Visualizing the dynamic interplay between the host and bacterial pathogen : a real-time study of renal infection

The human body is a complex entity capable of handling a variety of pathogenic challenges without developing disease symptoms. Sometimes, however, the pathogens are superior and the host immune system is unable to clear the infection. Alternatively, the host tissue suffers substantial damage during the inflammatory response, which may lead to serious physiological complications. The processes by which this is accomplished are widely dynamic and cannot be understood by studies utilizing simplified model systems containing one cell-type. Until recently the availability of techniques for studying the infection process inside a living organ has been limited.

By combining intravital multiphoton microscopy and bacterial genetics we have developed a technique facilitating real-time imaging of the progression of infection in a live animal. Microinjection of GFP+-expressing uropathogenic E. coli (UPEC) into tubules of exteriorized kidneys enabled spatial and temporal control of the experiment. The infection process was followed from the first interaction of bacteria with the epithelial lining, through the dynamic immune response, to the end stage of massive tissue destruction. This dynamic course of events was accomplished within 22 h, a time frame substantially shorter then previously appreciated. For the first time the localized immune cell recruitment was imaged in real-time. Within hours of infection the tubular and vascular integrity was compromized, with subsequent perivascular leakage and ischemic effects. We found UPEC forming multicellular communities on the tubular epithelial lining, indicative of biofilm formation. During later stages of infection, an intermediate filamentous phenotype of the bacteria was observed and UPEC was also found internalized in renal epithelial as well as endothelial cells.

This technique also allows a unique opportunity to study subtle and transient in vivo effects of bacterial virulence factors. We found a marked delay in the immune response triggered by a hemolysin (Hly) knock out UPEC strain in comparison to the isogenic wild type strain, even though the level of tissue destruction in the later stage of infection was similar. This is in correlation with the in vitro finding that sub-lytic concentrations of this bacterial exotoxin induces a pro-inflammatory response in renal epithelial cells, while high doses are lytic to both erythrocytes and nucleated cells. Hly is known to tightly interact with the bacterial outer membrane component lipopolysacharide (LPS) and we investigated whether this interaction is important for the dual concentration-dependent activities of Hly. Our findings show that LPS is essential for the pro-inflammatory signaling triggered by Hly, but not for the lytic activity. The Hly-LPS complex is targeted to lipid rafts in the host membrane through interaction with LBP and CD14, which represents a novel molecular mechanism for toxin delivery in bacterial pathogenesis.

The visualization techniques presented in this thesis can be applied to study the tissue response to a variety of bacterial pathogens. A detailed understanding of how the host responds to invading microbial pathogens and the in vivo importance of bacterial virulence factors is crucial for the development of new approaches to combat microbial infections.

List of scientific papers

I. Mansson LE, Melican K, Boekel J, Sandoval RM, Hautefort I, Tanner GA, Molitoris BA, Richter-Dahlfors A (2007). "Real-time studies of the progression of bacterial infections and immediate tissue responses in live animals." Cell Microbiol 9(2): 413-24. Epub 2006 Aug 1
https://pubmed.ncbi.nlm.nih.gov/16953802

II. Mansson LE, Melican K, Rodríguez-Martínez S, Sandoval RM, Shah BM, Tanner GA, Molitoris BA and Richter-Dahlfors A (2007). "UPEC exposed: real-time imaging reveals alternate lifestyles in vivo." (Submitted)

III. Mansson LE, Kjall P, Pellett S, Nagy G, Welch RA, Backhed F, Frisan T, Richter-Dahlfors A (2007). "Role of the lipopolysaccharide-CD14 complex for the activity of hemolysin from uropathogenic Escherichia coli." Infect Immun 75(2): 997-1004. Epub 2006 Nov 13
https://pubmed.ncbi.nlm.nih.gov/17101668