downloaded from on may 25, 2019 by guest file8 polymerization through the arp2/3 complex (6, 11, 14...

Requirement for formin-induced actin polymerization during spread of 1

Shigella 2

Jason E. Heindl, Indrani Saran†, Chae-ryun Yi, Cammie F. Lesser, Marcia B. Goldberg* 3

Division of Infectious Diseases, Massachusetts General Hospital and Harvard Medical 4

School, Cambridge, MA 02139 5

6

*Corresponding author: 7

Marcia B. Goldberg 8

Bacterial Pathogenesis 9

Massachusetts General Hospital 10

65 Landsdowne Street 11

Cambridge, MA 02139 12

Tel. 1-617-768-8740 13

Fax 1-617-768-8738 14

e-mail: [email protected] 15

†Current address: Yale University Graduate School of Arts and Sciences, Box 208323, 16

New Haven, CT 06520-8323. 17

Running title: Dia induced actin polymerization in Shigella spread 18

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00252-09 IAI Accepts, published online ahead of print on 19 October 2009

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Abstract 1

Actin polymerization in the cytosol and at the plasma membrane is locally regulated by 2

actin nucleators. Several microbial pathogens exploit cellular actin polymerization to 3

spread through tissue. Movement of the enteric pathogen Shigella flexneri both within 4

the cell body and from cell-to-cell depends on actin polymerization. During intercellular 5

spread, actin polymerization at the bacterial surface generates protrusions of the 6

plasma membrane, which are engulfed by adjacent cells. In the cell body, 7

polymerization of actin by Shigella sp is dependent on N-WASP activation of the Arp2/3 8

complex. Here, we demonstrate that, in contrast, efficient protrusion formation and 9

intercellular spread depends on actin polymerization that involves activation of the 10

diaphanous formin Dia. While the Shigella virulence protein, IpgB2, can bind and 11

activate Dia1 (3), its absence does not result in a detectable defect in Dia-dependent 12

protrusion formation or spread. The dependence on activation of Dia during S. flexneri 13

infection contrasts with the inhibition of this pathway observed during vaccinia infection. 14


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Introduction 1

During infection, several human bacterial pathogens enter into host cells and spread 2

through host tissues by moving directly from one cell into adjacent cells. These 3

microorganisms, including Shigella sp., Listeria monocytogenes (44), Rickettsia sp. 4

(43), Burkholderia sp. (21), and Mycobacterium marinum (42), induce the 5

polymerization of host actin into tails that propel them through the cell cytoplasm to the 6

cell periphery. Actin tail assembly in the cell body involves local activation of actin 7

polymerization through the Arp2/3 complex (6, 11, 14, 19, 27, 49). The Arp2/3 complex 8

initiates new filament assembly and crosslinks those filaments at 70° angles (28). At the 9

cell periphery, Shigella sp. push outwardly against the plasma membrane, creating a 10

membrane-bound cell extension ("protrusion") that extends tens of microns from the cell 11

surface and contains a bacterium at its tip (5). Contact of a protrusion tip with the 12

membrane of an adjacent cell is followed by its uptake into the adjacent cell by a 13

process that resembles macropinocytosis (20), leading to spread of the infection into 14

adjacent cells. 15

Although it is clear that actin assembly is required for formation of protrusions by 16

Shigella sp., the specific molecular mechanisms involved are poorly understood. 17

Shigella sp. frequently form protrusions in tissue culture cells at sites of focal adhesions 18

(30). The actin network at the base of protrusions contains filaments that are oriented in 19

parallel arrays, in contrast to the angled arrays of actin filaments that predominate in 20

actin tails associated with bacteria in the cell body (15), suggesting that actin nucleation 21

processes independent of the Arp2/3 complex may be involved in protrusion formation. 22


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Formins are ubiquitously expressed proteins that, like the Arp2/3 complex, initiate de 1

novo polymerization of actin (31, 36). In contrast to Arp2/3 complex-mediated actin 2

polymerization, formin-mediated actin polymerization leads to crosslinking of actin 3

polymers in parallel arrays (31, 36). Formins play critical roles in a variety of cytoskeletal 4

processes in different cell types, including cytokinesis, cell polarity, cell migration and 5

adhesion, and intracellular trafficking (13). At the cell membrane, the mammalian 6

diaphanous-related formins Dia1 and Dia2 function as effectors of the small GTPase 7

RhoA (1, 39, 48). RhoA plays a critical role in the generation of actin stress fibers that 8

attach at adherens junctions and focal adhesions. The localization of Dia1 and Dia2 at 9

sites of potential S. flexneri exit from the cell make them ideal candidates as mediators 10

of protrusion formation. Their potential role in this process is examined here. 11


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Materials and Methods 1

Bacterial Strains and Plasmids. The wild-type S. flexneri strain used in this study is 2

serotype 2a strain 2457T (23). The conditional virB mutant, 2457T virB::Tn5 pDSW206-3

Ptsc-virB, has been described previously (25). An isogenic ipgB2 mutant was generated 4

by deleting the coding sequence of ipgB2 and inserting a kanamycin cassette, using 5

phage λ Red recombinase-mediated homologous recombination (10). Following P1-6

mediated transduction of the kanamycin-resistant locus into a clean 2457T background, 7

the kanamycin cassette was removed using FLP recombinase to generate a non-polar 8

unmarked isogenic ipgB2 mutant (10). The lack of the ipgB2 coding sequence and the 9

kanamycin cassette and the maintenance of the flanking DNA sequences were verified 10

by PCR. Bacteria were grown in tryptic soy broth from individual colonies that were red 11

on agar containing Congo Red. 12

pCMV-Myc (Myc), pDsRed-Monomer-N1 (DsRed), pEGFP-C1, pEGFP-C2 (EGFP), and 13

pEGFP-N1 were from Clontech. pEGFP-C1-IpgB2 was created via the site-specific 14

Gateway (Invitrogen) recombination system. pEGFP-C1-Dia1 (16), encoding murine Dia 15

was the gift of Naoki Watanabe. pEGFP-N1-Dia1129-369 (DID-EGFP), encoding the 16

murine Dia1 DID, and pEGFP-N1-Dia1129-369 (A256D) (DID [A256D]-EGFP), encoding the 17

DAD-binding mutant of the murine Dia1 DID, EGFP-DID, and EGFP-DID (A256D) were 18

the gift of Henry N. Higgs. Murine and human Dia1 sequences are 86-88% identical and 19

murine and human Dia2 sequences are 84% identical. pMyc-RhoA (T19N) (RhoA 20

T19N) was the gift of Ralph R. Isberg, pGFPmut2 (8) the gift of Brendan Cormack, and 21

pSUPER-retro, pSUPER-retro-mDia1KD1, and pSUPER-retro-mDia1KD2 for RNAi to Dia1 22


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(35), the gifts of Leonidas Tsiokas. For experiments to assess protrusion formation 1

following depletion of Dia1 or Dia2, a mixture of pSUPER-retro-mDia1KD1 and pSUPER-2

retro-mDia1KD2 or Dharmacon siGENOME siRNA D-010347-01 was used to target Dia1 3

while V2HS_73202 shRNA (OpenBiosystems) or Dharmacon siGENOME siRNA D-4

018997-02-0002 was used to target Dia2. For plaque assays, siRNA to Dia1 was 5

performed using Dharmacon SMARTpool siRNA M-010347-02, with an siRNA that 6

targets gfp mRNA as a control. pDsRed-Monomer-N1-Dia1129-369 (DID-DsRed) and 7

pDsRed-Monomer-N1-Dia1129-369 (A256D) (DID [A256D]-DsRed) were generated by 8

cloning the XhoI-EcoRI fragments from DID-EGFP and DID(A256D)-EGFP, respectively, 9

into pDsRed-Monomer-N1. pCMV-Myc-Dia1129-369 (Myc-DID) and pCMV-Myc-Dia1129-369 10

(A256D) (Myc-DID [A256D]) were generated by cloning PCR-amplified DNA encoding the 11

indicated residues from EGFP-DID and EGFP-DID (A256D), respectively, into the SalI 12

and NotI sites of pCMV-Myc. pCMV-Myc-Dia1 (Myc-Dia1) was generated by cloning a 13

PCR-amplified Myc sequence into pEGFP-C1-Dia1. pCMV-Myc-IpgB2 (Myc-IpgB2) was 14

generated by cloning PCR-amplified DNA encoding the full coding sequence of IpgB2 15

into the EcoRI and NotI sites of pCMV-Myc. The sequence of primers used in PCR and 16

sequencing are available from the authors upon request. 17

Cell Culture and Transfection. PtK2 rat kangaroo kidney epithelial cells were 18

maintained in Dulbecco's modified Eagle's essential medium, supplemented with 0.1% 19

glucose and 10% fetal bovine serum, under humidified air containing 5% CO2 at 37°C. 20

HeLa cells were maintained under the same conditions in minimal essential medium 21

(MEM), supplemented with 10% fetal bovine serum. For the analysis of IpgB2 induction 22

of the formation of stress fibers, 3 x 106 HeLa cells were transfected by electroporation 23


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with EGFP and either Myc-IpgB2 or Myc at a ratio of 1:5 using a total of 6 ug of DNA in 1

300 uL serum-free MEM. Electroporations were performed using a Bio-Rad Gene 2

Pulser II electroporation system with a 4 mm cuvette at 0.250 kV and 950 uF. 50 uL 3

from each transfection was seeded onto acetone-rinsed coverslips in 2 mL of media and 4

incubated overnight at 37°C. Sixteen hr post-transfection, cells were fixed with 3.7% p-5

formaldehyde in cytoskeleton fix buffer (F buffer: 5 mM KCl, 137 mM NaCl, 4 mM 6

NaHCO3, 1.1 mM Na2HPO4, 0.4 mM KH2PO4, 2 mM MgCl2, 5 mM PIPES, 2 mM EGTA, 7

5.5 mM glucose, pH 7.2) and permeabilized with 0.5% Triton X-100 in F buffer. 8

Polymerized actin was labeled with Alexa Fluor 568 phalloidin (Invitrogen). Transfected 9

cells were identified by green fluorescence and were scored for increased stress fiber 10

formation relative to control cells. Results were similar for PtK2 and HeLa cells. 11

For the analysis of the co-localization of IpgB2 and Dia1, PtK2 cells were transfected by 12

electroporation with GFP-IpgB2 and either Myc-Dia1 or Myc at a ratio of 1:1, as 13

described above. Sixteen hr post-transfection, cells were fixed and permeabilized as 14

described above. Labeling of the Myc tag was performed using a monoclonal anti-Myc 15

antibody (Clontech) and a Texas Red-conjugated anti-mouse secondary antibody 16

(Jackson ImmunoResearch). 17

Bacterial Infection of Cells. For the analysis of the inhibition of protrusion formation by 18

the DID, PtK2 cells were transfected with EGFP, DID-EGFP, or DID (A256D)-EGFP and 19

seeded as described above. Sixteen hr post-transfection cells were infected with 20

exponential phase wildtype S. flexneri at a multiplicity of infection (MOI; bacteria to 21

cells) of 20 at 37 °C, as described (2). Following an initial invasion period of 1.5 hr, cells 22

were washed and the infection was allowed to continue for an additional 1.5 hr in the 23


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presence of 50 ug/mL gentamicin, which kills extracellular but not intracellular bacteria. 1

Cells were fixed with 3.7% p-formaldehyde in F buffer and permeabilized with 0.5% 2

Triton X-100 in F buffer. Polymerized actin was labeled with Alexa Fluor 568 phalloidin 3

(Invitrogen) and DNA was labeled with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen). 4

Transfected cells were identified by green fluorescence. Images were acquired of 5

transfected cells that were infected and were analyzed for total number of intracellular 6

bacteria, number of intracellular bacteria with actin tails, number of intracellular bacteria 7

in protrusions, and lengths of actin tails. Protrusions were defined as extensions of the 8

plasma membrane outside the normal contour of the plasma membrane that extended 9

more than a bacterial length, had a bacterium contained within the extension at its tip, 10

and in which the plasma membrane apposed the bacterium. The frequency of 11

protrusion formation was defined as the percentage of intracellular bacteria that were 12

within protrusions. None of the treatments had a significant effect on the number of 13

bacteria present within cells. Results were similar for PtK2 and HeLa cells. In each 14

experiment performed in this study, the confluency of cells in all conditions was similar. 15

For the comparison of protrusion formation by the ipgB2 mutant to that by the wildtype 16

strain, PtK2 cells were infected with either the wild-type or its isogenic non-polar 17

ipgB2::kan mutant at an MOI of 20, and infection was allowed to proceed as described 18

above. Cells were fixed and permeabilized as described above. Polymerized actin was 19

labeled with Alexa Fluor 568 phalloidin, and DNA was labeled with DAPI. Images of 20

infected cells were acquired as described below and were analyzed for total number of 21

intracellular bacteria, number of intracellular bacteria with actin tails, number of 22

intracellular bacteria in protrusions, and lengths of actin tails. 23


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For the analysis of the role of RhoA per se in Shigella protrusion formation, PtK2 cells 1

were transfected by electroporation with GFP and either dominant negative RhoA T19N 2

or Myc control plasmid at a ratio of 1:10 and seeded as described above. Sixteen hr 3

post-transfection, cells were infected with wildtype S. flexneri at an MOI of 10 at 37 °C. 4

Following an initial invasion period of 1.5 hr, cells were washed and the infection was 5

allowed to continue for an additional 1.5 hr in the presence of 50 ug/mL gentamicin. 6

Cells were fixed and permeabilized as described above. Polymerized actin was labeled 7

with Alexa Fluor 568 phalloidin and DNA was labeled with DAPI. Images were acquired 8

of transfected cells, identified by green fluorescence, that were infected, for three 9

independent experiments. Images were analyzed for total number of intracellular 10

bacteria and for number of intracellular bacteria in protrusions, defined as above. 11

To test whether inhibition of Dia had an effect on bacterial entry into cells, the efficiency 12

of bacterial entry was determined, essentially as described previously (41). PtK2 cells 13

were transfected with either EGFP or DID-EGFP and seeded as described above. 14

Sixteen hr post-transfection cells were infected with wildtype S. flexneri at an MOI of 15

100 at 37 °C. Following an initial invasion period of 30 minutes, cells were washed and 16

the infection was allowed to continue for an additional 30 minutes in the presence of 50 17

ug/mL gentamicin. Cells were fixed as described above, and DNA was labeled with 18

DAPI. Images of transfected cells, identified by green fluorescence, were acquired and 19

analyzed for the presence of intracellular bacteria. At least 30 transfected cells were 20

analyzed for each condition, in each of three independent experiments. 21

Two distinct assays were used to assess the efficiency of bacterial spread from one cell 22

into adjacent cells: (i) a spreading assay, which assesses spread during the first 3-4 hr 23


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of infection, and (ii) a plaque assay, which assesses spread during the first 48-72 hr of 1

infection. For the analysis of the inhibition of intercellular spread by the DID, a spreading 2

assay was used, because the DID-expressing vector could not be efficiently maintained 3

in the monolayer for the 72 hr required to set up and conduct a plaque assay (data not 4

shown). PtK2 cells were transfected by electroporation with DsRed, DID-DsRed, or DID 5

(A256D)-DsRed as described above, and were seeded at 70% to 90% confluency. 6

Sixteen hr post-transfection, cells were infected with wildtype S. flexneri carrying 7

pGFPmut2 at an MOI of 0.5 at 37°C; the low MOI was chosen so as to maximize the 8

likelihood that each infectious focus was the result of initial infection of only a single cell, 9

and not of multiple adjacent cells. Following an initial invasion period of 1 hr cells were 10

washed with fresh DMEM, supplemented with 10% fetal bovine serum, 100 ug/mL 11

ampicillin (to maintain pGFPmut2), and 50 ug/mL gentamicin, and the infection was 12

allowed to continue for an additional 2.5 hours. Cells were fixed and labeled with DAPI 13

as described above. Transfected cells were identified by red fluorescence. Images were 14

acquired of transfected cells that were highly likely to be the first cell within the field that 15

had been infected, based on (i) the low MOI, (ii) the presence of greater than 5-10-fold 16

more bacteria being present within that cell than were present in adjacent cells, and (iii) 17

the cell being centrally located within the focus of infected cells. Images were analyzed 18

for the number of cells within the focus that were infected. For each experimental 19

condition in each experiment, 6-20 infectious foci were analyzed. 20

For the analysis of intercellular spread of wildtype S. flexneri in HeLa cells depleted or 21

not depleted of Dia1 by RNAi or of the ipgB2 mutant compared to the wildtype strain in 22

untreated HeLa cells, a plaque assay was used. Confluent monolayers of cells grown in 23


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60 mm dishes or 6-well plates were infected at an MOI of 0.001 at 37 °C. For the RNAi-1

treated monolayers, the cells had been transfected with RNAi using HiPerFect (Qiagen) 2

48 hr prior to infection. An independent RNAi control was not used for this set of 3

experiments. Following an initial invasion period of 15 min to 1.5 hours, monolayers 4

were washed with fresh MEM and overlain with 0.5% agarose in DMEM, supplemented 5

with 10% fetal bovine serum, and 25 ug/mL gentamicin. Forty-eight hr later, monolayers 6

were stained with neutral red, and images of the infected monolayers were acquired 7

using an Epson Perfection 4990 Photo desktop scanner and Adobe Photoshop 8

Elements 2.0 software. The area of individual bacterial plaques within the monolayers 9

was measured using IPLab software (Scanalytics). 10

For the analysis of the dependence of Dia1 recruitment on the presence of IpgB2, HeLa 11

cells were transfected with Myc or Myc-Dia1 and seeded as described above. Sixteen 12

hr post-transfection cells were infected with wildtype S. flexneri or the ipgB2 mutant at a 13

MOI of 10, as described above. Following a 3 hr infection, cells were fixed, 14

permeabilized, and labeled with DAPI as described above. Labeling of the Myc tag was 15

performed as described above. 16

Depletion of Dia1 and Dia2. Transient depletion of Dia1 and Dia2 was performed in 17

HeLa cells. Dia1 mRNA levels were depleted using either pSUPER-retro-mDia1KD1 and 18

pSUPER-retro-mDia1KD2 shRNA vectors in a 1:1 combination or Dharmacon 19

siGENOME siRNA D-010347-01, while Dia2 RNA levels were reduced using either 20

V2HS_73202 shRNA vector (OpenBiosystems) or Dharmacon siGENOME siRNA D-21

018997-02-0002. For experiments in which Dia1 or Dia2 was depleted using shRNA 22

constructs, cells were seeded at a density of 5 x 105 cells per well in 6-well plates and 23


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allowed to recover overnight in MEM supplemented with 10% fetal bovine serum, non-1

essential amino acids, and penicillin-streptomycin solution. The following day 2 ug of the 2

appropriate shRNA vector(s) was added to the cells using the Arrest-In transfection 3

reagent (OpenBiosystems) according to the manufacturer’s protocol. Following a 40 hr 4

recovery, transfected cells were selected for 2 days by adding 1 ug/mL puromycin to the 5

medium. Selected cells were reseeded onto acetone-rinsed glass coverslips and 6

maintained in MEM supplemented with 10% fetal bovine serum, non-essential amino 7

acids, and 0.2 ug/mL puromycin. For experiments in which Dia1 or Dia2 was depleted 8

using siRNA, 5.7 x 105 cells were transfected in 6-well plates using HiPerFect. Sixteen 9

(Dia2) or 40 (Dia1) hours later, transfected cells were seeded onto glass coverslips and 10

allowed to recover overnight. Cells were infected, fixed, labeled, and imaged as 11

described above, except that an MOI of 10 was used for the infection. Images were 12

acquired of infected cells and analyzed as described above. For each experimental 13

condition in each experiment, 10 or more infected cells were analyzed. 14

Motility assay. Time-lapse microscopic imaging and determination of bacterial speed 15

were performed on semiconfluent monolayers of HeLa cells that had been transfected 16

16 hr prior with DID-GFP or GFP alone, as described above. Transfected cells were 17

infected with wild type strain 2457T (MOI 200), and images were recorded at one every 18

5 s for 5-min periods, as described previously (40). For each experimental condition in 19

each experiment, speeds were determined for 7 or more moving bacteria. 20

Western blot analysis. Levels of Dia1 and Dia2 in cells were determined by 21

immunoblotting whole cell protein preparations. Adherent cells from 2 wells of a 6-well 22

plate were washed with cold phosphate buffered saline (PBS) and lifted with 0.05% 23


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trypsin in MEM. Cells were recovered by centrifugation, washed twice with cold PBS, 1

and resuspended in 30 uL lysis buffer (50 mM HEPES, 4% SDS, 300 mM NaCl, 1 mM 2

EDTA, 5 ug/mL aprotinin, 5 ug/mL leupeptin, 1 ug/mL pepstatin A, pH 7.5). Lysates 3

were boiled for 5 minutes and incubated for 1 minute at room temperature. Then, 15 4

mM N-ethyl maleimide was added and samples were incubated for 5 minutes at room 5

temperature. Protein samples were diluted into SDS-PAGE loading buffer, 6

supplemented with 500 mM NaCl, 2 M urea, and 70 mM β-mercaptoethanol, were 7

separated on 7.5% polyacrylamide gels, and were transferred to nitrocellulose 8

membranes. Separate membranes were probed with antibodies raised against murine 9

Dia11-548 or Dia21-520, with secondary antibodies conjugated to horseradish peroxidase. 10

β-actin was detected using horseradish peroxidase-conjugated anti-β-actin antibody 11

(Sigma). Signal was detected using SuperSignal West Pico Chemiluminescent 12

Substrate (Thermo Scientific). 13

Immunolocalization of Dia1 and the DID. PtK2 cells were transfected by 14

electroporation with Myc-Dia1, Myc-DID, Myc-DID [A256D], or Myc alone, as described 15

above. Sixteen hr post-transfection, cells were infected with wildtype S. flexneri or the 16

conditional virB derivative of 2457T at an MOI of 20-100, as described above. For the 17

conditional virB strain, 0.1 mM IPTG was present in the growth media up until the 18

initiation of infection and was then either removed or maintained at the same 19

concentration for the duration of the infection. After 1.0-1.5 hr of initial invasion and 1.0-20

1.5 additional hr of infection in the presence of gentamicin, cells were fixed and 21

permeabilized as described above. Polymerized actin was labeled with BODIPY FL 22

phallacidin and DNA was labeled with DAPI. Labeling of the Myc tag was performed as 23


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described above. Results were similar for PtK2 and HeLa cells for infection with the 1

wildtype strain and were not compared for the conditional virB strain. 2

Microscopy and Data Analysis. Epifluorescence and phase microscopy was 3

performed using a Nikon Eclipse TE300 microscope equipped with Chroma Technology 4

filters and a Photometrics CoolSNAP HQ charge-coupled device camera (Roper 5

Scientific). Images were acquired using IPLab software. Color figures were assembled 6

by separately capturing images with each of the appropriate filter sets and digitally 7

pseudocoloring the images using Adobe Photoshop software. The statistical 8

significance of differences between experimental results was determined using the 9

Student's t test. 10


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Results and Discussion 1

Inhibition or depletion of Dia1 inhibits formation of plasma membrane protrusions 2

by S. flexneri 3

In resting cells, the diaphanous-related formins are present in an autoinhibited state 4

(13), in which the diaphanous inhibitory domain (DID) binds the diaphanous auto-5

regulatory domain (DAD) (Fig. 1A) (26). Isolated Dia1 DID (aa 129-369) inhibits actin 6

nucleation activity of Dia1 in vitro (26). We tested whether Dia1 was required for the 7

generation of plasma membrane protrusions by S. flexneri by comparing the percentage 8

of intracellular bacteria within protrusions in cells expressing DID-EGFP to that in cells 9

expressing EGFP alone. We performed these analyses in PtK2 cells, because their flat 10

morphology facilitates the identification of protrusions; similar results were obtained in 11

HeLa cells (data not shown). In the control infections, approximately 15-20% of bacteria 12

were found in plasma membrane protrusions at the time the analysis was performed (3 13

hr of infection). Expression of the DID led to a four-fold reduction in the formation of 14

protrusions by S. flexneri (p = 0.002, Fig. 1B and C, and Table 1), but had no significant 15

impact on the frequency of actin tails, length of actin tails, or speed of moving bacteria 16

within the cell body (Table 1). In cells expressing the DID, bacteria accumulated at the 17

cell periphery with small amounts of polymerized actin at the bacterial pole farthest from 18

the membrane, but were prevented from pushing out against the membrane (Fig. 1B, 19

arrowheads). Thus, expression of the DID had no significant effect on the movement of 20

bacteria in the cell body or on their accumulation at the cell periphery, but inhibited the 21

formation of protrusions by bacteria at the cell periphery. 22


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Not infrequently in cells expressing the DID, clusters of bacteria were found within 1

cellular projections that resemble retraction fibers (Fig. 2); these projections were 2

distinct from typical bacterial protrusions, both because they contained multiple bacteria 3

and because there were no actin tails associated with these bacteria. Bacteria within 4

protrusions are almost universally associated with actin tails. The presence of retraction 5

fibers in cells in which the RhoA pathway has been blocked is not unprecedented; RhoA 6

activation is thought to be required for retraction of the cell tail during cell migration (7). 7

Moreover, retraction fibers were observed to trail cells that were induced to migrate by 8

inhibition of RhoA activation of Dia1 by the vaccinia protein F11L (46). Thus, the 9

presence of structures resembling retraction fibers in cells expressing the DID construct 10

suggests that DID is indeed blocking the RhoA pathway by inhibiting the RhoA effectors 11

Dia1 and/or Dia2. 12

Entry of bacteria was not inhibited by expression of DID; instead, entry into cells 13

expressing DID was slightly but insignificantly increased over entry into cells expressing 14

GFP alone (61.3 ± 13.0% of cells expressing DID were infected, versus 40.0 ± 10.0% of 15

cells expressing GFP, p = 0.2). Therefore, the reduction in protrusion formation in cells 16

expressing DID is not due to less efficient entry of the bacteria into these cells. The 17

expression of a derivative of the DID that is defective in binding to the Dia1 DAD (A256D; 18

(34)) had no effect on bacterial protrusion formation (Fig. 1D and Table 1), indicating 19

that the inhibitory effect of the DID depends on its interaction with the DAD. 20

We tested whether reduction in Dia1 expression via interference RNA (RNAi) would 21

similarly inhibit this process. We performed these experiments in HeLa cells with RNAi 22

that targets human Dia. Depletion of Dia1 (Fig. 3B) led to a significant reduction in 23


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protrusion formation (p = 0.02, Fig. 4 and Table 2), confirming the role of Dia1 in this 1

process. Since autoinhibition due to the interaction of the DID with the DAD is common 2

to both Dia1 and Dia2, and the Dia1 DID binds the Dia1 DAD and the Dia2 DAD with 3

similar KD (47), we also tested whether Dia2 could contribute to protrusion formation. 4

Western blot analysis revealed that Dia1 is expressed at approximately equivalent 5

levels in HeLa and Ptk2 cells, and that Dia2 is also expressed in HeLa cells (Fig. 3A). 6

As in the DID inhibition experiments (described above), approximately 15-20% of 7

bacteria were found in plasma membrane protrusions of control cells at the time the 8

analysis was performed (3 hr of infection). Depletion of Dia2 led to a reduction in 9

protrusion formation that was comparable to that following depletion of Dia1 (p = 0.02, 10

Fig. 4 and Table 2), even though the depletion by RNAi was specific for each (Fig. 3B). 11

Depletion of both Dia1 and Dia2 together led to a slightly smaller reduction in the 12

efficiency of protrusion formation (Table 2), perhaps because, for reasons that are 13

unclear, the degree of depletion of Dia2 was not as marked as when it was depleted 14

alone (Fig. 3B). These results did not appear to be an artifact of the specific RNAi 15

construct, since Dia1 and Dia2 depletion with independent RNAi constructs that target 16

distinct sites on the mRNA gave similar reductions in protrusion formation: Dia1 17

depletion led to a reduction in protrusion formation of 40% (p = 0.03, Table 2) and Dia2 18

depletion led to a reduction in protrusion formation of 47% (p = 0.02, Table 2). Thus, 19

Dia1 and Dia2 are required by intracellular S. flexneri for the efficient formation of 20

plasma membrane protrusions. 21

Inhibition of Dia1 inhibits intercellular spread by S. flexneri 22


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Protrusions are thought to constitute a key step in Shigella spread. To test whether the 1

decrease in protrusion formation that occurs upon expression of the DID correlates with 2

a decrease in intercellular spread, we measured the efficiency of movement of S. 3

flexneri from an infected cell to adjacent uninfected PtK2 cells during the first 3.5 hr of 4

infection (Fig. 5A-C). We used an assay in which we could selectively analyze spread 5

from cells that express the transfection construct of interest (45), allowing us to compare 6

spread from cells expressing the DID to that from control cells. At 3.5 hr after initiation of 7

bacterial infection, the infected monolayers were fixed and stained, transfected cells that 8

were the initial site of bacterial entry were identified, and the percentage of intracellular 9

bacteria that had moved from the site of entry into adjacent cells was quantified (see 10

Materials and Methods). Spread from DID-DsRed-expressing cells was 3-fold 11

decreased versus spread from DsRed-expressing cells (p = 0.03), in a manner 12

dependent on DID binding to Dia (Table 3). Thus, DID inhibition of Dia reduces both 13

intercellular spread and protrusion formation. The observed correlation between 14

protrusion formation and spread suggests that the former is an important prerequisite to 15

the latter. 16

We extended these results by showing that depletion of Dia leads to a significant 17

reduction in spread of S. flexneri through a cell monolayer over 48 hr of infection. The 18

area of bacterial spread through monolayers of HeLa cells that had been depleted of 19

Dia1 by RNAi, as determined by plaque assay, was significantly reduced compared to 20

that through monolayers of cells treated with a control RNAi (area of spread 72 ± 5% of 21

control, p = 0.009; Fig. 5D). These results indicate that efficient intercellular spread of S. 22


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flexneri depends on Dia1 and that Dia1 functions in the step of bacterial movement from 1

an infected cell into adjacent cells. 2

Dia1 localizes to S. flexneri at the periphery of infected cells 3

Consistent with a role of Dia1 in S. flexneri protrusion formation, we found that Dia1 was 4

prominent in actin tails behind bacteria found in protrusions (Fig. 6A-C, arrows). On 5

bacteria within the body of the cytoplasm, the signal from Myc-Dia1 was seen as a thin 6

rim around the bacteria and tended to be more prominent on bacteria that were in the 7

periphery of the cell (Fig. 6A-B, arrowheads). The signal was also seen in a thin rim 8

around protrusions engulfed by adjacent cells (not shown), suggesting that Dia1 is 9

present in the membrane surrounding the bacterium within the engulfed protrusion. 10

Myc-DID also co-localized as a thin rim around S. flexneri at the periphery of the cell 11

(Fig. 6D, arrowheads), suggesting that Myc-DID localizes to these sites by binding Dia 12

and that it inhibits protrusion formation by blocking activation of Dia at these sites. Myc-13

DID (A256D), which is defective for binding Dia (34), also localized to the actin tails on 14

bacteria within protrusions, but was less prominent than Dia or the DID around bacteria 15

within the body of the cytoplasm (Fig. 6E), consistent with the observed decreased 16

binding of this mutant to Dia. Its presence in actin tails within the protrusions may be 17

due to a high concentration of Dia at these sites combined with the low affinity of DID 18

(A256D) for Dia or to an interaction with another protein within the protrusions. When 19

cells transfected with a vector expressing Myc alone were infected, the signal from Myc 20

showed no localization to bacteria or to bacterial actin tails in control cells transfected 21

with a vector expressing Myc alone (not shown). This pattern of DID localization is 22

consistent with its co-localization to sites of Dia activation by intracellular S. flexneri 23


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during protrusion formation and suggests that the DID acts locally at these sites to 1

inhibit Dia-dependent protrusion formation. 2

Many Shigella proteins that interact with host factors are secreted into the host 3

cytoplasm by the bacterial type III secretion system (12). To investigate a potential role 4

of type III secreted proteins in the recruitment of Dia to intracellular S. flexneri, we 5

infected cells expressing Myc-Dia1 with a S. flexneri derivative that conditionally 6

expresses the type III secretion machinery. In this strain, the global regulator of type III 7

secretion, VirB, is expressed under the control of an IPTG inducible promoter (38). 8

Since Shigella entry into cells depends on type III secretion, IPTG was included in the 9

media up until the time of infection, and then was either removed or maintained for the 10

duration of the infection. Under these conditions, VirB expression becomes 11

undetectable within 25 minutes of washing out the inducer (25). At 2 hours of infection, 12

the recruitment of Myc-Dia1 to intracellular S. flexneri was indistinguishable under VirB-13

non-expressing and VirB-expressing conditions (data not shown), suggesting that either 14

Dia recruitment is independent of type III secretion or is dependent on a bacterial 15

effector that is secreted early during the infection. 16

The formation of plasma membrane protrusions is independent of RhoA 17

In the stress fiber formation pathway, Dia functions as a downstream effector of 18

activated RhoA (18, 32, 46). Given the requirement of Dia activity for Shigella protrusion 19

formation, it was possible that Shigella activation of Dia during protrusion formation 20

might depend on RhoA. To test this, we compared the efficiency of protrusion formation 21

in PtK2 cells expressing a Myc-tagged dominant negative form of RhoA (RhoA T19N) to 22


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those expressing Myc alone. The expression of RhoA had no effect on protrusion 1

formation (17.3 ± 2.8% of bacteria were in protrusions in RhoA T19N transfected cells 2

versus 14.7 ± 2.0% of bacteria in Myc transfected cells, p = 0.5). The dominant negative 3

RhoA construct was highly expressed, as determined by western blot analysis, and the 4

expression of this construct had a dominant negative effect on stress fiber formation, 5

since the cells transfected with the RhoA T19N construct displayed substantially fewer 6

stress fibers than cells transfected in parallel with either a WT RhoA construct or a 7

constitutively active RhoA construct (data not shown). These results suggest that 8

activation of RhoA is not required for Shigella protrusion formation. 9

The stress fiber inducing Shigella effector IpgB2 is not required for protrusion 10

formation, intercellular spread, or recruitment of Dia1 11

Shigella proteins that are translocated into cells via the type III secretion system display 12

diverse effects on host cell processes that enhance pathogenesis. Shigella IpgB2, a 13

protein secreted by the type III secretion system, has been shown to bind cellular Dia1 14

in co-immunoprecipitation assays (3). In addition, expression of IpgB2 in mammalian 15

cells induces the formation of stress fibers (Fig. 7A-B and (3)). Together these findings 16

suggested that IpgB2 may modulate Dia1 to enhance S. flexneri protrusion formation 17

during intercellular spread. However, the role of IpgB2 in protrusion formation and 18

spread of Shigella sp. was not tested in this earlier study. 19

We tested whether S. flexneri lacking IpgB2 would be defective in protrusion formation 20

or intercellular spread. Bacterial spread, as assessed by the formation of plaques in 21

HeLa cell monolayers during 48 hr of infection, was similar for the wild type strain and 22


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an isogenic non-polar ipgB2 deletion mutant (diameter of spread: wild-type, 0.95 ± 0.17 1

mm [mean ± S.D.]; ipgB2 mutant, 0.87 ± 0.20 mm, Fig. 7). In addition, the frequency of 2

protrusion formation (wild-type, 39 ± 8% [mean ± S.D.]; ipgB2 mutant, 32 ± 15%) and 3

the mean length of the protrusions (wild-type, 5.0 ± 1.0 um [mean ± S.D.]; ipgB2 mutant, 4

4.6 ± 0.9 um) were similar for the two strains. Moreover, overexpression of IpgB2 by 5

transfection of cells expressing the DID did not rescue the efficiency of protrusion 6

formation (data not shown). 7

We examined whether the recruitment of Dia1 to intracellular bacteria was dependent 8

on IpgB2. We found that Dia1 co-localized with an ipgB2 mutant in a manner similar to 9

its co-localization with wildtype S. flexneri (Fig. 7D-E). In both cases, the signal from 10

Myc-Dia1 was prominent around bacteria at the periphery of the cell and in actin tails 11

within bacterial plasma membrane protrusions. The signal from HeLa cells expressing 12

Myc alone did not localize around intracellular bacteria (not shown), indicating that the 13

signal observed around the bacteria in cells expressing Myc-Dia1 was specific. Thus, 14

IpgB2 is not required for recruitment of Dia1 to intracellular bacteria. 15

Therefore, while IpgB2 is sufficient to induce stress fiber formation, likely as a result of 16

its described interaction with Dia1 (3), it is not essential for Dia recruitment, Dia1-17

dependent formation of protrusions, or intercellular spread by S. flexneri. Although 18

IpgB2 is not required for enhancing intercellular spread, it is possible that one or more 19

other Shigella protein may be functionally redundant with IpgB2 in this pathway. 20

Although we have no evidence that a redundant protein exists, functional redundancy is 21

common in bacterial type III secretion systems (50). 22


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Model of the role of Dia1 in Shigella protrusion formation. 1

The spread of intracellular Shigella from an infected cell into an adjacent cell has long 2

been thought to depend on the formation of actin-based bacterial protrusions from the 3

surface of the infected cell. Our findings constitute the first demonstration that activation 4

of Dia is required for the formation of actin-based cell surface projections and spread 5

during microbial infection. Moreover, the observation that intercellular spread is 6

significantly diminished when protrusion formation is inhibited establishes a direct 7

correlation between protrusion formation and intercellular spread. 8

The role of Dia1 in enhancing Shigella protrusion formation may reflect its function in 9

the maintenance and remodeling of the cellular cortical actin network. The cell cortex, 10

lying just beneath the plasma membrane, contains a dynamic and dense network of 11

actin filaments. The cortical actin network is continually remodeled, and its remodeling 12

is critical to the maintenance of cell shape and to cell motility. Actin polymerization in the 13

cell cortex is regulated by RhoA through its effectors Dia1 and Dia2 (1, 39, 48). Our 14

results may reflect a role of Dia in enhancing Shigella protrusion formation through 15

increased remodeling of the cortical actin network. Based on our findings, we suggest a 16

model in which Shigella directs the reorganization of cortical actin into an orientation 17

that is perpendicular to the plasma membrane and utilizes the force generated by the 18

formation of parallel arrays of polymerized actin to push out against the plasma 19

membrane. 20

In addition to promoting remodeling of the actin cortex, the role of Dia in protrusion 21

formation may also be due to a direct role in force generation at the plasma membrane. 22


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Formins generate 1.3 pN or more of force per actin filament (22). Forces of tens of pN 1

are thought to be required to enable a filopodium to protrude against the resistance of 2

the plasma membrane (9). Such a force could be generated by formins associated with 3

a bundle of actin filaments. To generate a plasma membrane protrusion, bacteria must 4

gather similar force using the resources of the cell. Our data are consistent with the 5

formin Dia1 being responsible for generating part or all of the force that is required in 6

this process during S. flexneri infection. 7

Shigella-induced actin polymerization in the cell body depends on localized activation of 8

the Arp2/3 complex by N-WASP at the bacterial surface (11, 14). Our results 9

demonstrate that whereas Dia is not required for actin tail assembly in the cell body, it is 10

required for the efficient formation of protrusions and intercellular spread. Moreover, 11

Dia1 recruitment to the bacteria is enriched at the cell periphery (Fig. 6 and Fig. 7D-E). 12

These findings indicate that when the bacteria reach the cell cortex or plasma 13

membrane, a switch likely occurs, leading to the activation of Dia-mediated actin 14

polymerization. This is similar to a switch that occurs during vaccinia virus infection. 15

Vaccinia move from the peri-Golgi region to the cell periphery by microtubule-based 16

motility (33); at the plasma membrane, viral particles switch from microtubule-based 17

motility to actin-based motility (29). How Shigella induce this switch and which bacterial 18

proteins are involved in this process will be the subject of further investigation. 19

The process of Shigella spread from one cell into an adjacent cell involves the uptake of 20

a plasma membrane bound bacterial protrusion by the adjacent cell in a process that 21

resembles macropinocytosis (37). It is possible that, in addition to playing a role in 22

Shigella protrusion formation, Dia is involved in the uptake of the protrusion by the 23


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25

adjacent cell. Our results do not directly address this. Moreover, although it is unknown 1

whether the uptake by the adjacent cell mechanistically mimics the initial entry of the 2

bacterium into cells from the extracellular milieu, our data on entry indicate that Dia has 3

little or no role in initial entry. 4

Distinct interactions with the RhoA-Dia pathway by different microorganisms. 5

In sharp contrast to the requirement for activation of Dia in S. flexneri spread is the 6

requirement for inhibition of the RhoA-Dia pathway for productive infection with vaccinia 7

virus. The vaccinia protein F11 inhibits the RhoA-Dia pathway and enhances vaccinia 8

release from cells (4, 46). Moreover, expression of constitutively active RhoA or Dia 9

inhibits viral particle accumulation at the cell periphery, actin tail formation on viral 10

particles at the cell periphery, and viral release from cells (4). 11

The observation that divergent effects on the RhoA-Dia pathway can lead to similar 12

outcomes, namely exit of microorganisms from the cell, suggests that the two 13

organisms have evolved distinct mechanisms for manipulating the cortical cytoskeleton 14

during spread. Specific differences in how the microorganisms move to the cell 15

periphery may be at the core of the differences in these mechanisms of exit. Vaccinia 16

virus moves to the cell periphery by microtubule-based motility (33), whereas Shigella 17

move to the periphery by polymerization of actin tails (5, 24). The findings of Arakawa et 18

al. (4) suggest that vaccinia inhibition of the RhoA-Dia pathway induces reorganization 19

of the cortical cytoskeleton that facilitates the movement of the virus through the cortical 20

cytoskeleton to the plasma membrane. Our data suggest a model in which, once 21

Shigella arrive at the plasma membrane via N-WASP dependent actin-based motility, 22


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their activation of the RhoA-Dia pathway enables Dia-dependent actin polymerization 1

and reorganization of the actin cortex in such a way as to enable the bacteria to push 2

outward from the cell surface and into adjacent cells. 3


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Acknowledgements 1

We thank D. Colon for technical assistance and B. Cormack, H.N. Higgs, R.R. Isberg, L. 2

Tsiokas, and N. Watanabe for providing reagents. This research was supported by 3

National Institutes of Allergy and Infectious Diseases grants AI052354 (to S.B. Snapper 4

and M.B.G.), AI073967 (to M.B.G.), and AI081724 (to M.B.G.), by a Harvey Fellowship 5

of the Mustard Seed Foundation (to J.E.H.), and by funds from the Executive 6

Committee on Research of the Massachusetts General Hospital (to M.B.G.). 7


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Salmonella inositol polyphosphatase acts in conjunction with other bacterial 22


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effectors to promote host cell actin cytoskeleton rearrangements and bacterial 1

internalization. Mol Microbiol 39:248-259. 2

3

4


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Figure Legends 1

Fig. 1. Inhibition of the formation of bacterial plasma membrane protrusions by 2

expression of the DID in cells. (A) Schematic drawing of Dia1 regulation. Left, open 3

conformation; right, autoinhibited conformation. DID, diaphanous inhibitory domain; *, 4

A256D substitution, which abrogates binding of the DID to the DAD; FH1, formin 5

homology 1 domain; FH2, formin homology 2 domain; DAD, diaphanous auto-regulatory 6

domain. Adapted from (17). (B-D) S. flexneri infection of PtK2 cells that have been 7

transfected with plasmids encoding DID-EGFP (B), EGFP only (control) (C), or DID 8

(A256D)-EGFP (D). Left panels, phase; second column, phalloidin labeling of 9

polymerized actin; third column, DAPI staining of bacterial and cellular DNA; right 10

panels, overlay of phalloidin (red) and DAPI (blue). Asterisks, transfected cells, 11

identified by GFP signal (not shown); arrowheads, bacteria at the cell periphery but not 12

forming protrusions; arrows, bacterial protrusions. Size bar, 20 um. Images are 13

representative. 14

Fig. 2. Long cellular extensions formed occasionally in DID-transfected cells. S. 15

flexneri infection of PtK2 cells that have been transfected with DID-EGFP (A) or EGFP 16

only (control) (B). Top panels, phase; second row, labeling of polymerized actin with 17

phalloidin; third row, DAPI staining of bacterial and cellular DNA; bottom panels, overlay 18

of phalloidin (red) and DAPI (blue). Asterisk, transfected cell, identified by GFP signal 19

(not shown); arrowheads, bacteria at the tip of a long cellular extension; arrows, 20

bacterial protrusions. Size bar (B, bottom panel), 20 um. Images are representative. 21


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Fig. 3. Expression of Dia1 and Dia2 and their reduction by siRNA. (A) Expression of 1

Dia1 and Dia2 in PtK2 and HeLa cells. (B) Reduction of expression of Dia1, Dia2, or 2

both in HeLa cells, using shRNA constructs specific to Dia1, Dia2, or a combination of 3

both constructs. Western blots with loading normalized, using antibodies specific to 4

Dia1 and Dia2; β actin levels confirmed loading. Molecular weight standards are 5

indicated in kilodaltons to the left of the blots. Results are representative of those 6

obtained in three or more independent experiments. 7

Fig. 4. Inhibition of the formation of bacterial plasma membrane protrusions by 8

depletion of Dia1 or Dia2 in HeLa cells. (A) Reduction of expression of Dia1 or Dia2 9

in HeLa cells, using siRNA specific to Dia1, Dia2, or a non-targeting control. Western 10

blots with loading normalized, using antibodies specific to Dia1 and Dia2; β actin levels 11

confirmed loading. Molecular weight standards are indicated in kilodaltons to the left of 12

the blots. Results are representative of those obtained in three or more independent 13

experiments. (B-D) S. flexneri infection of HeLa cells that have been reverse transfected 14

with siRNA specific to Dia1 (B), Dia2 (C), or a non-targeting control siRNA (D). Left 15

panels, phase; second column, phalloidin labeling of polymerized actin; third column, 16

DAPI staining of bacterial and cellular DNA; right panels, overlay of phalloidin (red) and 17

DAPI (blue). Arrowheads, bacteria at the cell periphery but not forming protrusions; 18

arrows, bacterial protrusions. Size bar, 20 um. Images are representative. 19

Fig. 5. Reduction of intercellular spread upon inhibition or depletion of Dia in 20

cells. Infection with GFP-expressing S. flexneri of PtK2 cells that have been transfected 21

with plasmids encoding DID-DsRed (A), DsRed only (B), or DID (A256D)-DsRed (C). Left 22


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panels, phase; second column, GFP; third column, DsRed; right panels, overlay of GFP 1

(green) and DsRed (red). Arrows, bacteria that have spread from the apparent primarily 2

infected cell into an adjacent cell. Size bar, 20 um. Images are representative. (D) Mean 3

area of spread of S. flexneri through HeLa cell monolayers, determined by plaque 4

assay, following RNAi to Dia1. Data are mean ± S.D. of results normalized to the area 5

of spread through the control RNAi-treated monolayer, from three independent 6

experiments. 7

Fig. 6. Localization of Dia1, DID, and DID (A256D) in S. flexneri infected cells. S. 8

flexneri infection of PtK2 cells that have been transfected with Myc-Dia1 (A-C), Myc-DID 9

(D), or Myc-DID (A256D) (E). (C) Higher magnification view of bacterial protrusions in 10

panel (A). The signal from cells transfected with Myc alone and labeled in a similar 11

fashion was weak and diffuse (not shown). Left panels, Myc; second panels, phalloidin 12

labeling of polymerized actin; third panels, DAPI staining of bacterial and cellular DNA; 13

fourth panels, overlay of Myc (red), phalloidin (green), and DAPI (blue); and right 14

panels, phase. Arrows, co-localization of Dia1 with bacterial actin tails within 15

protrusions. Arrowheads, co-localization of Dia (A-B) or DID (D) with bacteria at cell 16

periphery. Size bars: A-B and D-E (E, right panel), 20 um; C (right panel), 5 um. Images 17

are representative. 18

Fig. 7. IpgB2 effects on stress fibers and S. flexneri intercellular spread. (A-B) 19

IpgB2 induces stress fibers in cells. Stress fibers in cells co-transfected at a ratio of 1:20 20

with EGFP and Myc-IpgB2 (A) or Myc alone (B). Left panels, labeling of polymerized 21

actin with phalloidin; second panels, GFP fluorescence; third panels, phase; right 22

panels, higher magnification view of phalloidin staining of transfected cell. (C) ipgB2 S. 23


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flexneri are not defective in intercellular spread. Mean area of plaques formed by the 1

wildtype and ipgB2 mutant in HeLa cell monolayers. Data are mean ± S.D. of results 2

normalized to the wildtype strain from three or more independent experiments. (D-E) 3

Recruitment of Dia1 to intracellular S. flexneri is independent of IpgB2. HeLa cells that 4

have been transfected with Myc-Dia1 and subsequently infected with either wildtype (D) 5

or (E) ipgB2 S. flexneri. Left panels, Myc; middle panels, DAPI staining of bacterial and 6

cellular DNA; and right panels, phase. Upper images within each panel are focused on 7

the plane of the cell body and lower images within each panel are focused on 8

protrusions that are in a plane of focus just above the cell body. Arrows, bacteria in the 9

periphery of the cell body that co-localize with a strong signal from Myc-Dia1. 10

Arrowheads, bacterial protrusions that co-localize with a strong signal from Myc-Dia1. 11

Size bars, A-B (B, third and right panels) and D-E (E, right panel), 20 um. Images are 12

representative. 13 on January 9, 2020 by guesthttp://iai.asm

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Table 1. Inhibition of S. flexneri formation of plasma membrane protrusions in PtK2 cells 1

by the Dia1 DIDa 2

Bacteria in protrusions Bacteria within the cell body

Frequency

(%

control)

Length of

tails (um)

Frequency of

tail formation

(% control)

Length of

tails (um)

Speed of

moving

bacteria

(um/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 N.D.

EGFP alone 100b, c 5.1 ± 0.95 100 ± 12 3.5 ± 1.4d, e 7.1 ± 0.8f

a Data are mean ± S.D. of results from three independent experiments. Protrusions and 3

the frequency of protrusion formation were defined as described in Materials and 4

Methods. 5

b p = 0.002; c p = 0.6; d p = 0.8; e p = 0.3; f p = 0.1. 6

N.D., not done. 7


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Table 2. Inhibition of S. flexneri formation of plasma membrane protrusions in HeLa 1

cells by depletion of Dia1 or Dia2 RNAa 2

Bacteria in protrusions Bacteria within the cell body RNAi target

Frequency

(% control)

Length of

tails (um)

Frequency of

tail formation

(% control)

Length of tails

(um)

Dia1 42 ± 8b 6.1 ± 2.9g 92 ± 12h 3.3 ± 0.9i

Dia2 34 ± 9c 9.6 ± 3.4g 95 ± 27h 3.2 ± 0.5i

Dia1 &

Dia2

51 ± 10d 7.0 ± 4.0g 117 ± 49h 2.9 ± 1.0i

shRNA

vector only 100b, c, d 10.3 ± 1.5g 100h 4.1 ± 2.8i

Dia1 60 ± 13e N.D. N.D. N.D.

Dia2 53 ± 21f N.D. N.D. N.D.

siRNA

control 100e,f N.D. N.D. N.D.

a Data are mean ± S.D. of results from three independent experiments. Protrusions and 3

the frequency of protrusion formation were defined as described in Materials and 4

Methods. 5


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b, c p = 0.02; d p = 0.04; e p = 0.03; f p = 0.02; g, h, i compared to vector only, p > 0.5 for 1

each result. 2

N.D., not done. 3


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Table 3. Inhibition of S. flexneri intercellular spread by the Dia1 DIDa 1

Infection of adjacent cells

(number of cells)

DID-DsRed 0.5 ± 0.4b, c

DID(A256D)-DsRed 1.3 ± 0.2c

DsRed alone 1.5 ± 0.3b

a The efficiency of bacterial spread into adjacent cells during the initial 3-4 hr of 2

infection, assessed using a spreading assay (see Materials and Methods). Data 3

represent the number of cells in the infectious focus that are infected, excluding the 4

primarily-infected cell. Data are mean ± S.D. of results for 6-20 foci of infection per 5

experiment from three independent experiments. 6

b p = 0.03;, c p = 0.04. 7

8


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