Intracellular survival strategies: How Salmonella · PDF fileIntracellular survival...
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Intracellular survival strategies: How Salmonella evades the antimicrobial oxidative and nitrosative burst during infection of macrophages. Joris van der Heijden 1,2 , B. Brett Finlay 1,2,3 1 Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, V6T 1Z4, Canada 2 Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada, 3 Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada 4. In vitro monitoring of redox changes in S. Typhimurium 5. In vitro comparison of redox changes in S. Typhimurium and S. Typhi 6. Direct observation of redox changes during infection of cells The infection process of Salmonella Typhimurium and Salmonella Typhi is characterized by the survival and/or bacterial replication inside human phagocytic cells [1]. Phagocytic cells play a crucial role in the dissemination and persistence of infection. In order to survive inside phagocytic cells Salmonella needs to employ mechanisms to evade the attack of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The mechanisms that lead to evasion of ROS and RNS are currently unclear and the transient and highly dynamic nature of ROS and RNS make it difficult to study its effects on bacteria during infection [2]. Thus far it is impossible to measure changes in the intra- bacterial redox potential to understand how much oxidative/nitrosative stress is experienced by the invading bacteria during infection of phagocytes. In this study we use a redox sensitive Green Fluorescent Protein (roGFP) to monitor changes in the intra-bacterial redox potential during infection of phagocytic cells. 3. Redox sensitive GFP Figure 6. Visual comparison of the redox potential in S. Typhimurium and S. Typhi during infection of THP-1 cells. After infection of THP- 1 cells (MOI=50), cells were harvested at different time points. Intracellular bacteria were stained with Alexa660. After measurements were performed cells that were positive for Alexa660 were selected for analysis. The time post infection is indicated on the left hand side of the figures. On the right hand side: the normalized ratio as calculated by the AMNIS ImageStream shows the intra- bacterial redox potential that is calculated from the photo that is shown. Figure 3. Real-time measurement of redox changes inside Salmonella All ratio values presented are normalized to the max reduced and max oxidized values measured under the same conditions. The measurements in panel A, B and C are done with a fluorescent plate reader. S. Typhimurium is challenged by enzymatically produced superoxide, nitric oxide or hydrogen peroxide respectively. A. The arrow indicates a challenge with 0.1 U (light pink) or 0.2 U (dark pink) Xanthine Oxidase complemented with excess substrate. B. The arrow indicates a challenge with 5mM (light pink), 10mM (dark pink) or 20mM (dark red) SpermineNONOate. C. The arrow pointing up indicates a challenge with 50uM (light pink), 500uM (pink), 5mM (dark pink), 50mM (red) or 500mM (dark red) of H2O2. The arrow pointing down indicates the addition of 10mM DTT. D. Microscopic analysis of change in intra- bacterial redox potential after challenge with 5mM H2O2. E. Pictures of single bacterium that correspond to the numbers in panel D. Figure 2. Fluorescence excitation spectrum of roGFP2 Excitation spectrum of roGFP2 after full oxidation (blue line) and full reduction (red line) of the protein. Emission is followed at 510 nm. Figure 1. Conformational change in roGFP The formation of a reversible disulfide bond in roGFP is dependend on the redox potential. The two isoforms of roGFP have different excitation spectra. The ratio between roGFPox/roGFPred is a measure for the redox potential. A B C D This is the first and only system that can monitor real-time changes in the redox balance inside bacteria. With this system we were able to show in vitro redox changes that occur within a matter of minutes that have never been shown before. The system is sensitive to different sources of oxidative and nitrosative stress in a dose dependent manner. With this system we were able to look at the effect of pH, temperature, growth phase (stationary vs log) and the presence or absence of certain metals on bacterial defense mechanisms. We were able to compare WT S. Typhimurium with mutants deficient in an adequate stress response. In addition we analyzed differences in the stress response within the genera Salmonella and across species. The comparison between S. Typhimurium and S. Typhi showed a striking difference in the stress response after a challenge with H2O2. In contrast, the stress response after a challenge with NO showed a high degree of similarity. With the AMNIS ImageStream we can, for the first time, analyze the redox balance inside intracellular bacteria during infection of macrophages after exposure to the host generated oxidative and nitrosative burst. Differences in the stress response between S. Typhimurium and S. Typhi seem to translate to differences in virulent mechanisms during infection of macrophages. Thus far our results indicate that S. Typhi causes significantly less oxidative stress than S. Typhimurium after invasion of macrophages. 1 min 3 min 5 min 7 min 9 min D OX RED Wavelength [nm] 360 380 400 420 440 460 480 500 0 20 40 60 80 100 roGFP2 excitation spectrum Fluorescent intensity Time (min) 0 10 20 30 40 0.0 0.2 0.4 0.6 0.8 1.0 Time (min) 0 5 10 15 20 0.0 0.1 0.2 0.3 0.4 0.5 Normalized ratio 405/480nm Normalized ratio 405/480nm Challenge with H2O2 Microscopic analysis after challenge with H2O2 Time (min) 0 5 10 15 20 25 30 0.0 0.2 0.4 0.6 0.8 1.0 Time (min) 0 10 20 30 40 50 60 0.0 0.2 0.4 0.6 0.8 1.0 Normalized ratio 405/480nm Normalized ratio 405/480nm Challenge with Xanthine Oxidase (O 2 - ) Challenge with SpermineNONOate (NO) C D E 1 2 3 4 5 6 7 8 Time (min) 0 10 20 30 40 50 60 0.0 0.2 0.4 0.6 0.8 1.0 Time (min) 0 10 20 30 40 50 60 0.0 0.2 0.4 0.6 0.8 1.0 Figure 4. Comparison of redox potential between S. Typhimurium and S. Typhi. Experiments shown in panel A and B are done under conditions that mimic the intracellular environment A. The arrows indicate a challenge with 5mM H2O2. The intra- bacterial redox potential in S. Typhimurium is shown in red and S. Typhi is shown in blue. B. The arrow indicates a challenge with 5mM SpermineNONOate. C. and D. with the AMNIS ImageStream the intra- bacterial redox potential of S. Typhimurium or S. Typhi in PBS (red histogram) was compared to bacteria that were fully oxidized (grey histogram). The AMNIS ImageStream is an integrated system in which flow cytometry and microscopy are combined. Pictures of the analysis are shown in figure 5. S. Typhimurium redox potential with AMNIS ImageStream Redox potential in S. Typhimurium (red) and S. Typhi (blue) after challenge with H2O2 Redox potential in S. Typhimurium (red) and S. Typhi (blue) after challenge with NO Normalized ratio 405/480nm Normalized ratio 405/480nm Normalized frequency Normalized frequency S. Typhi redox potential with AMNIS ImageStream Normalized Ratio 405/488nm Normalized Ratio 405/488nm Figure 5. Visual comparison of the redox potential in S. Typhimurium and S. Typhi Photos of S. Typhimurium (left panel) and S. Typhi (right panel) bacteria are shown that visualize an increasing redox potential as measured by the AMNIS ImageStream. The brighfield channel is shown together with the fluorescent channels that show excitation at 488nm, 405 nm and a composite image of both 405 and 488 nm. From top to bottom the photo’s show in increasing redox potential. Develop a method to monitor changes in the intra-bacterial redox potential to examine the effect of ROS and RNS on Salmonella survival and virulence over the course of infection. Use this method to compare virulent strategies between bacterial pathogens and identify the mechanisms that are employed by either one of these pathogens. A B C D S. Typhimurium infection of THP-1 cells S. Typhi infection of THP-1 cells 7. Conclusions 2. Experimental goals 1. Introduction 8. References 1. J van der Heijden and BB Finlay, Future Microbiol, (2012) 7(6),1-19 2. JM Slauch, Microbiology ,(2011) 80, 580-583 For more information please contact [email protected]
Transcript of Intracellular survival strategies: How Salmonella · PDF fileIntracellular survival...
Intracellular survival strategies: How Salmonella evades the antimicrobial
oxidative and nitrosative burst during infection of macrophages. Joris van der Heijden1,2, B. Brett Finlay1,2,3
1Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, V6T 1Z4, Canada2Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada,
3Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
4. In vitro monitoring of redox changes in S. Typhimurium
5. In vitro comparison of redox changes in S. Typhimurium and S. Typhi
6. Direct observation of redox changes during infection of cells
The infection process of Salmonella Typhimurium andSalmonella Typhi is characterized by the survival and/orbacterial replication inside human phagocytic cells [1].
Phagocytic cells play a crucial role in the dissemination andpersistence of infection.
In order to survive inside phagocytic cells Salmonellaneeds to employ mechanisms to evade the attack ofreactive oxygen species (ROS) and reactive nitrogenspecies (RNS).
The mechanisms that lead to evasion of ROS and RNS arecurrently unclear and the transient and highly dynamicnature of ROS and RNS make it difficult to study its effectson bacteria during infection [2].
Thus far it is impossible to measure changes in the intra-bacterial redox potential to understand how muchoxidative/nitrosative stress is experienced by the invadingbacteria during infection of phagocytes.
In this study we use a redox sensitive Green FluorescentProtein (roGFP) to monitor changes in the intra-bacterialredox potential during infection of phagocytic cells.
3. Redox sensitive GFP
Figure 6. Visual comparison of the redox potential in S. Typhimurium and S. Typhi during infection of THP-1 cells. After infection of THP-1 cells (MOI=50), cellswere harvested atdifferent time points.Intracellular bacteriawere stained withAlexa660. Aftermeasurements wereperformed cells thatwere positive forAlexa660 wereselected for analysis.The time postinfection is indicatedon the left hand sideof the figures. On theright hand side: thenormalized ratio ascalculated by theAMNIS ImageStreamshows the intra-bacterial redoxpotential that iscalculated from thephoto that is shown.
Figure 3. Real-time measurement of redox changes inside Salmonella All ratio values presentedare normalized to the maxreduced and max oxidizedvalues measured under thesame conditions. Themeasurements in panel A, Band C are done with afluorescent plate reader. S.Typhimurium is challengedby enzymatically producedsuperoxide, nitric oxide orhydrogen peroxiderespectively. A. The arrowindicates a challenge with0.1 U (light pink) or 0.2 U(dark pink) XanthineOxidase complemented withexcess substrate. B. Thearrow indicates a challengewith 5mM (light pink),10mM (dark pink) or 20mM(dark red)SpermineNONOate. C. Thearrow pointing up indicatesa challenge with 50uM (lightpink), 500uM (pink), 5mM(dark pink), 50mM (red) or500mM (dark red) of H2O2.The arrow pointing downindicates the addition of10mM DTT. D. Microscopicanalysis of change in intra-bacterial redox potentialafter challenge with 5mMH2O2. E. Pictures of singlebacterium that correspondto the numbers in panel D.
Figure 2. Fluorescence excitation spectrum of roGFP2 Excitationspectrum ofroGFP2 afterfull oxidation(blue line)and fullreduction(red line) ofthe protein.Emission isfollowed at510 nm.
Figure 1. Conformational change in roGFPThe formation of areversible disulfidebond in roGFP isdependend on theredox potential.The two isoformsof roGFP havedifferent excitationspectra. The ratiobetweenroGFPox/roGFPred isa measure for theredox potential.
A B
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This is the first and only system that can monitor real-timechanges in the redox balance inside bacteria. With thissystem we were able to show in vitro redox changes thatoccur within a matter of minutes that have never beenshown before.
The system is sensitive to different sources of oxidativeand nitrosative stress in a dose dependent manner.
With this system we were able to look at the effect of pH,temperature, growth phase (stationary vs log) and thepresence or absence of certain metals on bacterialdefense mechanisms.
We were able to compare WT S. Typhimurium withmutants deficient in an adequate stress response. Inaddition we analyzed differences in the stress responsewithin the genera Salmonella and across species.
The comparison between S. Typhimurium and S. Typhishowed a striking difference in the stress response after achallenge with H2O2. In contrast, the stress response aftera challenge with NO showed a high degree of similarity.
With the AMNIS ImageStream we can, for the first time,analyze the redox balance inside intracellular bacteriaduring infection of macrophages after exposure to thehost generated oxidative and nitrosative burst.
Differences in the stress response between S.Typhimurium and S. Typhi seem to translate to differencesin virulent mechanisms during infection of macrophages.Thus far our results indicate that S. Typhi causessignificantly less oxidative stress than S. Typhimuriumafter invasion of macrophages.
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Figure 4. Comparison of redox potential between S.Typhimurium and S. Typhi.Experiments shown inpanel A and B are doneunder conditions thatmimic the intracellularenvironment A. The arrowsindicate a challenge with5mM H2O2. The intra-bacterial redox potential inS. Typhimurium is shown inred and S. Typhi is shown inblue. B. The arrowindicates a challenge with5mM SpermineNONOate.C. and D. with the AMNISImageStream the intra-bacterial redox potential ofS. Typhimurium or S. Typhiin PBS (red histogram) wascompared to bacteria thatwere fully oxidized (greyhistogram). The AMNISImageStream is anintegrated system in whichflow cytometry andmicroscopy are combined.Pictures of the analysis areshown in figure 5.
S. Typhimurium redox potential with AMNIS ImageStream
Redox potential in S. Typhimurium (red) and S. Typhi (blue) after challenge with H2O2
Redox potential in S. Typhimurium (red) and S. Typhi (blue) after challenge with NO
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Figure 5. Visual comparison of the redox potential in S. Typhimurium and S. Typhi Photos of S. Typhimurium (left panel) and S. Typhi (right panel) bacteria are shown that visualize an increasing redox potential as measured by the AMNIS ImageStream. The brighfield channel is shown together with the fluorescent channels that show excitation at 488nm, 405 nm and a composite image of both 405 and 488 nm. From top to bottom the photo’s show in increasing redox potential.
Develop a method to monitor changes in the intra-bacterialredox potential to examine the effect of ROS and RNS onSalmonella survival and virulence over the course ofinfection.
Use this method to compare virulent strategies betweenbacterial pathogens and identify the mechanisms that areemployed by either one of these pathogens.
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S. Typhimurium infection of THP-1 cells
S. Typhi infection of THP-1 cells
7. Conclusions
2. Experimental goals
1. Introduction
8. References1. J van der Heijden and BB Finlay, Future Microbiol, (2012) 7(6),1-19
2. JM Slauch, Microbiology ,(2011) 80, 580-583
For more information please contact [email protected]
