Elinor Shvartsman Presentation Summary Report (January 29, 2019)
Are FimH fimbriae a feasible target for drug antagonist/inhibition treatments of invasive
uropathogenic E. coli (UPEC) infections?
By: Elinor Shvartsman MMIC7050
Presentation Summary Report Lecturer: Dr. Denice Bay
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
Urinary Tract Infections and Host Factors Contributing to Infection:
UTIs are generally described as infections of the urinary tract including infections of the kidneys, bladder, ureters, and the urethra (Figure 1). Millions of individuals around the world develop urinary tract infections (UTIs) each year making UTIs some of the most prevalent bacterial infections (26, 9). UTIs are more prevalent in women, and an estimated 40% of women will experience at least one UTI in their life time (26).
UTIs occur when pathogenic bacteria invade the urinary tract and cause infection. Uropathogenic Escherichia coli (UPEC) cause most UTIs (26, 9). Other UTI causing agents include Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, Pseudomonas aeruginosa, and Candida, as well as other less prevalent bacterial and fungal pathogens (Figure 2) (26, 9). UTI syndromes include cystitis (lower UTI of the bladder), pyelonephritis (upper UTI of the kidneys), asymptomatic bacteriuria, urosepsis, and catheter associated UTIs (12). Common symptoms of UTI include dysuria (painful urination), fever, discomfort and frequent urination (12). UTIs are clinically categorized as either uncomplicated or complicated with uncomplicated UTIs occurring in normal non pregnant and non catheterized patients without any structural or functional abnormalities, whereas all other UTIs are classified as complicated (11). Urinary tract infection begin with contamination of the urethra or its surroundings with gut microbes (usually) which can then spread upwards to the bladder and cause disease by multiplying rapidly, forming biofilms, as well as causing inflammation and severe tissue damage via toxin and enzyme production. The bacteria can then continue moving upwards toward the kidney, and then cause systematic damage via dissemination through the blood by entering the renal vasculature and causing various complications (9) (Figure 1).
Women are particularly at risk for UTIs due to the close proximity of the urethtal opening to the anus (and therefore to the gut microbiome) and to the vaginal opening where various uropathogenic microorganisms reside (12). The bladder is closer to the urethral opening in women than in men, and the moist environment of the female periurethral space provides an optimal environment for bacterial growth(18). Other risk factors for UTIs include, having history of UTIs, sexual activity, condom or diaphragm usage, trauma or manipulation of the urinary tract such as that caused by catheterization, anatomic or genetic susceptibility markers, and diabetes or obesity (12).
Uropathogenic E. coli (UPEC):
Most UTIs (complicated and uncomplicated) are caused by E. coli which are Gram negative bacilli (rods). Some E. coli strains are found as commensals of the normal gut microbiota where they mostly remain harmless. However, some commensal E. coli strains may acquire and express virulence factors such as toxins, cell adhesion molecules such as pili and fimbriae, and iron acquisition systems by horizontal gene transfer (via conjugation, transduction and transformation) (26). UPEC are
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
classified as part of the extra-intestinal E. coli group, which corresponds to E. coli strains able to live in the gut harmlessly but with the ability to occupy other niches throughout the body such as the urinary tract to cause disease (3). UPEC phylotypes A, B1, B2, and D are the most notable and strains can be distinguished genetically from non-pathogenic strains by the presence or absence of specific virulence genes and pathogenicity islands which are genomic islands (horizontally transferred elements) present only in the pathogenic strains (4, 25).
Virulence and Invasiveness Factors of Uropathogenic E. coli:
Virulence and invasiveness factors are specialized molecules produced/expressed by pathogenic bacteria such as UPEC enabling them to replicate inside the host avoiding the host’s immune defenses and allowing UPEC to establish effective infection (6).
Common UPEC Virulence Factors: LPS is present in Gram negative bacteria such as UPEC. LPS acts as an important virulence factor as it stimulates toll like receptor 4 (TLR4) on host cells to activate innate immunity, which in turn results in inflammatory responses, as well as the perception of pain (23). LPS in UPEC strains also plays a role in detergent (such as bodily salts, and some antibiotics) resistance, and may be vital for acute colonization of the urothelium and the establishment of IBC reservoirs (2, 26). Another virulence factor found in UPEC are P- pili which are also known as Pap-pili because they are encoded by the pap (pyelonephritis associated pili) operon (15, 26). These pili are assembled by the chaperone usher pathway similar to type 1 pili and are each composed of six different subunits with PapG being the adhesin component that mediates adherence to cells of the urinary tract (26). The PapG subunit interacts with diagalactose components in P-blood group antigens and on uroepithelial cells (15, 26). P-type pili are an important virulence factor as they are encoded on pathogenicity islands, and are highly abundant in UPEC strains causing acute pyelonephritis (15). Curli fimbriae are fibrous surface appendages that facilitate biofilm formation and may also act as adhesins (5, 26). Curli fimbriae are considered an important virulence factor in UPEC associated cystitis (bladder infection) (5). Other non-piliated adhesins also exist and play important roles in bladder and kidney colonization (26). Flagella are also present in many clinical UPEC isolates and have suggested roles in adherence, dispersal and biofilm formation (26). UPEC may also secrete toxins such as α-hemolysin (encoded by the hlyA gene) which form pores causing lysis of host proteins including cytoskeletal proteins and proteins involved in inflammation and apoptosis (8). The α-hemolysin has also been specifically implicated in cell death of bladder epithelial cells resulting in cell exfoliation. Cell exfoliation is the removal of the surface epithelium cells and may benefit both the host as it is able to clear infected cells but also benefits UPEC and harms the host when UPEC can disseminate to other hosts and penetrate deeper into tissues of the urinary tract (26). UPEC require iron for various metabolic functions, and have developed specialized system to survive in the iron-limited environment of the urinary tract. Siderophores are iron binding molecules that
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
UPEC produce to sequester iron from the environment more efficiently (26). To better accommodate the iron limited environment, UPEC, especially ones in IBC, also exhibit upregulation of iron acquisition systems (26). The ability of E. coli to express multiple of these virulence factors (Figure 3) results in increased pathogenicity and various UTI complications. Additional UPEC phenotypes are being explored and it appears that UPEC strains can also be characterized by very rapid growth (10) via an unclear mechanism.
Type 1-pili: For UPEC to effectively colonize components of the urinary tract and cause infection they must be able to resist the body’s clearance and defence mechanisms including urination, antimicrobials, and immune system effector functions (20). This is often mediated by adhesins (19), which are special molecules expressed on the surface of the bacterial cells allowing them to effectively bind to cells within the urinary tract and resist clearance. UPEC possess type 1 pili, which are encoded by the fim operon and consist of multiple protein subunits including: FimA, FimG, FimF, and FimH (24, 26). UPEC type 1 pili are assembled using the chaperone-usher pathway which involves FimD as an outer membrane usher that functions to allow pilus subunits to form on the surface of the UPEC membrane. FimA then forms the thick helical rod (at the distal end of the pilus) followed by FimF and FimG subunits which form the adaptors connecting the rod to the FimH subunit, the actual adhesin (24, 26). The FimH pilus subunit contains a lectin domain which mediates binding to carbohydrates on the host cell surface, and a pilin domain connecting it to the adaptor subunits of the pilus. The urinary tract is lined at its distal end by a specialized epithelium called the uroepithelium or urothelium (13). Due to ability of the bladder to stretch and shrink as needed, BEC are transitional epithelial cells (9). BEC have three distinct layers, with an apical (lumen facing) layer with very large hexagonal umbrella cells (26). These umbrella cells express uroplakin proteins on their cell membrane at the apical (lumen facing) side (9). FimH lectin domain binds to mannosylated glycoproteins such as the ones coating uroplakins. FimH-mannosylated uroplakin binding allows UPEC to remain physically attached to uroepithelium, and also in some strains, appears to mediate cell invasion (9). FimH appears to mediate invasion by first interacting with surface integrins and activating cell signalling cascades (using Rho GTPases) which in turn lead to actin-dependent entry. Once inside the cytoplasm the invading bacterium can go multiple fates; it can either remain in vesicles or readily replicate in the cytoplasm to form biofilm like intracellular bacterial communities (IBC). UPEC may stimulate TLR-4 once inside the cells resulting in its expulsion from the cells, however IBC can avoid this host immune response by acting as quiescent (dormant) reservoirs that can be used to seed recurrent infections (9, 17, 22). This is why having previous history of UTIs makes one more susceptible for future UTIs.
FimH inhibitors and antagonists for the treatment of UPEC UTI:
FimH is considered a promising target for the development of therapeutics to treat UPEC UTIs. FimH plays a crucial role in adherence to urothelial surfaces of the
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
urinary tract and a potential role in invasion and thus initiation of infection. The prophylactic potential of FimH targeting has been shown in in-vivo studies in mice when UPEC strains lacking the type-1 pili (which uses FimH as the adhesin) were not able to establish infection (1). Furthermore, antibodies targeting the FimH adhesin were able to successfully prevent UPEC infection in a murine cystitis (bladder infection) model (16). The focus of FimH as a prophylactic and possibly a therapeutic target resulted in the development and discovery of FimH antagonists. FimH inhibitors/antagonists are compounds capable of preventing FimH adherence to cells by directly interacting with the mannose-lectin binding unit (Figure 4).Since FimH binds to mannose epitopes present on uroplakins, X-ray crystallography was used to guide design of mannose and mannose derivatives to act as potential FimH inhibitors (26) Studies of FimH-mannose interactions have revealed that the mannose binding pocket is predominantly constant in all UPEC strains, and that any changes to those residues, highly attenuate virulence of UPEC strains (7). In addition, FimH shows selectivity specifically to α-D-mannose, likely due to specific hydrogen bonds formed with this D-mannose anomer (21).
Kranjcec et al (14) studied the use of D-mannose prophylactically for prevention of recurrent UTIs in women with a history of UTIs. Kranjcec et al (14) showed that women in D-mannose arm showed a lower risk of recurrent UTI when compared to control arm over a six month period. Interestingly, prophylactic D-mannose consumption resulted in similar low risk for UTI recurrence as the nitrofurantoin (antibiotic) receiving group (Figure 6) (14). In addition, D-mannose treatment resulted in greater tolerability and reduced side-effects than the antibiotic treated subjects (14). Thus, D-mannose may be a promising UTI prevention method for women with recurrent or chronic UTI infections. This study is limited in that it was not blinded (14). Future investigations should include larger sample size and the assessment of D-mannose in male participants, and participants before catheterization.
To increase potency, stability and bioavailability of FimH antagonists, D-mannose derivatives such as small molecular weight mannosides (Figure 5) have been developed. These derviatives are designed based on the biochemical interactions between FimH and mannose and also the favourable interactions in the areas surrounding the mannose recognition pocket (21). This approach to drug design through the investigation of structural interactions, is known as “structure based design”, and resulted in the development of multiple improved mannose-derived FimH antagonists (21). Mannosides are showing therapeutic promise against UPEC infection as shown in mouse models. Cusumano et al (7) developed and optimized several mannoside compounds for increased bioavailability and fast-action for chronic UTIs using a mouse model. In these experiments several different mannosides differing in their side chain chemistries were tested. The researchers found better efficiency of some of their mannosides in treating chronic UTI in mice, when compared to standard trimethoprim/sulfamethoxazole antibiotic treatment(7). This is an important discovery, especially due to the rapid emergence of antibiotic resistant UPEC strains (7). In addition Cusumano et al, provides a
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
template for optimization of multiple mannose-dervied FimH antagonists, accounting for bioavailability, pharmacodynamics, efficacy and timeliness (7).
Benefits and Limitations of FimH antagonists for the treatment of UTI:
In an era where antibiotic resistance is at an all time high, alternative therapeutic and prophylactic drugs are needed to combat and prevent bacterial infections. Invasive bacterial infections such as UTIs caused by UPEC, are extremely common and targeting of essential virulence factors such as UPEC adhesion molecules has shown great promise as outlined above.
The main benefits of FimH antagonists are the potential to work against otherwise untreatable multi and pan- drug resistant UPEC strains, as they would bind to the bacteria and allow them to be washed away by the urine-preventing further infection and IBC formation. In addition, FimH antagonists such as α-D-mannose supplied as dietary supplements have a variety of other health benefits such as increasing the body’s inducing macrophage activation to fight disease (26). Furthermore, due to their specific binding to FimH, they would likely affect the microbiome less dramatically than would antibiotics. Another benefit of FimH antagonists is their cost-effectiveness, both in terms of overall production, and in terms of lowering the economic burden of both antibiotic resistance and UTI (26). As FimH inhibitors act to bind to FimH and not to directly harm the bacterium, they are not expected to result in “anti FimH antagonist resistance”-a huge benefit when compared to the currently used antibiotic treatments. In addition, trials report higher tolerability to FimH antagonist –D-mannose, with less side effects compared to antibiotics (14). Another benefit for FimH antagonists, is that it would work on UPEC in the gut as well, and other invasive E. coli expressing FimH adhesin, and thus would prevent future infections of other anatomical structures, even ones external to the urinary tract (26).
There are some limitations to using FimH antagonists for the treatment of UPEC infections. The major downside of this approach is that a FimH antagonist is only able to inhibit binding of bacteria present on the surface of cells and is not capable of clearing an already established quiescent IBC reservoirs, and thus does not provide a “cure” for recurring and chronic UTIs (26). Essentially, this means FimH antagonists would need to be consumed periodically event after the clearance of the current UTI to prevent recurrence. None the less, if the patient can tolerate FimH antagonists well, they would still be a better prophylactic/treatment option than antibiotics. Another caveat of FimH antagonists is that this approach can only target UPEC that use FimH adhesins, but would not be effective against those that use P-pilli for adherence, as P-pili bind to di-galactose components and not to mannose (15, 26). In addition, as FimH inhibitors simply clear the bacteria from the body, UPEC can still disseminate throughout the environment and cause disease.
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
Nonetheless with the development of new technologies, structure guided FimH inhibitor design is becoming more and more optimized, allowing the improvement of current product and thus most limitations may be overcome (21).
In conclusion, as most UPEC strains use FimH fimbriae to adhere and invade cells of the urinary tract, targeting FimH by drug antagonists shows promise as both a therapeutic to clear current infections, and a prophylactic measure to prevent recurrent UTIs (26).
References: 1) Abgottspon D, Ernst B. (2012) In vivo Evaluation of FimH Antagonists- A
Novel Class of Antimicrobials for the Treatment of Urinary Tract Infection. CHIMIA. 66(4):166-169. doi: 10.2533/chimia.2012.166
2) Anguiniga LM, Yaggie RE, Schaeffer AJ, Klumpp DJ. (2016) Lipopolysaccharide Domains modulate Urovirulence. Infection and Immunity. 84:3131-3140. Doi:10.1128/IAI.00315-16
3) Bien J, Sokolova O, Bozko P. (2012) Role of Uropathogenic Escherichia coli Virulence Factors in the Development of Urinary Tract Infection and Kidney Damage. International Journal of Nephrology. 2012: 681473. doi: 10.1155/2012/681473
4) Clermont O, Gordon D, Denamur E. (2015) Guide to the various phylogenetic classification schemes for Escherichia coli and the correspondence among schemes. Microbiology. 161:980-988. doi: 10.1099/mic.0.000063
5) Cordeiro MA. Werle CH, Milanes GP, Yano T. (2016) Curli fimbria: an Escherichia coli adhesin associated with human cystitis. Brazilian Journal of Microbiology. 47(2):414-416. 10.1016/j.bjm.2016.01.024
6) Cross AS. (2008) What is a virulence factor? Critical Care. 12(6):196. doi: 10.1186/cc7127
7) Cusumano CK, Pinkner JS, Han Z…et al. (2011) Treatment and Prevention of Urinary Tract Infection with Orally Active FimH Inhibitors. Science Translational Medicine. 3(109) 109ra115. doi: 10.1126/scitranslmed.3003021
8) Dhakal BK, Mulvey MA. (2012) The UPECPore Forming Toxin -Hemolysin αTriggers Proteolysis of Host Proteins to Disrupt Cell Adhesion, Inflammatory and Survival Pathways. Cell Host Microbe. 11(1):58-69. Doi: 10.1016/j.chom.2011.12.003
9) Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. (2015) Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nature Reviews in Microbiology. 13(5):269-84. doi: 10.1038/nrmicro3432
10)Forsynth VS, Ambruster CE, Smith SN, …et al. (2018) Rapid Growth of Uropathogenic Escherichia coli during Human Urinary Tract Infection (UTI). mBio. 9(2): e00186-18. doi:10.1128/mBio.00186-18
11)Foxman B. (2010) The epidemiology of urinary tract infection. Nature Reviews Urology. 7: 653-660.
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
12)Foxman B. (2014) Urinary tract infection syndromes. Infectious Disease Clinics of North America. 28(1):1-13. doi: 10.1016/j.idc.2013.09.003
13)Khandelwal P, Abraham SN, Apodaca G. (2009) Cell biology and physiology of the uroepithelium. American Journal of Physiology Renal Physiology. 297(6):F1477-F1501. doi: 10.1152/ajprenal.00327.2009
14)Kranjcec B, Papes D, Altarac S. (2014) D-mannose powder for prophylaxis of recurrent urinary tract infections in women: a randomized clinical trial. World Journal of Urology. 32:79-84 doi: 10.1007/s00345-013-1091-6
15)Lane MC, Mobley HLT. (2017) Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney. Kidney International. 72:19-25. doi: 10.1038/sj.ki.5002230
16)Langermann S, Palaszynski S, Barnhart M…, et al. (1997) Prevention of Mucosal Escherichia coli Infection by FimH-Adhesin-Based Systemic Vaccination. 276(5312):607-611
17)Luthje P, Brauner A. (2014) Virulence factors of uropathogenic E. coli and their interaction with the host. Advances in Microbial Physiology. 65: 337-72 doi: 10.1016/bs.ampbs.2014.08.006.
18)Minardi D, d’Anzeo G, Cantoro D, Conti A, Muzzonigro G. (2011) Urinary tract infections in women: etiology and treatment options. International Journal of General Medicine. 4:333-343. doi: 10.2147/IJGM.S11767
19)Mulvey MA, Schilling JD, Martines JJ, Hultgren SJ. (2000) Bad bugs and beleaugguered bladders: Interplay between uropathogenic Escherichia coli and innate host defenses. PNAS. 97(16):8829-8835
20)Mulvey MA. (2002) Adhesion and entry of uropathogenic Escherichia coli. Cellular Microbiology. 4(5): 257-271.
21)Mydock-McGrane LK, Hannan TJ, Janetka JW. (2017) Rational design strategies for FimH antagonists: new drugs on the horizon for urinary tract infection and Crohn’s disease. Expert opinion on drug discovery. 12(7): 711-731. doi: 10.1080/17460441.2017.1331216
22)Rosen DA, Hooton TM, Stamm WE, Humphrey PA, Hultgren SJ. (2007) Detection of Intracellular Bacterial Communities in Human Urinary Tract Infection. PLoS Medicine. 4(12): e329. doi: 10.1371/journal.pmed.0040329
23)Rosen JM, Klumpp DJ. (2014) Mechanisms of Pain from Urinary Tract Infection. International Journal of Urology. 21(01):26-32. doi: 10.1111/iju.12309
24)Schilling JD, Mulvey MA, Hultgren SJ. (2001) Structure and Function of Escherichia coli Type 1 Pili: New insight into the pathogenesis of Urinary Tract Infections. The Journal of Infectious Diseases. 183(1): S36-S40
25)Schmidt H, Hensel M. (2004) Pathogenicity Islands in Bacterial Pathogenesis. Clinical Microbiology Reviews. 17(1): 14-56. doi: 10.1128/CMR.17.1.14-56.2004
26)Terlizzi ME, Gribaudo G, and Maffei ME. (2017) UroPathogenic Escherichia coli (UPEC) Infections: Virulence Factors, Bladder Responses, Antibiotic and Non-antibiotic Antimicrobial Strategies. Frontiers in Microbiology. 8:1566 doi: 10.3389/fmicb.2017.01566
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
Appendix:
Figure 1. The urinary tract, and pathogenesis of UTIs. Source: Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. (2015) Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nature Reviews in Microbiology. 13(5):269-84. doi: 10.1038/nrmicro3432
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
Figure 2. Causative agents of complicated and uncomplicated urinary tract infections. Note the impact of UPEC in UTI. Source: Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. (2015) Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nature Reviews in Microbiology. 13(5):269-84. doi: 10.1038/nrmicro3432
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
Figure 3. Virulence factors associated with UPEC strains. Source: Terlizzi ME, Gribaudo G, and Maffei ME. (2017) UroPathogenic Escherichia coli (UPEC) Infections: Virulence Factors, Bladder Responses, Antibiotic and Non-antibiotic Antimicrobial Strategies. Frontiers in Microbiology. 8:1566 doi: 10.3389/fmicb.2017.01566
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
Figure 4. FimH antagonistic activity. Source: Abgottspon D, Ernst B. (2012) In vivo Evaluation of FimH Antagonists- A Novel Class of Antimicrobials for the Treatment of Urinary Tract Infection. CHIMIA. 66(4):166-169. doi: 10.2533/chimia.2012.166
Figure 5. Various FimH antagonist structures. Source: Terlizzi ME, Gribaudo G, and Maffei ME. (2017) UroPathogenic Escherichia coli (UPEC) Infections: Virulence Factors, Bladder Responses, Antibiotic and Non-antibiotic Antimicrobial Strategies. Frontiers in Microbiology. 8:1566 doi: 10.3389/fmicb.2017.01566
Elinor Shvartsman Presentation Summary Report (January 29, 2019)
Figure 6. Effect of Prophylactic powdered D-mannose administration of UTI recurrence as measured over a 6 month period. Source: Kranjcec B, Papes D, Altarac S. (2014) D-mannose powder for prophylaxis of recurrent urinary tract infections in women: a randomized clinical trial. World Journal of Urology. 32:79-84 doi: 10.1007/s00345-013-1091-6
