility).Each wrapping event is denoted by red pad-ding.Three lines represent the critical GUV size for full enclosing of bacteria at likely surface tensions γ.The shaded area shows the effect of uncertainty in effective Gb3content in a single vesicle at γ=800μJ ·m ?2(SI Materials and Methods ).For the right column,the osmolarity of the outside buffer was increased from 290to 550mOsm ·L ?1,whereas the inside buffer was kept at 290mOsm ·L ?1;the thereby reduced surface tension enhances wrapping at moderate GUV sizes.The theoretical curves show qualitative agreement with the observed statistics.

M I C R O B I O L O G Y

closing of the invaginated membrane cup to form an intracellular bacteria-containing vesicle.This hypothesis is supported by the fact that P.aeruginosa requires actin polymerization for complete entry into lung cells,as indicated by the reduced invasion into Arp2-knockdown cells (Fig.S6C ).

LecA/Gb3-Mediated Lipid Zipper Contributes Measurable Impact on the Cellular Invasion of P.aeruginosa .Because P.aeruginosa is capa-

ble of inducing a Gb3–LecA lipid zipper on GUVs (Fig.1)and cells (Fig.3)we quantified how the lipid zipper affects bacterial host-cell invasion efficiency.The P.aeruginosa PAO1ΔlecA mutant strain showed a 61.3%reduced invasion into H1299cells compared with

PAO1WT (Fig.4A ,“untreated ”).Next,we depleted PM Gb3levels by StxB-induced endocytosis of Gb3(32)(Fig.S9A and B ).StxB-mediated depletion of Gb3reduced the invasion of the WT strain into H1299cells by 69.0%(Fig.4A ,“StxB-treated ”)compared with untreated cells.No significant difference in invasion efficiency was observed with the ΔlecA strain on Gb3-depleted (“StxB-treated ”)and Gb3-containing (“untreated ”)H1299cells.Additionally,we confirmed by ectopic expression of Gb3in MDCKII cells that the invasion of PAO1WT correlated with the amount of PM-localized Gb3(Fig.4B ).Moreover,pretreatment of the bacteria with the LecA-binder para -nitrophenyl-α-D -galactopyranoside (PNPG)de-creased the invasiveness of PAO1WT by 68.6%(Fig.4A ,“PNPG-treated ”).PNPG selectively prevented LecA,but not StxB,binding to cells (Fig.S9C and D ).In contrast,cellular invasion by the ΔlecA strain was not affected by PNPG,because the invasion efficiency was at a similar level compared with the untreated ΔlecA strain.None of the tested conditions significantly affected the adhesion of P.aeruginosa PAO1WT or ΔlecA strains to host cells (Fig.4C ).These findings strongly suggest a major role of LecA and Gb3as internalization factors rather than adhesion factors.Moreover,the cholesterol dependency of the lipid zipper formation in GUVs

and

Fig.3.P.aeruginosa induces plasma membrane invaginations depending on LecA.(A )PAO1(green)-induced membrane invaginations (red)colocalize with actin (blue)in H1299cells during cellular entry.The white squared area is shown enlarged in the lower panel.(Scale bars,5μm and 2μm,respectively.)(B )Relative numbers of PM-engulfed PAO1WT and ΔlecA during cell entry stage as shown in A ,n =3independent experiments;mean ±SEM P value was calculated by two-tailed,paired t test.(C )Intensity profile across a membrane invagination in A (yellow line).(D )Actin polymerization is not required for PAO1-induced membrane invaginations.Bacteria still induce membrane invaginations (arrowheads)and are surrounded by host PM,either covered (zoom 1)or uncovered (zoom 2,recorded 0.5μm from above the focal plane shown in the overview)by actin in Arp2knockdown cells (see Fig.S6D for control siRNA-transfected cells).The white squared areas are shown enlarged in the lower panels.(Scale bars,10μm and 2.5μm,

respectively.)

Fig.4.LecA in conjunction with Gb3promotes efficient cellular invasion of P.aeruginosa .(A )Invasion of P.aeruginosa strains PAO1WT and PAO1ΔlecA into untreated or Gb3-depleted (StxB-treated)H1299cells,and with LecA inhibition (PNPG-treated).Data represent mean values of n ≥3in-dependent experiments.The P value for WT vs.ΔlecA was calculated by a two-tailed,paired t test.(B )Ectopic expression of Gb3in MDCKII cells enhances uptake of P.aeruginosa .Invasion of P.aeruginosa WT into untransfected MDCKII cells (wt)and Gb3-synthase-transfected MDCKII cells (clone 1and clone 2)was evaluated.Both clones differ in their Gb3ex-pression,which was analyzed by FACS after incubation with StxB-Alexa488(hatched bars).All data represent mean values ±SEM for n ≥3independent experiments with normalized StxB fluorescence and invasion,to MDCKII clone 2.The P values were calculated by a two-tailed,paired t test.(C )Ad-hesion of PAO1WT and ΔlecA to H1299cells according to the treatments as in A .All data represent mean values ±SEM of n ≥4independent experi-ments,normalized to the WT.(D ,Upper )Induced expression of lecA by IPTG in E.coli BL21(DE3)pET25pa1l detected by standard Western blot analysis.The parental,WT strain [BL21(DE3)]serves as control (P,bacterial pellet;S,culture supernatant).(D ,Lower )Invasion and adhesion of indicated E.coli strains to H1299cells.Invasion upon IPTG-induced expression of lecA is strongly enhanced.Data represent mean values of normalized invasion ±SEM for n ≥4independent experiments.For better visibility,adhesion was normalized to non-IPTG-treated E.coli BL21(DE3).

cells correlated with the efficiency of cellular invasion by PAO1 WT.Invasion in cholesterol-depleted cells was significantly de-creased by about70%compared with control cells(Fig.S10).This suggests an overall critical role of cholesterol for the lipid zipper in vitro and in cellulo.

To test the capacity of LecA as an invasion factor,we explored the invasiveness of a noninvasive E.coli strain upon induced expression of lecA by isopropylβ-D-1-thiogalactopyranoside (IPTG)(25).Remarkably,ectopic expression of lecA in E.coli BL21(DE3)transduced this strain into an invasive one,evi-denced by a significant increase of invasion into H1299cells of about340%compared with non-lecA-expressing E.coli strains (Fig.4D).Interestingly,adherence of lecA-expressing E.coli to H1299cells was drastically reduced(approximately20-fold) compared with the non-lecA-expressing E.coli strains(Fig.4D). Probably LecA functionally interferes with endogenous adher-ence factors of E.coli,which consequently prevents efficient adhesion to H1299cells.However,these findings highlight the dominant,invasin-like function of LecA.In summary,these data demonstrate that Gb3and LecA represent key factors for the cellular entry of P.aeruginosa into nonphagocytic cells. Discussion

By studying the interaction of P.aeruginosa with synthetic and cellular membranes we have identified a mechanism for bacterial entry that requires host GSLs and bacterial lectins but does not depend on actin polymerization for membrane engulfment.The lipid zipper highlights the particular importance of glycolipids in bacterial invasion processes.Numerous host-cell factors have been described to interact with P.aeruginosa to orchestrate cel-lular entry and infection of the bacterium.Therefore,it is in-triguing that only two interacting factors,the host-cell GSL Gb3 and the bacterial,GSL-binding lectin LecA are sufficient to in-duce the initial processes of the cellular entry of P.aeruginosa. GSLs are key components of lipid rafts and as such are in-volved in establishing signal transduction platforms in biological membranes(1,37).Cholesterol has been described to cluster GSLs in the external leaflet of the PM and thereby to stabilize lipid raft domains.We show that the lipid zipper mechanism depends on lipid raft components such as cholesterol and the GSL Gb3,whereas a nonraft lipid,1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC),does not directly promote the in-duction of the lipid zipper.Our results suggest that cholesterol stabilizes LecA-induced domains with a sufficiently high Gb3den-sity to trigger the lipid zipper,and finally the efficient cellular in-vasion.Therefore,our findings expand the lipid raft concept because(i)they introduce an autonomous role for GSLs(i.e.,Gb3) in triggering an endocytic event,uncoupled from cytoplasmic sig-naling processes,and(ii)consider raft domains to serve as mem-brane areas providing sufficiently high GSL densities for lipid zippering bacterial pathogens.

The effect of cholesterol on membrane stiffness is complex. Membrane stiffness is not necessarily decreased in cholesterol-depleted membranes.It could even be increased,at least in choles-terol-depleted cells(38).We found that cholesterol extraction on cells decreased the frequency of the lipid zipper,which could,in line with ref.38,point to an increase in membrane stiffness,impairing the lipid zipper.Following a study on DOPC vesicles,cholesterol has no(significant)effect on bending rigidity(39).However,we found a significantly lower occurrence of the lipid zipper in cho-lesterol-free GUVs.Interpreting our results in the context of the above-mentioned report(39),cholesterol might affect the lipid zipper on GUVs by mechanisms other than membrane stiffness. Studies of SV40entry engaging the GSL GM1for cellular entry (33)revealed that the chemical structure of the GSL(i.e.,fatty acid chain length and saturation)is critical for the uptake.Therefore,it will be interesting to investigate in future studies whether and how dif-ferent Gb3species affect the uptake of P.aeruginosa.

It is well established that the cellular uptake of Gram-negative bacterial pathogens(e.g.,Salmonella and Shigella species)critically depends on secreted bacterial effector proteins that induce a re-organization of the host cytoskeleton(40,41).The expression of

a type-III secretion system(T3SS)by P.aeruginosa and T3SS-and

T6SS-secreted effector proteins have been reported to be required

for the cellular invasion and are supposed to influence the growth-phase dependent cellular uptake of P.aeruginosa(11,42–45). However,our data suggest that initial membrane curvature and wrapping of the bacterium by the host PM depends neither on

a bacterial secretion system nor on secreted effector proteins. Furthermore,actin polymerization and an active endocytic ma-chinery are not required for the formation of PM invaginations,as suggested by actin inhibition experiments.The GUV experiments

and model calculations clearly demonstrate that the induction of membrane invaginations by P.aeruginosa through LecA–Gb3in-teractions is a thermodynamically favored,ATP-independent process that does not depend on cytosolic factors or on bacterial effectors.However,other bacterial and cellular effector proteins might be necessary to accelerate and/or finalize the uptake.In analogy to a previous report indicating that actin dynamics drive membrane scission on StxB-induced membrane invaginations(46),

actin could be actively involved in late stages of endocytic pro-cessing of PM invaginations.In addition,because P.aeruginosa induces membrane curvature,curvature-sensing Bin Amphiphysin Rvs.(BAR)domain-containing proteins(47)might be recruited to

the entry site and activate signaling pathways to promote the cel-

lular uptake of P.aeruginosa.

Moreover,our work demonstrates a previously unidentified pathophysiological relevance for LecA.So far,LecA has been re-

lated to bacterial adhesion and biofilm formation(20,48)and represents one of the virulence factors contributing to lung injury during infection(24).Our data identify LecA as an invasion factor

for P.aeruginosa,which might contribute to the dissemination of the pathogen within its host organism during infection.This is strongly supported by the finding that ectopic lecA expression induced an invasive phenotype of an originally noninvasive E.coli strain. Moreover,this finding shows that LecA does not need to interact

with specific P.aeruginosa factors to fulfill its invasive function. Inhibition of the LecA/Gb3-dependent pathway reduced host-

cell invasion by P.aeruginosa.Therefore,strategies to interfere

with the binding of LecA to Gb3could be the basis for new drugs

that efficiently prevent the dissemination and persistence of

P.aeruginosa.Moreover,many bacterial pathogens express lec-

tins on their cellular surface that recognize host GSLs(49).In

the context of our findings,it is possible that these pathogens use

GSL-dependent mechanisms to trigger their uptake by host cells, alternatively or synergistically to protein receptor-based entry pathways,such as the invasin-induced entry of Yersinia(50)or

the internalin-triggered pathway of Listeria spp.(51,52).

Clearly,the lipid zipper mechanism that drives bacterial in-vasion shares some common features with the GSL-mediated endocytosis of toxins(32)and viruses(33,53).Multivalent lectin–glycosphingolipid interactions trigger the formation of plasma membrane invaginations in the absence of cytosolic proteins(e.g.,

coat proteins and actin).However,for bacteria,in contrast to smaller particles such as toxins and viruses,the adhesion energy resulting from lectin-induced GSL clustering is already sufficient

to overcome the energy penalty for membrane bending as an initial step for their cellular uptake.

Materials and Methods

Detailed materials,methods,and the theoretical model are described in SI Materials and Methods.Briefly,GUVs were prepared as described(54).

H1299and MDCKII cells stably expressing marker proteins or Gb3synthase

were cultured in RPMI or DMEM supplemented with Geneticin and10%(vol/vol)

(5%vol/vol)FBS.Recombinant LecA-,lecA-,and non-lecA-expressing E.coli BL21

(DE3)were produced as reported(25).GFP tags and LecA mutants of

P.aeruginosa PAO1were produced as described in SI Materials and Methods. Invasion assays were performed as essentially described(11).GSL synthesis was inhibited by incubation of cells for3d with5μM PPMP.For LecA in-hibition,soluble LecA(2μg·mL?1)or bacteria were incubated for15min at 37°C with10mM PNPG.Gb3and cholesterol was depleted using5μg·mL?1 Cy3-labled StxB and10mM methyl-β-cyclodextrin,respectively.Actin poly-merization was inhibited by latrunculin A treatment(0.1μM)or knockdown of Arp2by siRNA.PMS were induced as described(36)and visualized by FM4-64dye.Cells,GUVs,and P.aeruginosa infection were imaged on a confocal microscope(Nikon Eclipse Ti-E with A1R confocal laser scanner,60×oil ob-jective,N.A.1.49).Statistical testing was performed with Excel with data of n≥3independent experiments using two-tailed,unpaired t test,unless stated otherwise.

ACKNOWLEDGMENTS.W.R.acknowledges support by the Excellence Initia-tive of the German Research Foundation(EXC294),the Ministry of Science, Research and the Arts of Baden-Württemberg(Az:33-7532.20),a starting grant from the European Research Council(Programme“Ideas,”ERC-2011-StG282105),the Excellence Initiative of the German Research Foundation (GSC-4,Spemann Graduate School).A.I.and A.A.acknowledge support from Neolect(ANR-11-BSV5-002),COST action BM1003,and Labex ARCANE(ANR-11-LABX-003).S.A.acknowledges support from the International Max Planck Research School for Molecular and Cellular Biology.

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Supporting Information Eierhoff et al.10.1073/pnas.1402637111

SI Materials and Methods

Lectin A Incubation and Inhibition and Depletion of Glycosphingolipids. Depletion of plasma membrane(PM)-localized globotriaosylcer-amide(Gb3)was achieved by30-min preincubation of cells at37°C with5μg·mL?1Cy3-labeled Shiga toxin B-subunit(StxB).For gly-cosphingolipid(GSL)depletion,cells were passaged for3d in the presence of5μM D-threo-l-phenyl-2-palmitoylarmino-3-morpholino-l-propanol(PPMP;Santa Cruz),a substrate analog of the gluco-sylceramide synthase(1).For lectin LecA inhibition,purified, Alexa488-conjugated LecA(2μg·mL?1)or bacteria were prein-cubated for15min at37°C with para-nitrophenyl-α-D-galacto-pyranoside(PNPG)(Sigma-Aldrich)at10mM final concentration and then incubated with cells for fluorescence microscopic analysis or invasion and adhesion assay,respectively.For con-firmation of Gb3depletion and LecA inhibition,cells were grown on coverslips incubated as described above with Cy3-labeled StxB and PNPG,respectively.Afterward,cells were washed twice with Dulbecco’s phosphate-buffered saline(DPBS), incubated with2μg·mL?1StxB-Alexa488or LecA-Alexa488for30 min at37°C.After incubation all cells were washed,fixed with4% paraformaldehyde and stained for actin by Phalloidin-ATTO647N (Sigma-Aldrich).

Generation of Stable Cell Lines.The human lung epithelial cell line H1299(American Type Culture Collection no.CRL-5803)was grown in RPMI medium,supplemented with10%FCS and L-glutamine at37°C and5%CO2.For cultivation of MDCKII cells we used DMEM supplemented with5%FCS and L-glutamine.For the generation of stable cell lines H1299cells were transfected with a GPI-mCherry or LifeAct-mCherry encoding plasmid and MDCKII cells were transfected with a Gb3-synthase encoding plasmid.Both plasmids additionally encode for a Geneticin resistance.For stable expression of GPI-mCherry and Gb3-synthase,positive clones were selected and continuously cultivated in RPMI or DMEM supple-mented with500μg·mL?1Geneticin,L-glutamine,and10%FCS (for H1299cells)or5%FCS(for MDCKII cells).For the detection of Gb3,MDCKII cells were trypsinized,washed once with DPBS,incubated for30min at37°C with StxB-Alexa488(12μg·mL?1),washed again twice with DPBS,and subjected to FACS.

GFP Tagging and Deletion of lecA in Pseudomonas aeruginosa.GFP-tagged P.aeruginosa PAO1WT and PAO1ΔlecA were con-structed as followed:PCR was used to generate a479-bp DNA fragment upstream of the lecA gene,with the(DellecAUp5A-CCCCGTGCCGGTTCGACCCCGGC,DellecAUp3GGTTGG-CAGGCCACCCCGTG)oligonucleotide pairs and a468-bp DNA fragment downstream of the lecA using the(DellecADn5-CAAGTTATCACCAAGCATGATTGATCTC,DellecADn3-CATGGCTTGGTGATAACTTGTCTCGGAAAA)oligonucle-otide pairs.The resulting DNA fragments were further used as templates for a second overlapping PCR run using a pair of ex-ternal oligonucleotides(DellecAUp5and DellecADn3),thus leading to a final approximate1.1-kb DNA fragment that was cloned into the pCR2.1vector.The resulting DNA fragment bearing appropriate sites,namely,BamHI/EcoRV,was further hydrolyzed and cloned into the suicide vector pKNG101.The recombinant plasmid was then mobilized into P.aeruginosa and the deletion mutant was selected on LB plates containing6% sucrose and streptomycin.The P.aeruginosa strains,the Escherichia coli donor strain harboring a plasmid containing miniTn7Δgfp (Gm cassette),and the two E.coli helper strains harboring pRK600and pUX-BF13were grown overnight at37°C in LB, in the presence of the required antibiotics.This protocol is similar to the three-partner mating but with a longer period of incubation at42°C for the recipient strain and overnight contact. P.aeruginosa stocks were prepared by inoculating LB–Miller medium containing60μg·mL?1Gentamicin with material of a single colony of P.aeruginosa grown on HiFluoro Pseudomonas Agar Base(Sigma-Aldrich),incubated overnight at37°C,mixed with glycerol(30%vol/vol),aliquoted,and stored at?80°C.For experiments,LB–Miller medium containing60μg·mL?1Genta-micin was inoculated with P.aeruginosa of a stock aliquot in-cubated at37°C on a Thermomixer(PeqLab)at650rpm for 16–20h to ensure that LecA was efficiently expressed(2).The growth kinetic of P.aeruginosa was recorded in a96-well plate containing LB medium by measurement of the OD at600nm in a Tecan Safire Plate Reader(Tecan).LecA expression was tested by standard Western blotting using LecA-specific,poly-clonal rabbit antibody.

Invasion and Adhesion Assay.Overnight cultures of bacteria were pelleted and resuspended in RPMI or DMEM for infection of H1299and MDCKII cells,respectively,containing additionally 1mM CaCl2and MgCl2.Bacteria were incubated for2h at37°C with cells(70–80%confluent)at a multiplicity of infection (MOI)of100.Cells were washed three times with DPBS.Ex-tracellular bacteria were inactivated by a2-h treatment of cells at 37°C with400μg·mL?1Amikacin sulfate(Sigma-Aldrich).Af-terward,cells were washed two times with DPBS and lysed with 0.25%(vol/vol)Triton X-100at37°C.Cell extracts were plated on Gentamicin-containing(60μg·mL?1)LB–Miller Agar plates and incubated over night at37°C.The next day bacterial colo-nies were counted.Invasion was calculated as percentage of Amikacin-survived bacteria compared with the total number of bacteria associated with Amikacin-untreated cells.For compar-ison purposes mean values of n≥3independent experiments were normalized to the invasion of untreated,WT bacteria.In-vasion and adhesion assays with lecA-and non-lecA-expressing E.coli strains were performed as described above.Instead of Amikacin,100μg·mL?1Gentamicin was used for1h at37°C. For adhesion assays cells were inoculated with bacteria at4°C for1h,afterward washed three times with cold DPBS,lysed,and plated as described for the invasion assay.

Inhibition of Actin Polymerization.Inhibition of actin polymeriza-tion was achieved by two complementary approaches:knockdown of actin-related protein2(Arp2)and treatment of H1299cells with latrunculin A(Sigma-Aldrich),a compound that binds to monomeric actin,thereby preventing actin polymerization. Knockdown of Arp2was achieved by transfecting5×105H1299 cells two times within3d with200pmol siRNA(Santa Cruz) using Lipofectamine2000(Invitrogen).The knockdown effi-ciency was tested by Western blot analysis using an Arp2-specific antibody(Santa Cruz).Three days posttransfection,cells were inoculated with P.aeruginosa PAO1WT for fluorescence mi-croscopy analysis or invasion assays as https://www.360docs.net/doc/cd9607008.html,trunculin A was applied at0.1μM final concentration15min before and during inoculation with P.aeruginosa PAO1WT.

Plasma Membrane Spheres.For plasma membrane spheres(PMS) studies,we used H1299cells stably expressing LifeAct-mCherry to visualize actin polymerization(3).PMS buffer contained 10μM(final concentration)of MG132(Sigma).Cells were

incubated with PMS buffer for13–16h at37°C.Subsequently, cells were incubated for1min at room temperature with FM4-64 Dye[N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl) hexatrienyl)pyridinium dibromide;Life Technologies]in PMS buffer(dilution:1:10,000)to visualize the PM.After replacement by fresh PMS buffer cells were inoculated with bacteria to per-form confocal microscopy.

Cholesterol Depletion.Cellular cholesterol was depleted by pre-incubation of cells at37°C with10mM methyl-β-cyclodextrin (MCD)for30min.Afterward,cells were washed once with DPBS and inoculated with P.aeruginosa PAO1WT to conduct invasion assays or microscopic analyses.

Microscopic Imaging.Cells,GUVs,and P.aeruginosa infection were imaged on a confocal microscope(Nikon Eclipse Ti-E with A1R confocal laser scanner,60×oil objective,N.A.1.49).Image acquisition and analysis was performed with NIS-Elements(Nikon). For visualization of cellular infection,H1299GPI-mCherry or H1299LifeAct-mCherry cells grown on glass coverslips or in CELLview dishes(Greiner)were inoculated for0.5and1h at 37°C,respectively,with P.aeruginosa with an MOI of100. Afterward,cells were washed once with DPBS,fixed,and stained for actin by phalloidin-ATTO647N(Sigma-Aldrich)and Arp2 using an Arp2-specific rabbit antibody(Santa Cruz)detected by an anti-rabbit Alexa405(Life Technologies)secondary antibody. Live cell imaging was performed at37°C by using an incubator stage mounted onto the microscope(Okolab).For visualization of GUVs,images were recorded using the resonant scanning mode of the A1R confocal.

GUV Preparation.GUVs were prepared by the electroformation technique at room temperature on indium-tin oxide(ITO)-coated slides.If not indicated otherwise all lipid preparations contain30 mol%cholesterol(Avanti Polar Lipids),0.25mol%DHPE-TR (Life Technologies),and indicated concentrations of Gb3(Matreya LLC).Additionally,lipid mixtures contain1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)(Avanti Polar Lipids)of the following concentrations:64.75mol%for5mol%Gb3-mixtures,68.75mol% for1mol%Gb3,69.65mol%for0.1mol%Gb3,69.25mol%for 0.5mol%Gb3,69.74mol%for0.01mol%Gb3,and69.75mol% for0mol%Gb3.Cholesterol-free GUVs contained94.75mol% DOPC,5mol%Gb3,and0.25mol%DHPE–Texas Red.Lipid mixtures were dissolved in chloroform(0.5mg·mL?1)and15μL was spread on the conductive faces of the ITO slides.After at least 2h of drying under vacuum,GUVs were grown in a290mOsm·L?1 sucrose solution by applying an alternating electric field from20 mV to1.1V for3h.Surface tension of GUVs was decreased by increasing the osmolarity of the outer GUV buffer,which was ad-justed to550mOsm·L?1.Bacteria were incubated with GUVs at room temperature and examined on the inverted confocal fluo-rescence microscope.

Physical Model.The mechanical Helfrich energy associated with an elastic membrane is,per unit area,

e=γ+2κH2;H=1

coseψT

dψ

d r

+sineψT

;[S1]

with surface tensionγ,bending rigidityκ,mean curvature H(4), and angleψand radius r as defined in Fig.2A.For a surface element2πr(s)d s the Helfrich energy and adhesion energy,as opposed to the unbound plane membrane,are with the adhesion energy per Gb3:

2κHesT2·2πresTd s=2πκ

RHesT

á2

~re~sTd~s

ebendingT

γe1?cosψT·2πresTd s=2πκ

~γe1?cosψT~re~sTd~s

esurface tensionT?eρ·2πresTd s=2πκ

?~e~ρ~re~sTd~s

eadhesion energyT:

Lipid?,the local Gb3surface densityρ,the cylinder radius R, and the geometry are as defined in Fig.2A.On the right-hand side we have rescaled the lengths by R,surfaces by2πR2(and surface densities by the inverse),γbyκR?2,and energies by2πκ. The cylinder surface fraction where the membrane and the rod-shaped particle are in contact is A=2πRh and,after rescaling, ~A=h=R=~h.We replace~A by~h and omit the tilde for rescaled quantities in the following.The rescaled free energy is

F mehT=

RHesT

á2

+γ

1?cosψesT

?eρehT

resTd s;[S2]

where the integral is carried out over the cylinder surface fraction h.The contribution of the free membrane part—the part not attached to the cylinder—cannot be treated analytically.How-ever,the contribution of this part is always very small,as we con-firmed by numerical integration(5).To make progress we neglect this part in what follows.In Fig.S4A we compare the full free energy with the approximation neglecting the free membrane part. After integration we obtain

F mehT=

>>>

>><

>>>

>>:

2?eρehT

+1γh2for?0≤h≤1

?eρehT+γ

+3?1γfor?1≤h≤L+1 h

2?eρehT

γeh?LT2+L

γ?3

for?L+1≤h≤L+2:

[S3] Considering N Gb3lipids(M DOPC lipids)on the vesicle with surface V from which n(m)lipids lie within the surface fraction h we can write the relations between areas and lipid numbers

V=Na+Mb;h=na+mb[S4]

with the surface areas a and b of Gb3and DOPC,respectively. The entropy of the noninteracting particles is proportional to the logarithm of the number of characteristic microstates:

Seh;ρT=ln

gen;m;N;MT

;g=

n+m

N?n+M?m

N?n

[S5]

with the local and average Gb3densitiesρ=n/h andσ=N/V. The entropy loss is

F leh;ρT=

k B Tà

SeρT?SeσT

=:K

SeρT?SeσT

:[S6]

Note that it does not matter whether the mixing entropy is derived in the microcanonical ensemble,as we do it here,or in the canon-ical https://www.360docs.net/doc/cd9607008.html,ing the Stirling formula and Eq.S4we obtain for the derivative

?F l

?ρ

log

bρ

1?aρ

log

beVσ?AρT

V?A?aeVσ?AρT

?log

b?a

bρ

+log

b?a

+V?A

beVσ?AρT

[S7]

By minimizing F l +F m for fixed h we obtain the local density ρ0.With the simplification b =a we can obtain a closed expression ?F m ?ρ+?F l

?ρ

=0?ρeσT=

νeσT??????????????????????????????????????????????

νeσT2?4Vha σαeα?1T

q 2ha eα?1T

[S8]

with α=exp (?/K )and ν(σ)=V +(α?1)(Va σ+h ).Further progress with these results turns out to be difficult (i.e.,analytical progress would be blocked and one would have to refer to nu-merical methods).However,the steric repulsion between the particle is only important for high local densities,that is,as long as the local density is low one could treat the lipids as point particles.Of course the local density is unknown a priori because it results from the minimization of the free energy.A possible route out of this dilemma is to approximate the lipids as point particles,calculate h ,and check subsequently the validity of the approximation.

In the following we derive the mixing entropy of point-like par-ticles in the canonical ensemble:Z eT ;V ;N T=1N ! V l N

;

F eT ;V ;N T=?k B T 2πκ

ln à

Z eT ;V ;N Tá;[S9]

with some length l .Again,we use the Stirling formula,add the free energies for the surface fractions h and V ?h ,subtract the free energy for ρ=σ,and obtain

F l eh ;ρT=Kh ρln ρσ +KV σ 1?h ρ

V σ

h ρV σ1?

h !

ρ=

σα

1+h

eα?1T

[S10]

Substituting ρinto Eqs.S3and S10the wrapping height fol-lows from

0=F m ′eh T+F l ′eh T=8

>>>>>>>>><>>>>>>>>>:?K σeα?1T1+eα?1Th =V +2+γh for 0≤h ≤1?

K σeα?1T1+eα?1Th =V +1

2+γfor 1≤h ≤L +1?K σeα?1T+2+γeh ?L Tfor

L +1≤h ≤L +2:

[S11]

To verify the validity of this result,we numerically calculated the full free energy (including the numerically evaluated free mem-brane part in Eq.S2and the free energy for the lattice gas in Eq.S6)for different surface densities σ.Fig.S4A depicts the good agreement between the simplified energies for point par-ticles,the corrected energy for the lattice gas,and the full free energy.For small densities,the contribution of the free mem-brane part dominates the deviations,whereas the saturating den-sity of the lattice gas (Fig.S4C )becomes important for increas-ing densities.It can be seen that the approximations done only introduce a minor error.Further,we show in Fig.S4B the relative error of the local density ρP ,if one treats the lipids as point particles and ignores the steric repulsion,in compar-ison with the saturating local densities ρfor the lattice gas.The relative error is in the relevant regime always smaller than 5%,which justifies the point-particle approximation.

Critical GUV Diameters for Full Wrapping.For larger vesicles,en-

tropy loss becomes less important owing to the larger surface reservoir.By solving Eq.S11with respect to the diameter of the vesicle,we achieve an expression for the minimal or critical di-ameter d c for which the bacteria is at least wrapped up to a height h as a function of the system parameters.For the case of full wrapping (h =L +2)and of wrapping just up to the lower boundary of the spherical cap (h =L +1),one obtains

d c eh =L +1T=R

???????????????????????????????????????????????

18eα?1T

K σeα?1T=e0:5+γT?1

s [S12]d c eh =L +2T=R

?????????????????????????????????????????????

20eα?1T

K σeα?1T=e2+2γT?1

s :[S13]

Owing to the dynamics of the GUV system and resultant technical challenges we were unable to clearly resolve the extent of wrap-ping.In addition,the global Gb3concentrations on the vesicles might vary owing to lipid heterogeneities during GUV prepara-tion.The resulting uncertainty regime of the critical vesicle diam-eter is shown in Fig.2D .The lower boundary of the uncertainty regime was calculated by using d c (h =L +1)with 40%increased Gb3concentration and the upper boundary by using d c (h =L +2)with 40%decreased Gb3concentration.The surface tension was fixed to γ=800μJ/m 2in both cases.

Values.The calculations were carried out at room temperature

(T =293K).The bending rigidity for typical phospholipid bilayers is κ=20k B T (see refs.6and 7).The surface tension of the GUVs lies within γ=10?6to 10?3J/m 2(8).Microcalorimetry titration shows that the free energy gain owing to the binding of Gb3(αGal1-4βGal1-4Glc)to LecA (PA-IL)is ?ΔG =5.6kcal/mol (9),which is equivalent to an adhesion energy ?≈9.6k B T per lipid.

In GUV experiments,the bilayer contained 65mol%DOPC and 5mol%Gb3.The area per lipid of Gb3and DOPC in a bilayer is a =80?2(10)and b =67.4?2(11),respectively.Thus,the surface concentration of Gb3was σ≈105μm ?2on average and may not surpass 1.25nm ?2.

The fraction of glycosphingolipids in the plasma membrane is believed to be ～1–5%of all lipids.For Gb3,0.1–0.5%seems plausible,so we expect the average surface density of Gb3within the range σ=1,400?7,400μm ?2.

Clustering of Gb3Lipids.During the wrapping process of the

bacteria Gb3lipids are recruited to the wrapping region and thereby enhance the adhesion energy and subsequently the wrapping height.The local density of Gb3lipids is shown in Fig.S4C as a function of the global Gb3density σand the diameter of the GUV.Owing to the recruiting mechanism bacteria are fully wrapped at a lower global Gb3density compared with the case of immobile lipids (Fig.2C ).This effect is more pronounced for larger vesicles,because the Gb3lipid reservoir is larger compared with smaller vesicles.Although the clustering of Gb3lipids is important for the wrapping process,the local density does not saturate before the bacteria are fully wrapped.This can be seen by comparing Fig.2C and Fig.S4C .For example,a global Gb3density of σ=0.001nm ?2results for a GUV of 50-μm diameter (red curve)in a fully wrapped bacteria,whereas the local density is significantly increased but still well below the sat-uration limit.

Simian Virus40.Although we treated the density of(mobile)LecA on bacteria as an unknown but nonlimiting factor,the number of glycolipid receptors on the SV40virus capsids and its geometry are well known.To compare our results to the invagination of virus capsids,we assume for simplicity that the local concentration of Gb3is as large as the receptor density,so the adhesion effect is maximal.We thus minimize F l+F m with respect to h for a constant local densityρto calculate the ratio of wrapping height h to capsid radius R:

h R =

ρe?2κ

γ?1=

ρe?2

γ?1=:eZ?2T~γ?1;[S14]

where?is the adhesion energy per glycolipid andρthe local glycolipid concentration.The size R determines the strength of the influence of the bending rigidityκon the wrapping of the capsid.

We calculateρby dividing the number of receptors[72for the capsids(8)]by the surface4πR2.The radii and adhesion energies (see ref.8)for the receptor monomers,pentamers,and capsids as well as the derived quantities are given in Table S1.

All densities are far below the upper boundary for Gb3,ρmax=1.25nm?2.Because Z<2for the monomers and pentamers,Eq.S14has no positive solution for h,so without mechanisms other than described here no invagination would occur.The capsids will be just fully wrapped wheneZ?2T~γ?1= 2?~γ?1≈2:3?γ=3·10?5?J=m2.Invaginations on GUVs in-duced by SV40virus-like particles have been observed even with surface tensions in the order of10?3J/m2(see ref.8),so other mechanisms have to be considered for the capsids as well.

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Fig.S1.Characteristics of P.aeruginosa PAO1WT andΔlecA mutant.Growth kinetics of P.aeruginosa PAO1WT andΔlecA cultures at37°C.PAO1WT and ΔlecA exhibit the same growth kinetics.(Inset)Overnight cultures of P.aeruginosa PAO1WT andΔlecA were tested for LecA production by standard Western blot analysis,verifying the lack of LecA synthesis in theΔlecA mutant.

Fig.S2.Representative images of GUVs inoculated with P.aeruginosa PAO1WT or ΔlecA mutant.(A –C )Images of GUVs as shown in Fig.1containing 5mol%Gb3inoculated with P.aeruginosa PAO1WT or ΔlecA .(D and E )In A and B ,a tether-like structure is visible between the membrane-wrapped bacterium and the outer GUV membrane (white arrowhead).Although PAO1ΔlecA bacteria bound either horizontally or vertically to Gb3-containing GUVs,they do not induce membrane invaginations,as observed frequently for the WT strain.(F )In most cases PAO1ΔlecA bacteria did not bind to Gb3-containing GUVs.(Scale bars,A –C ,Upper ,D ,and F ,5μm;A –C ,Lower ,and E ,2.5μm.)

Fig.S3.Decrease in membrane tension facilitates the lipid zipper.Membrane tension of GUVs containing indicated Gb3concentrations was decreased by exposure of GUVs to a hyperosmolar,external buffer solution (550mOsm ·L ?1outside buffer vs.290mOsm ·L ?1inside buffer).Decrease of surface tension leads to an increase of GUVs showing membrane-wrapped bacteria.Relative numbers of GUVs containing membrane-wrapped P.aeruginosa PAO1WT are shown.Numbers of invaginated GUVs were normalized to number of GUVs bound by bacteria.Bars represent mean values ±SEM of n ≥3independent experiments.In total ≥100GUVs were analyzed (46GUVs at 550mOsm ·L ?1).

0.0

0.20.40.60.81.01.21

10100

1000104

L o c a l d e n s i t y i n n m

Average Gb3surface density σin μm

Wrapping height h in μm

Fig.S4.(A )Numerically calculated full free energy (including the numerically evaluated free membrane part in Eq.S2and the free energy for the lattice gas in Eq.S6)for different surface densities σ.The perpendicular black lines show the minimum of the free energy.At these points the difference between the numerically calculated free energy and the used approximations are always minute,justifying the validity of the simplifications made.(B )Relative error of the local density ρP ,for point particles (ignoring steric repulsion)in comparison with the saturating local densities ρfor the lattice gas.The relative error is always smaller than 5%in the relevant regime,which justifies the point-particle approximation.(C )Local Gb3density as a function of the global density σon the vesicle for different GUV diameters.Although the mobility of the Gb3density leads to significantly enhanced local densities,the bacteria are always fully wrapped before the local density goes into saturation (compare with Fig.2C ).

Fig.S5.Glycosphingolipid expression is a prerequisite for efficient P.aeruginosa uptake.(A ,Left )Inhibition of glucosylceramide synthase by its substrate analog PPMP resulted in an 80%reduced invasiveness of P.aeruginosa .Internalization of P.aeruginosa PAO1WT into untreated and PPMP-treated H1299cells as measured by the invasion assay.(A ,Right )Adhesion of P.aeruginosa PAO1WT to untreated or PPMP-treated H1299cells.All data represent mean values ±SEM for n ≥3experiments normalized to the WT.(B )Inhibition of glucosylceramide synthase by PPMP prevents binding of StxB-Cy3,which selectively binds to the glucosylceramide-derived GSL Gb3,to H1299cells,representing the inhibition of GSL synthesis in H1299cells by PPMP (Lower ).TM,transmission.(Scale bar,10μm.)

Fig.S6.P.aeruginosa induces negative PM curvature independently of actin polymerization.(A )GFP-tagged P.aeruginosa WT (PAO1-GFP)bound to an H1299cell,which stably expresses GPI-mCherry as a PM marker.The lower panel represents a zoom of the squared area of the upper panel.Note that the PM is bent at the bacterial pole region at the cellular adhesion site,colocalizing with actin (arrowheads).Images were recorded from cells inoculated for 1h with P.aeruginosa at MOI ～100.For better visualization of the curved PM,the contrast of the images in the lower panel was adjusted.(Scale bars,10μm and 2.5μm,respectively.)(B )RNAi-mediated knockdown of Arp2by siRNA was confirmed by Western blotting of uninfected H1299cells.In total,Arp2was reduced by 72%as quantified by densiometric analysis of Western blots compared with the level of Akt as a loading control (n =3,mean ±SEM).(C )Invasion of Arp2-depleted H1299cells by P.aeruginosa WT was significantly reduced by about 66%.The reduction of invasion for the lecA mutant (ΔlecA )by 75%was even more pronounced (n =4independent experiments,mean ±SEM,P value calculated by two-tailed,paired t test).These observations show that actin polymerization is in general crucial for a subset of P.aeruginosa cells to efficiently enter host cells.However,the initial steps of membrane invagination (Fig.3A )do not require actin polymerization-dependent processes.(D )Control cells,corresponding to Arp2-depleted cells shown in Fig.3D .Cells were transfected with scrambled siRNA and recorded with the same laser power and gains as the Arp2siRNA-transfected cells.The same type of membrane invaginations (arrowhead)induced by P.aeruginosa WT as observed in untransfected (Fig.3A )and Arp2-depleted H1299cells (Fig.3D )is visible.Images were recorded from cells inoculated with P.aeruginosa for 1h at MOI ～100.(Scale bars,10μm and 2.5μm,respectively.)(E )Complementary to the Arp2knockdown approach we assessed the de-pendency of membrane invaginations on actin polymerization processes,which we inhibited by latrunculin A (0.1μM).In latrunculin A-treated H1299cells P.aeruginosa is localized in PM invaginations (arrowheads)at 1h postinoculation (MOI ～100).The same type of localization can be seen in untreated cells (Fig.3A )where bacteria are also engulfed by the host cell membrane.Actin-covered (zoom 1)as well as uncovered invaginations (zoom 2)were observed.Therefore,actin polymerization is not essential for membrane wrapping and invaginations.(Scale bars,10μm and 2.5μm,respectively.)

Fig.S7.Cholesterol depletion significantly reduces lipid zipper on H1299cells.Cells expressing GPI-mCherry as a PM marker were cholesterol-depleted by treatment with10mM MCD for30min or left untreated.Afterward,cells were infected for1h at37°C with P.aeruginosa PAO1WT.PM-engulfed bacteria per cell

of untreated and MCD-treated cells were counted.Values represent mean values±SEM of n=4independent experiments with>500bacteria analyzed in total.

Fig.S8.P.aeruginosa induces membrane invaginations in PMS.(A )PMS were induced in H1299cells stably expressing the actin polymerization marker LifeAct-mCherry.Actin was only visible in the cell body but was excluded in the PMS.PAO1WT inoculated with PMS-containing cells induced a membrane invagination in the PMS,which was not covered by actin (arrowhead).Membrane was visualized by FM4-64dye.(B )Example of another PMS induced from a H1299cell,which shows a bacterial cell engulfed by the membrane of the PMS in the upper part of the cell body (“top ”).Interestingly,the invaginated membrane is connected via a tether-like structure with the outer membrane of the PMS (arrowhead)as already observed in GUVs (Fig.S2B ).The lower part (“bottom ”)shows actin in the remaining cell body.(Scale bars,10μm and 5μm.)

Fig.S9.Control of Gb3depletion and LecA inhibition.(A)PM-localized Gb3content of H1299cells is significantly decreased by pretreatment with StxB-Cy3as indicated by a strong reduction of StxB-Alexa488uptake.(B)Decreased PM-localized Gb3in H1299cells results in significantly reduced LecA binding and uptake compared with untreated(control)cells.Furthermore,this indicates that Gb3is a receptor for LecA.For comparison,untreated and StxB-Cy3–treated cells were imaged with equal laser power and gain settings for StxB-Alexa488and LecA-Alexa488,respectively.(Scale bars,20μm.)(C)PNPG selectively inhibits LecA uptake.Binding and uptake of LecA-Alexa488(C)and StxB-Alexa488(D)after treatment of H1299cells with PNPG was visualized.PNPG significantly impairs LecA

but not StxB uptake.Images of Alexa488fluorescence were recorded with equal laser power and gain settings.TM,transmission.(Scale bars,10μm.)

Fig.S10.Cholesterol depletion significantly reduces host cell invasion by P.aeruginosa.Invasion of PAO1WT into cholesterol-depleted H1299cells was quantified.Values represent mean values±SEM of n=4independent experiments.

Table https://www.360docs.net/doc/cd9607008.html,parison to SV40capsid proteins

Parameter Monomer Pentamer Capsid Large spheres

r,nm51250250

?2k B T10k B T10k B T10k B T

ρ 1.6×10?3nm?2 5.5×10?4nm?2 2.3×10?3nm?2 2.3×10?3nm?2

Z Z=0.008Z=0.04Z?2=0.86Z?2=70

~γ0.03?320.001?1.3