salmonella enterica serovar typhimurium ( s. typhimurium ) is a facultative intracellular pathogen that has developed powerful strategies to infect the gastrointestinal tract . this bacteria uses various routes to invade the gut epithelial barrier : penetration through intestinal epithelial cells ( iecs ) , invasion of peyer s patches via m cells , and paracellular access to the lamina propria by breaching tight junctions or following capture by dendritic cells ( watson and holden , 2010 ) . salmonella is able to interact directly with the brush border of iecs to promote internalization ( finlay and falkow , 1990 ) . microbial invasion of iecs requires extensive , though transient , host cytoskeleton modifications at the bacterial entry site , leading to dramatic changes in the architecture of the brush border . actin rearrangements play a central role in this process of bacterial uptake ( ly and casanova , 2007 ) . however , rapid subsequent cytoskeleton disassembly is necessary to return the membrane structure to normal . fast actin depolymerization is critical for actin - dependent processes , but it is not clear whether this is a crucial point during bacterial invasion . an array of bacterial factors enables interaction with and active invasion of the intestinal epithelium through iecs ( agbor and mccormick , 2011 ) . a type - three secretion system is used to inject these effector proteins into the cytosol of host cells in order to induce rapid actin polymerization and membrane ruffling ( ly and casanova , 2007 ) . the bacterial actin - binding proteins ( abps ) sipa and sipc promote the formation of actin filaments at the bacterial entry site and prevent filament disassembly by host factors , such as the abps gelsolin and cofilin ( hayward and koronakis , 1999 ; zhou et  al . sope / sope2 and sopb activate rho - gtpases , such as rac1 and cdc42 , to favor the constitution of a highly branched actin network ( hardt et  al . , 1998 ; hong and miller , 1998 ; steele - mortimer et  al . , 2000 after internalization , the actin cytoskeleton returns to its steady state through the inactivation of rac and cdc42 by the bacterial gtpase - activating protein sptp ( fu and galn , 1999 ) . this effector is also a tyrosine phosphatase that re - establishes host cell integrity by inhibiting the phosphorylation of host proteins , such as vimentin ( murli et  al . , 2001 ) . therefore , both actin polymerization and depolymerization need to occur simultaneously to enable actin filaments formation and relocation . during development , iecs differentiate into tightly adherent polarized cells that form a physical and physiological barrier , protecting the host from infection . at their apical surface , these cells present microvilli that increase the cell surface area and  favor absorptive and secretory functions . their modifications depend on abps that can polymerize , bundle , depolymerize , sever , or cap actin filaments . among these abps , villin is an abundant epithelial actin - modifying protein present in the brush border and responsible for the establishment of the structure and function of the microvilli ( revenu et  al . , 2012 ) . indeed , villin itself can cap , sever , nucleate , or bundle actin filaments ( khurana and george , 2008 ) . villin activities are regulated not only by posttranslational modifications , including tyrosine phosphorylation ( tomar et  al . , 2004 ) , but also by phosphatidylinositol 4,5-bisphosphate ( pip2 ) or lyso - phosphatidic acid ( lpa ) ( khurana and george , 2008 ) . moreover , actin binding by villin depends on intracellular calcium concentrations ( bretscher and weber , 1980 ; kumar et  al . , 2004a ; kumar and khurana , 2004 ) . at high calcium concentrations , the severing function of villin is activated . however , severing can also be established when calcium concentrations are low if villin is phosphorylated , inducing a decrease in the pool of f - actin . the ligand - binding properties of villin are mechanistically important for the crosstalk between signaling cascades and actin remodeling in iecs , suggesting villin is also an important regulatory target . it has been demonstrated in  vivo that villin knockout mice display a normal brush border with functional microvilli , indicating that other proteins may have similar functions on actin organization . however , during stress conditions that modify calcium concentrations , such as dss treatment , the lack of villin impacts cell reorganization and plasticity ( ferrary et  al . , 1999 ) . these results suggest that villin plays an important role in actin dynamics in response to physiological or pathological stimuli . it has been shown that villin is recruited at the interaction site with the parasites cryptosporidium parvum ( forney et  al . , 1999 ) and entamoeba histolytica ( lauwaet et  al . , 2003 ) , and at the entry site of listeria monocytogenes ( temm - grove et  al . , 1994 ) . moreover , this protein is required in  vivo for shigella flexneri infection , as villin knockout mice are resistant to gut invasion by shigella ( athman et  al . , 2005 ; fernandez et  al . , in particular , it has been shown that villin - severing activity is necessary for shigella motility inside host cells ( revenu et  al . , 2007 ) . however , the actual involvement of this activity in pathogenic adhesion and invasion is not known . shigella invades the intestinal epithelium through m cells or by opening paracellular junctions , contrary to salmonella , which interacts directly with the brush border of the iecs . salmonella is therefore a more appropriate model to study the involvement of villin in bacterial invasion . here we investigated a possible role for villin in salmonella invasion of the intestinal epithelium . we examined the effects of the downregulation of villin expression during salmonella infection in tc7 cells . using a streptomycin mouse model of enterocolitis ( hapfelmeier and hardt , 2005 ) , we also analyzed the impact of villin on salmonella invasion in  vivo . through a combination of in  vitro and in  vivo approaches , our data indicate that the absence of villin , which is normally located at the entry site of salmonella , leads to a decrease in apical invasion of iecs , reflected in reduced inflammation and mucosal destruction in the infected intestinal mucosa . dedicated type - iii bacterial secreted effectors account for the modulation of villin activities through phosphorylation , particularly its severing activity . our results therefore demonstrate an important role for villin during the initial steps of apical entry of salmonella . upon reaching the membrane , salmonella induce cytoskeleton rearrangements to assemble specific actin - rich structures called ruffles , which are associated with the entry sites of the bacteria . as villin is expressed and properly distributed only in differentiated iecs , we used a cellular model that enables an endogenous apical expression of this protein after differentiation ( i.e. , human colonic tc7 cells ) . in differentiated cells , villin was located at the apex of the cells and colocalized with the actin cytoskeleton in the microvilli ( figure  1a ) . within salmonella - infected cells , when ruffles were formed , villin concentrated mainly around the bacterial bodies in association with actin ( figures 1b and 1c ) . however , villin did not colocalize with salmonella - containing vacuoles inside the host cytoplasm ( data not shown ) . confocal microscopy revealed the existence of an upper layer of villin not associated with actin , though seeming to form the ruffle ( figure  1d ) . in a second layer , this organized distribution suggests that villin could play a key role in leading ruffle assembly during initial salmonella invasion . to gain further insight into the functional significance of villin during salmonella infection , villin expression was silenced through stable transduction with recombinant lentiviruses expressing shrnas targeting two independent sites in the villin mrna ( i.e. , sh - vil1 and sh - vil2 ) . western blots of stably transduced tc7 cells were performed after differentiation and showed a decrease in villin production ( figure  s1a available online ) . immunofluorescence was performed to study the morphology of villin knockdown cells ( figure  2a ) . microvilli were terminally differentiated as observed by scanning electron microscopy ( sem ) ( figure  2d , panel d ) . to study the effects of villin knockdown on bacterial entry ,  control and villin knockdown cells were infected with s.  typhimurium , and invasion was measured by gentamicin assays ( figure  2b ) . the noninvasive prgh mutant was used as a control . a significant decrease in the number of cfu was observed 1  hr postinfection ( p.i . ) in villin knockdown cells compared to control cells . indeed , salmonella invasion was reduced by 44% with sh - vil1 ( p  < 0.01 ) and 49% with sh - vil2 ( p  < 0.001 ) . as the two villin - specific shrnas had comparable effects in all assays , only the data obtained with sh - vil1 are presented . to confirm previous results , the number of intracellular bacteria per cell was determined by confocal microscopy following washing and bacterial staining ( figure  2c ) . villin knockdown was associated with a significant decrease in the number of internalized bacteria ( p  < 0.001 ) . to further explore the role of villin during salmonella invasion , as shown in figure  2d ( panels b and c ) , sh - scr cells supported the formation of ruffles with a characteristic doughnut - like structure . these ruffles displayed a particular radial symmetry and were formed mainly around single bacteria . in contrast , in sh - vil1 cells , a major modification in ruffle morphology was observed ( figure  2d , panels e and f ) . they lost their normal , symmetrical structure and appeared disorganized , with many aberrant protrusions and an apparent defect in the fusion of membrane extensions . salmonella invasion is dependent upon actin polymerization to generate the forces required to invade iecs . the recruitment of host - cell proteins to salmonella - induced ruffles was therefore examined by immunofluorescence . although villin - knockdown notably decreased salmonella invasion , this did not lead to a defect in actin recruitment per se , as we could still observe actin at the entry site of salmonella ( figure  3a ) . however , confocal microscopy revealed that this actin recruitment showed a broader base at the entry site and did spread much more extensively in sh - vil1 cells . to decipher if villin knockdown could produce effects on the actin cytoskeleton , we quantified the impact on actin polymerization during salmonella invasion . cells were infected , and staining of fixed cells was performed with phalloidin to visualize the f - actin content in the invasion ruffles . only ruffles containing one single bacterium were taken into account to avoid any potential artifacts . using imaris software , the diameter and the volume of 100 invasion sites were measured ( figure  3b ) . we observed radial symmetric recruitment of actin in the ruffles with an average diameter of 3.396  m and a range of 2.124.94  m in sh - scr cells . in contrast , ruffles in sh - vil1 cells exhibited a more diffuse staining , with an average diameter of 4.065  m and a range of 2.579.08  m . the knockdown of villin induced an increase of the f - actin content in the invasion ruffles compared to sh - scr cells ( figure  3b ) . the actin volume content in the ruffles was significantly higher in the absence of villin , with a range of 7.410103  m in control cells and 16.3126  m in sh - vil1 cells . consequently , villin influences actin remodeling at the entry sites and is therefore required for regulation of the early membrane - associated processes . to confirm that villin has a role in modulating actin dynamics during salmonella infection , we assayed f- and g - actin contents . after separation by ultracentrifugation , f- and g - actin contents were measured via western blot and quantified by densitometric scanning ( figure  3c ) . in sh - scr cells , the f - actin content was constant during the course of the infection , in agreement with published data ( higashide et  al . , this indicates that the total cellular quantity of f - actin is controlled during salmonella infection , even though massive amounts of f - actin accumulate at the entry site of the bacteria . in uninfected sh - vil1 cells , a decrease in the f - content was observed with a reorganization of the f - actin , whereas the g - actin content was not affected . this is consistent with previously published data ( friederich et  al . , 1989 ; thus , although sh - vil1 cells did not exhibit abnormal morphologies , the f - actin content of the cells was perturbed . interestingly , salmonella infection leads to a significant increase in the amount of f - actin but not g - actin . there is a strong imbalance of the f - actin ratio in the absence of villin , indicating that this protein is essential to equilibrating f - actin content during invasion . therefore , these results demonstrate that villin plays an important role in maintaining the steady - state level of f - actin during salmonella infection . we tested the effect of villin downregulation on salmonella invasion in the absence of bacterial effectors known to regulate the actin cytoskeleton ( figure  4a ) . in sh - scr cells , the absence of sipa , sipc , sptp , or sope / sope2/sopb / sipa leads to a significant decrease in salmonella invasion . in sh - vil1 cells , the inactivation of sipc or sope / sope2/sopb / sipa leads to a stronger decrease in the invasion compared to control cells , whereas sipa or sptp did not induce such a decrease , indicating that these two proteins could be putative regulators of villin activities . sipa is an abp that inhibits actin turnover by displacing host abps from f - actin . this effector protects f - actin from severing , therefore enabling its polymerization ( zhou et  al . salmonella mutants lacking sipa entered less efficiently into control cells . in agreement with published results , numerous ruffles were observed on sh - scr cells ( figure  4c , middle panel ) . the sipa mutant induced the formation of projections that contain actin at the base of the ruffle , but mainly villin in the  apical part ( figure  4c , middle panel ) , exacerbating what was observed with wild - type ( wt ) salmonella ( figure  4c , upper  panel ) . these projections were asymmetrical and highly extended at late time points ( figure  4c , lower panel ) . these observations indicate that the first steps in induction of membrane ruffling are not affected by the loss of sipa . as sipa counteracts the severing activity of adf and cofilin , we tested whether or not it would be the case with villin . to investigate a potential interplay between sipa and villin , in  vitro assays of actin dynamics were performed ( figure  4d ) . villin protein ( vill ) or a mutated form for the severing activity domain ( villsev ) were purified and added to rh - labeled f - actin . whereas the wt protein induced actin severing , the mutated form did not modify f - actin structure . in contrast , when sipa was added simultaneously with the wt villin , actin severing induced by villin was inhibited . previous studies have shown that the c - terminal region of sipa contains a functional domain responsible for its actin - modulating activity ( amino acids 460 to 685 ) and that the minimal region to promote this activity is sipa514 - 657 . moreover , it has been described that the amino acid residues k635 and e637 are necessary for g - actin polymerization ( li et  al . , 2013 ) . we first decided to test if this minimal domain could be sufficient to inhibit villin - severing activities . we observed that similarly to sipa , sipa514 - 657 counteracted the severing activity of villin ( figure  4d ) , contrary to the shorter isoform sipa514 - 640 that does not retain this activity . the sipa514 - 657 isoform that is not able to polymerize g - actin has also lost the ability to block villin functions , indicating that the capacity of sipa to polymerize g - actin is necessary to counterbalance villin - severing activities . the identification of villin as a putative target for the tyrosine phosphatase sptp prompted us to examine potential villin targeting by this bacterial effector . interestingly , the site of invasion is a region of intensive phosphorylation activity ( figure  5b ) . cells were infected with wt s.  typhimurium or its isogenic sptp mutant , and villin phosphorylation was examined during the course of infection . tyrosine phosphorylated proteins were immunoprecipitated , and villin was detected by western blotting ( figure  5c ) . an increase in villin phosphorylation was observed 15  min p.i . with the wt strain , followed by a decrease 30  min p.i . in the absence of the bacterial phosphatase sptp , villin phosphorylation was observed earlier during the course of infection , indicating that sptp prevents villin phosphorylation at the beginning of the invasion process . to ensure that sptp phosphatase activity is responsible for this increase , cells were infected with a strain expressing a phosphatase - inactive full - length mutant of sptp ( sptp ) ( figure  5c ) . in this context , villin phosphorylation was also modified with a strong increase observed within 5  min p.i . , confirming that the phosphatase activity of sptp inhibits villin phosphorylation during the first steps of infection . as tyrosine phosphorylation of villin can modify its binding activity to f - actin in  vitro , as well as its actin nucleation and severing activities ( zhai et  al . , 2001 ) , we therefore tested if villin phosphorylation in infected cells modified its binding to actin . we took advantage of the sptp mutant that exhibits a significant increase in villin phosphorylation early during the invasion process . a coimmunoprecipitation experiment was performed to detect potential modifications in actin binding to villin during the course of infection . as shown in figure  5d , with the sptp mutant , we could detect an increase in actin coimmunoprecipitated with villin , indicating that villin phosphorylation affects its actin binding capacities . the tyrosine kinase c - src regulates the phosphorylation of villin in  vitro and in cells under physiological conditions ( mathew et  al . , 2008 ) . interestingly , c - src phosphorylation occurred during salmonella infection ( figure  5d ) and appeared even more important in the absence of the phosphatase sptp , indicating that this bacterial effector regulates c - src activities . to test if this kinase could be responsible for villin phosphorylation during salmonella infection , their interaction was first tested by  coimmunoprecipitation . villin was immunoprecipitated and c - src coimmunoprecipitation was visualized by western blot ( figure  5e ) . with salmonella . to confirm that c - src is responsible for villin phosphorylation upon infection , the activity of this kinase was inhibited with pp2 , an inhibitor for src - family kinases that downregulates psrc-416 ( figure  5f ) whereas a strong increase in villin phosphorylation was observed in the absence of pp2 when cells were infected with sptp , this was not observed when c - src was inactivated with the drug ( figure  5 g ) . these results indicate altogether that this kinase is responsible for villin phosphorylation during salmonella infection , but also sptp controls villin phosphorylation by c - src through psrc-416 regulation . to assess the function of villin during host infection by s.  typhimurium , age- and sex - matched villin ( vil ) and villin ( vil ) mice were orally infected with s.  typhimurium . villin knockout mice are viable , fertile and have normal lifespans ( ferrary et  al . , 1999 ) . infection of vil mice with s.  typhimurium resulted in severe enteritis with typical hallmarks of inflammation , including pronounced edema in the submucosa , disruption of the crypt architecture , loss of goblet cells , epithelial erosion , and infiltration of polymorphonuclear neutrophils ( figure  6a ) . already at 24  hr p.i . the epithelial lining from vil mice was more destroyed with diffused desquamation ; the lumen was filled with dead epithelial cells and the sub - mucosa was oedematous . to determine if these effects were due to a differential colonization of the intestinal tissues by the bacteria , the bacterial load of salmonella in the small intestine was determined at 24  hr . at day1 p.i . , vil mice did not exhibit a reduced bacterial load in the intestine ( figure  6b ) . to better characterize any effect on luminal colonization or the intestinal tissue , immunofluorescence staining was performed on tissue sections to precisely determine the location of bacteria ( figure  6c ) . the intestinal lumen of vil mice was heavily colonized by bacteria , with little tissue invasion , in contrast with vil mice that had pronounced bacterial invasion of the mucosal tissue . these results clearly indicate that s.  typhimurium invades intestinal tissues of vil mice more efficiently . finally , invasion of the intestinal mucosa by salmonella was linked to an early acute proinflammatory response . in vil , the reduced histological changes correlated with a decreased expression of proinflammatory cytokine genes , such as il-6 and il-1 , as assessed by rt - qpcr at 24 and 48  hr p.i . several clues tend to indicate that actin - severing activities could play an important role during salmonella infection . second , the two identified bacterial regulators seem to control severing events . moreover , villin - severing activity is essential for the apical remodeling during epithelial restitution after damage ( ubelmann et  al . , 2013 ) . therefore , we decided to test the importance of villin - severing activity in  vivo during the first steps of salmonella invasion . we used transgenic mice that are knockout for villin but re - express a wild - type ( tgvillinwt ) or a severing mutated form ( tgvillinsev ) in the intestinal cells after induction with tamoxifen ( ubelmann et  al . , 2013 ) . tgvillinwt mice exhibited significant pathological changes compared to tgvillinsev mice at the level of the epithelial lining ( figure  7a ) . immunofluorescence staining performed on small intestine samples indicated that salmonella can be found in close contact with the epithelium in both cases , but intracellular bacteria were only found in tgvillinwt ( figure  7b ) . finally , intestinal mucosa invasion by salmonella was linked to an early acute proinflammatory response in the presence of villin wt in the epithelial cells ( figure  7c ) . therefore , the animals lacking the severing activity of villin in the intestinal epithelium are more resistant to salmonella infection . considering the complex and highly organized cytoskeletal structure of the apical brush border , one expects that its subversion precedes and participates with bacterial invasion of polarized iecs by pathogens , including salmonella . taking into account its developmental and functional characteristics , villin was an obvious target candidate for a pathogen s subversion of the brush border . our results demonstrate that villin is required for apical invasion of iecs by salmonella and necessary for early membrane - associated processes and for optimal ruffle assembly by balancing the steady - state level of actin . villin - severing activities can promote brush border dissociation and microvilli remodeling upon salmonella adhesion , leading to a better engagement of the membrane by the bacteria . this is in agreement with published data indicating that treatment with cytochalasin d disrupts actin and unexpectedly leads to an increase in salmonella invasion of polarized caco-2 and ht-29 cells , but not of hela cells ( wells et  al . , 1998 ) . membrane ruffling is due to intense polymerization events that produce strong mechanical actin - based forces . however , these forces have to be constrained and highly driven to promote efficient and targeted invasion . the f - actin content is not modified during salmonella invasion , suggesting that a tight control exists to balance between simultaneous actin polymerization and depolymerization activities that relax the mechanical pressure induced by the ruffle and to allow directional protrusions ( higashide et  al . , 2002 ) . interestingly , when microvilli differentiate , actin polymerization appears sufficient to create a force that overcomes resistance of the apical membrane to bending ( mogilner and rubinstein , 2005 ) . therefore the length of the microvilli may largely depend on the length and rigidity of actin filaments , and therefore on actin polymerization capacities . it has been previously shown that salmonella invasion is more efficient in polarized cells than in nonpolarized cells ( lorkowski et  al . , 2014 ) , and according to our results , indeed , polymerization activities are supported by several bacterial factors during salmonella infection , leading to the formation of protrusions . the size of these protrusions is limited by villin - severing activities , enabling the formation of a structured network of actin filaments that constitute the ruffle . similar dominant concentrations of villin on actin have been demonstrated at the leading edge of migrating cells ( nusrat et  al . in absence of villin , the polymerization of filaments is not controlled , leading to the formation of abnormal extensions resembling giant microvilli , as observed by sem . interestingly , these aberrant ruffles are similar to those observed in nonpolarized cells such as hela cells , indicating that villin is a key factor in the invasion of differentiated cells . moreover , our work indicates that villin recruitment to dynamic zones of bacterial entry sites to sever and depolymerize actin filaments can promote the creation of barbed ends to serve as new nuclei to further promote polymerization . in parallel , depolymerization enables the increase of the local pool of g - actin that becomes available for further polymerization . in the absence of a structured actin network , we could also hypothesize that membrane protein localization can be affected , such as cytoskeletal proteins that are supposed to interact with actin to promote bacterial internalization . in particular , severing activities of villin are obvious targets and potentially temporally and spatially regulated to permit ruffle resolution and membrane refolding following bacterial internalization . we demonstrated a mechanism through which membrane ruffling involves a time - dependent phosphorylation of villin . changes in the phosphorylation status of villin are early and rapid events that are prone to complex regulations that allow efficient entry , but they also appear to be simultaneous at the scale of a single ruffle . we have shown that the kinase c - src is responsible for transient villin phosphorylation during the infection . villin phosphorylation is known to activate its severing activities and therefore may be important to promote organized membrane ruffling at the leading edge . however , a rapid dephosphorylation cascade induced by the bacterial factor sptp follows this phosphorylation wave . this certainly diminishes villin - severing capacities , thus enabling the reconstruction of microvilli after invasion of iecs . moreover , the increase of phosphorylation observed with the sptp mutant does not persist , indicating that either other factors are involved in villin dephosphorylation or villin phosphorylation is highly transient and does not persist over the time of infection . we identified that villin phosphorylation regulates villin binding to actin . at first sight , this is not in agreement with published results that showed that villin phosphorylation decreases the binding activity of villin for f - actin in  vitro ( zhai et  al . , 2001 ) . however , in our experiment , we were not able to distinguish between f- and g - actin . as villin can bind both types of actin , we therefore can not exclude that villin phosphorylation could favor the binding of one type of molecule over the other in infected cells to promote invasion . this modification in actin binding may be highly localized and participate in the construction of a precise but dynamic actin network . a decrease in actin binding is essential for the last steps of the entry process to inhibit the actin - modifying protein villin , thus enabling the resolution of apical membrane alterations . moreover , altering villin phosphorylation could modify its ligand binding abilities and therefore modify its localization close to the membrane . our results indicate that sipa - bound f - actin is resistant to villin - induced depolymerization . this inhibition may result in the accumulation of f - actin at the entry site to favor ruffle formation and bacterial entry . sipa may support actin assembly even though the free g - actin content is below the critical concentration that is physiologically necessary for actin polymerization . this indicates that , although villin - severing activities are locally inhibited by sipa to promote polymerization , villin is still active at the global cell level to renew the pool of free g - actin . it has been demonstrated that salmonella entry induces intracellular calcium release ( ruschkowski et  al . , 1992 ) , which is  susceptible to activate villin - severing activities . furthermore , it would be interesting to test how lpa and pip2 binding could affect villin activities toward actin . indeed , it has been shown that the association of villin with lpa inhibits its actin regulatory functions ( tomar et  al . , 2009 ) , whereas association of villin with pip2 inhibits actin depolymerization and enhances actin cross - linking by villin ( kumar et  al . , 2004b ) . the bacterial effector sopb is known to hydrolyze pip2 to pi5p during salmonella invasion ( mason et  al . , 2007 ) . salmonella ruffles are enriched in pip2 in the outer region , but not in the invaginating region . in the absence of sopb , pip2 is not hydrolyzed , and fission of the invaginating membranes is delayed due to stronger rigidity of the membrane ( terebiznik et  al . , 2002 ) . we hypothesize that pip2 can recruit and sequester villin in association with actin at the membrane during salmonella infection and that the hydrolysis of pip2 by sopb participates in membrane invagination by activating villin - severing activities and modifying actin structure . other specific host abps may be redundant to promote ruffling and brush border reparation during bacterial invasion . the abps cofilin / adf and gelsolin are also present in the iecs and are potential targets during salmonella invasion . cofilin and adf depolymerizing activities play a major role in bacterial entry ( dai et  al . , 2004 ) . in fact , these proteins are first dephosphorylated during infection by the host phosphatase slingshot and then rephosphorylated by the host lim kinase 1 in order to activate them . however , cofilin is not present in the brush border ( ashworth et  al . , 2004 ) , and all experiments were performed in nonpolarized hela or hek-293 cells ( dai et  al . , 2004 ) . similarly , gelsolin function during salmonella entry has been studied in  vitro , showing that it is primarily necessary for actin severing , and that its activity is inhibited by sipa , similar to villin inhibition . nevertheless , gelsolin is expressed not in the microvilli of the brush border but in the terminal web , displaying a different pattern from that reported for villin . this emphasizes that , even if these abps are functionally similar to villin and are potentially recruited during bacterial invasion , they may have different impact on salmonella entry due to their various locations and alternative regulations in the course of infection ( yin et  al . , 1981 ) . they could only be involved in later steps of the invasion process , after the initiation of entry by the microvillus - resident villin . consequently , in the context of the subversion of the brush border by salmonella , villin appears as the primary target for the regulation of the actin cytoskeleton remodeling . tc7 cells , a subclone of caco-2 cells , were maintained in dmem 4.5 g / l of glucose ( life technologies ) with 1% penicillin - streptomycin , 1% neaa , and 20% heat - inactivated fetal bovine serum ( fbs ) at 37c in 10% co2 . cells were seeded at 1   10 cells per well in 12-well tissue culture plates containing or not coverslips and maintained as differentiated monolayers for 2128  days . for pp2 treatment ( p0042 , sigma ) , cells are incubated 2  hr prior infection with the inhibitor ( 10  m ) . bacteria were cultured o / n at 37c with shaking in luria broth medium , pelleted at 5,000   g for 5  min , washed in pbs , and resuspended in dmem complemented with hepes 50  mm . the inoculum was diluted to the requested moi and added to the cells at 37c . s.  typhimurium strains were isogenic derivatives of the naturally streptomycin - resistant wt strain sl1344 ( hoiseth and stocker , 1981 ) . the strains sl1344 sipa , sipc , sptp , and sope - sope2-sopb - sipa ( called 4 in this study ) were also used ( hapfelmeier and hardt , 2005 ) as well as the wt strain sb300 and the isogenic mutants sptp and sptp ( murli et  al . , 2001 ) . differentiated cells were washed with dmem , and salmonella were added directly to the cells ( moi 20 ) . after infection , cells were fixed with pfa 3.8% for 15  min at rt and washed with pbs . cells were permeabilized for 20  min in 0.1% triton x-100 , and saturation was performed with 3% bsa , 5% fbs in pbs for 30  min . the coverslips were then incubated with the appropriate primary antibody for 1  hr at rt . the coverslips were washed in pbs , incubated with the secondary antibody for 1  hr , washed again in pbs , and incubated with dapi for 2  min . coverslips were mounted onto glass slides with prolong gold antifade reagent ( life technologies ) . images were collected using a confocal microscope with a 60 , 1.4 na oil immersion objective ( sp5 leica ) . all images were processed and pseudocolored using imagej ( national institutes of health ) or imaris software . wild - type ( vil ) and isogenic villin knockout mice ( vil ) ( ferrary et  al . , 1999 ) were bred in a c57bl/6j background . tgvillinwt and tgvillinsev mice were obtained after tamoxifen treatment as previously described ( ubelmann et  al . , briefly , mice , including controls , were injected intraperitoneally with tamoxifen ( 50  g / g of animal body weight ) on two consecutive days / week for a total of 4  weeks . all mice were kept in a specific pathogen - free area . for bacterial infection , streptomycin - pretreated mice ( 20  mg / animal ) were infected by gavage with o / n cultures of salmonella sl1344 ( 5   10 bacteria in 100  l ) . briefly , mice were euthanized , and tissue samples were removed for bacterial quantification and weighted . samples were homogenized using a precellys homogenizer ( ozyme ) for 2   15  s at a frequency of 5,000  rpm . each experiment was repeated three times  with a minimum of three animals per group . all animal experiments were carried out under approval by the use committee of pasteur institute and by the french ministry of agriculture ( ethic committee protocol number : 2013 - 0028 ) . the mean   sem from at least three independent experiments is shown in figures .
summarysalmonella invasion of intestinal epithelial cells requires extensive , though transient , actin modifications at the site of bacterial entry . 
 the actin - modifying protein villin is present in the brush border where it participates in the constitution of microvilli and in epithelial restitution after damage through its actin - severing activity . 
 we investigated a possible role for villin in salmonella invasion . 
 the absence of villin , which is normally located at the bacterial entry site , leads to a decrease in salmonella invasion . 
 villin is necessary for early membrane - associated processes and for optimal ruffle assembly by balancing the steady - state level of actin . 
 the severing activity of villin is important for salmonella invasion in  vivo . 
 the bacterial phosphatase sptp tightly regulates villin phosphorylation , while the actin - binding effector sipa protects f - actin and counterbalances villin - severing activity . 
 thus , villin plays an important role in establishing the balance between actin polymerization and actin severing to facilitate the initial steps of salmonella entry .