Manipulating flagellar gene regulation in uropathogenic Escherichia coli to explore its effect(s) on the urothelial proinflammatory response
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2024-02
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Newcastle University
Abstract
Urinary tract infections (UTIs) are a global problem and the major pathogen associated with such infections is Uropathogenic Escherichia coli (UPEC). UTIs are classified according to the site of infection: cystitis describes a bladder infection and pyelonephritis a kidney infection. If not treated symptomatic UTIs can lead to bacteraemia, sepsis and death. An additional condition is asymptomatic bacteriuria (ASB), which mainly affects older and/or immunocompromised populations. ASB is associated with UPEC colonising the lower urinary tract (UT) without causing symptoms and often underlies symptomatic infections.
A panel of clinical uro-associated E. coli isolates recovered from cystitis, pyelonephritis, ASB and bacteraemia patients were characterised for motility and their ability to induce an innate response in urothelial cells, determined by the induction of NF-κB and/or IL-8 synthesis. Data suggested a mix of motile and non-motile strains, but the motility patterns and abilities of the motile strains to induce an innate response were very variable. The aim of this thesis was to understand the factors underpinning this variation.
Lower UT tissues defend themselves from bacterial assaults via innate mechanisms involving urothelial receptors with the most important being Toll-like Receptor 5 (TLR5). TLR5 detects the bacterial flagellar subunit, flagellin and activation results in the synthesis of host defence agents and bacterial killing. Hence, flagellar appear key in inducing the urothelial host defences. Synthesising flagella is an energy-consuming process initiated only when motility is advantageous. This led to the hypothesis that growth fitness is important in the recognition of uro-associated E. coli by urothelial cells.
Chapter 3 describes experiments, measuring bacterial doubling times, and comparing the growth fitness of motile and non-motile clinical isolates. The motile (M) strain mean doubling time was 17.4±3.0 min versus 19.7±2,5 min for the non-motile (NM) strains (P=0.011). However, the range of doubling times was similar (M: 13.9-26.9 min; NM: 14.7-25.3min). These latter data argued against fitness being a primary driving factor in the variation of host recognition. To examine if variation linked to flagellar abundance strains were engineered, using pSE_flhDC to hyperexpress flagellar. The mutant strains showed increased motility and innate responses (2-fold) indicating that variability in the host responses linked to fluctuations in bacterial flagellar numbers and subsequent motility.
Chapter 4 confirmed these data by constructing and using ΔflhDC and ΔclpP mutants, which resulted in either a loss of flagellar or unregulated flagellar synthesis. Six strains were analysed NCTC10418, CFT073, 3408, MG1655, 5469 and 5489. To quantify flagellar numbers the FlgE of three strains NCTC10418, CFT073 and 3408 was replaced with FlgEA240C and the mutated cells stained with Alexa Fluor 488 maleimide. These cells were examined using fluorescence microscopy. Visualisation of the FlgEA240C foci of the ΔclpP mutants supported increases in flagellar numbers as well as an increase in the bacterial population carrying flagellar. These data further supported the concept that variability in the bladder host response is linked to population heterogeneity and flagellar abundance of the invading uropathogens.
Data presented in Chapters 3 and 4 related to bacteria cultured in enriched media, which physiologically does not reflect the UT environment. Experiments were therefore performed using three strains, 3408, 5469 and 5489 and their ΔflhDC and ΔclpP mutants grown in artificial urine media (AUM) at pH 5.5 (acid urine) and pH 6.5 (alkaline urine). Data showed growth in AUM was associated with a reduced motility and bladder innate response, and that pH did not impact these data.
In summary results have shown that uro-associated E. coli isolates, generally, regulate flagellar synthesis to allow colonisation or infection, but appear to exploit population heterogeneity to prevent recognition by urothelial cell TLR5 receptors and bacterial killing.
Description
Uropathogenic Escherichia coli (UPEC) are responsible for the majority of uncomplicated and complicated urinary tract infections (UTIs) suffered by patients worldwide (Flores-Mireles et al., 2015). Clinical treatments at present are limited to antibiotics and current National Institute for Health and Care Excellence (NICE) and Scottish Intercollegiate Guidelines Network (SIGN) treatment guidelines for UTIs recommend either Nitrofurantoin or Trimethoprim as first-line antibiotics (NICE, 2021). A consequence of such treatments is antimicrobial resistance (AMR) with resistance reported in 5 and >30% of uro-associated E. coli isolates respectively (Carter et al., 2023; Vallee et al., 2023). This resistance is driving global antibiotic stewardship programmes and the need for further understanding of UPEC/urothelial interactions that will help underpin the development of new UTI therapies.
A further concern is that over 25% of all UTI cases, particularly in females, are associated with recurrent infections. The success of UPEC, particularly in females, links to its motility and ability to ascend the urethra, which is shorter in females, into the bladder (Hickling et al., 2015). While flagella underpin UPEC motility this property can be costly as animal and bird tissues have evolved an innate defence mechanism to detect the flagellar filament subunit, flagellin. In humans, the recognition mechanism involves a membrane bound receptor known as Toll-like Receptor-5 (TLR5). TLR5 activation leads to the rapid release of inflammatory cytokines such as interleukin-8 (IL-8) that recruit macrophages and neutrophils to the site of infection, and host antimicrobial agents that directly kill bacteria. To further our understanding of UPEC/urothelial interactions this thesis describes experiments interrogating the interactions between UPEC flagella and the urothelial host responses (Ali et al., 2017).
UTIs affect 150 million people around the world every year (O'Brien et al., 2016). UTIs are commonly associated with Escherichia coli, originating in the gut, contaminating the lower urinary tract and represent one of the most frequent reasons for those suffering a bacterial infection consulting their health care provider (Foxman, 2002). Women are particularly affected with data suggesting that during their lifetime up to 50% of women will experience at least one case of UTI (Foxman et al., 2003; Micali et al., 2014). While 11% of women over the age of 18 will suffer a case of UTI annually (Foxman & Brown, 2003), UK data suggests up to 57% of these experience three or more episodes, which clinically, is referred to as suffering from recurrent UTIs (rUTIs) (Butler et al., 2015). Recurrent UTIs are defined as two UTI episodes in six months or more than three episodes in 12 months (Sihra et al., 2018), with these repeated infections linking either to a relapse i.e., re-infection with the same strain or reinfection with a different microbe or strain (Kodner & Thomas Gupton, 2010). While rUTIs are debilitating for the patient, these infections also carry significant economic consequences, not only through lost productivity, due to absence from work, but also through the healthcare costs associated with treating the infections. Such costs were estimated at $3.5 billion in the USA, $1.5 billion in the European Union (Sihra et al., 2018), and $26 million is the cost of only primary care in the UK in 1995 (Callan et al., 2014).
UTIs can be classed clinically as either symptomatic or asymptomatic. Symptomatic episodes are characterized by urinary urgency, frequency, pain during urination (dysuria), urine being contaminated by blood and the detection of > 104 CFU/mL microorganisms in a single voided midstream urine. If not treated, symptomatic UTIs can, particularly in older populations and the immunocompromised, lead to bacteraemia, sepsis, and death (Gonzalez & Schaeffer, 1999). In contrast, asymptomatic bacteriuria (ASB) infections are defined as the presence of bacteria in urine in the absence of clinical symptoms. Many individuals presentwith asymptomatic bacteriuria, but particularly older populations, where up to 15% have been
reported to be characterized by persistent bacteriuria (Ariathianto, 2011).
UTIs are also classified according to the localization of the infection. Urethritis describes an infection of the urethra, cystitis describes a bladder infection and pyelonephritis is the term used when a patient’s kidneys are infected (Figure 1.1) (Stamm & Hooton, 1993).
Keywords
TLR5, UPEC, flagellin, urinary tract infection, bacterial motility, flhDC, IL-8, clpP