Maintaining a Sterile Urinary Tract: The Role of Antimicrobial Peptides

Review Article

Maintaining a Sterile Urinary Tract: The Role of Antimicrobial Peptides Ased S. M. Ali, Claire L. Townes, Judith Hall and Robert S. Pickard* From Newcastle University, Newcastle upon Tyne, United Kingdom

Purpose: The normally sterile urinary tract is constantly challenged by microbial invasion leading to a high prevalence of isolated, recurrent and catheter associated urinary tract infection. The continuous emergence of bacterial resistance following overuse of traditional antibiotics requires the urgent development of alternative treatment strategies. The involvement of innate immune mechanisms in host defense is an emerging field of microbiological research with recent work focusing on the urinary tract. We performed a comprehensive literature review to establish the current level of knowledge concerning the role of innate immunity and specifically antimicrobial peptides within the human urinary tract. Materials and Methods: A systematic review of the literature was performed by searching PubMed® from January 1988 to September 2008. Electronic searches were limited to the English language using the key words antimicrobial, peptide and urinary. Reference lists from relevant reviews were hand searched and appropriate articles were retrieved. The proceedings of conferences held in the last 2 years by the American Urological Association, European Association of Urology and British Association of Urological Surgeons were also searched. Results: Several defensive mechanisms have evolved in response to the threat of urinary infection, comprising physical factors and innate immune responses characterized by the expression of antimicrobial peptides. Antimicrobial peptides are small (less than 10 kDa), cationic and amphipathic peptides of variable length, sequence and structure with broad spectrum killing activity against a wide range of microorganisms including gram-positive and gram-negative bacteria. Several antimicrobial peptides have been identified in the urinary tract, and the amount and type of antimicrobial peptides expressed vary according to tissue source and disease state. These differences may reflect altered levels of innate response and, hence, susceptibility to infection. Antimicrobial peptides are already being exploited therapeutically for skin and endovascular catheter infection, and prospects for useful application in the urinary tract are emerging. Conclusions: Although investigation of antimicrobial peptide function in the human urinary tract is at an early stage, it is clear that there is considerable potential for the future design of novel therapeutic strategies. More knowledge is needed concerning the pathway of involvement of antimicrobial peptides in the maintenance of urinary tract sterility and the ways in which this is altered during active infection.

Abbreviations and Acronyms AMP ⫽ antimicrobial peptides HNP ⫽ human neutrophil peptide IL ⫽ interleukin TLR ⫽ Toll-like receptors UPEC ⫽ uropathogenic Escherichia coli UTI ⫽ urinary tract infection Submitted for publication October 30, 2008. * Correspondence: Surgical and Reproductive Sciences (SARS), Faculty of Medical Sciences, 3rd Floor, William Leech Building, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, United Kingdom (telephone: ⫹44 (0)191 2137139; FAX: ⫹44 (0)191 2137127; e-mail: r.s. [email protected]).

Key Words: urinary tract infections; antimicrobial cationic peptides; immunity, innate

0022-5347/09/1821-0021/0 THE JOURNAL OF UROLOGY® Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 182, 21-28, July 2009 Printed in U.S.A. DOI:10.1016/j.juro.2009.02.124

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ANTIMICROBIAL PEPTIDES AND URINARY TRACT STERILITY

THE normally sterile urinary tract is constantly challenged by microbial invasion chiefly via retrograde urethral spread. Several defensive mechanisms have evolved in response to this threat comprising physical factors such as urine flow, pH and ionic composition together with innate immune response characterized by the constitutive or inducible expression of antimicrobials. On the other hand, adaptive cellular and humoral immune elements are not thought to have a defensive role but are obviously crucial in combating established infection. Interest in endogenous molecular defense has been stimulated by increasing concern regarding the overuse of antibiotics with the expectation that enhancement of natural mechanisms will be an exciting novel therapeutic avenue in the management of UTI. We focus on a group of molecules called antimicrobial peptides whose expression by neutrophils and epithelia has been highly conserved throughout evolution. Such peptides have a key role in microbial surveillance by maintaining commensal flora and suppressing pathogens. We summarize the current knowledge concerning the occurrence and putative actions of AMPs in the urinary tract, and discuss emerging therapeutic use.

THE IMPORTANCE OF URINARY TRACT INFECTION UTI is one of the most common bacterial infections encountered in clinical practice. An estimated 150 million cases of UTI occur annually worldwide, resulting in more than £4 billion (€5 billion, $8 billion) of health care expenditure.1 Women are most commonly affected with an estimated mean incidence of 0.5 to 0.7 episode a year, while other higher risk groups include children, the elderly, people with structural abnormalities of the urinary tract and those having urinary tract intervention such as catheter insertion.2 The occurrence and severity of UTI can be related to genetic, hormonal and behavioral factors together with the virulence of the invading organism, chiefly uropathogenic Escherichia coli.2 Although these infections rarely cause long-term damage, they are associated with a high burden of troublesome symptoms and health care costs, and they contribute significantly to the problem of nosocomial infection. Current treatment with preventive behavioral advice and intermittent or long-term antibiotics3 provides limited symptom relief and encourages the emergence of resistant organisms. Consequently there is a real need for alternative therapies.4

CLASSIFICATION OF ANTIMICROBIAL PEPTIDES Antimicrobial peptides are a ubiquitous component of innate immunity expressed by neutrophils or ep-

ithelial cells either constitutively or via induction by pathogens. AMPs consist of 15 to 45 amino acid residues and most have an overall positive charge. This cationic state is a key functional attribute common to most AMPs, and it results from numerous arginine and lysine residues. However, a few AMPs are rich in glutamic and aspartic acids, and have a negative charge. AMPs can be categorized into 4 groups according to amino acid composition, structure and size using nuclear magnetic resonance with more than 100 different entities described throughout the animal kingdom (Appendix 1).

EVIDENCE FOR ANTIMICROBIAL ACTIVITY In vitro and in vivo models provide compelling evidence that AMPs protect against a range of microorganisms including bacteria, enveloped viruses, fungi and some protozoa.5 Much of the evidence relates to lower order animals that lack adaptive immunity and, hence, are totally reliant on innate mechanisms such as AMP expression for survival. Translation to human physiology can be difficult since genome location, structure and activation from pro-peptides differ from other species, even mammals such as rodents.

MECHANISMS OF ACTION Although the exact way in which AMPs kill microorganisms is not fully understood, it is known that their distinct structure, containing a charged, hydrophilic and a noncharged, lipophilic segment, facilitates cell membrane disruption. Predilection for microbial rather than native cell membranes may relate to differing arrangements of charged and nonionic lipid components of the membrane bilayer.6,7 Bacterial membranes tend to be rich in negatively charged lipids with phospholipid head groups such as phosphatidylglycerol on the outer surface, encouraging attachment of cationic peptides. In contrast, mammalian membranes have mainly neutral lipids such as phosphatidylcholine and contain cholesterol, both of which tend to discourage cationic peptide attack.8 Many studies have demonstrated that the bacteriocidal activity of AMPs relates to their secondary structure, charge and hydrophobic nature which govern interaction with cell membranes.9,10 Several models of this interaction have been proposed. The barrel and stave model suggests that ␣-helical peptides form channels by boring through the bacterial cell membrane using the hydrophobic domain and then bind together like the vertical staves of a barrel with their hydrophilic domains orientated to the cell interior. Polymerization increases the size and number of membrane pores resulting in cell lysis.11 With

ANTIMICROBIAL PEPTIDES AND URINARY TRACT STERILITY

the carpet model the peptides coat the outer bacterial cell membrane in a carpet-like fashion, and interaction between the hydrophobic domains and the lipid bilayer causes it to deform and disintegrate.9,12 Another possible mechanism of cell lysis is the toroidal pore model in which AMPs encourage membrane permeabilization by acting as bridges between the exterior and cytoplasmic membrane layers. Interaction between the membrane lipids and peptides forms a channel through which ions can escape, upsetting homeostasis and eventually leading to cell lysis.13 Other mechanisms for AMP activity under investigation include activation of autolysis as indicated by studies of bovine seminal plasmin8 as well as nonlytic mechanisms such as inhibition of protein synthesis, degradation of proteins required for DNA replication, and interference with the transport and energy metabolism of bacterial cells.14 It is perhaps not surprising that a number of resistance mechanisms have been developed by certain bacteria to protect against AMPs, including altered cell surface charge, active efflux and peptide inactivation by trapping or protease digestion.15 Consideration of these potential inactivating mechanisms will be an important part of harnessing the therapeutic potential of AMPs.

ANTIMICROBIAL PEPTIDES IN THE URINARY TRACT To date 5 AMPs have been identified in the genitourinary tract (fig. 1 and table).

␣-Defensins Defensins, characterized by a 15 to 20 amino acid sequence including 6 cysteine residues, have received the most attention in terms of human AMP research. The ␣-defensins HNP1 to 4 are primarily found in neutrophils where they provide nonoxidative microbicidal activity. A clinical study revealed an 8-fold increase in urinary HNP1 in patients with chronic pyelonephritis compared to that in normal controls and patients with glomerulonephritis.16 The increase in HNP1 correlated with similar changes in urinary levels of the cytokine IL-8 as well as the leukocyte count. However, no microbiological information was provided in that study. HNP1 to 3 have been shown to be increased in vaginal washings in women during episodes of vaginitis/cervicitis compared to those in healthy controls.17 In that study HNP content was 2-fold higher in women with mixed or group B streptococcal infections, while that in women with chlamydial infection did not differ from that in controls. The expression and function of epithelial human ␣-defensins HD5 and 6 have been reported mostly in the small intestine where they are secreted by Paneth cells

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into the intestinal crypts. Research findings indicate a key role in maintaining the numerical and species balance of intestinal flora together with a protective function of maintaining stem cell activity.18 Preliminary work has demonstrated HD5 expression in the female urogenital tract with highest concentrations identified in the vaginal epithelium and associated secretions. HD5 is thought to form an intrinsic component of the innate immune defense system against infection by chlamydia with its expression modulated by hormonal and proinflammatory factors.19 We recently reported localization studies indicating that HD5 is also expressed in the urinary tract, particularly in the kidneys and upper ureters.20

␤-Defensins ␤-Defensins are widely expressed throughout human epithelia. Although more than 30 gene loci are believed to exist,21 predominantly clustered on chromosome 8p23, only a few human ␤-defensins (HBD1 to 4) have been studied in depth. Expression of a variety of HBDs has been demonstrated throughout the urinary tract by protein and RNA molecular identification techniques but their functional role remains uncertain. Apart from their characterized antimicrobial activity ␤-defensins have also been implicated in iron metabolism and tumor surveillance.22 In the kidney HBD1 has been shown to be constitutively expressed by epithelial cells lining the loop of Henle, distal tubule and collecting duct.23 Despite this expression, urine levels of HBD1 are insufficient to lyse invading bacteria. It may be that there is significantly increased HBD1 at the luminal surface of the tubular epithelium which, in conjunction with other antimicrobials, provides a fast acting antimicrobial coating, destroying microbes that manage to gain attachment and, thus, preventing invasion. One could think of this layer as an antimicrobial paint protecting the epithelial surface beneath. In contrast, HBD2 is not expressed in normal kidneys but is induced by chronic renal infection, suggesting that it is involved as a second line defense, perhaps in cooperation with other induced mediators such as cytokines.24 The most convincing evidence that defensins have a direct role in the defense of the kidney against infection is provided by studies using knockout mouse models. A study of animals with deletion of the murine HBD1 gene (Defb1 ⫺/⫺) showed a 30% increase in bacteriuria compared to that in wild-type mice (Defb1 ⫹/⫹).25 The same group that demonstrated HBD1 secretion in the kidney also showed HBD1 expression in the female urogenital tract (endocervix, ectocervix, vagina) and isolated peptides from vaginal lavage.18 More recently expression of HBD1 to 4 mRNA has been detected in endometrium and it has been

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ANTIMICROBIAL PEPTIDES AND URINARY TRACT STERILITY

A

B

Figure 1. A, AMPs identified in kidneys and ureter. B, locations where AMPs have been identified in female genitourinary tract

shown that each defensin has a unique temporal expression profile.26,27 HBD1 and 3 are expressed at highest levels during the secretory phase, which is similar to the pattern of expression for the ␣-defensin HD5.15 In contrast, HBD2 mRNA expression shows a dramatic peak during menstruation while HBD4 is expressed mainly in the proliferative phase. There are significant copy number polymorphisms in the HBD gene locus and mRNA concen-

tration correlates with copy number. Therefore, it is possible that variations in amino acid content and structure of expressed peptides could affect host antimicrobial defense capability and may increase susceptibility to disease. This contention is supported by findings in patients with Crohn’s disease affecting the colon, where the gene copy number of HBD2 is lower than normal.28 In addition to copy number, intragenic single nucleotide polymorphisms have

ANTIMICROBIAL PEPTIDES AND URINARY TRACT STERILITY

Main AMPs in the urinary or genital tract Peptide Length

Structural Features

␣-Defensin Gram-pos, gram5 (HD5) neg, viral

35

␤-Defensin Gram-pos, gram1 (HBD1) neg

36

␤-Defensin Gram-pos, gram2 (HBD2) neg

41

Cathelicidin Gram-pos, gram(LL-37) neg, viral Hepcidin Gram-pos, gram(LEAP1) neg, fungal

37

␤-Sheet with 3 disulfide bonds ␤-Sheet with 3 disulfide bonds Combined helical and ␤-sheet ␣-Helix

AMP

Antimicrobial Activity

25

␤-Sheet with 4 disulfide bonds

Net % Hydrophobic Charge Residue 5

40

5

36

7

36

6

35

4

52

been reported. It is tempting to speculate that such genetic polymorphisms may be linked to a predisposition to UTI. Cathelicidin The human cathelicidin LL37 is an ␣-helical AMP expressed on all epithelial surfaces and by circulating neutrophils.29 Its transcription is determined by a single gene, cAMP, situated at 3p21 and its expression in most epithelial sites is induced by local injury or infection. There is limited but convincing evidence that cathelicidin has a role in human urinary defense against bacterial invasion. A recent report on the expression of cathelicidin in the human kidney indicated that it is induced to high levels when a microbial presence is detected in the urinary tract.30 In support of this finding the normally low urinary levels of LL-37 were increased in children with UTI. The cathelicidin found in urine is thought to be of epithelial origin as levels do not correlate with urinary leukocyte count, which is supported by localization studies on renal biopsies revealing continuous synthesis of LL-37 by tubular epithelium with subsequent release into the lumen. The inducibility of LL-37 gene expression was demonstrated by exposure of renal tissue to UPEC. In these experiments cathelicidin mRNA increased to maximum levels within 5 minutes and continued at supranormal levels for up to 2 hours, accompanied by a surge in peptide secretion. These findings suggest that the cathelicidin defense mechanism is designed to facilitate an immediate and sustained response to bacterial threat. The same study provided further evidence for the role of cathelicidin using a mouse model to investigate the activity of the murine cathelicidin homologue, CRAMP.30 A large inoculum of UPEC was used to overwhelm the defenses of the lower urinary tract and induce severe pyelonephritis. The results

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showed that in early infection cathelicidin was secreted by renal tubular epithelium while in more advanced stages it was also released from leukocytes, suggesting a dual source for urinary cathelicidin in the mouse. Deletion of the CRAMP gene in knockout mice resulted in greater, more rapid bladder colonization and invasion as well as higher rates of ascending infection. Although these results require confirmation by others, they provide a powerful rationale for the possible therapeutic exploitation of LL-37 in the treatment or prophylaxis of UTI. Hepcidin Hepcidin was recently isolated from human urine by Park et al as a novel peptide.31 Independently Krause et al discovered the same peptide from plasma ultrafiltrate and named it liver expressed antimicrobial peptide-1.32 Hepcidin does not show sequence similarity to any of the known antimicrobial peptides, but structurally resembles the defensin family because of the 4 disulfide bridges in its tertiary structure. It is translated in the liver as an 84 amino acid pre-pro-peptide. After processing and excretion through the kidneys a 25 amino-acid peptide (hepc-25) is the predominant form in the urine but shorter peptides (hepc-22 and hepc-20) are also detected.31 Hepcidin is active against gram-positive and gram-negative bacteria as well as yeasts.32 Soon after its discovery researchers found that hepcidin production in mice increased in conditions of iron overload and inflammation. Genetically modified mice engineered to over express hepcidin died shortly after birth with severe iron deficiency, supporting a central role in iron regulation. Hepcidin was also found to be linked to anemia of inflammation.33 Researchers studied tissue from 2 patients with liver tumors with a severe microcytic anemia unresponsive to iron supplementation. The tumor tissue appeared to be overproducing hepcidin and contained large quantities of hepcidin mRNA. Surgically removing the tumors cured the anemia. Together these discoveries suggest that hepcidin has dual functions. In addition to its direct antimicrobial activity, it also acts as an iron regulatory hormone, reducing the amount of iron available for pathogens.

PROTEINS WITH ANTIMICROBIAL ACTIVITY Although not primarily within the scope of this review, it should be noted that several larger proteins have been identified as important in the innate defense of the urinary tract. Perhaps the most researched of these is the Tamm-Horsfall protein, a glycoprotein secreted by epithelia lining the loop of Henle which is present at high concentrations in the urine. It binds to uropathogenic bacteria, particularly UPEC, prevent-

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ANTIMICROBIAL PEPTIDES AND URINARY TRACT STERILITY

ing epithelial adhesion.34 Tamm-Horsfall protein knockout mice have been shown to clear E. coli less rapidly than wild-type mice35 and to have chronic bladder wall inflammation suggestive of persistent infection. The proteins lactoferrin and lipocalin also show antimicrobial activity through the sequestering of iron. Lipocalin knockout mice were more susceptible to systemic infection from E. coli.36 Lactoferrin is expressed in the distal collecting tubules and deposited on the luminal surface. It inhibits bacterial growth by chelation of iron and membrane damage using mechanisms similar to AMPs but does not appear to be induced by active infection.37

Toll - like receptor

Adapter molecules (MyD88, Tirap Trif, TramMyD88 )

IRAK

TRAF6

INTERACTION WITH OTHER IMMUNE PATHWAYS A key aspect of innate immunity is the ability to escalate the response in line with the severity of the threat. Current understanding suggests that this amplification is mediated through at least 3 mechanisms comprising cell-surface receptors such as tolllike receptors and protease activated receptors, cytoplasmic pattern recognition molecules such as NOD1 and NOD2, and direct chemo-attraction of antigen presenting T lymphocytes. Of particular importance in the urinary tract are TLR4, which is activated by bacterial lipopolysaccharide, and TLR5, which is activated by flagellum. Once activated these receptors signal through nuclear factor ␬B and other transcription factors to drive the continued expression and secretion of defensins accompanied by cytokines, particularly IL-6 and IL-8 (fig. 2).38

THERAPEUTIC PROSPECTS The therapeutic potential of AMPs to prevent or treat infection has been clear since the discovery of their bactericidal properties but development has been given added impetus by the realization of increasing adverse effects and short-term usefulness of traditional exogenous antibiotics. A number of possible strategies have been put forward to harness these biomolecules, and a few have reached phase II and phase III clinical trials. Use of such agents to combat infection requires a degree of caution since these are powerful biomolecules that will cause native cell death if applied in high concentration, which in itself has therapeutic potential for cancer treatment.39 Topical Therapy The most straightforward method of treatment with AMPs would be by direct application to the site of action. Following successful phase II studies the topical agent omiganan pentahydrochloride 1% gel (MX226, Migenix Inc., Vancouver, British Columbia, Can-

NFκB Transcription activation leading to AMP & cytokine secretion Nucleus Figure 2. TLR simplified intracellular signaling pathway. Activation of cell surface TLR recruits cytoplasmic adapter molecules to propagate signal. These proteins, identified as MyD88, Tirap (Mal), Trif and TramMyD88, interact with TLR through Toll/IL-1 receptor domain and in turn engage serine-threonine kinase (IRAK) through a death domain. Signal transduction factors such as TRAF6 carry signal through series of phosphorylations until nuclear factor ␬B is ultimately released to enter nucleus where it can activate transcription of appropriate immune response genes resulting in production of AMPs and cytokines.

ada) derived from bovine indolicidin was tested in phase III clinical trials as a coating and prophylactic gel to reduce colonization and infection of central venous catheters.40 The results were a 49% decrease in local catheter site infections and a 21% decrease in catheter colonization. Other agents such as frog derived magainin have been trialed for use in skin infections against gram-positive organisms, and the activity of gel preparation containing cecropin analogues against Chlamydia trachomatis has been demonstrated in vitro.41 Oral Therapy The small peptide structure of AMPs creates difficulty in designing active agents for oral administration. However, oral use of the antimicrobial protein lactoferrin B effectively decreased infection and inflammation in the mouse urinary tract after infection with E. coli.42 Therapeutic Induction of AMP Expression An alternative possibility for using AMPs therapeutically would be to induce or augment their natural

ANTIMICROBIAL PEPTIDES AND URINARY TRACT STERILITY

production.43 To this end a number of groups have demonstrated enhanced expression of AMP genes in a variety of epithelial cells using specific nutrients, particularly vitamins. An example relevant to the urinary tract is induction of the cathelicidin LL-37 by vitamin D and other short chain fatty acids.44,45 In humans the cathelicidin gene has a vitamin D binding site.46 This binding site can be activated by epithelial attachment of bacteria to TLR2 and vitamin D synthesis can be stimulated by bacterial lipopolysaccharide.47 These pathways provide potential sites for pharmacological intervention to enhance LL-37 expression. The benefits of this approach are indirectly supported by the observation that innate immune function is attenuated in vitamin D deficient individuals.34 In the distal colon complex carbohydrates are converted by bacteria into short chain fatty acids that provide energy and stimulate expression of cathelicidin by the epithelium. It has been shown in a rabbit model that colonic expression of cathelicidin (CAP-18) can be induced by treatment with oral

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sodium butyrate during early phases of shigella infection, and results in reduced fecal bacterial load, decreased severity of clinical illness and quicker recovery.48

FUTURE DIRECTIONS Although exploration of AMP function in the human urinary tract is at an early stage, we highlight several areas encouraging the development and trial of therapeutic agents. However, a better understanding of the involvement of AMPs in the maintenance of urinary tract sterility is required (Appendix 2). As some of these basic issues begin to be clarified, strategies and possible agents for managing UTI can be tested in vitro and clinically as part of the drive to decrease antibiotic use. Perhaps the clearest and most immediate possibility is the development of urinary catheter coatings to reduce the risk of catheter associated UTI, which is a major health care problem in societies with aging populations.49

APPENDIX 1 Classes of Antimicrobial Peptides Group Linear cationic ␣-helical Cationic peptides enriched for specific amino acids Cationic peptides enriched for specific amino acids Anionic peptides

Examples Cecropins, magainins, cathelicidin Apidaecins, drosocins, indolicidin, PR-39, histatin Defensins, hepcidin, protegrins Maximin, dermicidin

Structure

Amino Acid Content

Human Forms

Linear helical

No cysteine residues

LL-37

Linear extended helical

Rich in certain amino acids, eg proline, arginine or typtophan, but no cysteine residues Cysteine residues

Histatin

␤-Sheet structures that are stabilized by 2 or 3 intramolecular disulfide bonds Small peptides with 1 intramolecular disulfide bond, characteristically near the C-terminus

Rich in glutamic and aspartic acids

␣ and ␤-Defensins, hepcidin Dermicidin

APPENDIX 2 Research Priorities for Urinary Tract AMP ● ● ● ●

Catalog AMP profile in urinary tract distinguishing constitutive and inducible agents Development of standardized techniques to isolate, quantify and measure the activity of urinary AMPs Clinical studies to determine whether individuals prone to UTI have altered AMP profile Development of in vitro and animal models to investigate molecular regulation of AMPs and testing of therapeutic induction agents

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