Keira Melican

Uropathogenic Escherichia coli P and Type 1 fimbriae act in synergy in a living host to facilitate renal colonization leading to nephron obstruction.

The progression of a natural bacterial infection is a dynamic process influenced by the physiological characteristics of the target organ. Recent developments in live animal imaging allow for the study of the dynamic microbe-host interplay in real-time as the infection progresses within an organ of a live host. Here we used multiphoton microscopy-based live animal imaging, combined with advanced surgical procedures, to investigate the role of uropathogenic Escherichia coli (UPEC) attachment organelles P and Type 1 fimbriae in renal bacterial infection. A GFP+ expressing variant of UPEC strain CFT073 and genetically well-defined isogenic mutants were microinfused into rat glomerulus or proximal tubules. Within 2 h bacteria colonized along the flat squamous epithelium of the Bowmans capsule despite being exposed to the primary filtrate. When facing the challenge of the filtrate flow in the proximal tubule, the P and Type 1 fimbriae appeared to act in synergy to promote colonization. P fimbriae enhanced early colonization of the tubular epithelium, while Type 1 fimbriae mediated colonization of the center of the tubule via a mechanism believed to involve inter-bacterial binding and biofilm formation. The heterogeneous bacterial community within the tubule subsequently affected renal filtration leading to total obstruction of the nephron within 8 h. Our results reveal the importance of physiological factors such as filtration in determining bacterial colonization patterns, and demonstrate that the spatial resolution of an infectious niche can be as small as the center, or periphery, of a tubule lumen. Furthermore, our data show how secondary physiological injuries such as obstruction contribute to the full pathophysiology of pyelonephritis.

Bacterial infection-mediated mucosal signalling induces local renal ischaemia as a defence against sepsis.

Ascending urinary tract infections can cause extensive damage to kidney structure and function. We have used a number of advanced techniques including multiphoton microscopy to investigate the crucial early phases of uropathogenic Escherichia coli induced pyelonephritis within a living animal. Our results reveal a previously undescribed innate vascular response to mucosal infection, allowing isolation and eradication of the pathogen. The extremely rapid host response to mucosal infection was highlighted by the triggering of a cascade of events within 3–4 h. Epithelial signalling produced an increase in cellular O2 consumption and affected microvascular flow by clotting, causing localized ischaemia. Subsequent ischaemic damage affected pathophysiology with actin re‐arrangement and epithelial sloughing leading to paracellular bacterial movement. A denuded tubular basement membrane is shown to hinder immediate dissemination of bacteria, giving the host time to isolate the infection by clotting. Suppression of clotting by heparin treatment caused fatal urosepsis. Clinically these findings may be relevant in antibiotics delivery in pyelonephritis patients and to the use of anticoagulants in sepsis.

Real‐time studies of the progression of bacterial infections and immediate tissue responses in live animals

By combining intravital multiphoton microscopy and bacterial genetics we have developed a technique enabling real‐time imaging of bacterial proliferation and tissue responses in a live animal. Spatial and temporal control of the infection process was achieved by microinjecting GFP+‐expressing uropathogenic Escherichia coli (UPEC) into tubules of exteriorized kidneys in live rats. GFP+ was introduced in the clinical UPEC strain CFT073 as a single‐copy chromosomal gene fusion. Within hours, bacterial colonization was accompanied by marked ischaemic effects, perivascular leakage, loss of tubular integrity and localized recruitment of immune cells. The pathophysiology was altered in response to an isogenic bacterial strain lacking the exotoxin haemolysin, revealing the subtle and temporal roles of bacterial virulence factors in vivo. Microdissection and RNA extraction of the injected nephron allowed molecular analysis of prokaryotic and eukaryotic gene expression. The techniques described here can be applied to study the integrated cell communication evoked by a variety of bacterial pathogens, assisting in the design of strategies to combat bacterial infections.

Adhesion of Neisseria meningitidis to Dermal Vessels Leads to Local Vascular Damage and Purpura in a Humanized Mouse Model

Septic shock caused by Neisseria meningitidis is typically rapidly evolving and often fatal despite antibiotic therapy. Further understanding of the mechanisms underlying the disease is necessary to reduce fatality rates. Postmortem samples from the characteristic purpuric rashes of the infection show bacterial aggregates in close association with microvessel endothelium but the species specificity of N. meningitidis has previously hindered the development of an in vivo model to study the role of adhesion on disease progression. Here we introduced human dermal microvessels into SCID/Beige mice by xenografting human skin. Bacteria injected intravenously exclusively associated with the human vessel endothelium in the skin graft. Infection was accompanied by a potent inflammatory response with the secretion of human inflammatory cytokines and recruitment of inflammatory cells. Importantly, infection also led to local vascular damage with hemostasis, thrombosis, vascular leakage and finally purpura in the grafted skin, replicating the clinical presentation for the first time in an animal model. The adhesive properties of the type IV pili of N. meningitidis were found to be the main mediator of association with the dermal microvessels in vivo. Bacterial mutants with altered type IV pili function also did not trigger inflammation or lead to vascular damage. This work demonstrates that local type IV pili mediated adhesion of N. meningitidis to the vascular wall, as opposed to circulating bacteria, determines vascular dysfunction in meningococcemia.

Progression of bacterial infections studied in real time--novel perspectives provided by multiphoton microscopy.

The holy grail of infection biology is to study a pathogen within its natural infectious environment, the living host. Advances in in vivo imaging techniques have begun to introduce the possibility to visualize, in real time, infection progression within a living model. In this review we detail the current advancements and knowledge in multiphoton microscopy and how it can be related to the field of microbial infections. This technology is a new and very valuable tool for in vivo imaging, and using this technique it is possible to begin to study various microbes within their natural infectious environment – the living host.

Vascular colonization by Neisseria meningitidis

Bacterial infection of human vasculature can lead to unregulated systemic activation of coagulation and innate immunity and rapidly becomes life threatening. Neisseria meningitidis is a vascular pathogen that causes fatal sepsis and meningitis. Post-mortem histological analysis of tissues from individuals infected with N. meningitidis show large bacterial aggregates in close association with the vascular wall of small vessels. The ability of this bacterium to colonize blood vessel endothelium is likely to impact its capacity to both multiply in the blood stream and reach the brain. This process will be referred to as vascular colonization. Recent work has described a number of early steps in N. meningitidis vascular colonization, from attachment to proliferation and dissemination, focusing on the bacterial-host interaction.

Multiphoton imaging of host-pathogen interactions.

Studying the events that occur when a pathogen comes into contact with its host is the basis of the field of infection biology. Over the years, work in this area has revealed many facets of the infection process, including attachment, invasion and colonization by the pathogen, and of the host responses, such as the triggering of the immune system. Recent advancements in imaging technologies, such as multiphoton microscopy (MPM), mean that the field is in the process of taking another big leap forward. MPM allows for cellular‐level visualization of the real‐time dynamics of infection within the living host. The use of live animal models means that all the interplaying factors of an infection, such as the influences of the immune, lymphatic and vascular systems, can be accounted for. This review outlines the developing field of MPM in pathogen‐host interactions, highlighting a number of new insights that have been ‘brought to light’ using this technique.

Nondestructive, real-time determination and visualization of cellulose, hemicellulose and lignin by luminescent oligothiophenes

Enabling technologies for efficient use of the bio-based feedstock are crucial to the replacement of oil-based products. We investigated the feasibility of luminescent conjugated oligothiophenes (LCOs) for non-destructive, rapid detection and quality assessment of lignocellulosic components in complex biomass matrices. A cationic pentameric oligothiophene denoted p-HTEA (pentamer hydrogen thiophene ethyl amine) showed unique binding affinities to cellulose, lignin, hemicelluloses, and cellulose nanofibrils in crystal, liquid and paper form. We exploited this finding using spectrofluorometric methods and fluorescence confocal laser scanning microscopy, for sensitive, simultaneous determination of the structural and compositional complexities of native lignocellulosic biomass. With exceptional photostability, p-HTEA is also demonstrated as a dynamic sensor for real-time monitoring of enzymatic cellulose degradation in cellulolysis. These results demonstrate the use of p-HTEA as a non-destructive tool for the determination of cellulose, hemicellulose and lignin in complex biomass matrices, thereby aiding in the optimization of biomass-converting technologies.

Real-time optotracing of curli and cellulose in live Salmonella biofilms using luminescent oligothiophenes

Extracellular matrix (ECM) is the protein- and polysaccharide-rich backbone of bacterial biofilms that provides a defensive barrier in clinical, environmental and industrial settings. Understanding the dynamics of biofilm formation in native environments has been hindered by a lack of research tools. Here we report a method for simultaneous, real-time, in situ detection and differentiation of the Salmonella ECM components curli and cellulose, using non-toxic, luminescent conjugated oligothiophenes (LCOs). These flexible conjugated polymers emit a conformation-dependent fluorescence spectrum, which we use to kinetically define extracellular appearance of curli fibres and cellulose polysaccharides during bacterial growth. The scope of this technique is demonstrated by defining biofilm morphotypes of Salmonella enterica serovars Enteritidis and Typhimurium, and their isogenic mutants in liquid culture and on solid media, and by visualising the ECM components in native biofilms. Our reported use of LCOs across a number of platforms, including intracellular cellulose production in eukaryotic cells and in infected tissues, demonstrates the versatility of this optotracing technology, and its ability to redefine biofilm research.

Microbial pathogenesis revealed by intravital microscopy: pros, cons and cautions

Intravital multiphoton imaging allows visualization of infections and pathogenic mechanisms within intact organs in their physiological context. Today, most organs of mice and rats are applicable to in vivo or ex vivo imaging, opening completely new avenues for many researchers. Advances in fluorescent labeling of pathogens and infected cells, as well as improved small animal models for human pathogens, led to the increased application of in vivo imaging in infectious diseases research in recent years. Here, we review the latest literature on intravital or ex vivo imaging of viral and bacterial infections and critically discuss requirements, benefits and drawbacks of applied animal models, labeling strategies, and imaged organs.