Protective vascular coagulation in response to bacterial infection of the kidney is regulated by bacterial lipid A and host CD147

Bacterial infection of the kidney leads to a rapid cascade of host protective responses, many of which are still poorly understood. We have previously shown that following kidney infection with uropathogenicEscherichia coli (UPEC), vascular coagulation is quickly initiated in local perivascular capillaries that protects the host from progressing from a local infection to systemic sepsis. The signaling mechanisms behind this response have not however been described. In this study, we use a number ofin vitro andin vivo techniques, including intravital microscopy, to identify two previously unrecognized components influencing this protective coagulation response. The acylation state of the Lipid A of UPEC lipopolysaccharide (LPS) is shown to alter the kinetics of local coagulation onsetin vivo. We also identify epithelial CD147 as a potential host factor influencing infection-mediated coagulation. CD147 is expressed by renal proximal epithelial cells infected with UPEC, contingent to bacterial expression of the α-hemolysin toxin. The epithelial CD147 subsequently can activate tissue factor on endothelial cells, a primary step in the coagulation cascade. This study emphasizes the rapid, multifaceted response of the kidney tissue to bacterial infection and the interplay between host and pathogen during the early hours of renal infection

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 Bowman's 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

The physiological and microbiological response to renal UPEC infection

The pathological outcome of a bacterial infection depends on the interplay between the host s defences and the virulence arsenal of the pathogen. Appreciation of this interplay is crucial to the understanding of pathogenesis and the development of efficient clinical treatments. In this thesis we wanted to study the dynamics of the early stages of renal bacterial infection. While sacrificial models and in vitro experimentation has given us a wealth of information, they lack the spatial and temporal resolution required to follow the crucial first hours of infection. To overcome this we developed a multiphoton based live animal infection model which allows for the visualization of infection progression in real-time. This model allows for cellular resolution visualisation of events occurring in the live kidney with the influences of the all physiological factors such as the blood, nervous, hormonal and immune systems intact. Our model utilizes micropuncture techniques to directly infuse bacteria into the renal tubules allowing for a well defined time-frame of infection. What we achieved was a unique insight into the rapid physiological responses to infection. Physiological responses described in this thesis include ischemic and obstruction injuries. These injuries are both related to dynamic physiological functions for which real-time live imaging is particularly suitable. Within 3-4 hours of the first bacterial interaction, epithelial signalling lead to activation of the clotting cascade and shut-down of local peri-tubular capillaries. The clotting response was shown to be crucial to isolate the infection and prevent sepsis. A rapid and dramatic drop in local tissue oxygen tension was also recorded with the combination resulting in a local ischemic injury. This infection-induced ischemia resulted in the characteristic cellular actin and integrin re-arrangements, but lacked a re-perfusion stage, instead resulting in localised tissue damage. We also investigated the effect of bacterial infection on renal filtration, revealing total nephron obstruction within 8 h. Other physiological responses seen include the infiltration of immune cells including both neutrophils and other unidentified mononuclear cells. This work shows that the full pathophysiology of pyelonephritis is a combination of numerous physiological injuries. Investigating the microbiological response to infection revealed that certain virulence factors affected the kinetics of both bacterial colonisation and the host response. Expression of the exotoxin α- haemolysin was shown to induce a more rapid host vascular response. A synergistic interaction between the adhesion factors P and Type-1 was shown to facilitate optimal kidney colonisation. P fimbriae were important for bacterial-epithelial interaction and in withstanding the sheer stress of filtrate flow, while Type 1 fimbriae expression becomes pertinent as the bacterial community expands into the lumen. This heterogeneous population allowed for the formation of an epithelial anchored biofilm which contributes to renal obstruction. Our work reveals new findings from both the physiological and microbiological responses to renal UPEC infection. These findings were made possible by the development and utilisation of the multiphoton based live-animal imaging model. It is hoped that as these types of live models become more integrated into infection biology awareness of these dynamic interplays will allow for improved treatment regimes

Nitrate Metabolism Modulates Biosynthesis of Biofilm Components in Uropathogenic Escherichia coli and Acts as a Fitness Factor During Experimental Urinary Tract Infection

To successfully colonize a variety of environments, bacteria can coordinate complex collective behaviors such as biofilm formation. To thrive in oxygen limited niches, bacteria's versatile physiology enables the utilization of alternative electron acceptors. Nitrate, the second most favorable electron acceptor after oxygen, plays a prominent role in the physiology of uropathogenic Escherichia coli (UPEC) and is abundantly found in urine. Here we analyzed the role of extracellular nitrate in the pathogenesis of the UPEC strain CFT073 with an initial focus on biofilm formation. Colony morphotyping in combination with extensive mutational, transcriptional, and protein expression analyses of CFT073 wild-type and mutants deficient in one or several nitrate reductases revealed an association between nitrate reduction and the biosynthesis of biofilm extracellular matrix components. We identified a role for the nitrate response regulator NarL in modulating expression of the biofilm master regulator CsgD. To analyze the role of nitrate reduction during infection in vivo, we tested wild-type CFT073 and a nitrate reductase null mutant in an ascending urinary tract infection (UTI) model. Individually, each strain colonized extensively, suggesting that nitrate reduction is expendable during UTI. However, during competitive co-infection, the strain incapable of nitrate reduction was strongly outcompeted. This suggests that nitrate reduction can be considered a non-essential but advantageous fitness factor for UPEC pathogenesis. This implies that UPEC rapidly adapts their metabolic needs to the microenvironment of infected tissue. Collectively, this work demonstrates a unique association between nitrate respiration, biofilm formation, and UPEC pathogenicity, highlighting how the use of alternative electron acceptors enables bacterial pathogens to adapt to challenging infectious microenvironments

Vancomycin-Loaded Microneedle Arrays against Methicillin-Resistant Staphylococcus Aureus Skin Infections

Skin and soft tissue infections (SSTIs) caused by methicillin‐resistant Staphylococcus aureus (MRSA) are a major healthcare burden, often treated with intravenous injection of the glycopeptide antibiotic vancomycin (VAN). However, low local drug concentration in the skin limits its treatment efficiency, while systemic exposure promotes the development of resistant bacterial strains. Topical administration of VAN on skin is ineffective as its high molecular weight prohibits transdermal penetration. In order to implement a local VAN delivery, microneedle (MN) arrays with a water‐insoluble support layer for the controlled administration of VAN into the skin are developed. The utilization of such a support layer results in water‐insoluble needle shafts surrounded by drug‐loaded water‐soluble tips with high drug encapsulation. The developed MN arrays can penetrate the dermal barriers of both porcine and fresh human skin. Permeation studies on porcine skin reveal that the majority of the delivered VAN is retained within the skin. It is shown that the VAN‐MN array reduces MRSA growth both in vitro and ex vivo on skin. The developed VAN‐MN arrays may be extended to several drugs and may facilitate localized treatment of MRSA‐caused skin infections while minimizing adverse systemic effects

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

<div><p>Septic shock caused by <em>Neisseria meningitidis</em> is typically rapidly evolving and often fatal despite antibiotic therapy. Further understanding of the mechanisms underlying the disease is necessary to reduce fatality rates. <em>Postmortem</em> samples from the characteristic <em>purpuric</em> rashes of the infection show bacterial aggregates in close association with microvessel endothelium but the species specificity of <em>N. meningitidis</em> has previously hindered the development of an <em>in vivo</em> 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 <em>purpura</em> 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 <em>N. meningitidis</em> were found to be the main mediator of association with the dermal microvessels <em>in vivo</em>. 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 <em>N. meningitidis</em> to the vascular wall, as opposed to circulating bacteria, determines vascular dysfunction in meningococcemia.</p> </div

Human skin graft.

<p>(A) 21 days post graft of human skin onto SCID/Beige mouse. (B) Histology of grafted human skin showing dermal/epidermal border (arrow); (H&E). (C) Graft interface (dotted line) between human (Hu) and mouse (Ms) skin (H&E). (D) Human endothelium in the grafted skin labeled with <i>Ulex europaeus agglutinin</i> lectin (UEA, red). Cell nuclei are labeled with DAPI (blue). (E) Junction between human (UEA lectin, red) and mouse (msCD31, green) vessels at the graft border. (F) Frame from Movie S1. Intravital microscopy showing perfusion of human vessels labeled with UEA lectin (red). Blood plasma is labeled with 150 kDa FITC-dextran (green). Black silhouettes within the flow are red blood cells.</p

Inflammatory signaling following infection.

<p>(A) Human IL-6 concentrations in serum of mice grafted with human skin and injected with either PBS or <i>N. meningitidis</i>. MOM, control mice grafted with mouse skin and infected for 24 h. (B) Human IL-8 concentrations from serum of mice as in (A). (C) Neutrophil infiltration (Ly6G/Ly6C, red) at 24 h post infection, a few bacteria can be seen (green) in the inflamed area. Cell nuclei stained with DAPI (blue). (D) Neutrophil infiltration (red) at 6 h post infection. (E) <i>N. meningitidis</i> (green) phagocytosed by a neutrophil (red), 6 h post infection.</p