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The Multivalent Adhesion Molecule SSO1327 plays a key role in Shigella
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sonnei pathogenesis
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Supporting Information
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Contains Figures S1-S7, Tables S1-S2.
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Rasha Y. Mahmoud1, 3, Daniel Henry Stones2, Wenqin Li1, Mohamed Emara3, Eldomany R A3,
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Depu Wang4, Yili Wang5, Anne Marie Krachler2, Jun Yu1,*
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Strathclyde Institute of Pharmacy and Biomedical Sciences (SIPBS), University of Strathclyde,
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Glasgow, UK
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Institute of Microbiology and Infection, School of Biosciences, University of Birmingham,
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Edgbaston, B15 2TT Birmingham, UK
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Department of Microbiology and Immunology, Faculty of Pharmacy, Helwan University, Cairo,
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Egypt
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The center of Translational Medicine, The First Affiliated Hospital, and
Institute for Cancer Research, School of Basic Medical Science, Health Science Center, Xi’an
Jiao Tong University, Xi’an, China
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*
Correspondence to: Jun Yu (jun.yu@strath.ac.uk)
SSO1327 is an adhesin required for S. sonnei pathogenesis
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Figure S1. S. sonnei MAM SSO1327 organization and construction of deletion and
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complementation strains. S. sonnei MAM SSO1327 domain organization. The protein consists
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of an N-terminal hydrophobic region of 41 residues (H) and seven putative MCE domains (MCE)
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(A). Genetic organization and context of Shigella Multivalent Adhesion Molecule orthologs.
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Genetic context of MAM orthologs in S. sonnei (SSO1327), S. boydii (SBO1249), S. dysenteriae
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(SDY1985) and the pseudogene (cross) in S. flexneri (Sf1391) was visualized using the ShiBASE
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database for comparative genomics (Yang et al, 2006). MAM orthologs are highlighted in red
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SSO1327 is an adhesin required for S. sonnei pathogenesis
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and insertion sequences (Is) are indicated by bars (B). Deletion of SSO1327 from S. sonnei
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20071599 using the method of Dadsenko and Wanner method (Datsenko and Wanner, 2000),
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(C). Construction of the kanamycin cassette containing strain (MAM-kan), removal of the kan
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cassette to give the non-polar SSO1327 deletion strain (MAM), and complementation with
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MAM-His was confirmed by PCR, using primers c and d (Table S1), (D and E).
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Figure S2. Analysis of -lactamase activity for S. sonnei reporter strains. S. sonnei wild type
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(negative control), wild type or MAM containing chromosomal ipgD-bla fusions, or wild type
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containing pGEM T-Easy (ampR, bla secretion, but not translocation) were tested for -
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lactamase activity. Pure colonies for each strain were picked, suspended in PBS and directly
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applied to nitrocefin discs. Discs were imaged after incubation at 22 °C for 30 minutes. Red
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coloration indicates -lactamase activity.
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SSO1327 is an adhesin required for S. sonnei pathogenesis
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Figure S3. S. sonnei infection causes apoptosis in G. mellonella larval hemocytes. Total DNA
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was isolated from mock-infected and S. sonnei wild type infected, or mock-infected and S. sonnei
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ΔMAM larvae and analyzed for chromatin fragmentation by agarose gel electrophoresis, as
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described previously (Zychlinsky et al., 1994).
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Figure S4. MAM beads protect G. mellonella larvae against S. sonnei infection. G.
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mellonella larvae were injected with 106 CFU S. sonnei wild type and buffer (black squares), or
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mixtures containing 106 CFU S. sonnei and 25 (empty triangles), 19 (black triangles), 12.5 (black
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circles) or 6 M (empty circles) MAM beads. Mortality rates over 5 days were analyzed using
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Kaplan-Meier survival curves.
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SSO1327 is an adhesin required for S. sonnei pathogenesis
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Figure S5. MAM beads do not interfere with TTSS effector production or secretion. S.
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sonnei wild type was grown to mid-log phase under inducting conditions and treated with buffer
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(wt), control GST beads (wt+cont) or MAM beads (wt+MAM). Cell lysates and culture
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supernatants were analyzed for the presence of type III system secreted effectors by SDS-PAGE
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and Western Blotting with antibodies against IpaB and IpaC.
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SSO1327 is an adhesin required for S. sonnei pathogenesis
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Figure S6. Effect of deoxycholate on S. sonnei virulence in vivo. Guinea pig eyes were
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inoculated with 108 CFU of wild type S. sonnei cultured in L-broth (top panels) or L-broth
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containing 2.5 mM of DOC (bottom panels). Full keratoconjunctivitis was developed within 3
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days in all animals inoculated with bacteria cultured in L-broth. Mild conjunctivitis was observed
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within 3 days in all guinea pigs inoculated with bacteria cultured in L-broth containing 2.5 mM
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DOC. Results shown are representative of experiments carried out in 3 animals. (B) Effect of
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DOC on S. sonnei virulence in the G. mellonella larvae model. 10 larvae were included in each
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group. Bacteria were cultured in L-broth or L-broth containing 2.5 mM DOC overnight. 105 CFU
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from cultures of S. sonnei were injected to each larvae of the experimental groups. Survival was
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scored for up to 5 days and analyzed using Kaplan-Meier survival curves.
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SSO1327 is an adhesin required for S. sonnei pathogenesis
2.5
w ild type
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∆MAM
∆icsA
OD600nm
∆MAM∆icsA
1.5
1
0.5
0
00:00 01:00 02:00 02:30 03:00 03:30 04:00 04:30 05:00
Time (h)
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Figure S7. Growth curves of S. sonnei strains. Fresh overnight cultures were sub-cultured with
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1:100 dilutions in L-broth, and incubated at 37 oC with shaking at 200 rpm. Samples were taken
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at indicated time intervals to measure optical density (OD) at 600 nm.
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SSO1327 is an adhesin required for S. sonnei pathogenesis
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Table S1. Primers used in this study.
Gene
name
MAM
purpose
primers
Deletion
MAM
Sequencing
MAM
Cloning
His-Tag
icsA
Deletion
icsA
Sequencing
icsA
Cloning
icsA
Cloning
dsbA
Deletion
dsbA
Sequencing
dsbA
Cloning
His-Tag
Primers a,b
MAM_F 5’ATGCACATGAGTCAGGAAACGCCCGCTTCGACGACTGAAGCG
CAGATTAAATGTGTAGGCTGGAGCTGCTTCG-3’
MAM_R 5’TTATTTGGGAAGCGCAGTACCCCATTCACGCCACTCTTTCGGT
TCACTTTCTATGGGAATTAGCCATGGTCC-3
Primers c,d
MAM_seq_F 5’-CACATGAGTCAGGAAACGCC-3’
MAM_seq_R 5’- CCCCATTCACGCCACTCTTT-3’
Primers e,f
MAM_Clon-F 5’- ATGCACATGAGTCAGGAAACG-3’
MAM_Clon-R 5’TTAGTGGTGATGGTGATGATGTTTGGGAAGCGCAGTACC-3’
Primers g,h
IcsA_F 5’ATGAATCAAATTCACAAATTTTTTTGTAATATGACCCAATGTT
CACAGGGG TGTGTAGGCTGGAGCTGCTTCG-3’
IcsA_ R 5’TCAGAAGGTATATTTCACACCCAAAATACCTTGGGTGTCTCTG
TAACTGTT TATGGGAATTAGCCATGGTCC-3’
Primers i,j
IcsA -seq-F 5’-ATGAATCAAATTCACAAATT-’3
IcsA -seq-R 5’-TCAGAAGGTATATTTCACAC-’3
Primers k,l
IcsA -Clon-F 5’- CCCGAATTCTCGAACATATAGCTTTCCCCC-’3
IcsA -Clon-R 5’CCCGTCGACGAAGGTATATTTCACACCCAAAAT-’3
Primers k_Pst, l_Pst
k_Pst: 5’- CCCTGCAGTCGAACATATAGCTTTCCCCC-3’
l-Pst: 5’- CCCTGCAGTCAGAAGGTATATTTCACACCCAAAAT-3’
Primers m,n
dsbA_F5’ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTT
TAGCGTTTAGCGCATGTGTAGGCTGGAGCTGCTTCG-3
dsbA_R5’TTATTTTTTCTCGGACAGATATTTCACTGTATCAGCAT
ACTGCTGAACAAATATGGGAATTAGCCATGGTCC’3
Primers o,p
dsbA -seq-F 5’ATGAAAAAGATTTGGCTGGC’3
dsbA -seq-R 5’TTATTTTTTCTCGGACAGAT’3
Primers q,r
dsbA -clon-F 5’ATGAAAAAGATTTGGCTGGC’3
SSO1327 is an adhesin required for S. sonnei pathogenesis
ipgD
pKD4
pKD46
icsA
MAM
cycG
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dsbA -clon-R
5’TTAGTGGTGATGGTGATGATGTTTTTTCTCGGACAGAT’3
Gene-fusion Primers s,t
TEM-fusion_F 5’ATAGGGGACTCAAAAATATGGAATATGGTGAAAGGGTATTCG
TCATTTGTACTGGTGAAAGTAAAAGATGCT-3’
TEM-fusion_R 5’GATACCCAAGGCTCGGCAAATAACATCTGCTAAATCTTCCATA
TATTCCTCTTACCAATGCTTAATCAGTGAG-3’
PCR
Primers u,v
pkD4_F 5’- GGGAGATCTGTGTAGGCTGGAGCTGCTTC-3’
pkD4_R 5’- GGGAGATCTCATATGAATATCCTCCTTA-3’
PCR
Primers w, x
pkD46_inv_1 5’-GGGAGATCTTTATTGTCTCATGAGCGG-3’
pkD46_inv_2 5’-GGGAGATCTCTCATGACCAAAATCCCTTA-3’
RT-PCR
Primers y,z
IcsA-RT-F 5’CTAGGCATGGGGTAAAAGCA’3
IcsA-RT-R 5’ CGGTTGCCTATCTGGTGATT’3
RT-PCR
Primers y1,z1
MAM-RT-F 5’ GGTATTGGTACGCCTGTGCT’3
MAM-RT-R 5’ TTAAAGGTGCCGGTTTTCAC’3
Primers y2,z2
RT-PCR
cycG-RT-F 5’ CATCGCGATTATGCCCAGAG’3
cycG-RT-R 5’ GTGAGCGTACCGTCAATCAC’3
SSO1327 is an adhesin required for S. sonnei pathogenesis
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Table S2. Strains used in this study.
Strain
Shigella sonnei strain
20071599
S. sonnei ΔMAM
S. sonnei ΔMAM+pMAMHis
S. sonnei ΔicsA
S. sonnei ΔicsA+pIcsA
S. sonnei ΔMAM/ΔicsA
S. sonnei ΔMAM/ΔicsA+
pMI
S. sonnei wild type:ipgD-bla
S. sonnei ΔMAM:ipgD-bla
S. sonnei ∆dsbA+pDsbA
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Description
Wild type strain used in this study.
Source
(Xu et al.)
SSO1327 (MAM) deletion strain
The ΔMAM mutant complemented with
pGEM®-T Easy (Promega) expressing MAM
gene fused to C-terminal 6xHis tag; ampR
icsA deletion mutant
icsA deletion mutant complemented with pET28a
expressing icsA; kanR
MAM and icsA double deletion mutant
The double mutant strain was complemented
with cloned MAM and icsA in pGEM®-T-Easy
vector
Wild type strain carrying a chromosomal ipgDbla in frame fusion on the chromosome.
The ∆MAM mutant carrying an ipgD-bla in
frame fusion on the chromosome.
dsbA deletion mutant carrying cloned wild type
dsbA gene with C-terminal 6Xhis-tag in
pGEM®-T-Easy vectors, ampR
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