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.

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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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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-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 jorisvdh@msl.ubc.ca