Accepted Article
Received Date : 28-Jan-2014 Revised Date : 05-May-2014 Accepted Date : 06-May-2014 Article type
: Research Letter
Editor
: Klaus Hantke
Enterohemorrhagic Escherichia coli OmpT regulates outer membrane vesicle biogenesis. Veena Premjani*, Derek Tilley∫, Samantha Gruenheid¶, Hervé Le Moual¶, and John A. Samis*‡
*Applied Biosciences Program, Faculty of Science, University of Ontario Institute of Technology, Oshawa, ON, Canada. ∫ School of Engineering Technology and Applied Science, Centennial College, Toronto, ON, Canada. ¶ Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada. ‡ Medical Laboratory Science Program, Faculty of Health Sciences, University of Ontario Institute of Technology, Oshawa, ON, Canada. Correspondence: John A. Samis, PhD, Faculty of Health Sciences, University of Ontario Institute of Technology, Oshawa, ON. L1H7K4. Canada. Tel: +1-905-721-8668 ext. 3760; FAX: +1-905-721-3179 email: [email protected] Keywords: Protease, Gram-negative bacteria, lipid and protein content. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12463 This article is protected by copyright. All rights reserved.
Accepted Article
Running Title: OmpT Regulates EHEC OMV Biogenesis.
Abstract Enterohemorrhagic Escherichia coli (EHEC) infection from food or water often results in severe
diarrheal disease and is a leading cause of death globally. Outer membrane vesicles (OMVs) secreted from E. coli induce lethality in mice. The omptin outer membrane protease OmpT from E. coli inactivates antimicrobial peptides and may enhance colonization of the uroepithelium; but its precise function remains unclear. Given OmpT is an outer membrane protease, we hypothesized it may have a role in OMV biogenesis. To further characterize the effect of OmpT on OMV production, a genetic approach using wild type, an ompT deletion mutant and an ompT over-expressing construct in EHEC was employed. ompT gene deletion markedly decreased OMV production and stainable lipid but increased vesicle diameter. Conversely, ompT overexpression profoundly increased OMV biogenesis but decreased stainable lipid, protein content, and vesicle diameter. Alterations in EHEC ompT gene expression has an impact on the biogenesis, composition, and size of OMVs. Changes in ompT gene expression may dynamically alter OMV formation, composition, and diameter in response to different host environments and contribute to cell-free intercellular communication to enhance bacterial growth and survival.
Introduction Gram-negative bacteria, including Escherichia coli (E. coli) constitutively produce outer membrane vesicles (OMVs) which are spherical bilayered proteolipids with an average diameter
This article is protected by copyright. All rights reserved.
Accepted Article
of approximately 20-200nm (Kuehn & Kesty, 2005). OMVs are comprised of outer membrane proteins and lipids, periplasmic proteins, lipopolysaccharide, and RNA and DNA (Lee et al., 2007). Although OMVs may function in bacterial growth and survival, transfer of nucleic acids and proteins, virulence factor delivery, and alteration of the host inflammatory and immune response (Mashburn-Warren & Whiteley, 2006); their precise role(s) during the pathogenesis of Gram-negative infections is currently unclear. Cell surface and secreted proteases have important functions in evading the host acquired and innate immune systems and as virulence factors during bacterial infections. Microbial proteases hydrolyze and inactivate host antibodies (Mistry & Stockley, 2011), complement proteins (Honda-Ogawa et al., 2013), and antimicrobial peptides (Thomassin et al., 2012). Bacterial
proteases also degrade host fibrin (Yeo et al., 2011) and extracellular matrix proteins (Ruggiero et al., 2013) which retard pathogen spread during infection. The omptin family of proteases are present in a variety of Gram-negative bacteria and have high amino acid sequence homology (Haiko et al., 2009). Omptins fold into a conserved β-barrel with
the active site exposed to the environment (Vandeputte-Rutten et al., 2001). Omptins require lipopolysaccharide as a cofactor for activity and have a cleavage preference between dibasic amino acids with Arg or Lys in the P1 and P’1 positions (Kukkonen & Korhonen 2004). The omptins Pla from Yersinia pestis and PgtE from Salmonella enterica activate host plasminogen to plasmin and inactivate the major host plasmin inhibitor, α2-antiplasmin and the major host inhibitor of plasminogen activation, plasminogen activator inhibitor 1 (Kukkonen et al., 2001;
Lahteenmaki et al., 2005; Haiko et al., 2010). PgtE of S. enterica has also been shown to activate
host matrix metalloproteases (Ramu et al., 2008). The action of Pla and PgtE would, via
This article is protected by copyright. All rights reserved.
Accepted Article
engagement of the host plasmin and matrix metalloprotease systems, be expected to disrupt the fibrin and extracellular matrix barriers to enhance microbial survival and growth. The E. coli outer membrane protease omptin OmpT has historically been considered a ‘housekeeping’ protease towards endogenous microbial and extracellular proteins (Haiko et al., 2009). There is currently no significant association of the presence and expression of OmpT with specific clinical isolates and pathogenic strains of E. coli (Kukkonen & Korhonen 2004). Further, although our previous work has shown OmpT inactivates host antimicrobial peptides (Thomassin et al., 2012) and others have shown OmpT may enhance E. coli colonization of the
uroepithelium (Hui et al., 2010); its precise function(s) in the pathogenesis of E. coli infections is presently unclear. Enterohemorrhagic Escherichia coli (EHEC) is a water- or food-borne pathogen that causes severe diarrheal disease (Nataro & Kaper 1998). EHEC cause attaching and effacing lesions that erode intestinal microvilli and promote bacterial attachment to host cell membranes (Golan et al.,
2011). Given OmpT is an outer membrane protease, we hypothesized it may have a role in the formation and/or determining lipid or protein composition of OMVs. Using a genetic approach, we demonstrate here that the presence of OmpT, like other outer membrane and periplasmic proteins, has a profound impact on the formation, composition, and diameter of OMVs.
Materials and Methods Bacterial strains and growth conditions The E. coli O157:H7 strains used in this study are listed in Table 1 (Thomassin et al., 2012).
EHEC cells were grown at 37oC with aeration at 200 rpm in Minimal A medium (60mM This article is protected by copyright. All rights reserved.
Accepted Article
K2HPO4, 33mM KH2PO4, 7.5mM ammonium sulphate, 1.7mM trisodium citrate, pH 7.0 with 2mg ml-1 glucose, 60μg ml-1 tryptone, 1mM MgSO4). EHEC cells were grown in Minimal A
medium rather than a more complex protein medium (ie LB broth) to reduce potential interference of medium proteins with immunoblotting and electrophoresis and silver staining. Liquid medium was supplemented with chloramphenicol for the ompT over-expressing strain (30 µg ml-1). Preparation of outer membrane vesicles EHEC cells were grown in Minimal A medium (10 ml) with/without chloramphenicol (30 µg ml1
) for 12-14 h at 37oC with aeration at 200 rpm. The cultures were subcultured into 1 L of fresh
Minimal A medium and incubated at 37oC without aeration for 16-24 h to an optical density at 600nm of 0.5. The cultures were centrifuged at 10,000 x g for 30 min at 4oC and supernatants
were filtered through 0.22µm vacuum filters (Sarstedt) to remove any bacteria. The pH of the supernatants was adjusted to 7.4 and concentrated approximately 33-fold (1 L to 30 ml) with a 100kDa molecular weight cutoff membrane using a tangential flow filtration capsule (Pall Life Sciences) at room temperature. Samples were further concentrated by 3-fold (30 ml to 10 ml) using 100kDa molecular weight cutoff centrifugal filters (Millipore) at 1,800 x g at 4oC for 10
min. OMVs were pelleted by ultracentrifugation at 150,000 x g (Hitachi Koki) at 4oC for 1.5 h and were resuspended in 250 μl of 20mM HEPES, 150mM NaCl, pH 7.4. OMVs were stored in sealed cryovials at -80oC and thawed/refrozen samples were not used.
Preparation of cell lysates EHEC cells were grown in Minimal A medium (1 ml) with/without chloramphenicol (30 µg ml1
) with aeration at 200 rpm to an optical density at 600nm of 0.5 and centrifuged at 10,000 x g
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for 30 min at 4oC. The pellets were resuspended in 100 μl of 20mM HEPES, 150mM NaCl, pH 7.4 and sonicated on ice for 10 min with a Dismembranator (Fisher). Cell lysates were stored in sealed cryovials at -80oC and thawed/refrozen samples were not used.
Protein assay of cell lysates and outer membrane vesicles The protein concentration of cell lysates and OMVs was determined with bicinchoninic acid using bovine serum albumin (Fisher) as the standard. Samples were read at 562nm using microplate reader (Molecular Devices) after correction with 20mM HEPES, 150mM NaCl, pH 7.4. Standard curves of absorbance at 562nm vs bovine serum albumin concentration were used to interpolate the protein concentrations of cell lysates and OMVs. Transmission electron microscopy Copper grids (Electron Microscopy Sciences) were loaded with OMVs (10 μl) for 1 min and excess volume was removed with filter paper. Grids were floated on 2 mg ml-1 uranyl acetate for
30 s, blotted with filter paper and air dried for 1 min. Specimens were examined by transmission electron microscopy (FEI Company, Model Technai) with an accelerating voltage of 200kV at 100,000 x magnification (Mount Sinai Hospital, Toronto). The number of OMVs/field and size distributions of OMVs were measured using magnification software (Orbicule). Imunoblotting for OmpT in cell lysates and outer membrane vesicles Immunoblotting for determination of OmpT protein expression in EHEC cell lysates and OMVs employed a rabbit anti-CroP IgG as described in our previous study (Thomassin et al., 2012).
Cell lysates and OMVs were added to Loading dye (62.5mM Tris pH 6.8, 125 mg ml-1 glycerol, 20mg ml-1 SDS, 250 μg ml-1 bromophenol blue, 20 μg ml-1 mercaptoethanol), heated at 95oC for
This article is protected by copyright. All rights reserved.
Accepted Article
5 min, and electrophoresed on 10% polyacrylamide under denaturing conditions (Sambrook et al., 1989) with prestained molecular weight standards (BioRad) in 25mM Tris, 192mM glycine, 1 mg ml-1 SDS, pH 8.3 for 40 min at 80V and 90 min at 100V. Protein was electroblotted onto polyvinylidene membranes (Millipore) at 35V in 25mM Tris HCl, 192mM glycine, 500 μg ml-1
SDS, 100 μl ml-1 methanol, pH 8.3 for 16 h at 4oC. Membranes were blocked with Tris-Buffered Saline-Tween (20mM Tris HCl, 500mM NaCl, 250 μl ml-1 Tween-20, pH 7.4) containing 50 mg
ml-1 non-fat dry milk powder for 1 h at room temperature. OmpT on the blots was detected with a rabbit polyclonal anti-CroP IgG (Comparative Medicine Animal Resources Centre, McGill University, 1:10,000 dilution), goat anti-rabbit IgG IgG conjugated with horse radish peroxidase (Sigma, 1:5,000 dilution), chemiluminescence reagents (PerkinElmer), and XOMAT film
(Kodak) according to the manufacturer’s instructions. The intensity of the OmpT band on the film in the lysates and OMVs was determined by densitometry (Coral Photo Paint). Sodium dodecylsulfate gel electrophoresis and silver staining EHEC cell lysates and OMVs were fractionated by SDS polyacrylamide gel electrophoresis and
the gels were silver stained (Merril et al., 1981). Lysates and OMVs were added to an equal
volume of Loading Dye, heated at 95oC for 5 min and loaded on 4-20% polyacrylamide Criterion gradient gels with prestained molecular weight standards (BioRad). The gels were electrophoresed in 25mM Tris HCl, 192mM glycine, 1 mg ml-1 SDS, pH 8.3 at 150V for 1.5 h.
Gels were microwaved in 500 μl ml-1 methanol and 100 μl ml-1 ethanol for 1.5 min, distilled
water for 2 min, 100µM dithiothreitol for 2 min, 1 mg ml-1 silver nitrate for 1.5 min, washed
twice with distilled water, and finally with 30 mg ml-1 sodium carbonate and 500 μl ml-1
formaldehyde until the lipid and protein bands were visible. Reactions were stopped with 2.3M
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Accepted Article
citric acid and the gels were washed with distilled water and stored in 300 μg ml-1 carbonate at 4oC until photography. Statistical Analysis All experiments were performed on three independent occasions in duplicate. Statistical comparisons of variable means were made with t-tests using Sigma Plot 12.0 (San Jose) with significance set at p≤0.05.
Results The EHEC strains used here are listed in Table 1. Our previous study characterized the antimicrobial peptide susceptibility to EHEC OmpT-dependent cleavage and inactivation using these strains (Thomassin et al., 2012). To determine the effect of OmpT on OMV biogenesis and/or composition, an ompT deletion mutant and the ompT over-expressing plasmid in the ompT deletion mutant were used in conjunction with the wild type EHEC strain (E. coli O157:H7). Effect of OmpT gene expression on EHEC outer membrane vesicle production OMVs were isolated with tangential flow and centrifugal centrifugation, ultracentrifugation and analyzed with transmission electron microscopy. Growth curves of these strains from three independent experiments performed in duplicate in Minimal A medium were not significantly different from each other (Figures 1B, D and F; ANOVA Holm-Sidak method; p=0.142). This indicates that OMVs were isolated from the same volume of CFU/ml from all three strains and that any variations in growth ability between the three strains did not contribute to the numbers of OMVs isolated. Wild type EHEC produced a significantly greater number of OMVs than the ompT deletion mutant and the ompT over-expressing strain produced a significantly larger
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Accepted Article
number of OMVs than both the wild type EHEC and the ompT deletion mutant (Figures 1A, C, E). The ompT over-expressing strain produced approximately 40-fold more OMVs than wild type and approximately 300-fold more OMVs than the ompT deletion mutant and these differences were highly significant (p
Received Date : 28-Jan-2014 Revised Date : 05-May-2014 Accepted Date : 06-May-2014 Article type
: Research Letter
Editor
: Klaus Hantke
Enterohemorrhagic Escherichia coli OmpT regulates outer membrane vesicle biogenesis. Veena Premjani*, Derek Tilley∫, Samantha Gruenheid¶, Hervé Le Moual¶, and John A. Samis*‡
*Applied Biosciences Program, Faculty of Science, University of Ontario Institute of Technology, Oshawa, ON, Canada. ∫ School of Engineering Technology and Applied Science, Centennial College, Toronto, ON, Canada. ¶ Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada. ‡ Medical Laboratory Science Program, Faculty of Health Sciences, University of Ontario Institute of Technology, Oshawa, ON, Canada. Correspondence: John A. Samis, PhD, Faculty of Health Sciences, University of Ontario Institute of Technology, Oshawa, ON. L1H7K4. Canada. Tel: +1-905-721-8668 ext. 3760; FAX: +1-905-721-3179 email: [email protected] Keywords: Protease, Gram-negative bacteria, lipid and protein content. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12463 This article is protected by copyright. All rights reserved.
Accepted Article
Running Title: OmpT Regulates EHEC OMV Biogenesis.
Abstract Enterohemorrhagic Escherichia coli (EHEC) infection from food or water often results in severe
diarrheal disease and is a leading cause of death globally. Outer membrane vesicles (OMVs) secreted from E. coli induce lethality in mice. The omptin outer membrane protease OmpT from E. coli inactivates antimicrobial peptides and may enhance colonization of the uroepithelium; but its precise function remains unclear. Given OmpT is an outer membrane protease, we hypothesized it may have a role in OMV biogenesis. To further characterize the effect of OmpT on OMV production, a genetic approach using wild type, an ompT deletion mutant and an ompT over-expressing construct in EHEC was employed. ompT gene deletion markedly decreased OMV production and stainable lipid but increased vesicle diameter. Conversely, ompT overexpression profoundly increased OMV biogenesis but decreased stainable lipid, protein content, and vesicle diameter. Alterations in EHEC ompT gene expression has an impact on the biogenesis, composition, and size of OMVs. Changes in ompT gene expression may dynamically alter OMV formation, composition, and diameter in response to different host environments and contribute to cell-free intercellular communication to enhance bacterial growth and survival.
Introduction Gram-negative bacteria, including Escherichia coli (E. coli) constitutively produce outer membrane vesicles (OMVs) which are spherical bilayered proteolipids with an average diameter
This article is protected by copyright. All rights reserved.
Accepted Article
of approximately 20-200nm (Kuehn & Kesty, 2005). OMVs are comprised of outer membrane proteins and lipids, periplasmic proteins, lipopolysaccharide, and RNA and DNA (Lee et al., 2007). Although OMVs may function in bacterial growth and survival, transfer of nucleic acids and proteins, virulence factor delivery, and alteration of the host inflammatory and immune response (Mashburn-Warren & Whiteley, 2006); their precise role(s) during the pathogenesis of Gram-negative infections is currently unclear. Cell surface and secreted proteases have important functions in evading the host acquired and innate immune systems and as virulence factors during bacterial infections. Microbial proteases hydrolyze and inactivate host antibodies (Mistry & Stockley, 2011), complement proteins (Honda-Ogawa et al., 2013), and antimicrobial peptides (Thomassin et al., 2012). Bacterial
proteases also degrade host fibrin (Yeo et al., 2011) and extracellular matrix proteins (Ruggiero et al., 2013) which retard pathogen spread during infection. The omptin family of proteases are present in a variety of Gram-negative bacteria and have high amino acid sequence homology (Haiko et al., 2009). Omptins fold into a conserved β-barrel with
the active site exposed to the environment (Vandeputte-Rutten et al., 2001). Omptins require lipopolysaccharide as a cofactor for activity and have a cleavage preference between dibasic amino acids with Arg or Lys in the P1 and P’1 positions (Kukkonen & Korhonen 2004). The omptins Pla from Yersinia pestis and PgtE from Salmonella enterica activate host plasminogen to plasmin and inactivate the major host plasmin inhibitor, α2-antiplasmin and the major host inhibitor of plasminogen activation, plasminogen activator inhibitor 1 (Kukkonen et al., 2001;
Lahteenmaki et al., 2005; Haiko et al., 2010). PgtE of S. enterica has also been shown to activate
host matrix metalloproteases (Ramu et al., 2008). The action of Pla and PgtE would, via
This article is protected by copyright. All rights reserved.
Accepted Article
engagement of the host plasmin and matrix metalloprotease systems, be expected to disrupt the fibrin and extracellular matrix barriers to enhance microbial survival and growth. The E. coli outer membrane protease omptin OmpT has historically been considered a ‘housekeeping’ protease towards endogenous microbial and extracellular proteins (Haiko et al., 2009). There is currently no significant association of the presence and expression of OmpT with specific clinical isolates and pathogenic strains of E. coli (Kukkonen & Korhonen 2004). Further, although our previous work has shown OmpT inactivates host antimicrobial peptides (Thomassin et al., 2012) and others have shown OmpT may enhance E. coli colonization of the
uroepithelium (Hui et al., 2010); its precise function(s) in the pathogenesis of E. coli infections is presently unclear. Enterohemorrhagic Escherichia coli (EHEC) is a water- or food-borne pathogen that causes severe diarrheal disease (Nataro & Kaper 1998). EHEC cause attaching and effacing lesions that erode intestinal microvilli and promote bacterial attachment to host cell membranes (Golan et al.,
2011). Given OmpT is an outer membrane protease, we hypothesized it may have a role in the formation and/or determining lipid or protein composition of OMVs. Using a genetic approach, we demonstrate here that the presence of OmpT, like other outer membrane and periplasmic proteins, has a profound impact on the formation, composition, and diameter of OMVs.
Materials and Methods Bacterial strains and growth conditions The E. coli O157:H7 strains used in this study are listed in Table 1 (Thomassin et al., 2012).
EHEC cells were grown at 37oC with aeration at 200 rpm in Minimal A medium (60mM This article is protected by copyright. All rights reserved.
Accepted Article
K2HPO4, 33mM KH2PO4, 7.5mM ammonium sulphate, 1.7mM trisodium citrate, pH 7.0 with 2mg ml-1 glucose, 60μg ml-1 tryptone, 1mM MgSO4). EHEC cells were grown in Minimal A
medium rather than a more complex protein medium (ie LB broth) to reduce potential interference of medium proteins with immunoblotting and electrophoresis and silver staining. Liquid medium was supplemented with chloramphenicol for the ompT over-expressing strain (30 µg ml-1). Preparation of outer membrane vesicles EHEC cells were grown in Minimal A medium (10 ml) with/without chloramphenicol (30 µg ml1
) for 12-14 h at 37oC with aeration at 200 rpm. The cultures were subcultured into 1 L of fresh
Minimal A medium and incubated at 37oC without aeration for 16-24 h to an optical density at 600nm of 0.5. The cultures were centrifuged at 10,000 x g for 30 min at 4oC and supernatants
were filtered through 0.22µm vacuum filters (Sarstedt) to remove any bacteria. The pH of the supernatants was adjusted to 7.4 and concentrated approximately 33-fold (1 L to 30 ml) with a 100kDa molecular weight cutoff membrane using a tangential flow filtration capsule (Pall Life Sciences) at room temperature. Samples were further concentrated by 3-fold (30 ml to 10 ml) using 100kDa molecular weight cutoff centrifugal filters (Millipore) at 1,800 x g at 4oC for 10
min. OMVs were pelleted by ultracentrifugation at 150,000 x g (Hitachi Koki) at 4oC for 1.5 h and were resuspended in 250 μl of 20mM HEPES, 150mM NaCl, pH 7.4. OMVs were stored in sealed cryovials at -80oC and thawed/refrozen samples were not used.
Preparation of cell lysates EHEC cells were grown in Minimal A medium (1 ml) with/without chloramphenicol (30 µg ml1
) with aeration at 200 rpm to an optical density at 600nm of 0.5 and centrifuged at 10,000 x g
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Accepted Article
for 30 min at 4oC. The pellets were resuspended in 100 μl of 20mM HEPES, 150mM NaCl, pH 7.4 and sonicated on ice for 10 min with a Dismembranator (Fisher). Cell lysates were stored in sealed cryovials at -80oC and thawed/refrozen samples were not used.
Protein assay of cell lysates and outer membrane vesicles The protein concentration of cell lysates and OMVs was determined with bicinchoninic acid using bovine serum albumin (Fisher) as the standard. Samples were read at 562nm using microplate reader (Molecular Devices) after correction with 20mM HEPES, 150mM NaCl, pH 7.4. Standard curves of absorbance at 562nm vs bovine serum albumin concentration were used to interpolate the protein concentrations of cell lysates and OMVs. Transmission electron microscopy Copper grids (Electron Microscopy Sciences) were loaded with OMVs (10 μl) for 1 min and excess volume was removed with filter paper. Grids were floated on 2 mg ml-1 uranyl acetate for
30 s, blotted with filter paper and air dried for 1 min. Specimens were examined by transmission electron microscopy (FEI Company, Model Technai) with an accelerating voltage of 200kV at 100,000 x magnification (Mount Sinai Hospital, Toronto). The number of OMVs/field and size distributions of OMVs were measured using magnification software (Orbicule). Imunoblotting for OmpT in cell lysates and outer membrane vesicles Immunoblotting for determination of OmpT protein expression in EHEC cell lysates and OMVs employed a rabbit anti-CroP IgG as described in our previous study (Thomassin et al., 2012).
Cell lysates and OMVs were added to Loading dye (62.5mM Tris pH 6.8, 125 mg ml-1 glycerol, 20mg ml-1 SDS, 250 μg ml-1 bromophenol blue, 20 μg ml-1 mercaptoethanol), heated at 95oC for
This article is protected by copyright. All rights reserved.
Accepted Article
5 min, and electrophoresed on 10% polyacrylamide under denaturing conditions (Sambrook et al., 1989) with prestained molecular weight standards (BioRad) in 25mM Tris, 192mM glycine, 1 mg ml-1 SDS, pH 8.3 for 40 min at 80V and 90 min at 100V. Protein was electroblotted onto polyvinylidene membranes (Millipore) at 35V in 25mM Tris HCl, 192mM glycine, 500 μg ml-1
SDS, 100 μl ml-1 methanol, pH 8.3 for 16 h at 4oC. Membranes were blocked with Tris-Buffered Saline-Tween (20mM Tris HCl, 500mM NaCl, 250 μl ml-1 Tween-20, pH 7.4) containing 50 mg
ml-1 non-fat dry milk powder for 1 h at room temperature. OmpT on the blots was detected with a rabbit polyclonal anti-CroP IgG (Comparative Medicine Animal Resources Centre, McGill University, 1:10,000 dilution), goat anti-rabbit IgG IgG conjugated with horse radish peroxidase (Sigma, 1:5,000 dilution), chemiluminescence reagents (PerkinElmer), and XOMAT film
(Kodak) according to the manufacturer’s instructions. The intensity of the OmpT band on the film in the lysates and OMVs was determined by densitometry (Coral Photo Paint). Sodium dodecylsulfate gel electrophoresis and silver staining EHEC cell lysates and OMVs were fractionated by SDS polyacrylamide gel electrophoresis and
the gels were silver stained (Merril et al., 1981). Lysates and OMVs were added to an equal
volume of Loading Dye, heated at 95oC for 5 min and loaded on 4-20% polyacrylamide Criterion gradient gels with prestained molecular weight standards (BioRad). The gels were electrophoresed in 25mM Tris HCl, 192mM glycine, 1 mg ml-1 SDS, pH 8.3 at 150V for 1.5 h.
Gels were microwaved in 500 μl ml-1 methanol and 100 μl ml-1 ethanol for 1.5 min, distilled
water for 2 min, 100µM dithiothreitol for 2 min, 1 mg ml-1 silver nitrate for 1.5 min, washed
twice with distilled water, and finally with 30 mg ml-1 sodium carbonate and 500 μl ml-1
formaldehyde until the lipid and protein bands were visible. Reactions were stopped with 2.3M
This article is protected by copyright. All rights reserved.
Accepted Article
citric acid and the gels were washed with distilled water and stored in 300 μg ml-1 carbonate at 4oC until photography. Statistical Analysis All experiments were performed on three independent occasions in duplicate. Statistical comparisons of variable means were made with t-tests using Sigma Plot 12.0 (San Jose) with significance set at p≤0.05.
Results The EHEC strains used here are listed in Table 1. Our previous study characterized the antimicrobial peptide susceptibility to EHEC OmpT-dependent cleavage and inactivation using these strains (Thomassin et al., 2012). To determine the effect of OmpT on OMV biogenesis and/or composition, an ompT deletion mutant and the ompT over-expressing plasmid in the ompT deletion mutant were used in conjunction with the wild type EHEC strain (E. coli O157:H7). Effect of OmpT gene expression on EHEC outer membrane vesicle production OMVs were isolated with tangential flow and centrifugal centrifugation, ultracentrifugation and analyzed with transmission electron microscopy. Growth curves of these strains from three independent experiments performed in duplicate in Minimal A medium were not significantly different from each other (Figures 1B, D and F; ANOVA Holm-Sidak method; p=0.142). This indicates that OMVs were isolated from the same volume of CFU/ml from all three strains and that any variations in growth ability between the three strains did not contribute to the numbers of OMVs isolated. Wild type EHEC produced a significantly greater number of OMVs than the ompT deletion mutant and the ompT over-expressing strain produced a significantly larger
This article is protected by copyright. All rights reserved.
Accepted Article
number of OMVs than both the wild type EHEC and the ompT deletion mutant (Figures 1A, C, E). The ompT over-expressing strain produced approximately 40-fold more OMVs than wild type and approximately 300-fold more OMVs than the ompT deletion mutant and these differences were highly significant (p
