Real-time analysis of progression of bacterial infections in organs of live animals -
Bacterial infection is a complex array of actions both from invading bacteria and the defending host. Over the years these interactions have been studied in more and more complex models. Data have been obtained from mono-cultures, co-cultures, explants, and in vivo sacrificial models. The relationship between data from these different types of experimental set-ups is non-trivial. Although essentially correct information is obtained within a given context, the sum of reductionistic models do not necessarily represent the complete picture of what happens in the live animal.
A complete list of publications for the described project can be found here
Figure 1 illustrates the problem above, the sum of the results from the two different mono-culture experiments A and B are not the same as the results obtained in the co-culture experiment C, while the processes in an animal cannot be described by A, B, and C in any combination. Richter-Dahlfors et al. Curr Opin Microbiol 2012. For a larger view, click the image.
Development of an intravital infection model in the live animal
Bacterial infection of the rat kidney was chosen as the model system. Using microinfusion, spatial and temporal control can be achieved.
Figure 2. One kidney is exposed on the sedated rat. GFP-expressing Uropathogenic E. coli bacteria are slowly infused into the proximal tubule of one selected nephron. For a discussion on choice of promoter and construction of the bacterial strain, click here. For a larger view, click the image.
Figure 3. The rat is placed on the inverted two-photon-microscope and the infection is followed. After six to ten hours, the rat is stiched up and returned to its cage. The infection site can then be studied again at a later time point. For a larger view, click the image.
Click on the image below to see a movie of the blood flow in real time.
Figure 4. Real-time imaging of an E. coli infected nephron. The bacterium is green, the blood flow is red and the epithelial cells of the infected tubule are blue. Epithelial cells in uninfected tubules are faint green. a) One hour after injection of bacteria, a few bacteria has not been flushed out by the flow and managed to adhere to the epithelial cells at the tubule wall (arrow). b) The same area five hours post injection. The few initial bacteria have now multiplied and filled up the tubule. Note that there are no bacteria in the uninjected tubule or in the blood vessel. c) 22 hours post injection. The infection is cleared and the tissue is scarred. d) A nearby uninjected tubule is still unaffected after 22 hours. For a larger view, click the image.
The conclusion from the results obtained from this intra-vital model of an infection in a live animal, is that the time span of the infection is surprisingly short. This conclusion could only be drawn since both time and space for the infection starting point is so well controlled. Månsson et al. Cell Microbiol 2007.
For a full description of the intravital imaging method see Choong et al. Meth Enzymol 2012.
Ischaemia as a defense against sepsis
The course of infection is not only fast, the blood flow in the adjacent blood vessel seems to be shut off already five hours post injection (Fig 4b). The cause for this was investigated. It was hypothesized that the bacterial infection in the tubule induces trans-epithelial and trans-endothelial signalling to the blood vessel where clots are formed. This in turn would stop the blood flow.
Figure 5. Bacteria are green, the blood flow is red, epithelial cells in the infected tubule are blue. It was found that already 2.5 hours post injection of bacteria into the tubule, accumulation of what seems to be platelets occur in the adjacent blood vessel (arrow and arrow head). This result was substantiated by the finding that genes in the clotting cascade are upregulated. Note that all this happen before the bacteria have spread to the blood stream. Scale bar = 30 μm.
In additional experiments heparin was added to inhibit clot formation, without exception all rats died from massive sepsis. Bacteria had breached the epithelium and spread to the blood stream and the rest of the body. For a larger view, click the image.
From these results it was suggested that the infection induces local ischaemia as a concequence of clot formation. This will encapsulate the bacteria and stop them from dessimination via the blood to the rest of the body. The sacrifice of the infected nephron will save the life of the animal. Melican et al. Cell Microbiol 2008.
The concept of tissue microbiology
P and Type 1 fimbriae facilitate kidney colonization
Adhesins are appendages protruding from the bacterium facilitating adherence to different types of surfaces. Two well studied Escherichia coli adhesins implied to be important at different stages of urinary tract infections are P and Type 1 fimbriae. In this work we used the intravital model for infection to study the effect of these fimbriae on kidney colonization, pyelonephritis.
Figure 7. Bacteria are green, the blood flow red, and epithelial cells in the infused tubule are blue. The course of infection for bacteria lacking Type 1 fimbriae was followed. It was found that at early time points the infection looks normal but already at five hours it is clear that the bacteria have difficulties to establish themselves in the middle of the tubule. Scale bar 30 = μm. For a larger view, click the image.
Figure 8. The course of infection for a bacterium lacking P fimbriae was followed. It was found that the colonization kinetics was slowed down, bacteria were not visible until after eight hours post infusion. When studied in detail it could be seen that the bacteria behaved like a cloud not attached to the epithelium. This cloudlike mass is moving with the flow in the tubule. The time span between Fig 8a and 8b is 70 s, in a) is a line depicting the trail of the bacterial cloud. For a larger view, click the image.
Click on the image below to see the cloud of bacteria lacking P fimbriae move in the proximal tubule.
From these results we conclude that P fimbriae are needed for attachment to epithelial cells while Type 1 fimbriae mediates attachment between bacteria. Melican et al. PLoS Pathogens 2011.
Transcriptomic analysis of early infection suggests a rapid inter-organ communication
The intra-vital infection model was combined with a transcriptome analysis of the infected nephron.
Figure 9. Samples were withdrawn from the infected site of animals sacrificed at three, five, and eight hours, respectively. Gene expression was quantified by microarray analysis and the result is shown from left to right. Red dots are measurements where the increase in expression was considered significant. For a larger view, click the image.
Figure 10. After statistical evaluation of the data, 59 annotated genes were found to be upregulated at eight hours post infection. The relevance of this was verified by making a gene ontology analysis and it was found that all genes could be classified as linked to infection or inflammation. For a larger view, click the image.
After some data mining it was found that there is a dynamic expression of some genes when the five hour sample is compared to the eight hour sample. Not only was it found that a group of genes upregulated at eight hours are stimulated already at five hours, it was also shown that a subset of genes are upregulated at five hours and not at eight hours.
Our data set was compared to other published studies performed with Gram negative bacteria at early time points, with local delivery to an otherwise sterile organ, we could define 80 genes upregulated as a 'General tissue response to early local becterial infections'. It was found that 25% of these genes are regulated in response to Interferon γ. This notion was investigated further and it was shown that the infections elicicit a rapid inter-organ signalling between the kidney and the spleen.
In conclusion, this study shows that a hypothesis-free approach in combination with our intra-vital infection model can identify new components of the early immune response. Boekel et al. BMC Genomics 2011.
To view an animated summary of our results so far, click on the black box below.
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