www.sciencemag.org/cgi/content/full/330/6002/390/DC1 Supporting Online Material for Salmonella Pathogenesis and Processing of Secreted Effectors by Caspase-3 C. V. Srikanth, Daniel M. Wall, Ana Maldonado-Contreras, Haining Shi, Daoguo Zhou, Zachary Demma, Karen L. Mumy, Beth A. McCormick* *To whom correspondence should be addressed. E-mail: [email protected]Published 15 October 2010, Science 330, 390 (2005) DOI: 10.1126/science.1194598 This PDF file includes: Materials and Methods Figs. S1 to S6 Tables S1 and S2 References
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Published 15 October 2010, Science 330, 390 (2005) DOI: 10.1126/science.1194598
This PDF file includes:
Materials and Methods Figs. S1 to S6 Tables S1 and S2 References
1
Supporting online material for
Salmonella pathogenesis and processing of secreted effectors by caspase-3
C.V. Srikanth1,2*, Daniel M. Wall1,3,*, Ana Maldonado-Contreras2, Haining Shi1, Daoguo Zhou 4,Zachary Demma 2, Karen L. Mumy1,2, and Beth A. McCormick1,2
1 Department of Pediatric Gastroenterology and Nutrition, Harvard Medical School and
Massachusetts General Hospital, Boston, MA 02129, USA.
2 Department of Molecular Genetics and Microbiology, University of Massachusetts Medical
School, 55 Lake Avenue North, Worcester, MA 01655, USA.
3 Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life
Sciences, University of Glasgow, G12 8QQ, UK.
4 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907
* Proteins reported having differentfunctions related to the N and C-terminus or specific protein region
Analysis of the primary acid amino sequence. Thecaspase-3 motifs are highlighted in red.
Figure Legends to Supplemental Data
Fig. S1. Time course of caspase-3 activation. T84 cells were infected with either wild-
type S. Typhimurium (SL1344) or an isogenic invasion deficient strain (vv341) over a
four-hour time course. Immunoblots were performed with anti-caspase-3 antibody (Cell
Signaling Technology). A-Casp3 represents the activated form of the enzyme.
Fig. S2. Analysis of PMN infiltration. A) PMN infiltration into the proximal colon was
quantified by tissue myeloperoxidase activity (MPO) in mice following a 48 hour
infection with the ∆SipA, ∆SipA/pSipA, and ∆SipA/pCSM-SipA strains. MPO activity
was measured using the Mouse MPO ELISA test kit from Cell Sciences. Data are
expressed as the mean ± SD and represent groups of five mice. (*), P < 0.01 when the
data set for a given infection was compared to the wild-type (SL1344) S. Typhimurium.
B) Fluorescently stained proximal colon sections at 20x magnification. Sections were
stained with DAPI (blue) and PMNs were stained using a FITC-labeled antibody (green)
specific for the PMN surface markers Ly-6G and Ly-6C. Shown is a comparison between
the SipA complemented strain (∆SipA/pSipA) and the caspase-3 site mutant
complemented strain (∆SipA/pCSM-SipA). (C) Bone-marrow-derived macrophages from
wild-type and caspase-3-/- (caspase-3 KO) mice were infected (at a multiplicity of
infection of 10) and the bacterial colony forming units (cfu) were determined 4 hour post
infection.
Fig. S3. Pharmacologic inhibition of caspase-3. A) The effect of the specific caspase-3
inhibitor on S . Typhimurium-induced PMN transepithelial migration across T84
monolayers. Prior to infection, T84 monolayers were exposed to buffer only (black bars),
or the caspase-3 inhibitor for 2 hour. (B) The effect of the specific caspase-1 inhibitor on
S. Typhimurium-induced PMN transepithelial migration across T84 monolayers. PMN
migration across uninfected T84 monolayers in the presence of 1 µM PMN
chemoattractant (fMLP), or buffer served as the positive and negative controls,
respectively. The data are expressed as the mean ± SD of triplicate samples and represent
one of at least three independent experiments performed with similar results. (CE), cell
equivalents, (*), P < 0.01.
Fig. S4. Quantitative RT-PCR analysis of caspase-3 expression. Caspase-3 mRNA
production in response to S. Typhimurium infection was monitored over the first 2 hour
of infection. Caspase-3 mRNA levels were normalized to GAPDH transcript levels and
the normalized values of the Salmonella-infected versus the control uninfected cells were
used to calculate and plot caspase-3 mRNA relative expression. The data are expressed as
the mean ± SD of triplicate samples and represent one of at least three independent
experiments performed with similar results.
Fig. S5. The effect of the exogenous addition of caspase-3 on S. Typhimurium-induced
PMN transepithelial migration across T84 monolayers. During S. Typhimurium wild-type
infection, T84 monolayers were exposed to buffer (black bars), caspase-3 (20 µM; white
bars), or caspase-1 (20 µM; gray bars) for 1 hour. PMN migration across uninfected T84
monolayers in the presence of 1 µM PMN chemoattractant (fMLP), or buffer only served
as the positive and negative controls, respectively. The data are expressed as the mean ±
SD of triplicate samples and represent one of at least three independent experiments
performed with similar results. (CE), cell equivalents, (*), P < 0.05.
Fig. S6: Cell lysis in response to infection with S. Typhimurium. Lactate dehydrogenase
(LDH) activity in the supernatant of T84 infected cells was measured over 4 hour as an
indicator of cell lysis in response to infection. Wild-type S. Typhimurium (SL1344) and
the SipA negative mutant strain SipA- and HilA-, an invasion deficient strain, were used
to infect T84 cells for 1 hour and LDH release was quantified using an In Vitro Toxicity
Assay kit (Sigma-Aldrich). One percent Triton and HBSS+ buffer were used as positive
and negative controls, respectively. The data are expressed as the mean ± SD of triplicate
samples and represent one of at least three independent experiments performed with
similar results.
References:
1. K. Dharmsathaphorn, J. L. Madara, Methods Enzymol 192, 354 (1990).2. J. L. Madara et al., J Clin Invest 89, 1938 (Jun, 1992).3. D. M. Wall et al., Cell Microbiol 9, 2299 (Sep, 2007).4. C. A. Lee, S. Falkow, Proc Natl Acad Sci U S A 87, 4304 (Jun, 1990).5. B. A. McCormick, S. P. Colgan, C. Delp-Archer, S. I. Miller, J. L. Madara, J Cell
Biol 123, 895 (Nov, 1993).6. C. A. Parkos, C. Delp, M. A. Arnaout, J. L. Madara, J Clin Invest 88, 1605 (Nov,
1991).7. M. Barthel et al., Infect Immun 71, 2839 (May, 2003).8. C. C. Chen, S. Louie, B. McCormick, W. A. Walker, H. N. Shi, Infect Immun 73,
5468 (Sep, 2005).9. R. C. Burns et al., Gastroenterology 121, 1428 (Dec, 2001).10. F. Loher et al., J Pharmacol Exp Ther 305, 549 (May, 2003).11. T. R. Brummelkamp, R. Bernards, R. Agami, Science 296, 550 (Apr 19, 2002).12. B. A. McCormick, B. A. Stocker, D. C. Laux, P. S. Cohen, Infect Immun 56, 2209