Uropathogenic Escherichia coli colonization of the urinary tract: pathways to chronicity

Abstract

Chronically recurrent urinary tract infection (rUTI) caused most frequently by uropathogenic strains of Escherichia coli (UPEC) are common infections and cause considerable healthcare burdens. Repeated reintroduction of bacteria into the bladder, such as from sexual intercourse, increases the risk of recurrence and chronicity. Studies have sought to define the complex host-pathogen interactions that occur during chronic rUTI, however many areas of mouse rUTI modelling remain incompletely defined. Interleukin 17A (IL-17A) has important roles in the control of acute UTI and may have important roles in the control of chronic rUTI. This thesis aimed to develop and characterise a mouse model of UPEC superinfection and use this to investigate the roles of IL-17A in the pathogenesis and control of chronic rUTI. A mouse model of superinfection was characterised in BALB/c mice, a previously considered resistant mouse strain for chronic rUTI, using a 2 x 108 CFU infectious dose of UPEC strains EC958 (a multidrug-resistant cystitis strain) or CFT073 (a urosepsis and pyelonephritis strain) 24 hours apart. EC958 exhibited significantly higher bacteriuria loads than CFT073, significantly higher bacterial burdens in the kidney after 28 days of infection, and a higher incidence of chronicity, defined as 104 CFU/ml urine at all collection timepoints and in the bladder after 28-days of infection. In IL-17A-/- mice, CFT073 exhibited a significantly higher incidence of chronicity, and, similar to EC958, exhibited kidney bacterial loads compared to WT BALB/c mice. CFT073 was able to induce IL-17A production in BALB/c WT mice, and both strains induced IL-17A production at day 10 and day 28 post-superinfection, which correlated with bladder bacterial load at day 28 post-superinfection. Plasma collected at 24h after superinfection with EC958 was used in a multiplex assay to reveal interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) as predictors of chronic bacteriuria and bladder infection. IL-6 was significantly associated with chronic kidney infection. Immunohistochemistry (IHC) identified chronic kidney infection in BALB/c and IL-17A-/- mice was characterised by neutrophilia, regardless of mouse background and UPEC strain. Mixed cellular infiltrates of T cells and macrophages were evident in 28-day chronically infected kidneys, with significantly higher levels of macrophages in BALB/c WT mice compared to IL-17A-/- mice. Flow cytometry analysis of cellular response to superinfection with EC958 at day 10 and day 28 showed bladder responses were dominated by lymphocytes such as T cells and natural killer cells. Chronically infected mice had a significantly higher level of neutrophils in the kidneys at day 10 and day 28 post-superinfection compared with controls. Intracellular cytokine staining revealed that IL-17A is produced from both  T cells and CD4+ T cells in the bladder and the kidney, with significantly more IL-17+ cells at day 10 in resolved mice compared with PBS control mice, and a switch in the trend at day 28 with significantly more IL-17A+ cells in chronically-infected mice compared with resolved and PBS control mice. Several CFT073 bacterial mutants were investigated in superinfection experiments to explore any potential effects of UPEC haemolysin and flagella on colonisation and IL-17A induction, and considering virulence differences between EC958 and CFT073. Hemolysin was not associated with chronicity, however hyperflagellated CFT073 caused significantly higher bacterial burden in IL-17A-/- mice but not WT mice. These findings suggest a possible nexus between flagella expression and IL-17A-mediated clearance. In whole blood killing assays, lipopolysaccharide and capsule mutants of EC958 and CFT073 were not attenuated and no differences were observed in the killing capacity of blood from WT mice versus IL-17A-/- mice. EC958 and CFT073 grew in mouse blood but were killed in human blood. In summary, the research contained within this Thesis shows that EC958 is more virulent than CFT073 in a BALB/c model of superinfection, and in ex vivo mouse blood growth. IL-6 and MCP-1 are predictive cytokines for chronic rUTI in mice. IL-17A is essential to promote the control of chronic rUTI. Chronically infected mice exhibit significant T-cell derived IL-17A production that, while important for the control of infection, might also contribute to immunopathology and chronicity. This research represents the first in-depth characterisation of the cellular response to superinfection. IL-17A-mediated infection may be a response to UPEC flagella. Collectively, this body of work represents a significant and original contribution of knowledge into the host response and pathogenesis of chronic rUTI due to UPEC.