Tissue microbiology : an integrated approach for intravital studies of host-pathogen interactions

A bacteria infection in the living host is the summation of a myriad of interactions. Reductionistic and sacrificial experiment models cannot replicate this complexity. The Tissue Microbiology approach overcomes this by allowing the live host and pathogen to control all facets of the infection model. This enabled the discovery of many novel aspects of the pathophysiology of infection. In this thesis, we were drawn into the study of inter-organ communication networks and how they are activated during an infection. Earlier studies linked transcriptomic changes at the spleen to the kidney infection but did not show how the preceding inter-organ communication had occurred. Using the intravital infection model for experimentation and intravital imaging to guide the design of Cellular Microbiology models, we uncovered the presence of sensory afferent nerves in the renal cortex and demonstrated their unique role in sensing and communicating infections between kidney and spleen already a few hours after infection was initiated. We also showed a link between the E. coli toxin α-haemolysin and splenic expression of IFN-γ, which is mediated by epithelial eATP signaling. This work expands on the growing field of Nervous Driven Immunity, and demonstrated the versatility of sensory afferents in infection sensing and initiating systemic responses by swift long distance communication.

Bacteria colonizing the host microenvironment adopt a multicellular lifestyle to resist efforts of innate immunity and early-induced innate responses. While showing all indications of being a biofilm, no method exists to verify or study this in vivo. To overcome this we developed an optical method for continuous in situ analysis of biofilms. This was based on luminescent conjugated oligothiophenes (LCOs), a non-toxic small molecular fluorophore. This chameleon like oligomers allowed specific detection of Salmonella extracellular matrix (ECM) curli fibers and cellulose via a target specific optical signature, and enabled dynamic tracking of their formation in a variety of growth models. Named Optotracing, this technology uncovered a rare glimpse of cellulose in intracellular bacteria, in eukaryotic cells and infected tissues.

Optotracing proved to be a versatile trans-disciplinary platform and an Enabling technology. The unique affinity for bacteria produced cellulose was transferrable to the study of lignocellulose feedstock. p-HTEA, a pentameric cationic LCO, was highly effective for rapid, non-destructive, detection and quantification of cellulose, lignin, hemicelluloses, across materials of a variety of physical states and chemical compositions. Optotracing, as a method for material analysis, could be easily performed on spectrophotometers and fluorescence microscopes to provide visual maps of the surface topography of lignocellulose materials. Continuous monitoring of enzymatic reactions such as cellulolysis was also possible. Investigating the mechanism of how LCOs detect cellulose revealed a 2-step process of selective binding and target specific reporting. As an example, h-FTAA selectively bond β-configured, but not α-configured glucose polysaccharide. Each β-configured glucose polysaccharide induces an identifiable target specific optical signature of h-FTAA that is determined by qualities such as saccharide length, bonding positions and projecting R-groups. This 2-step mechanism allowed for high-resolution imaging of cellulose structures in plant tissues, with virtually no cross-reactivity with the myriad of other carbohydrates present. Optotracing also allowed for high-resolution imaging and cellulose specific ‘Carbohydrate anatomical mapping’ of plant tissues.

This thesis contributes to better understanding of host communication networks in sensing and responding to infections, and produced new technology and tools that is likely to improve the research of biofilms and cellulose materials. We envision that future development of work enclosed in this thesis will continue to produce novel tools that will accelerate the study of bacteria related diseases in humans, microbe lifestyles and plant-based materials.

List of scientific papers

I. Choong FX, Steiner SE, Schulz A, Bas D, Sandoval RM, Molitoris BA, Melican K, Svensson C, Richter-Dahlfors A. Nervous driven immune response during early pyelonephritis. [Manuscript]

II. Choong FX, Bäck M, Fahlén S, Melican K, Rhen M, Nilsson KPR, Richter-Dahlfors A. Real-time optotracing of curli and cellulose in live Salmonella biofilms using luminescent oligothiophenes. NPJ Biofilms and Microbiomes. 2016 16024.
https://doi.org/10.1038/npjbiofilms.2016.24

III. Choong FX, Bäck M, Steiner SE, Melican K, Nilsson KPR, Edlund U, Richter-Dahlfors A. Nondestructive, real-time determination and visualization of cellulose, hemicellulose and lignin by luminescent oligothiophenes. Scientific Reports. 2016 (6):35578.
https://doi.org/10.1038/srep35578

IV. Choong FX, Bäck M, Schulz A, Nilsson KPR, Edlund U, Richter-Dahlfors A. Carbohydrate anatomical mapping and molecular identification of glucans using cellulose specific oligothiophenes. [Manuscript]