Cell Host & Microbe, Volume 15

Supplemental Information

IcsA Is a Shigella flexneri Adhesion Regulated by the Type III Secretion System and Required for

Pathogenesis

Anna Brotcke Zumsteg, Christian Goosmann, Volker Brinkmann, Renato Morona, and Arturo Zychlinsky

Table S1 Strain List, related to Figures 1 and 3.

All strains are isogenic mutants in the M90T background using lambda red recombination to introduce antibiotic resistance cassettes as described in (Ranallo et al., 2006). The sequence corresponding to priming sites 1 and 2 in pKD3 and pKD4 are underlined. pMW211 is a kind gift from Dr. Dirk Bumann and is described in reference (Sorensen et al., 2003).

Table S2 Plasmid List, related to Figures 1 and 3.

Plasmid Source Primers Notes on Construction pUC19 Invitrogen pBR322 (May and

Morona, 2008)

pBAD/Myc-his A

Invitrogen

ipaD pUC19 This study ipaD_F_BamHI GCGCGCggatccatgaatataacaactctgac ipaD_R_EcoRI GCGCGCgaattcatggcgcacctcagaaatgg

ipaD was inserted into the BamHI and EcoRI sites in pUC19.

pIcsA (May and Morona, 2008)

IcsA inserted into pBR322. See original paper for description.

icsA pBAD This study IcsA_NcoI_F GCGCGCccatggatcaaattcacaaatttttttgt IcsA_XhoI_R GCGCGCctcgagtgaaggtatatttcacaccca

IcsA was inserted into the NcoI and XhoI sites in pBAD/Myc-his. Stop codon was included to prevent fusion with the myc and his tags.

spa33pBAD ” spa33_NcoI_F GCGCGCccatggtaagaattaaacattttgacgct spa33_HindIII_R_ GCGCGCaagcttttactcctttaccatccaagaac

Spa33 was inserted into the NcoI and HindIII sites in pBAD/Myc-his. Stop codon was included to prevent fusion with the myc and his tags.

IcsA-HA pUC19

” IcsA_F_BamHI GCGCGCggatcccggtttgaagcagactatcata HA_tag_R_PstI GCGCGCctgcagagcgtaatctggaacatcgtatgggtagctcatctgttttgattcttga HA_tag_F_PstI GCGCGCctgcagtacccatacgatgttccagattacgctaatcaagagtctactcaaat IcsA_R_EcoRI GCGCGCgaattctgcaggacatcaacacgccct

IcsA was amplified in 2 pieces using IcsA F and HA tag R or HA tag F and IcsA R, with primers that incorporate the HA tag. Ends were digested using the indicated restriction enzymes and then the two pieces were ligated together with digested pUC19. Insertion of tag was confirmed by sequencing. HA tag is inserted at aa position 748, 10 residues before the IcsP cleavage site.

pIcsA library (Named KMRM1-67)

(May and Morona, 2008)

Described in May and Morona, 2008. Amino acid insertions are randomly distributed throughout the alpha domain of IcsA and were created using Mutation Generation System (Finnzymes).

pIcsAadhesion- This study KMRM5 (pIcsA i386) and KMRM44 (pIcsA i148) were digested with PsiI, two generate two pieces.

The small 750 bp fragment from KMRM5 was ligated with the large 8.36 kb fragment from KMRM44, resulting in a plasmid which contained both insertions, which was confirmed by sequencing.*

Cloning was carried out using standard methods and restriction sites within the primer sequence are underlined.

* The insertions in IcsAadhesion- are unlined in the following sequence, with 12 bp of upstream and downstream sequence for reference:

i148 ATAATCAATCAATGCGGCCGCAATCAAGGCATGATTCTT i386 TCACTATCAGGTTGCGGCCGCACAGGTAACAAATTCACT

Table S3, related to Figures 1 and 3. Adhesion rate of S. flexneri mutants Adhesion rates are defined as fold adhesion ≤1 (-), ≤5 (+), ≤10 (++), >10 (+++), relative to the wild-type untreated sample during HeLa assays.

Strain Adhesion Phenotype BS176 - WT - Δspa33 - ΔipaC - ΔipaB + ΔipaD ++ ΔipaDΔspa33 - ΔipaBCDAΔmxiE +++ ΔipaDΔspa32 + ΔipaDΔspa15 ++ ΔipaDΔipgB1 + ΔipaDΔipgB2 ++ ΔipaDΔipgD + ΔicsA - ΔipaDΔicsA -

Figure S1, related to Figures 1 and 3 ΔipaB and ΔipaD mutants hyper-adhere to host cells when

quantified via microscopy and hyper-adherence is independent of invasion related early effectors

and Spa32 A) Hyper-adherence was quantified via microscopy using the indicated Shigella strains

expressing DsRed. Data are the mean +/- SEM of bacteria/HeLa cell and are combined from two

independent assays. Significance was calculated using a T test and p values are: ***<0.001. (B) SEM of

the ΔipaD and ΔipaDΔspa32 mutants. Note the extra long T3SS needles which are characteristic of the

ΔipaDΔspa32 mutant. (C) Adhesion rate at 15 min. of S. flexneri mutants during HeLa infections.

Adhesion rate is calculated as the output/input x100 and data are the mean +/- SEM of at least 3

independent experiments. D) Secreted proteins from overnight culture supernatants from the indicated

strains. E) Adhesion rate at 15 min. of S. flexneri mutants during Caco-2 infections. Adhesion rate is

calculated as the output/input x100 and normalized to the wild-type. Data are the mean +/- SEM of at

least 3 independent experiments.

Figure S2, related to Figure 4 LPS is not altered in hyper-adherent ΔipaB and ΔipaD strains LPS

was isolated using the small scale hot phenol extraction method described in (Apicella, 2008) from

overnight cultures of wild-type, Δspa33, ΔipaB and ΔipaD strains. LPS was run on a 12% SDS-PAGE gel

and stained using Emerald ProQ (Invitrogen).

Figure S3, related to Figure 4 An HA tagged IcsA construct can fully complement mutants for

adhesion and invasion Adhesion (A, B, C, E) and invasion (D) rate of S. flexneri and E. coli BL21

strains during HeLa infections. Adhesion and invasion rates are calculated as the output/input x100. The

experiment was performed three times and data are the mean +/- SEM from one representative

experiment. Note that the Δspa33 mutant can be partially complemented in trans by expressing of spa33

from an inducible promoter (E).

Figure S4, related to Figure 5 Deoxycholate treatment activates IcsA-dependent adhesion and this is

independent of mxiE and ospE1/2. (A) HeLa cells infected for 15 minutes with the indicated strains

grown in the presence or absence of 2.5mM DOC. Bacteria are in red, actin in green, and nuclei are in

blue. Images were taken using a 40X objective. (B) Hyper-adherence was quantified via microscopy

using the indicated Shigella strains expressing DsRed grown in the presence or absence of 2.5mM DOC.

Data are the mean +/- SEM of bacteria/HeLa cell and are combined from two independent assays.

Significance was calculated using a T test and p values are: ****<0.0001. (C) Adhesion rate of S. flexneri

strains during HeLa infections at 15 minutes. Adhesion rate is calculated as the output/input x100 and

then normalized to the wild-type untreated sample. D) Limited proteolysis assays using Δspa33ΔicsA

and ΔicsA complemented with IcsA-HA pUC19 grown in the presence of 2.5mM DOC. Two

additional independent experiments are shown to demonstrate the reproducibility of the assay.

Aliquots were taken at 0, 1, 3,10, 30 and 60 minutes post cleavage with 34nM NE and IcsA

degradation was assessed by western blot using an anti-HA antibody. NE resistant fragments are

indicated by a (*).

Supplemental References:

Apicella, M.A. (2008). Isolation and characterization of lipopolysaccharides. Methods Mol Biol 431, 3-13. May, K.L., and Morona, R. (2008). Mutagenesis of the Shigella flexneri autotransporter IcsA reveals novel functional regions involved in IcsA biogenesis and recruitment of host neural Wiscott-Aldrich syndrome protein. J Bacteriol 190, 4666-4676. Ranallo, R.T., Barnoy, S., Thakkar, S., Urick, T., and Venkatesan, M.M. (2006). Developing live Shigella vaccines using lambda Red recombineering. FEMS Immunol Med Microbiol 47, 462-469. Sorensen, M., Lippuner, C., Kaiser, T., Misslitz, A., Aebischer, T., and Bumann, D. (2003). Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria. FEBS Lett 552, 110-114.

% a

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CScale bar = 200 m

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Figure S1

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Figure S2

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Figure S3

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0 1 10 30 60 0 1 10 30 60 icsAspa33 icsA

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Strains expressing IcsA-HA and detected using anti-HA antibody

+ DOC

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+ DOC

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Figure S4