INFECTION AND IMMUNITY, Jan. 2010, p. 193–203 Vol. 78, No. 10019-9567/10/$12.00 doi:10.1128/IAI.00252-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Requirement for Formin-Induced Actin Polymerizationduring Spread of Shigella flexneri�

Jason E. Heindl, Indrani Saran,† Chae-ryun Yi, Cammie F. Lesser, and Marcia B. Goldberg*Division of Infectious Diseases, Massachusetts General Hospital and Harvard Medical School,

Cambridge, Massachusetts 02139

Received 4 March 2009/Returned for modification 8 April 2009/Accepted 4 October 2009

Actin polymerization in the cytosol and at the plasma membrane is locally regulated by actin nucleators.Several microbial pathogens exploit cellular actin polymerization to spread through tissue. The movement ofthe enteric pathogen Shigella flexneri, both within the cell body and from cell to cell, depends on actinpolymerization. During intercellular spread, actin polymerization at the bacterial surface generates protru-sions of the plasma membrane, which are engulfed by adjacent cells. In the cell body, polymerization of actinby Shigella spp. is dependent on N-WASP activation of the Arp2/Arp3 complex. Here we demonstrate that, incontrast, efficient protrusion formation and intercellular spread depend on actin polymerization that involvesactivation of the Diaphanous formin Dia. While the Shigella virulence protein IpgB2 can bind and activate Dia1(N. M. Alto et al., Cell 124:133-145, 2006), its absence does not result in a detectable defect in Dia-dependentprotrusion formation or spread. The dependence on the activation of Dia during S. flexneri infection contrastswith the inhibition of this pathway observed during vaccinia virus infection.

During infection, several human bacterial pathogens enterhost cells and spread through host tissues by moving directlyfrom one cell into adjacent cells. These microorganisms, in-cluding Shigella spp., Listeria monocytogenes (44), Rickettsiaspp. (43), Burkholderia spp. (21), and Mycobacterium marinum(42), induce the polymerization of host actin into tails thatpropel them through the cell cytoplasm to the cell periphery.Actin tail assembly in the cell body involves local activation ofactin polymerization through the Arp2/Arp3 (Arp2/3) complex(6, 11, 14, 19, 27, 49). The Arp2/3 complex initiates new fila-ment assembly and cross-links those filaments at 70° angles(28). At the cell periphery, Shigella spp. push outwardly againstthe plasma membrane, creating a membrane-bound cell exten-sion (“protrusion”) that extends tens of micrometers from thecell surface and contains a bacterium at its tip (5). Contact ofa protrusion tip with the membrane of an adjacent cell isfollowed by its uptake into the adjacent cell by a process thatresembles macropinocytosis (20), leading to the spread of theinfection into adjacent cells.

Although it is clear that actin assembly is required for theformation of protrusions by Shigella spp., the specific molecu-lar mechanisms involved are poorly understood. Shigella spp.frequently form protrusions in tissue culture cells at sites offocal adhesions (30). The actin network at the base of protru-sions contains filaments that are oriented in parallel arrays, incontrast to the angled arrays of actin filaments that predomi-nate in actin tails associated with bacteria in the cell body (15),suggesting that actin nucleation processes independent of theArp2/3 complex may be involved in protrusion formation.

Formins are ubiquitously expressed proteins that, like theArp2/3 complex, initiate de novo polymerization of actin (31,36). In contrast to Arp2/3 complex-mediated actin polymeriza-tion, formin-mediated actin polymerization leads to cross-link-ing of actin polymers in parallel arrays (31, 36). Formins playcritical roles in a variety of cytoskeletal processes in differentcell types, including cytokinesis, cell polarity, cell migrationand adhesion, and intracellular trafficking (13). At the cellmembrane, the mammalian Diaphanous-related formins Dia1and Dia2 function as effectors of the small GTPase RhoA (1,39, 48). RhoA plays a critical role in the generation of actinstress fibers that attach at adherens junctions and focal adhe-sions. The localization of Dia1 and Dia2 at sites of potentialShigella flexneri exit from the cell makes them ideal candidatesas mediators of protrusion formation. Their potential role inthis process is examined here.

MATERIALS AND METHODS

Bacterial strains and plasmids. The wild-type S. flexneri strain used in thisstudy is serotype 2a strain 2457T (23). The conditional virB mutant, 2457TvirB::Tn5/pDSW206-Ptsc-virB, has been described previously (25). An isogenicipgB2 mutant was generated by deleting the coding sequence of ipgB2 andinserting a kanamycin cassette via phage � Red recombinase-mediated homol-ogous recombination (10). Following P1-mediated transduction of the kanamy-cin-resistant locus into a clean 2457T background, the kanamycin cassette wasremoved using FLP recombinase to generate a nonpolar, unmarked isogenicipgB2 mutant (10). The lack of the ipgB2 coding sequence, the lack of thekanamycin cassette, and the maintenance of the flanking DNA sequences wereverified by PCR. Bacteria were grown in tryptic soy broth from individual colo-nies that were red on agar containing Congo red.

pCMV-Myc (carrying Myc), pDsRed-Monomer-N1 (carrying DsRed), andpEGFP-C1, pEGFP-C2, and pEGFP-N1 (carrying enhanced green fluorescentprotein [EGFP]) were obtained from Clontech. pEGFP-C1-IpgB2 was createdvia the site-specific Gateway (Invitrogen) recombination system. pEGFP-C1-Dia1 (16), encoding murine Dia1, was a gift from Naoki Watanabe. pEGFP-N1-Dia1129-369 (DID-EGFP), encoding the murine Dia1 Diaphanous inhibitory do-main (DID), and pEGFP-N1-Dia1129-369(A256D) [DID(A256D)-EGFP],encoding the Diaphanous autoregulatory domain (DAD)-binding mutant of themurine Dia1 DID, EGFP-DID, and EGFP-DID(A256D) were gifts from HenryN. Higgs. Murine and human Dia1 sequences are 86 to 88% identical, and

* Corresponding author. Mailing address: Bacterial Pathogenesis,Massachusetts General Hospital, 65 Landsdowne Street, Cambridge,MA 02139. Phone: (617) 768-8740. Fax: (617) 768-8738. E-mail:mgoldberg1@partners.org.

† Present address: Yale University Graduate School of Arts andSciences, Box 208323, New Haven, CT 06520-8323.

� Published ahead of print on 19 October 2009.

193

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

murine and human Dia2 sequences are 84% identical. pMyc-RhoA(T19N) [en-coding Myc-RhoA(T19N)] was a gift form Ralph R. Isberg; pGFPmut2 (8) wasa gift from Brendan Cormack; and pSUPER-retro, pSUPER-retro-mDia1KD1,and pSUPER-retro-mDia1KD2, for interfering RNA (RNAi) for Dia1 (35), weregifts from Leonidas Tsiokas. For experiments to assess protrusion formationfollowing depletion of Dia1 or Dia2, a mixture of pSUPER-retro-mDia1KD1 andpSUPER-retro-mDia1KD2 or Dharmacon siGENOME small interfering RNA(siRNA) D-010347-01 was used to target Dia1, while V2HS_73202 short hairpinRNA (shRNA) (OpenBiosystems) or Dharmacon siGENOME siRNAD-018997-02-0002 was used to target Dia2. For plaque assays, RNA interferencewith Dia1 was performed using Dharmacon SMARTpool siRNA M-010347-02,with an siRNA that targets gfp mRNA as a control. pDsRed-Monomer-N1-Dia1129-369 (DID-DsRed) and pDsRed-Monomer-N1-Dia1129-369(A256D)[DID(A256D)-DsRed] were generated by cloning the XhoI-EcoRI fragmentsfrom DID-EGFP and DID(A256D)-EGFP, respectively, into pDsRed-Mono-mer-N1. pCMV-Myc-Dia1129-369 (Myc-DID) and pCMV-Myc-Dia1129-369

(A256D) [Myc-DID(A256D)] were generated by cloning PCR-amplified DNAencoding the indicated residues from EGFP-DID and EGFP-DID(A256D), re-spectively, into the SalI and NotI sites of pCMV-Myc. pCMV-Myc-Dia1 (carry-ing Myc-Dia1) was generated by cloning a PCR-amplified Myc sequence intopEGFP-C1-Dia1. pCMV-Myc-IpgB2 (carrying Myc-IpgB2) was generated bycloning PCR-amplified DNA encoding the full coding sequence of IpgB2 into theEcoRI and NotI sites of pCMV-Myc. The sequences of primers used in PCR andsequencing are available from the authors upon request.

Cell culture and transfection. PtK2 rat kangaroo kidney epithelial cells weremaintained in Dulbecco’s modified Eagle’s essential medium (DMEM), supple-mented with 0.1% glucose and 10% fetal bovine serum, under humidified aircontaining 5% CO2 at 37°C. HeLa cells were maintained under the same con-ditions in minimal essential medium (MEM) supplemented with 10% fetal bo-vine serum. For analysis of the induction of formation of stress fibers by IpgB2,3 � 106 HeLa cells were transfected by electroporation with EGFP and eitherMyc-IpgB2 or Myc at a ratio of 1:5 using a total of 6 �g of DNA in 300 �lserum-free MEM. Electroporations were performed using a Bio-Rad GenePulser II electroporation system with a 4-mm-diameter cuvette at 0.250 kV and950 �F. A 50-�l portion from each transfection was seeded onto acetone-rinsedcoverslips in 2 ml of medium and was incubated overnight at 37°C. Sixteen hoursposttransfection, cells were fixed with 3.7% p-formaldehyde in cytoskeleton fixbuffer [F buffer, comprising 5 mM KCl, 137 mM NaCl, 4 mM NaHCO3, 1.1 mMNa2HPO4, 0.4 mM KH2PO4, 2 mM MgCl2, 5 mM piperazine-N,N�-bis(2-ethane-sulfonic acid) (PIPES), 2 mM EGTA, and 5.5 mM glucose (pH 7.2)] and werepermeabilized with 0.5% Triton X-100 in F buffer. Polymerized actin was labeledwith Alexa Fluor 568 phalloidin (Invitrogen). Transfected cells were identified bygreen fluorescence and were scored for increased stress fiber formation relativeto that of control cells. The results for PtK2 and HeLa cells were similar.

For analysis of the colocalization of IpgB2 and Dia1, PtK2 cells were trans-fected by electroporation with GFP-IpgB2 and either Myc-Dia1 or Myc at a ratioof 1:1, as described above. Sixteen hours posttransfection, cells were fixed andpermeabilized as described above. Labeling of the Myc tag was performed usinga monoclonal anti-Myc antibody (Clontech) and a Texas red-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch).

Bacterial infection of cells. For analysis of the inhibition of protrusion forma-tion by the DID, PtK2 cells were transfected with EGFP, DID-EGFP, orDID(A256D)-EGFP and were seeded as described above. Sixteen hours post-transfection, cells were infected with exponential-phase wild-type S. flexneri at amultiplicity of infection (MOI) (ratio of bacteria to cells) of 20 at 37°C, asdescribed previously (2). Following an initial invasion period of 1.5 h, cells werewashed, and the infection was allowed to continue for an additional 1.5 h in thepresence of 50 �g/ml gentamicin, which kills extracellular but not intracellularbacteria. Cells were fixed with 3.7% p-formaldehyde in F buffer and were per-meabilized with 0.5% Triton X-100 in F buffer. Polymerized actin was labeledwith Alexa Fluor 568 phalloidin (Invitrogen), and DNA was labeled with 4�,6-diamidino-2-phenylindole (DAPI; Invitrogen). Transfected cells were identifiedby green fluorescence. Images of transfected cells that were infected were ac-quired and were analyzed for the total number of intracellular bacteria, thenumber of intracellular bacteria with actin tails, the number of intracellularbacteria in protrusions, and the lengths of actin tails. A protrusion was definedas an extension of the plasma membrane outside the normal contour of theplasma membrane that extended more than a bacterial length, that contained abacterium at its tip, and in which the plasma membrane apposed the bacterium.The frequency of protrusion formation was expressed as the percentage ofintracellular bacteria that were within protrusions. None of the treatments had asignificant effect on the number of bacteria present within cells. Results for PtK2

and HeLa cells were similar. In each experiment performed in this study, theconfluence of cells was similar under all conditions.

For the comparison of protrusion formation by the ipgB2 mutant with that bythe wild-type strain, PtK2 cells were infected with either the wild type or itsisogenic nonpolar ipgB2::kan mutant at an MOI of 20, and infection was allowedto proceed as described above. Cells were fixed and permeabilized as describedabove. Polymerized actin was labeled with Alexa Fluor 568 phalloidin, and DNAwas labeled with DAPI. Images of infected cells were acquired as describedbelow and were analyzed for the total number of intracellular bacteria, thenumber of intracellular bacteria with actin tails, the number of intracellularbacteria in protrusions, and the lengths of actin tails.

For analysis of the role of RhoA per se in Shigella protrusion formation, PtK2cells were transfected by electroporation with GFP and either dominant negativeRhoA(T19N) or the Myc control plasmid at a ratio of 1:10 and were seeded asdescribed above. Sixteen hours posttransfection, cells were infected with wild-type S. flexneri at an MOI of 10 at 37°C. Following an initial invasion period of1.5 h, cells were washed, and the infection was allowed to continue for anadditional 1.5 h in the presence of 50 �g/ml gentamicin. Cells were fixed andpermeabilized as described above. Polymerized actin was labeled with AlexaFluor 568 phalloidin, and DNA was labeled with DAPI. Images were acquired oftransfected cells, identified by green fluorescence, that were infected, for threeindependent experiments. Images were analyzed for the total number of intra-cellular bacteria and the number of intracellular bacteria in protrusions, definedas described above.

To test whether inhibition of Dia had an effect on bacterial entry into cells, theefficiency of bacterial entry was determined, essentially as described previously(41). PtK2 cells were transfected with either EGFP or DID-EGFP and wereseeded as described above. Sixteen hours posttransfection, cells were infectedwith wild-type S. flexneri at an MOI of 100 at 37°C. Following an initial invasionperiod of 30 min, cells were washed, and the infection was allowed to continuefor an additional 30 min in the presence of 50 �g/ml gentamicin. Cells were fixedas described above, and DNA was labeled with DAPI. Images of transfectedcells, identified by green fluorescence, were acquired and analyzed for the pres-ence of intracellular bacteria. At least 30 transfected cells were analyzed for eachcondition, in each of three independent experiments.

Two distinct assays were used to assess the efficiency of bacterial spread fromone cell into adjacent cells: (i) a spreading assay, which assesses spread duringthe first 3 to 4 h of infection, and (ii) a plaque assay, which assesses spread duringthe first 48 to 72 h of infection. For analysis of the inhibition of intercellularspread by the DID, a spreading assay was used, because the DID-expressingvector could not be efficiently maintained in the monolayer for the 72 h requiredto set up and conduct a plaque assay (data not shown). PtK2 cells were trans-fected by electroporation with DsRed, DID-DsRed, or DID(A256D)-DsRed asdescribed above and were seeded at 70% to 90% confluence. Sixteen hoursposttransfection, cells were infected with wild-type S. flexneri carrying pGFPmut2at an MOI of 0.5 at 37°C; the low MOI was chosen so as to maximize thelikelihood that each infectious focus was the result of the initial infection of onlya single cell, and not of multiple adjacent cells. Following an initial invasionperiod of 1 h, cells were washed with fresh DMEM supplemented with 10% fetalbovine serum, 100 �g/ml ampicillin (to maintain pGFPmut2), and 50 �g/mlgentamicin, and the infection was allowed to continue for an additional 2.5 h.Cells were fixed and labeled with DAPI as described above. Transfected cellswere identified by red fluorescence. Images were acquired of transfected cellsthat were highly likely to be the first cell within the field that had been infected,based on (i) the low MOI, (ii) the presence of �5- to 10-fold more bacteria inthat cell than in adjacent cells, and (iii) the central location of the cell within thefocus of infected cells. Images were analyzed for the number of cells within thefocus that were infected. For each experimental condition in each experiment, 6to 20 infectious foci were analyzed.

For analysis of the intercellular spread of wild-type S. flexneri in HeLa cellsdepleted or not depleted of Dia1 by RNAi, or for analysis of the intercellularspread of the ipgB2 mutant compared to that of the wild-type strain in untreatedHeLa cells, a plaque assay was used. Confluent monolayers of cells grown in60-mm-diameter dishes or 6-well plates were infected at an MOI of 0.001 at37°C. For the RNAi-treated monolayers, the cells had been transfected withRNAi using HiPerFect (Qiagen) 48 h prior to infection. No independent RNAicontrol was used for this set of experiments. Following an initial invasion periodof 15 min to 1.5 h, monolayers were washed with fresh MEM and overlaid with0.5% agarose in DMEM supplemented with 10% fetal bovine serum and 25�g/ml gentamicin. Forty-eight hours later, monolayers were stained with neutralred, and images of the infected monolayers were acquired using an EpsonPerfection 4990 Photo desktop scanner and Adobe Photoshop Elements soft-

194 HEINDL ET AL. INFECT. IMMUN.

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

ware (version 2.0). The area of individual bacterial plaques within the monolay-ers was measured using IPLab software (Scanalytics).

For analysis of the dependence of Dia1 recruitment on the presence of IpgB2,HeLa cells were transfected with Myc or Myc-Dia1 and were seeded as describedabove. Sixteen hours posttransfection, cells were infected either with wild-type S.flexneri or with the ipgB2 mutant at an MOI of 10, as described above. Followinga 3-h infection, cells were fixed, permeabilized, and labeled with DAPI as de-scribed above. Labeling of the Myc tag was performed as described above.

Depletion of Dia1 and Dia2. Transient depletion of Dia1 and Dia2 was per-formed in HeLa cells. Dia1 mRNA levels were depleted using either pSUPER-retro-mDia1KD1 and pSUPER-retro-mDia1KD2 shRNA vectors in a 1:1 combi-nation or Dharmacon siGENOME siRNA D-010347-01, while Dia2 RNA levelswere reduced using either the V2HS_73202 shRNA vector (OpenBiosystems) orDharmacon siGENOME siRNA D-018997-02-0002. For experiments in whichDia1 or Dia2 was depleted using shRNA constructs, cells were seeded at adensity of 5 � 105 per well in 6-well plates and were allowed to recover overnightin MEM supplemented with 10% fetal bovine serum, nonessential amino acids,and penicillin-streptomycin solution. The following day, 2 �g of the appropriateshRNA vector(s) was added to the cells by using the Arrest-In transfectionreagent (OpenBiosystems) according to the manufacturer’s protocol. Followinga 40-h recovery, transfected cells were selected for 2 days by addition of 1 �g/mlpuromycin to the medium. Selected cells were reseeded onto acetone-rinsedglass coverslips and were maintained in MEM supplemented with 10% fetalbovine serum, nonessential amino acids, and 0.2 �g/ml puromycin. For experi-ments in which Dia1 or Dia2 was depleted using siRNA, 5.7 � 105 cells weretransfected in 6-well plates using HiPerFect. Sixteen (Dia2) or 40 (Dia1) hourslater, transfected cells were seeded onto glass coverslips and allowed to recoverovernight. Cells were infected, fixed, labeled, and imaged as described above,except that an MOI of 10 was used for the infection. Images of infected cells wereacquired and analyzed as described above. For each experimental condition ineach experiment, 10 or more infected cells were analyzed.

Motility assay. Time lapse microscopic imaging and determination of bacterialspeed were performed on semiconfluent monolayers of HeLa cells that had beentransfected 16 h earlier with DID-EGFP or EGFP alone, as described above.Transfected cells were infected with wild-type strain 2457T (MOI, 200), andimages were recorded at a rate of 1 every 5 s for 5-min periods, as describedpreviously (40). For each experimental condition in each experiment, speedswere determined for 7 or more moving bacteria.

Western blot analysis. Levels of Dia1 and Dia2 in cells were determined byimmunoblotting of whole-cell protein preparations. Adherent cells from 2 wellsof a 6-well plate were washed with cold phosphate-buffered saline (PBS) andlifted with 0.05% trypsin in MEM. Cells were recovered by centrifugation,washed twice with cold PBS, and resuspended in 30 �l lysis buffer (50 mMHEPES, 4% sodium dodecyl sulfate [SDS], 300 mM NaCl, 1 mM EDTA, 5 �g/mlaprotinin, 5 �g/ml leupeptin, 1 �g/ml pepstatin A [pH 7.5]). Lysates were boiledfor 5 min and incubated for 1 min at room temperature. Then 15 mM N-ethylmaleimide was added, and samples were incubated for 5 min at roomtemperature. Protein samples were diluted into SDS-polyacrylamide gel electro-phoresis loading buffer supplemented with 500 mM NaCl, 2 M urea, and 70 mM�-mercaptoethanol, were separated on 7.5% polyacrylamide gels, and weretransferred to nitrocellulose membranes. Separate membranes were probed withantibodies raised against murine Dia11-548 or Dia21-520 and with secondary an-tibodies conjugated to horseradish peroxidase. �-Actin was detected using ahorseradish peroxidase-conjugated anti-�-actin antibody (Sigma). Signal wasdetected using SuperSignal West Pico chemiluminescent substrate (Thermo Sci-entific).

Immunolocalization of Dia1 and the DID. PtK2 cells were transfected byelectroporation with Myc-Dia1, Myc-DID, Myc-DID(A256D), or Myc alone, asdescribed above. Sixteen hours posttransfection, cells were infected with wild-type S. flexneri or the conditional virB derivative of 2457T at an MOI of 20 to 100,as described above. For the conditional virB strain, 0.1 mM isopropyl-�-D-thio-galactopyranoside (IPTG) was present in the growth medium until the initiationof infection, when it was either removed or maintained at the same concentrationfor the duration of the infection. After 1.0 to 1.5 h of initial invasion and 1.0 to1.5 additional hours of infection in the presence of gentamicin, cells were fixedand permeabilized as described above. Polymerized actin was labeled withBODIPY FL phallacidin, and DNA was labeled with DAPI. Labeling of the Myctag was performed as described above. Results for infection with the wild-typestrain were similar for PtK2 and HeLa cells; results for infection with theconditional virB strain were not compared.

Microscopy and data analysis. Epifluorescence and phase-contrast microscopywere performed using a Nikon Eclipse TE300 microscope equipped withChroma Technology filters and a Photometrics CoolSNAP HQ charge-coupled

device camera (Roper Scientific). Images were acquired using IPLab software.Color figures were assembled by separately capturing images with each of theappropriate filter sets and digitally pseudocoloring the images using AdobePhotoshop software. The statistical significance of differences between experi-mental results was determined using Student’s t test.

RESULTS AND DISCUSSION

Inhibition or depletion of Dia1 inhibits the formation ofplasma membrane protrusions by S. flexneri. In resting cells,the Diaphanous-related formins are present in an autoinhib-ited state (13), in which the Diaphanous inhibitory domain(DID) binds the Diaphanous autoregulatory domain (DAD)(Fig. 1A) (26). Isolated Dia1 DID (amino acids 129 to 369)inhibits the actin nucleation activity of Dia1 in vitro (26). Wetested whether Dia1 was required for the generation of plasmamembrane protrusions by S. flexneri by comparing the percent-age of intracellular bacteria within protrusions in cells express-ing DID-EGFP to that in cells expressing EGFP alone. Weperformed these analyses in PtK2 cells, because their flat mor-phology facilitates the identification of protrusions; similar re-sults were obtained with HeLa cells (data not shown). In thecontrol infections, approximately 15 to 20% of bacteria werefound in plasma membrane protrusions at the time the analysis

FIG. 1. Inhibition of the formation of bacterial plasma membraneprotrusions by expression of the DID in cells. (A) Schematic drawingof Dia1 regulation. (Left) Open conformation; (right) autoinhibitedconformation. DID, diaphanous inhibitory domain; *, the A256D sub-stitution, which abrogates binding of the DID to the DAD; FH1,formin homology 1 domain; FH2, formin homology 2 domain; DAD,diaphanous autoregulatory domain. (Adapted from reference 17 withpermission of the publisher.) (B to D) S. flexneri infection of PtK2 cellsthat have been transfected with plasmids encoding DID-EGFP (B),EGFP only (control) (C), or DID(A256D)-EGFP (D). From left toright, panels show phase-contrast microscopy, phalloidin labeling ofpolymerized actin, DAPI staining of bacterial and cellular DNA, andan overlay of phalloidin labeling (red) and DAPI staining (blue).Asterisks, transfected cells, identified by a GFP signal (not shown);arrowheads, bacteria at the cell periphery that are not forming pro-trusions; arrows, bacterial protrusions. Bar, 20 �m. Images arerepresentative.

VOL. 78, 2010 Dia-INDUCED ACTIN POLYMERIZATION IN SHIGELLA SPREAD 195

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

was performed (3 h of infection). Expression of the DID led toa fourfold reduction in the formation of protrusions by S.flexneri (P � 0.002) (Fig. 1B and C; Table 1) but had nosignificant impact on the frequency or length of actin tails or onthe speed of moving bacteria within the cell body (Table 1). Incells expressing the DID, bacteria accumulated at the cellperiphery, with small amounts of polymerized actin at thebacterial pole farthest from the membrane, but were preventedfrom pushing out against the membrane (Fig. 1B, arrowheads).Thus, expression of the DID had no significant effect on themovement of bacteria in the cell body or on their accumulationat the cell periphery but inhibited the formation of protrusionsby bacteria at the cell periphery.

Not infrequently, in cells expressing the DID, clusters ofbacteria were found within cellular projections that resembleretraction fibers (Fig. 2); these projections were distinct from

typical bacterial protrusions, both because they contained mul-tiple bacteria and because there were no actin tails associatedwith these bacteria. Bacteria within protrusions are almostuniversally associated with actin tails. The presence of retrac-tion fibers in cells in which the RhoA pathway has beenblocked is not unprecedented; RhoA activation is thought tobe required for retraction of the cell tail during cell migration(7). Moreover, retraction fibers have been observed to trailcells that were induced to migrate by inhibition of RhoA ac-tivation of Dia1 by the vaccinia virus protein F11L (46). Thus,the presence of structures resembling retraction fibers in cellsexpressing the DID construct suggests that the DID is indeedblocking the RhoA pathway by inhibiting the RhoA effectorsDia1 and/or Dia2.

The entry of bacteria was not inhibited by expression of theDID; instead, entry into cells expressing DID was slightly butinsignificantly increased over entry into cells expressing GFPalone (61.3% � 13.0% of cells expressing the DID were in-fected, versus 40.0% � 10.0% of cells expressing GFP only;P � 0.2). Therefore, the reduction in the rate of protrusionformation in cells expressing the DID is not due to less-effi-cient entry of the bacteria into these cells. The expression of aderivative of the DID that is defective in binding to the Dia1DAD [DID(A256D)] (34) had no effect on bacterial protrusionformation (Fig. 1D; Table 1), indicating that the inhibitoryeffect of the DID depends on its interaction with the DAD.

We tested whether a reduction in Dia1 expression via RNAiwould similarly inhibit this process. We performed these ex-periments on HeLa cells with RNAi that targets human Dia.Depletion of Dia1 (Fig. 3B) led to a significant reduction inprotrusion formation (P � 0.02) (Fig. 4 and Table 2), confirm-ing the role of Dia1 in this process. Since autoinhibition due tothe interaction of the DID with the DAD is common to bothDia1 and Dia2, and the Dia1 DID binds the Dia1 DAD andthe Dia2 DAD with similar KD (equilibrium dissociation con-stants) (47), we also tested whether Dia2 could contribute toprotrusion formation. Western blot analysis revealed that Dia1is expressed at approximately equivalent levels in HeLa andPtk2 cells and that Dia2 is also expressed in HeLa cells (Fig.3A). As in the DID inhibition experiments (described above),approximately 15 to 20% of bacteria were found in plasmamembrane protrusions of control cells at the time the analysiswas performed (3 h of infection). Depletion of Dia2 led to a

TABLE 1. Inhibition by the Dia1 DID of the formation of plasma membrane protrusions in PtK2 cells by S. flexneria

DID

Bacteria in protrusions Bacteria within the cell body

Frequency(% of control)

Length of tails(�m)

Frequency oftail formation(% of control)

Length of tails(�m)

Speed of movingbacteria

(mm/min)

DID-EGFP 24 � 3b 4.9 � 0.46 100 � 57 2.2 � 1.2d 8.5 � 0.9f

DID(A256D)-EGFP 121 � 30c 5.3 � 1.1 114 � 14 2.4 � 0.70e NDEGFP alone 100 5.1 � 0.95 100 � 12 3.5 � 1.4 7.1 � 0.8

a Data are mean results from three independent experiments � SDs. Protrusions and the frequency of protrusion formation are defined in Materials and Methods.ND, not done.

b P � 0.002 for comparison to EGFP alone.c P � 0.6 for comparison to EGFP alone.d P � 0.8 for comparison to EGFP alone.e P � 0.3 for comparison to EGFP alone.f P � 0.1 for comparison to EGFP alone.

FIG. 2. Long cellular extensions formed occasionally in DID-trans-fected cells. Images show infection by S. flexneri of PtK2 cells that havebeen transfected with DID-EGFP (A) or EGFP only (control) (B).Asterisks, transfected cells, identified by GFP signals (not shown);arrowheads, bacteria at the tip of a long cellular extension; arrows,bacterial protrusions. Bar, 20 �m. Images are representative.

196 HEINDL ET AL. INFECT. IMMUN.

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

reduction in protrusion formation that was comparable to thatfollowing depletion of Dia1 (P � 0.02) (Fig. 4 and Table 2),even though the depletion by RNAi was specific for each (Fig.3B). Depletion of both Dia1 and Dia2 together led to a slightlysmaller reduction in the efficiency of protrusion formation(Table 2), perhaps because, for reasons that are unclear, thedegree of depletion of Dia2 was not as marked as when it wasdepleted alone (Fig. 3B). These results did not appear to be anartifact of the specific RNAi construct, since depletion of Dia1and depletion of Dia2 with independent RNAi constructs thattarget distinct sites on the mRNA gave similar reductions inprotrusion formation: Dia1 depletion led to a 40% reduction(P � 0.03), and Dia2 depletion led to a 47% reduction (P �0.02), in protrusion formation (Table 2). Thus, Dia1 and Dia2are required by intracellular S. flexneri for the efficient forma-tion of plasma membrane protrusions.

FIG. 3. Expression of Dia1 and Dia2 and their reduction byshRNA. (A) Expression of Dia1 and Dia2 in PtK2 and HeLa cells.(B) Reduction of expression of Dia1, Dia2, or both in HeLa cells byusing shRNA constructs specific to Dia1, Dia2, or a combination ofboth constructs. Western blot analysis, with loading normalized, usedantibodies specific to Dia1 and Dia2; �-actin levels confirmed loading.Molecular size standards (in kilodaltons) are given to the left of theblots. Results are representative of those obtained in three or moreindependent experiments.

FIG. 4. Inhibition of the formation of bacterial plasma membrane protrusions by depletion of Dia1 or Dia2 in HeLa cells. (A) Reduction ofexpression of Dia1 or Dia2 in HeLa cells by using a siRNA specific to Dia1 or Dia2. A nontargeting siRNA was used as a control. Western blotanalysis, with loading normalized, used antibodies specific to Dia1 and Dia2; �-actin levels confirmed loading. Molecular size standards (inkilodaltons) are given to the left of the blots. Results are representative of those obtained in three or more independent experiments. (B to D)S. flexneri infection of HeLa cells that had been reverse transfected with a siRNA specific to Dia1 (B) or Dia2 (C), or with a nontargeting controlsiRNA (D). Arrowheads, bacteria at the cell periphery that are not forming protrusions; arrows, bacterial protrusions. Bar, 20 �m. Images arerepresentative.

VOL. 78, 2010 Dia-INDUCED ACTIN POLYMERIZATION IN SHIGELLA SPREAD 197

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

Inhibition of Dia1 inhibits intercellular spread by S. flexneri.The formation of protrusions is thought to constitute a key stepin Shigella spread. To test whether the decrease in protrusionformation that occurs upon expression of the DID correlateswith a decrease in intercellular spread, we measured the effi-ciency of movement of S. flexneri from an infected cell toadjacent uninfected PtK2 cells during the first 3.5 h of infection(Fig. 5A to C). We used an assay with which we could selec-tively analyze the spread from cells that express the transfec-tion construct of interest (45), allowing us to compare thespread from cells expressing the DID to that from control cells.At 3.5 h after the initiation of bacterial infection, the infectedmonolayers were fixed and stained; transfected cells that werethe initial site of bacterial entry were identified; and the per-centage of intracellular bacteria that had moved from the siteof entry into adjacent cells was quantified (see Materials andMethods). The spread from DID-DsRed-expressing cells(0.5 � 0.4 infected cell in the infectious focus, excluding theprimarily infected cell) was threefold lower than the spreadfrom DsRed-expressing cells (1.5 � 0.3 infected cells) (P �0.03), in a manner dependent on DID binding to Dia. Thespread from cells expressing DID(A256D)-DsRed (1.3 � 0.2infected cells) was significantly different from the spread from

TABLE 2. Inhibition by depletion of Dia1 or Dia2 RNA of theformation of plasma membrane protrusions in

HeLa cells by S. flexneria

RNAi and target

Bacteria in protrusions Bacteria within the cellbody

Frequency(% of

control)

Length oftails (�m)

Frequencyof tail

formation(% of

control)

Length oftails (�m)

shRNADia1 42 � 8b 6.1 � 2.9f 92 � 12f 3.3 � 0.9f

Dia2 34 � 9b 9.6 � 3.4f 95 � 27f 3.2 � 0.5f

Dia1 and Dia2 51 � 10c 7.0 � 4.0f 117 � 49f 2.9 � 1.0f

Vector only 100 10.3 � 1.5 100 4.1 � 2.8

siRNADia1 60 � 13d ND ND NDDia2 53 � 21e ND ND NDControl 100 ND ND ND

a Data are mean results from three independent experiments � SDs. Protru-sions and the frequency of protrusion formation are defined in Materials andMethods. ND, not done.

b Significantly different from the value for the vector only (P � 0.02).c Significantly different from the value for the vector only (P � 0.04).d Significantly different from the value for the control (P � 0.03).e Significantly different from the value for the control (P � 0.02).f Not significantly different from the value for the vector only (P � 0.5).

FIG. 5. Reduction of intercellular spread upon inhibition or depletion of Dia in cells. (A through C) Infection with GFP-expressing S. flexneriof PtK2 cells that had been transfected with a plasmid encoding DID-DsRed (A), DsRed only (B), or DID(A256D)-DsRed (C). From left to right,images show phase-contrast microscopy, GFP labeling, DsRed labeling, and an overlay of GFP (green) and DsRed (red) labeling. Arrows indicatebacteria that have spread from the apparent primarily infected cell into an adjacent cell. Bar, 20 �m. Images are representative. (D) Mean areaof spread of S. flexneri through HeLa cell monolayers, determined by a plaque assay, following treatment with RNAi for Dia1. Data are meanresults, normalized to the area of spread through the control RNAi-treated monolayer, from three independent experiments. Error bars, SDs.

198 HEINDL ET AL. INFECT. IMMUN.

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

DID-DsRed-expressing cells (P � 0.04). (Values are means �standard deviations [SDs] for 6 to 20 foci of infection perexperiment, from three independent experiments). Thus, DIDinhibition of Dia reduces both intercellular spread and protru-sion formation. The observed correlation between protrusionformation and spread suggests that the former is an importantprerequisite for the latter.

We extended these results by showing that depletion of Dialeads to a significant reduction in the spread of S. flexnerithrough a cell monolayer over 48 h of infection. The area ofbacterial spread through monolayers of HeLa cells that hadbeen depleted of Dia1 by RNAi, as determined by a plaqueassay, was significantly smaller than that through monolayersof cells treated with a control RNAi (area of spread, 72% �5% of the control area; P � 0.009) (Fig. 5D). These resultsindicate that efficient intercellular spread of S. flexneri dependson Dia1 and that Dia1 functions in the step of bacterial move-ment from an infected cell into adjacent cells.

Dia1 localizes to S. flexneri at the peripheries of infectedcells. Consistent with a role for Dia1 in protrusion formationby S. flexneri, we found that Dia1 was prominent in actin tailsbehind bacteria found in protrusions (Fig. 6A to C, arrows).On bacteria within the body of the cytoplasm, the signal fromMyc-Dia1 was seen as a thin rim around the bacteria; thissignal tended to be more prominent on bacteria that were inthe periphery of the cell (Fig. 6A and B, arrowheads). Thesignal was also seen in a thin rim around protrusions engulfedby adjacent cells (not shown), suggesting that Dia1 is present inthe membrane surrounding the bacterium within the engulfedprotrusion. Myc-DID also colocalized as a thin rim around S.flexneri at the periphery of the cell (Fig. 6D, arrowheads),suggesting that Myc-DID localizes to these sites by binding Diaand that it inhibits protrusion formation by blocking the acti-vation of Dia at these sites. Myc-DID(A256D), which is defec-tive in the binding of Dia (34), also localized to the actin tailson bacteria within protrusions but was less prominent than Dia

FIG. 6. Localization of Dia1, DID, and DID(A256D) in S. flexneri-infected cells. Images show S. flexneri infection of PtK2 cells that had beentransfected with Myc-Dia1 (A to C), Myc-DID (D), or Myc-DID(A256D) (E). (C) Higher-magnification view of bacterial protrusions in panel A.The signal from cells transfected with Myc alone and labeled in a similar fashion was weak and diffuse (not shown). From left to right, panels showMyc labeling; phalloidin labeling of polymerized actin; DAPI staining of bacterial and cellular DNA; an overlay of Myc (red), phalloidin (green),and DAPI (blue); and phase-contrast microscopy. Arrows indicate colocalization of Dia1 with bacterial actin tails within protrusions; arrowheadsindicate colocalization of Dia1 (A and B) or DID (D) with bacteria at the cell periphery. Bars, 20 �m (E) and 5 �m (C). The scale of panels A,B, and D corresponds to the bar in panel E. Images are representative.

VOL. 78, 2010 Dia-INDUCED ACTIN POLYMERIZATION IN SHIGELLA SPREAD 199

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

or the DID around bacteria within the body of the cytoplasm(Fig. 6E), consistent with the observed decrease in the bindingof this mutant to Dia. The presence of Myc-DID(A256D) inactin tails within the protrusions may be due to a high concen-tration of Dia at these sites, combined with the low affinity ofDID(A256D) for Dia, or to an interaction with another proteinwithin the protrusions. When cells transfected with a vectorexpressing Myc alone were infected, the signal from Mycshowed no localization to bacteria or to bacterial actin tails(not shown). This pattern of DID localization is consistent withits colocalization to sites of Dia activation by intracellular S.flexneri during protrusion formation and suggests that the DIDacts locally at these sites to inhibit Dia-dependent protrusionformation.

Many Shigella proteins that interact with host factors aresecreted into the host cytoplasm by the bacterial type III se-cretion system (12). To investigate a potential role of type IIIsecreted proteins in the recruitment of Dia to intracellular S.flexneri, we infected cells expressing Myc-Dia1 with an S. flex-neri derivative that conditionally expresses the type III secre-tion machinery. In this strain, the global regulator of type IIIsecretion, VirB, is expressed under the control of an IPTG-inducible promoter (38). Since the entry of Shigella into cellsdepends on type III secretion, IPTG was included in the me-dium until the time of infection and then was either removedor maintained for the duration of the infection. Under theseconditions, VirB expression becomes undetectable within 25min of washout of the inducer (25). At 2 h of infection, therecruitment of Myc-Dia1 to intracellular S. flexneri was indis-tinguishable under non-VirB-expressing and VirB-expressingconditions (data not shown), suggesting that Dia recruitmenteither is independent of type III secretion or is dependent ona bacterial effector that is secreted early during infection.

The formation of plasma membrane protrusions is indepen-dent of RhoA. In the stress fiber formation pathway, Dia func-tions as a downstream effector of activated RhoA (18, 32, 46).Given that Dia activity is required for Shigella protrusion for-mation, it was possible that Shigella activation of Dia duringprotrusion formation might depend on RhoA. To test this, wecompared the efficiency of protrusion formation in PtK2 cellsexpressing a Myc-tagged dominant negative form of RhoA[RhoA(T19N)] to that in cells expressing Myc alone. The ex-pression of RhoA had no effect on protrusion formation[17.3% � 2.8% of bacteria were in protrusions in RhoA(T19N)-transfected cells versus 14.7% � 2.0% of bacteria in Myc-transfected cells (P � 0.5)]. The dominant negative RhoAconstruct was highly expressed, as determined by Western blotanalysis, and the expression of this construct had a dominantnegative effect on stress fiber formation: cells transfected withthe RhoA(T19N) construct displayed substantially fewer stressfibers than cells transfected in parallel with either a wild-typeor a constitutively active RhoA construct (data not shown).These results suggest that activation of RhoA is not requiredfor Shigella protrusion formation.

The stress fiber-inducing Shigella effector IpgB2 is not re-quired for protrusion formation, intercellular spread, or re-cruitment of Dia1. Shigella proteins that are translocated intocells via the type III secretion system display diverse effects onhost cell processes that enhance pathogenesis. Shigella IpgB2,a protein secreted by the type III secretion system, has been

shown to bind cellular Dia1 in coimmunoprecipitation assays(3). In addition, expression of IpgB2 in mammalian cells in-duces the formation of stress fibers (Fig. 7A and B) (3). To-gether these findings suggested that IpgB2 may modulate Dia1to enhance S. flexneri protrusion formation during intercellularspread. However, the role of IpgB2 in protrusion formation by,and spread of, Shigella spp. was not tested in this earlier study.

We tested whether S. flexneri lacking IpgB2 would be defec-tive in protrusion formation or intercellular spread. Bacterialspread, as assessed by the formation of plaques in HeLa cellmonolayers during 48 h of infection, was similar for the wild-type strain and an isogenic nonpolar ipgB2 deletion mutant(the diameter of spread, given as the mean � SD, was 0.95 �0.17 mm for the wild type and 0.87 � 0.20 mm for the ipgB2mutant [Fig. 7]). In addition, the frequencies of protrusionformation (means � SDs, 39% � 8% for the wild type and32% � 15% for the ipgB2 mutant) and the lengths of theprotrusions (means � SDs, 5.0 � 1.0 �m for the wild type and4.6 � 0.9 �m for the ipgB2 mutant) were similar for the twostrains. Moreover, overexpression of IpgB2 by transfection ofcells expressing the DID did not rescue the efficiency of pro-trusion formation (data not shown).

We examined whether the recruitment of Dia1 to intracel-lular bacteria was dependent on IpgB2. We found that Dia1colocalized with an ipgB2 mutant in a manner similar to itscolocalization with wild-type S. flexneri (Fig. 7D and E). Inboth cases, the signal from Myc-Dia1 was prominent aroundbacteria at the periphery of the cell and in actin tails withinbacterial plasma membrane protrusions. The signal from HeLacells expressing Myc alone did not localize around intracellularbacteria (not shown), indicating that the signal observedaround the bacteria in cells expressing Myc-Dia1 was specific.Thus, IpgB2 is not required for the recruitment of Dia1 tointracellular bacteria.

Therefore, while IpgB2 is sufficient to induce stress fiberformation, likely as a result of its previously described inter-action with Dia1 (3), it is not essential for Dia recruitment,Dia1-dependent formation of protrusions, or intercellularspread by S. flexneri. Although IpgB2 is not required for en-hancing intercellular spread, it is possible that one or moreother Shigella proteins may be functionally redundant withIpgB2 in this pathway. Although we have no evidence that aredundant protein exists, functional redundancy is common inbacterial type III secretion systems (50).

Model of the role of Dia1 in Shigella protrusion formation.The spread of intracellular Shigella from an infected cell intoan adjacent cell has long been thought to depend on the for-mation of actin-based bacterial protrusions from the surface ofthe infected cell. Our findings constitute the first demonstra-tion that activation of Dia is required for the formation ofactin-based cell surface projections and for spread during mi-crobial infection. Moreover, the observation that intercellularspread is significantly diminished when protrusion formation isinhibited establishes a direct correlation between protrusionformation and intercellular spread.

The role of Dia1 in enhancing Shigella protrusion formationmay reflect its function in the maintenance and remodeling ofthe cellular cortical actin network. The cell cortex, lying justbeneath the plasma membrane, contains a dynamic and densenetwork of actin filaments. The cortical actin network is con-

200 HEINDL ET AL. INFECT. IMMUN.

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

tinually remodeled, and its remodeling is critical to the main-tenance of cell shape and to cell motility. Actin polymerizationin the cell cortex is regulated by RhoA through its effectorsDia1 and Dia2 (1, 39, 48). Our results may reflect a role of Dia

in enhancing Shigella protrusion formation through increasedremodeling of the cortical actin network. Based on our find-ings, we suggest a model in which Shigella directs the reorga-nization of cortical actin into an orientation that is perpendic-

FIG. 7. Effects of IpgB2 on stress fibers and S. flexneri intercellular spread. (A and B) IpgB2 induces stress fibers in cells. Micrographs showstress fibers in cells cotransfected at a ratio of 1:20 with EGFP and Myc-IpgB2 (A) or Myc alone (B). From left to right, panels show labeling ofpolymerized actin with phalloidin, GFP fluorescence, phase-contrast microscopy, and higher-magnification views of phalloidin staining of atransfected cell. (C) The S. flexneri ipgB2 mutant is not defective in intercellular spread. Bar graph shows mean areas of plaques formed by thewild type (WT) and the ipgB2 mutant in HeLa cell monolayers. Data are means � SDs of results normalized to those for the wild-type strain fromthree or more independent experiments. (D and E) Recruitment of Dia1 to intracellular S. flexneri is independent of IpgB2. HeLa cells that hadbeen transfected with Myc-Dia1 and were subsequently infected with either wild-type S. flexneri (D) or the ipgB2 mutant (E) are shown. From leftto right, panels show Myc labeling, DAPI staining of bacterial and cellular DNA, and phase-contrast microscopy. Within each panel, upper imagesare focused on the plane of the cell body and lower images are focused on protrusions that are in a plane of focus just above the cell body. Arrowsindicate bacteria in the periphery of the cell body that colocalize with a strong signal from Myc-Dia1. Arrowheads indicate bacterial protrusionsthat colocalize with a strong signal from Myc-Dia1. Bars, 20 �m. Images are representative.

VOL. 78, 2010 Dia-INDUCED ACTIN POLYMERIZATION IN SHIGELLA SPREAD 201

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

ular to the plasma membrane and utilizes the force generatedby the formation of parallel arrays of polymerized actin to pushout against the plasma membrane.

In addition to promoting remodeling of the actin cortex, therole of Dia in protrusion formation may also be due to a directrole in force generation at the plasma membrane. Forminsgenerate 1.3 pN or more of force per actin filament (22).Forces of tens of pN are thought to be required to enable afilopodium to protrude against the resistance of the plasmamembrane (9). Such a force could be generated by forminsassociated with a bundle of actin filaments. To generate aplasma membrane protrusion, bacteria must gather similarforce using the resources of the cell. Our data are consistentwith a model in which the formin Dia1 is responsible forgenerating part or all of the force that is required in thisprocess during S. flexneri infection.

Shigella-induced actin polymerization in the cell body de-pends on localized activation of the Arp2/3 complex by N-WASP at the bacterial surface (11, 14). Our results demon-strate that whereas Dia is not required for actin tail assemblyin the cell body, it is required for the efficient formation ofprotrusions and for intercellular spread. Moreover, Dia1 re-cruitment to the bacteria is enriched at the cell periphery (Fig.6 and Fig. 7D and E). These findings indicate that when thebacteria reach the cell cortex or plasma membrane, a switchlikely occurs, leading to the activation of Dia-mediated actinpolymerization. This is similar to a switch that occurs duringvaccinia virus infection. Vaccinia virus moves from the peri-Golgi region to the cell periphery by microtubule-based mo-tility (33); at the plasma membrane, viral particles switch frommicrotubule-based motility to actin-based motility (29). HowShigella induces this switch and which bacterial proteins areinvolved in this process will be the subject of further investi-gation.

The process of Shigella spread from one cell into an adjacentcell involves the uptake of a plasma membrane-bound bacterialprotrusion by the adjacent cell in a process that resemblesmacropinocytosis (37). It is possible that, in addition to playinga role in Shigella protrusion formation, Dia is involved in theuptake of the protrusion by the adjacent cell. Our results donot directly address this. Moreover, although it is not knownwhether the uptake by the adjacent cell mechanistically mimicsthe initial entry of the bacterium into cells from the extracel-lular milieu, our data on entry indicate that Dia has little or norole in initial entry.

Distinct interactions with the RhoA-Dia pathway by differ-ent microorganisms. In sharp contrast to the requirement foractivation of Dia in S. flexneri spread is the requirement forinhibition of the RhoA-Dia pathway for productive infectionwith vaccinia virus. The vaccinia virus protein F11 inhibits theRhoA-Dia pathway and enhances the release of vaccinia virusfrom cells (4, 46). Moreover, expression of constitutively activeRhoA or Dia inhibits viral particle accumulation at the cellperiphery, actin tail formation on viral particles at the cellperiphery, and viral release from cells (4).

The observation that divergent effects on the RhoA-Diapathway can lead to similar outcomes, namely, the exit ofmicroorganisms from the cell, suggests that the two organismshave evolved distinct mechanisms for manipulating the corticalcytoskeleton during spread. Specific differences in how the

microorganisms move to the cell periphery may be at the coreof the differences in these mechanisms of exit. Vaccinia virusmoves to the cell periphery by microtubule-based motility (33),whereas Shigella moves to the periphery by polymerization ofactin tails (5, 24). The findings of Arakawa et al. (4) suggestthat vaccinia virus inhibition of the RhoA-Dia pathway inducesa reorganization of the cortical cytoskeleton that facilitates themovement of the virus through the cortical cytoskeleton to theplasma membrane. Our data suggest a model in which, onceShigella arrives at the plasma membrane via N-WASP-depen-dent actin-based motility, its activation of the RhoA-Dia path-way enables Dia-dependent actin polymerization and reorga-nization of the actin cortex in such a way as to enable thebacteria to push outward from the cell surface and into adja-cent cells.

ACKNOWLEDGMENTS

We thank D. Colon for technical assistance and B. Cormack, H. N.Higgs, R. R. Isberg, L. Tsiokas, and N. Watanabe for providing re-agents.

This research was supported by National Institutes of Allergy andInfectious Diseases grants AI052354 (to S. B. Snapper and M.B.G.),AI073967 (to M.B.G.), and AI081724 (to M.B.G.), by a Harvey Fel-lowship from the Mustard Seed Foundation (to J.E.H.), and by fundsfrom the Executive Committee on Research of the MassachusettsGeneral Hospital (to M.B.G.).

REFERENCES

1. Alberts, A. S., N. Bouquin, L. H. Johnston, and R. Treisman. 1998. Analysisof RhoA-binding proteins reveals an interaction domain conserved in het-erotrimeric G protein beta subunits and the yeast response regulator proteinSkn7. J. Biol. Chem. 273:8616–8622.

2. Ally, S., N. J. Sauer, J. J. Loureiro, S. B. Snapper, F. B. Gertler, and M. B.Goldberg. 2004. Shigella interactions with the actin cytoskeleton in the ab-sence of Ena/VASP family proteins. Cell. Microbiol. 6:355–366.

3. Alto, N. M., F. Shao, C. S. Lazar, R. L. Brost, G. Chua, S. Mattoo, S. A.McMahon, P. Ghosh, T. R. Hughes, C. Boone, and J. E. Dixon. 2006.Identification of a bacterial type III effector family with G protein mimicryfunctions. Cell 124:133–145.

4. Arakawa, Y., J. V. Cordeiro, S. Schleich, T. P. Newsome, and M. Way. 2007.The release of vaccinia virus from infected cells requires RhoA-mDia mod-ulation of cortical actin. Cell Host Microbe 1:227–240.

5. Bernardini, M. L., J. Mounier, H. d’Hauteville, M. Coquis-Rondon, and P. J.Sansonetti. 1989. Identification of icsA, a plasmid locus of Shigella flexnerithat governs bacterial intra- and intercellular spread through interaction withF-actin. Proc. Natl. Acad. Sci. U. S. A. 86:3867–3871.

6. Breitbach, K., K. Rottner, S. Klocke, M. Rohde, A. Jenzora, J. Wehland, andI. Steinmetz. 2003. Actin-based motility of Burkholderia pseudomallei in-volves the Arp 2/3 complex, but not N-WASP and Ena/VASP proteins. Cell.Microbiol. 5:385–393.

7. Clark, K., M. Langeslag, C. G. Figdor, and F. N. van Leeuwen. 2007. MyosinII and mechanotransduction: a balancing act. Trends Cell Biol. 17:178–186.

8. Cormack, B. P., R. H. Valdivia, and S. Falkow. 1996. FACS-optimizedmutants of the green fluorescent protein (GFP). Gene 173:33–38.

9. Dai, J., and M. P. Sheetz. 1999. Membrane tether formation from blebbingcells. Biophys. J. 77:3363–3370.

10. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromo-somal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad.Sci. U. S. A. 97:6640–6645.

11. Egile, C., T. P. Loisel, V. Laurent, R. Li, D. Pantaloni, P. J. Sansonetti, andM. F. Carlier. 1999. Activation of the CDC42 effector N-WASP by theShigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complexand bacterial actin-based motility. J. Cell Biol. 146:1319–1332.

12. Galan, J. E., and H. Wolf-Watz. 2006. Protein delivery into eukaryotic cellsby type III secretion machines. Nature 444:567–573.

13. Goode, B. L., and M. J. Eck. 2007. Mechanism and function of formins in thecontrol of actin assembly. Annu. Rev. Biochem. 76:593–627.

14. Gouin, E., C. Egile, P. Dehoux, V. Villiers, J. Adams, F. Gertler, R. Li, andP. Cossart. 2004. The RickA protein of Rickettsia conorii activates theArp2/3 complex. Nature 427:457–461.

15. Gouin, E., H. Gantelet, C. Egile, I. Lasa, H. Ohayon, V. Villiers, P. Gounon,P. J. Sansonetti, and P. Cossart. 1999. A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigellaflexneri, and Rickettsia conorii. J. Cell Sci. 112:1697–1708.

202 HEINDL ET AL. INFECT. IMMUN.

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

16. Higashida, C., T. Miyoshi, A. Fujita, F. Oceguera-Yanez, J. Monypenny, Y.Andou, S. Narumiya, and N. Watanabe. 2004. Actin polymerization-drivenmolecular movement of mDia1 in living cells. Science 303:2007–2010.

17. Higgs, H. N. 2005. Formin proteins: a domain-based approach. Trends Bio-chem. Sci. 30:342–353.

18. Hotulainen, P., and P. Lappalainen. 2006. Stress fibers are generated by twodistinct actin assembly mechanisms in motile cells. J. Cell Biol. 173:383–394.

19. Jeng, R. L., E. D. Goley, J. A. D’Alessio, O. Y. Chaga, T. M. Svitkina, G. G.Borisy, R. A. Heinzen, and M. D. Welch. 2004. A Rickettsia WASP-likeprotein activates the Arp2/3 complex and mediates actin-based motility. Cell.Microbiol. 6:761–769.

20. Kadurugamuwa, J. L., M. Rohde, J. Wehland, and K. N. Timmis. 1991.Intercellular spread of Shigella flexneri through a monolayer mediated bymembranous protrusions and associated with reorganization of the cytoskel-etal protein vinculin. Infect. Immun. 59:3463–3471.

21. Kespichayawattana, W., S. Rattanachetkul, T. Wanun, P. Utaisincharoen,and S. Sirisinha. 2000. Burkholderia pseudomallei induces cell fusion andactin-associated membrane protrusion: a possible mechanism for cell-to-cellspreading. Infect. Immun. 68:5377–5384.

22. Kovar, D. R., and T. D. Pollard. 2004. Insertional assembly of actin filamentbarbed ends in association with formins produces piconewton forces. Proc.Natl. Acad. Sci. U. S. A. 101:14725–14730.

23. Labrec, E. H., H. Schneider, T. J. Magnani, and S. B. Formal. 1964. Epi-thelial cell penetration as an essential step in the pathogenesis of bacillarydysentery. J. Bacteriol. 88:1503–1518.

24. Lett, M. C., C. Sasakawa, N. Okada, T. Sakai, S. Makino, M. Yamada, K.Komatsu, and M. Yoshikawa. 1989. virG, a plasmid-coded virulence gene ofShigella flexneri: identification of the virG protein and determination of thecomplete coding sequence. J. Bacteriol. 171:353–359.

25. Leung, Y., S. Ally, and M. B. Goldberg. 2008. Bacterial actin assembly requiresToca-1 to relieve N-WASP autoinhibition. Cell Host Microbe 3:39–47.

26. Li, F., and H. N. Higgs. 2005. Dissecting requirements for auto-inhibition ofactin nucleation by the formin, mDia1. J. Biol. Chem. 280:6986–6992.

27. Moreau, V., F. Frischknecht, I. Reckmann, R. Vincentelli, G. Rabut, D.Stewart, and M. Way. 2000. A complex of N-WASP and WIP integratessignalling cascades that lead to actin polymerization. Nat. Cell Biol. 2:441–448.

28. Mullins, R. D., J. A. Heuser, and T. D. Pollard. 1998. The interaction ofArp2/3 complex with actin: nucleation, high affinity pointed end capping, andformation of branching networks of filaments. Proc. Natl. Acad. Sci. U. S. A.95:6181–6186.

29. Newsome, T. P., N. Scaplehorn, and M. Way. 2004. SRC mediates a switchfrom microtubule- to actin-based motility of vaccinia virus. Science 306:124–129.

30. Prevost, M. C., M. Lesourd, M. Arpin, F. Vernel, J. Mounier, R. Hellio, andP. J. Sansonetti. 1992. Unipolar reorganization of F-actin layer at bacterialdivision and bundling of actin filaments by plastin correlate with movementof Shigella flexneri within HeLa cells. Infect. Immun. 60:4088–4099.

31. Pruyne, D., M. Evangelista, C. Yang, E. Bi, S. Zigmond, A. Bretscher, and C.Boone. 2002. Role of formins in actin assembly: nucleation and barbed-endassociation. Science 297:612–615.

32. Ridley, A. J., and A. Hall. 1992. The small GTP-binding protein rho regulatesthe assembly of focal adhesions and actin stress fibers in response to growthfactors. Cell 70:389–399.

33. Rietdorf, J., A. Ploubidou, I. Reckmann, A. Holmstrom, F. Frischknecht, M.Zettl, T. Zimmermann, and M. Way. 2001. Kinesin-dependent movement onmicrotubules precedes actin-based motility of vaccinia virus. Nat. Cell Biol.3:992–1000.

34. Rose, R., M. Weyand, M. Lammers, T. Ishizaki, M. R. Ahmadian, and A.Wittinghofer. 2005. Structural and mechanistic insights into the interactionbetween Rho and mammalian Dia. Nature 435:513–518.

35. Rundle, D. R., G. Gorbsky, and L. Tsiokas. 2004. PKD2 interacts andco-localizes with mDia1 to mitotic spindles of dividing cells: role of mDia1 inPKD2 localization to mitotic spindles. J. Biol. Chem. 279:29728–29739.

36. Sagot, I., A. A. Rodal, J. Moseley, B. L. Goode, and D. Pellman. 2002. Anactin nucleation mechanism mediated by Bni1 and profilin. Nat. Cell Biol.4:626–631.

37. Sansonetti, P. J., J. Mounier, M. C. Prevost, and R. M. Mege. 1994. Cadherinexpression is required for the spread of Shigella flexneri between epithelialcells. Cell 76:829–839.

38. Schuch, R., R. C. Sandlin, and A. T. Maurelli. 1999. A system for identifyingpost-invasion functions of invasion genes: requirements for the Mxi-Spa typeIII secretion pathway of Shigella flexneri in intercellular dissemination. Mol.Microbiol. 34:675–689.

39. Seth, A., C. Otomo, and M. K. Rosen. 2006. Autoinhibition regulates cellularlocalization and actin assembly activity of the diaphanous-related forminsFRL and mDia1. J. Cell Biol. 174:701–713.

40. Shere, K. D., S. Sallustio, A. Manessis, T. G. D’Aversa, and M. B. Goldberg.1997. Disruption of IcsP, the major Shigella protease that cleaves IcsA,accelerates actin-based motility. Mol. Microbiol. 25:451–462.

41. Shibata, T., F. Takeshima, F. Chen, F. W. Alt, and S. B. Snapper. 2002.Cdc42 facilitates invasion but not the actin-based motility of Shigella. Curr.Biol. 12:341–345.

42. Stamm, L. M., J. H. Morisaki, L. Y. Gao, R. L. Jeng, K. L. McDonald, R.Roth, S. Takeshita, J. Heuser, M. D. Welch, and E. J. Brown. 2003. Myco-bacterium marinum escapes from phagosomes and is propelled by actin-based motility. J. Exp. Med. 198:1361–1368.

43. Teysseire, N., C. Chiche-Portiche, and D. Raoult. 1992. Intracellular move-ments of Rickettsia conorii and R. typhi based on actin polymerization. Res.Microbiol. 143:821–829.

44. Tilney, L. G., and D. A. Portnoy. 1989. Actin filaments and the growth,movement, and spread of the intracellular bacterial parasite, Listeria mono-cytogenes. J. Cell Biol. 109:1597–1608.

45. Tran Van Nhieu, G., C. Clair, R. Bruzzone, M. Mesnil, P. Sansonetti, and L.Combettes. 2003. Connexin-dependent inter-cellular communication in-creases invasion and dissemination of Shigella in epithelial cells. Nat. CellBiol. 5:720–726.

46. Valderrama, F., J. V. Cordeiro, S. Schleich, F. Frischknecht, and M. Way.2006. Vaccinia virus-induced cell motility requires F11L-mediated inhibitionof RhoA signaling. Science 311:377–381.

47. Wallar, B. J., B. N. Stropich, J. A. Schoenherr, H. A. Holman, S. M. Kitchen,and A. S. Alberts. 2006. The basic region of the diaphanous-autoregulatorydomain (DAD) is required for autoregulatory interactions with the diapha-nous-related formin inhibitory domain. J. Biol. Chem. 281:4300–4307.

48. Watanabe, N., P. Madaule, T. Reid, T. Ishizaki, G. Watanabe, A. Kakizuka,Y. Saito, K. Nakao, B. M. Jockusch, and S. Narumiya. 1997. p140mDia, amammalian homolog of Drosophila diaphanous, is a target protein for Rhosmall GTPase and is a ligand for profilin. EMBO J. 16:3044–3056.

49. Welch, M. D., A. Iwamatsu, and T. J. Mitchison. 1997. Actin polymerizationis induced by Arp2/3 protein complex at the surface of Listeria monocyto-genes. Nature 385:265–269.

50. Zhou, D., L. M. Chen, L. Hernandez, S. B. Shears, and J. E. Galan. 2001. ASalmonella inositol polyphosphatase acts in conjunction with other bacterialeffectors to promote host cell actin cytoskeleton rearrangements and bacte-rial internalization. Mol. Microbiol. 39:248–259.

Editor: J. B. Bliska

VOL. 78, 2010 Dia-INDUCED ACTIN POLYMERIZATION IN SHIGELLA SPREAD 203

on June 26, 2020 by guesthttp://iai.asm

.org/D

ownloaded from