HARRY L. T. MOBLEY,1* MICHAEL S. DONNENBERG,2 AND ERIN C. HAGAN1
Infections in the urinary tract range in severity from asymptomatic bacteriuria to urosepsis, which can be fatal. E. coli is the predominant cause of the entire spectrum of UTI, accounting for more than 80% of community-acquired infections. Uncomplicated UTIs include cystitis infections in adult women who are not pregnant and do not suffer from structural or neurological dysfunction (17). Cystitis is a clinical diagnosis presumed to represent an infection of the bladder. Cystitis is often defined by the presence of ≥103 bacteria/ml in a midstream, clean-catch urine sample from a patient with symptoms including dysuria, urinary urgency, and increased frequency (79, 373). Of women visiting physicians for a UTI, 95% do so for symptoms of cystitis (81). It is estimated that 40% of adult women will experience symptoms of cystitis during their lifetime, and E. coli will be identified as the etiologic agent in 75 to 80% of these cases. Forty to fifty percent of women will have more than one UTI.
The serious clinical syndrome of acute pyelonephritis is less common than cystitis, and it is also most often caused by strains of E. coli (372). This infection in one or both kidneys usually results from the ascent of organisms from the bladder via the ureter, and is distinguished from other UTIs clinically and pathologically, and by characteristics of the causative organisms (374). The patient with acute pyelonephritis classically presents with the triad of fever, flank pain, and bacteriuria with or without diaphoresis, rigors, abdominal or groin pain, and nausea and vomiting. Laboratory studies show leukocytosis, bacteriuria, pyuria, diminished renal concentrating ability, and elevated C-reactive protein (204). On pathological examination, wedge-shaped areas of inflammation containing predominantly polymorphonuclear leukocytes (PMNs) extend from papillae to cortex; tubules are filled with PMNs, and necrosis of proximal tubular epithelial cells is evident. Glomeruli tend to be spared, even in areas of intense inflammation (128). Localized inflammation may coalesce to form a renal abscess and perinephric abscess and pyonephrosis may result, in particular, in the presence of urinary stones or outflow obstruction (300). The infection may spread beyond the urinary tract if bacteria enter the bloodstream. Indeed, 12% of patients with pyelonephritis have bacteremia, which can accentuate the severity of pyelonephritis infections and, if left untreated, may be lethal (79, 144).
Studies suggest that up to 95% of all UTIs develop by an ascending route of infection (17), meaning that infection begins with colonization of the periurethral area, followed by an upward progression to infect the bladder, and, in some cases, continued progression of the bacteria through the ureters to infect the kidneys. In extremely rare instances, the urinary tract can be seeded when bacteria from distant sites lodge in the kidney via the bloodstream. This occurs more often with microorganisms other than E. coli, such as Staphylococcus aureus (220).
The first step in the development of an ascending UTI is the colonization of the distal urethra and introitus by fecal bacteria arising from the host's intestinal microbiota. Colonization is likely influenced by a number of factors, including competition from resident flora, host factors such as pre- or postmenopausal hormonal state, and the ability to specifically adhere to urethral epithelial cells (17). Once bacteria have colonized, the periurethral area, they are easily introduced into the bladder, an event that is facilitated by sexual intercourse. Women are infected more often both because of a short urethra and the proximity of the urethral opening to the anus (132).
Bacteria are normally prevented from entering the ureter and ascending to the kidneys in a retrograde fashion by a one-way vesicoureteral valve that resists the high pressure that develops in the bladder as it expands and contracts during voiding. A bladder infection itself, with accompanying inflammation, can lead to vesicoureteral reflux. Reflux may also be due to congenital or acquired valve defects.
Bacteria that ascend the ureter can disperse into the renal pelvis and deep into the renal parenchyma through open ducts at the tips of the papillae. In these instances, patients may develop acute pyelonephritis, an acute suppurative inflammation of the kidney. Bacteria in renal tubules are perilously close to the bloodstream, because they must cross only one epithelial cell (e.g., proximal tubular epithelium) and one endothelial cell in the wall of the capillary to reach a venue for bacteremia and potential systemic spread.
After an initial episode of cystitis, there is a risk of approximately 25% that a woman will have a second symptomatic episode within 6 months (88). Five studies in which an attempt was made to determine whether such recurrent episodes represent infections with the same strain as the initial episode have yielded highly variable results, with same-strain episodes making up anywhere from 25 to 100% of the cases (reviewed in reference 161). At present, the reservoir for reinfection cannot be discriminated among intestinal colonization, vaginal colonization (161), or intracellular bacteria residing within transitional epithelial cells (258). Several pathogen-related factors predispose women to recurrent UTI, including periurethral bacterial colonization and the presence of certain E. coli virulence factors. Host behavioral risk factors include voiding dysfunction, high intercourse frequency, and oral contraceptive and spermicide use (82).
Strong correlations have been made between the presence of specific genetic determinants and the urovirulence of particular E. coli strains, often leading to the identification of potential virulence factors. Mannose-resistant hemagglutinin, or P fimbria, has frequently been associated with pyelonephritogenic E. coli, with P fimbriae-positive strains accounting for 74 to 100% of pyelonephritis isolates (91, 157, 273, 369). Similarly, expression of both P and F fimbriae is detected more frequently in pyelonephritis isolates than in either cystitis or fecal strains (14). In contrast, several studies have found that the presence of type 1 fimbriae does not correlate with strain virulence; type 1-fimbriated clones are equally common among pyelonephritis, cystitis, and fecal strains (219, 369). Nevertheless, type 1 fimbrial genes are almost always present in uropathogenic isolates (157). Hemolytic activity has also been associated with uropathogenicity. Hemolysin determinants were found in approximately 75% of pyelonephritis isolates (91, 273), 43% of urosepsis isolates (157), and 85% of virulent urine isolates (247). Thus, the relative risks of pyelonephritis strains expressing hemolysin or F1C fimbriae, compared with fecal strains, are ~2.5 and ~12, respectively, while the relative risk of the same strains expressing type 1 fimbriae is ~0.9 (62).
Genes that encode potential virulence factors are commonly clustered within regions of DNA termed PAIs. PAIs were first defined by Hacker and colleagues to include DNA regions greater than 30 kb that are associated with pathogenic organisms and that harbor virulence-associated genes not commonly found in the genome of fecal E. coli (109). In addition, PAIs are frequently inserted within or adjacent to tRNA genes, have a G + C content different from the genome DNA, and include the presence of mobility genes including transposons, insertion elements, and intact or degraded bacteriophage genomes. Some PAIs have been proven to be unstable and can be deleted under specific environmental conditions or as the result of site-specific recombination (242). All of these characteristics suggest that PAIs have been inserted in the chromosome of UPEC via a lateral transfer mechanism (25, 56, 109). Similarity between the structures and content of distinct PAIs support this premise (57).
The complete genomic sequences of three UPEC strains has now revealed that each has numerous PAIs encoding many genes of unknown function (Fig. 1). In fact, the 68-kb GI-CFT073-selC and 22-kb PAI VII536 (selU) encode primarily hypothetical genes with uncharacterized functions. Like the asnT-associated PAI IV536/UTI89 (PAI-CFT073-asnT), which encodes yersiniabactin, some islands are highly conserved among these UPEC isolates, while others appear to be much less prevalent, if not strain-specific. Even among “conserved” PAIs, heterogeneity is often observed between strains, with smaller insertions and deletions occurring within otherwise identical islands. Moreover, virulence genes present on a specific PAI in one strain may be found on distinct PAIs or chromosomal locations in other strains.
The current dogma of bacterial pathogenesis identifies adherence, colonization, avoidance of host defenses, and damage to host tissues as events vital for achieving bacterial virulence (83). As the first step in this process, adherence plays a prominent role. Bacteria use many different surface structures by which they bind to targets on the host cell surface. Some of these structures can be observed by electron microscopy to project as rigid or flexible rods from the surface of the bacteria. The words fimbriae, derived from the Latin term meaning threads or fringe, and pili, Latin for hair, are used to describe these organelles. Alternatively, some related but thinner, wiry structures known as fibrillae and others that may have no detectable morphology are commonly referred to as afimbrial adhesins
UPEC strains express a number of different pili and related adhesins including P, F1C, S, M, Dr, and type 1 fimbriae (153, 269, 270, 288, 371). These adhesins bind different host receptors, and as a result are believed to target different areas within the urinary tract (153, 371). Fimbriae were originally subdivided into two different groups based on whether or not they could cause agglutination of erythrocytes from various mammals and, if so, whether or not this hemagglutination is blocked by mannose (32, 65, 66, 74, 102). In particular, hemagglutination of guinea pig erythrocytes by type 1 fimbriae is inhibited by mannose, and, therefore, these pili are defined as mannose sensitive (153).
Type 1 fimbriae.
Type 1 fimbriae have been definitively shown to be virulence factors in a murine model of E. coli UTI (19, 48, 103, 338). However, the genes responsible for type 1 fimbriae are found among almost all strains of E. coli and many other genera within the family Enterobacteriaceae and thus cannot alone account for the urovirulence of a small subset of such strains. The ubiquitous presence of the type 1 fimbrial genes may be explained in part by evidence that they are required for colonization of the oropharynx as a prelude to intestinal colonization (279). Type 1 fimbrial expression is primarily under the control of an invertible element that contains the promoter responsible for transcription of the major structural subunit. Cystitis and pyelonephritis isolates have been observed to differ in the control of the invertible element orientation and the expression of type 1 fimbriae during the course of an infection (349). Recent research has focused on the role of type 1 fimbriae in the invasion of host cells and host immune responses, the potential cross talk between the control of type 1 fimbriae and other fimbriae types, the effect of flow on fimbrial adhesion (262, 356), and the development of a vaccine directed against type 1 fimbriae (216, 217).
Type 1 fimbriae are on average 0.5 to 2 μm in length with a diameter of 7 nm and a center axial hole 0.2 to 0.25 nm in diameter (33). At the distal end of the fimbria, a fibrillar structure, which contains the FimH adhesin responsible for binding and hemagglutination, projects roughly an additional 16 nm in length. A type 1 fimbriated bacterium typically expresses 500 fimbriae per cell, which accounts for about 8% of the total protein content of the bacterium at any given time.
Type 1 fimbriae are encoded by an operon consisting of nine separate genes present on the chromosome in the following order: fimB, fimE, fimA, fimI, fimC, fimD, fimF, fimG, and fimH (138, 141, 323) (Fig. 2). The operon is transcribed as at least three separate transcripts, one for encoding the main structural subunit of the fimbriae (fimA), and two others responsible for the adhesin (fimH) and accessory proteins necessary for assembly of the fimbriae (153).
The FimA polypeptides polymerize into a helix composed of 3–1/8 FimA proteins per turn to form the shaft of the fimbriae (33). The FimH adhesin binds to a variety of glycolipids and glycoproteins containing mannosides that are widely distributed on epithelial surfaces of humans including uroplakin receptors that coat transitional epithelial cells of the bladder (2, 3, 109, 143, 198, 382). With use of atomic force microscopy, cryo-electron microscopy, and a FimCH complex, it was determined that FimH binds to uroplakin Ia on the six inner domains of the 16-nm urothelial plaque particle (245, 246, 395). Recently, potent inhibitors of FimH receptor binding have been identified and include butyl α-D-mannoside and other aryl mannosides (30). Collectively, the products of the fimG and fimF genes combine with the FimH adhesin to form the fimbrillar tip at the end of the fimbriae (165). FimC is a periplasmic chaperone protein with seven antiparallel β-sheets forming an immunoglobulin-like fold responsible for stabilizing the fimbrial components as they are transported from the cytoplasmic membrane to the site of fimbriae construction on the outer membrane (164). An additional FimC β-sheet completes an immunoglobulin fold in each fimbrial protein subunit in a process termed donor-strand complementation that prevents premature interactions between fimbrial subunits (46). The FimD protein has been shown to function as an outer membrane usher, which recognizes the chaperone-fimbrial subunit complexes, and assists in localizing them to the proper area for fimbriae construction (185). Further details of the chaperone-usher pathway of fimbrial assembly are provided in Chapter 150 of the last print edition of Escherichia coli and Salmonella: Cellular and Molecular Biology.
Expression of type 1 fimbriae undergoes phase variation. Individual bacterial cells either express the fimbriae over their entire surface or do not express any of these fimbriae at all. This phase variability of type 1 fimbriae is controlled at the transcriptional level by the previously mentioned invertible stretch of DNA, which is acted upon by the FimB and FimE recombinases (1). The σ70 promoter, responsible for transcription of the major fimbrial subunit FimA, is located within this invertible DNA element (69, 278). The 314-bp invertible element is flanked on both ends by inverted DNA repeats of 9 bp in length; these repeat sequences are essential for proper inversion (240). The FimB and FimE recombinases have also been shown to bind to specific sites on the DNA that flank either side of the inverted repeats. Two recently identified UPEC-specific recombinases, IpuA and IpbA (FimX), also mediate FimB/FimE-independent switching of the invertible element (34, 116).
The orientation of the invertible element and, subsequently, the expression of type 1 fimbriae, is believed to be controlled by a variety of environmental factors. For example, low pH and high osmolarity favor a shift in the invertible element to the phase-off orientation (328). Different molecular elements, including temperature and the presence of branched-chain amino acids or sialic acid, influence the FimB and FimE recombinases and contribute to the dynamic switching behavior demonstrated by E. coli, especially UPEC strains (70, 92) (Fig. 3). Three regulatory proteins, leucine responsive protein (LRP), integration host factor (IHF), and the histone-like nucleoid structuring protein (H-NS), have been shown to affect the switching of the invertible element and type 1 fimbrial expression by binding to DNA sequences around and within the invertible element region, thus assisting or blocking the switching actions of the recombinases (93). Environmental conditions can also affect the orientation of the invertible element by selecting for bacteria that express or fail to express the fimbriae. The invertible element in the ON orientation, and subsequent expression of type 1 fimbriae, occurs in a majority of strains when E. coli are grown statically in nutrient broth. Alternatively, when the bacteria are cultured on solid nutrient agar, the invertible elements are primarily in the OFF orientation and type 1 fimbriae are not expressed (142, 184).
The ability to modulate the invertible element has been studied in a collection of E. coli fecal, cystitis, and pyelonephritis isolates under various in vitro growth conditions that selected for or against switching (104). Under these defined conditions, fecal and cystitis isolates were able to switch a greater percentage of their invertible elements into the “ON” orientation when compared with pyelonephritis isolates. Indeed, when representative strains, isolated from cases of pyelonephritis and cystitis, were compared as to the orientation of their invertible elements during experimental infections of CBA mice, significant differences were observed (222). The majority of invertible elements from cystitis isolates recovered from the urine of mice, had invertible elements switched into the “ON” position, while only a small percentage of pyelonephritis isolates maintained the switch in the “ON” position.
Although the invertible element itself is not a virulence factor, the ability to phase vary type 1 fimbriae at specific times during infection is probably important. Mutations constructed in the invertible element generated strains with the element locked permanently in either the “ON” (Locked-ON) or “OFF” (Locked-OFF) orientation. In the pyelonephritis isolate CFT073, Locked-OFF mutants were attenuated throughout the urinary tract specifically at 24 hours postinoculation (hpi), while Locked-ON mutants colonized at higher levels than wild type (103). However, in the cystitis isolate F11 (Table 2), the Locked-OFF mutant was attenuated throughout the experiment (24 to 168 hpi) and the Locked-ON strain did not show a colonization advantage over wild type (338).
Hultgren and colleagues used high-resolution electron microscopy to demonstrate binding of the tips of type 1 fimbriae to the hexagonal arrays of the bladder cell surface glycoprotein uroplakin in vivo (256). They also noted apoptosis and loss of surface umbrella cells while the bacteria seemed to resist this shedding by invading underlying cells (256). From these observations, they proposed a model in which UPEC use type 1 pili to attach to the bladder mucosa, induce apoptosis of host cells, and facilitate invasion and colonization of the damaged tissue. Evidence has also been presented demonstrating that kps-encoded capsular polysaccharide genes are downregulated upon type 1 fimbriae binding to a mannose substrate, suggesting a cross talk between the two gene clusters (327).
Paradoxically, the flow of urine may actually facilitate binding via type 1 fimbriae. Bacteria expressing these pili adhere to mannose in flow chambers more strongly as the flow increases (356). Molecular modeling suggests that the shear force results in a conformational change in the ligand-binding pocket of FimH. Indeed, small changes in amino acid sequence in the binding pocket of FimH can dramatically influence binding (339, 340, 341).
An understanding of fimbrial structure and biogenesis was advanced by the cloning of the genes encoding P fimbriae by Hull and colleagues (138). Sequencing of the smallest DNA fragment capable of encoding fimbriae revealed 11 genes. Normark and Hultgren advanced these studies to elucidate a model of fimbrial biogenesis that is currently applicable to a wide range of enterobacterial fimbriae (59, 140, 201, 202, 229, 263, 264, 367). Details of the chaperone-usher pathway can be found in Chapter 150 of the last print edition of Escherichia coli and Salmonella: Cellular and Molecular Biology.
Following the initial correlation of the presence of P fimbria and virulent uropathogenic strains, a number of investigators confirmed and extended this work (63, 114, 148, 154, 160, 171, 219, 273, 317, 368, 370, 380). In an analysis of published, controlled studies, Donnenberg and Welch reported that E. coli from otherwise healthy patients with pyelonephritis are six times as likely to possess P fimbriae as strains from the feces of controls (62). Approximately 80% of such strains have P fimbriae. There is also a strong relationship between the severity of the infection and the prevalence of P fimbriae. Studies of humans with UTI demonstrating expression of P fimbriae by E. coli in urine (183) and antibodies to P fimbriae in serum (54) indicate that E. coli produce and display P fimbriae in vivo in infected patients.
Despite the strong association between the expression of P fimbriae and the ability to cause UTI, only two reports have appeared in which true isogenic P-fimbrial mutants of virulent clinical isolates have been constructed for testing in animal models of infection (253, 303). In a study by Mobley and colleagues, CBA mice (n = 100) were challenged transurethrally with 105, 106, 107, or 109 CFU of strains CFT073 or a P fimbria-negative mutant. After one week, no substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration (253). It was concluded that adherence by P fimbriae of uropathogenic E. coli strain CFT073 plays, at best, a subtle role in the development of acute pyelonephritis in the CBA mouse model. Roberts and colleagues studied the effect of a papG mutation on colonization of the bladder and on the development of pyelonephritis in cynomolgus monkeys. While they found no difference in colonization between the wild-type and mutant strain after bladder inoculation, they found that the wild-type strain colonized the urinary tract for significantly longer periods than the mutant strain after instillation into the ureter. Furthermore, there was evidence of loss of kidney function and mass on the inoculated side only in recipients of the wild-type strain (303). Although no mutant complemented with a copy of the wild-type allele was tested, this study provides the best evidence available that P fimbriae contribute to the pathogenesis of pyelonephritis. However, the fact that the recipients of the mutant strain also had renal colonization and the fact that not all strains that cause UTI express P fimbriae indicate that these organelles are dispensable for infection. Clearly, P-fimbrial genes (pap) are preferentially found in urovirulent strains, may serve as a marker for the presence of additional genes that confer virulence, and themselves contribute to virulence.
Strains that carry P fimbriae can be categorized by which of three PapG adhesins are expressed at the tip of the fimbria. PapG I was the first for which the gene was cloned but is not common in human isolates, PapG II is the most common of the adhesins, and PapG III is found in some cystitis isolates. The three adhesins recognize slightly different portions of the Gal-Gal disaccharide-containing glycosphingolipids (348). A related pilus, denoted Pix, was identified in UPEC strain X2194. Although the genes were homologous to pap genes, the pilus did not bind to the P blood group antigen or other known receptors (228). Also present in pyelonephritis strain 536, the distribution and contribution of Pix to infection has not been extensively studied.
In humans, P fimbriae trigger a mucosal response to infection by UPEC. To demonstrate this, an asymptomatic bacteriuria strain, capable of infecting humans, was transformed with a plasmid expressing P fimbria. When volunteers were infected with this recombinant strain, elevations of urinary interleukin-6 (IL-6) and interleukin-8 (IL-8) and neutrophils were observed, while loss of P-fimbrial expression returned cytokine levels to normal (383). When a ΔpapG construct was introduced into the parent ABU strain, the resulting nonadhesive strain failed to trigger these responses (24).
The Dr adhesin family is composed of fimbrial and afimbrial structures on the surface of E. coli that bind to the Dr blood group antigen (267), a portion of the decay-acceleration factor, which is a membrane protein that prevents cell lysis by complement (266, 268). Within the urinary tract, Dr adhesins bind to bladder epithelium and type IV collagen on basement membranes (379). These adhesins are present in a minority of UTI strains; however, pooled data indicate that the genes for members of the Dr family are more prevalent among pyelonephritis and cystitis strains than among fecal control strains of E. coli (62). The presence of Dr adhesins has been associated with invasion of epithelium. Specifically, domains of DraD and DraE have been demonstrated as necessary for invasion (52, 389, 390), and other chaperone-subunit interactions have recently been elucidated (290). Some intriguing data suggest that the interaction of these fimbriae with their host receptor is quite persistent, because intravenously administered purified Dr adhesins bound for months to the mesangium of rat kidneys (243). Furthermore, these adhesins appear to have a role in UPEC pathogenesis, because a Dr-positive E. coli strain but not its Dr-negative isogenic mutant caused a disease pathologically similar to chronic tubulointerstitial nephritis in a mouse model of ascending UTI. Compared with wild type, the Dr mutant strain was deficient for persistent kidney colonization, a difference that was reversed upon complementation with the cloned gene (99).
F1C and S fimbriae.
Other potential adhesins.
The genome sequence of pyelonephritis strain CFT073 revealed 13 distinct fimbrial gene clusters. One such cluster encodes Auf fimbria expressed during exponential growth and shown by reverse transcriptase PCR to be expressed in the urine of experimentally infected mice (36). Antisera from infected mice react with recombinant AufA. No hemagglutination of cell-binding properties have yet to associated with this chaperone-usher-assembled fimbria, and no effect of deleting the fimbrial operon on colonization of the murine urinary tract could be detected (36).
A family of afimbrial adhesins, encoded by related gene clusters that belong to the usher-chaperone family, confer adhesion to tissue culture cells but do not form recognizable fimbriae (182, 209). Although some members of the Afa family have been isolated from UTI strains, no role in the pathogenesis of UTI has been established.
The iron-regulated gene homolog adhesin (Iha) is found more often in UPEC (38 to 74%) than in fecal isolates (14 to 22%) (155). Recombinant Iha from strain CFT073 conferred upon nonadherent E. coli ORN172 the ability to adhere to cultured T-24 human uroepithelial cells. CFT073 and its double pap mutant (P fimbria-negative) each outcompeted their respective iha deletion mutants in mice 48 h after transurethral challenge, suggesting a role in infection (155). However, the effect of complementation of the mutants was not reported. Moreover, the results of these studies are complicated by further work that showed that Iha also functions as a receptor for the siderophore enterobactin (221). Thus, it is likely that the colonization deficiencies of the iha mutants are not solely due to defective adherence, but also impaired iron acquisition.
UPEC produce three classes of proteins that may be regarded as toxins: hemolysin, cytotoxic necrotizing factor 1 (CNF-1), and the secreted autotransporter toxins. Additional secreted factors with cytotoxic activity have also been described.
Expression of the hemolysin operon is positively regulated by RfaH, which is also a positive regulator of LPS synthesis (21). Inactivation of the rfaH gene resulted in a 10-fold decrease in the hemolytic activity of UPEC strain 536 (260). The PAI-associated tRNA gene leuX, which encodes leucyl-tRNA, has also been shown to modulate the expression of hemolysin in strain 536 (57, 58).
Once secreted, hemolysin requires calcium to assume its functional tertiary structure and to bind to the host cell membrane (27, 28, 227). Hemolysin appears to insert into the outer leaflet of the host cell membrane, rather than forming a pore through the membrane as originally proposed (342). The receptor for the toxin is as yet unidentified.
At high doses, hemolysin is cytotoxic, not only to erythrocytes, but also to a variety of nucleated cell types including leukocytes (42), fibroblasts (41), and, more relevant to UTI, uroepithelial cells (146, 252). Hemolysin is also suggested to play a role in the invasion of the renal parenchyma by destruction of the epithelial barrier. In vitro, a hemolytic strain penetrated a monolayer of proximal renal tubular cells in higher numbers than a nonhemolytic mutant of the same strain (359). In addition, sublytic concentrations of hemolysin have been shown to induce oscillations of the intracellular concentration of Ca2+ in renal epithelial cells. These oscillations are associated with production of IL-6 and IL-8 (210, 281, 366). In vivo, hemolysin can stimulate production of cytokines by this mechanism, which can lead to inflammatory responses. While a receptor for the hemolytic activity of the toxin has not been reported, the sublytic calcium-modulating effect seems to involve binding of a complex composed of hemolysin and LPS to the LPS receptor protein CD14 (233). In contrast, CD14 and LPS play no known role in hemolysis. That hemolysin is produced in vivo is supported by a study showing that antihemolysin antibodies increase during natural human UTI (329).
Presumably through its effects on secretion of chemokines, hemolysin may be involved in the rapid innate immune response to E. coli UTI in vivo. In a real-time dual-photon fluorescence microscopy study involving microinjection of bacteria into the proximal tubule of exteriorized kidneys from live rats, wild-type hemolytic UPEC strain CFT073 was observed to multiply rapidly, recruit inflammatory cells, and cause rapid capillary obstruction and massive local tissue destruction (234). An isogenic hlyA mutant caused similar changes, but they were delayed several hours compared with the wild-type strain.
The role of hemolysin in UTI has proven difficult to elucidate. Initial genetic evidence demonstrated that hemolysin is a virulence factor for ExPEC infections. Introduction of the hemolysin operon from UTI strain J96 into a nonhemolytic fecal strain conferred virulence in a rat peritonitis model. This phenotype could be complemented, because mutation of the hemolysin genes by transposon insertion abolished virulence (377). Support for the involvement of hemolysin in UPEC urinary tract colonization, however, initially rested on studies that did not use true isogenic mutant strains. In one such study, the addition of the hly operon on a plasmid rendered a nonhemolytic UPEC able to cause more kidney damage and higher mortality than the parental strain in a murine model of ascending UTI (272). In other studies, reintroduction of the hly operon on a high-copy-number plasmid into a strain deleted of pathogenicity islands encoding hemolysins contributed to restoration of virulence in a rat model of ascending UTI (236), and loss of the PAIs of UPEC strain 536 carrying hemolysin genes resulted in complete loss of virulence in the kidneys of intravenously challenged mice (75). Because deletion of two pathogenicity islands eliminates many other genes besides hemolysin genes, the results of this study are difficult to interpret.
Recently, definitive evidence for the role of hemolysin was presented by assessing the ability of isogenic hemolysin mutants to cause UTI in a mouse model of infection (335). While a hlyA mutant was able to colonize both the bladder and kidneys to wild-type levels in either independent or coinfections, histological differences were observed. Early in infection, the hlyA mutant induced significantly reduced epithelial damage and bladder hemorrhage than did the wild-type strain. This defect could be complemented by introduction of hlyA, thus satisfying Molecular Koch's postulates for hemolysin (335). More details on hemolysin and type I secretion can be found in EcoSal Chapter The Escherichia coli Hemolysin.
Numerous studies suggest a potential role for CNF-1 in the pathogenesis of UPEC. Treatment of HEp-2 cells with CNF-1 enabled them to internalize latex beads and noninvasive bacteria (78). In addition, CNF-1 induced effacement of intestinal microvilli, reduced PMN transmigration in intestinal T84 epithelial cells (130), and increased permeability in polarized intestinal cell monolayers (94). CNF-1 was also shown to increase adherence of PMNs to T84 monolayers and decrease their phagocytic effect (131). Furthermore, CNF-1 causes apoptosis in the 5637 bladder cell line, a phenomenon that might explain the exfoliation of the bladder epithelial cells after infection with UPEC (244). Although these effects, together, suggest that CNF-1 may play a role in UTI, an isogenic mutant of cnf1 of the highly virulent CFT073 strain was no different from its wild-type parent UPEC strain CFT073 in its ability to colonize or cause inflammation in a murine model of UTI (150). However, subsequent mixed-infection studies with strain C189 demonstrated that the CNF1-positive parent was recovered in higher numbers than the CNF1-negative mutant in urine, bladder, and kidneys (298). Furthermore, this effect was reversed upon complementation. In addition, CNF1-positive strains consistently showed deeper, more extensive inflammation (298) and were better able to resist killing by fresh human neutrophils and modulate their function (53).
Pic and Tsh—Prototypic pyelonephritis strain CFT073 produces at least two additional autotransporters that have been characterized (126, 284). Culture supernatants contained polypeptides of 105 and 110 kDa that were identified as Pic and Tsh, respectively. Pic has serine protease activity, typical of SPATE proteins, whereas Tsh does not. pic was found in 31% of pyelonephritis isolates and 7% of fecal strains; tsh was identified in 63% of pyelonephritis strains and 33% of fecal strains. pic and tsh mutants were not significantly attenuated when evaluated by competition experiments in the CBA murine model of ascending UTI (126). Beyond Sat, Pic, and Tsh, seven additional autotransporters predicted by the genome sequence await further characterization (284, 376).
In addition to these classic toxins, recent studies have identified other proteins secreted by UPEC that have cytotoxic activity. A group of large nonribosomal peptide synthases (NRPS) and polyketide synthases (PKS) produced by B2 E. coli strains were recently found to induce DNA double-stranded breaks in cultured epithelial cells (265). E. coli expressing these enzymes activated the eukaryotic DNA damage pathway, arresting the cell cycle at the G2/M transition. Located on a 54-kb genomic island designated the pks island (PAI VI536), the genes encoding these enzymes are present in 53% of ExPEC strains and 34% of commensal strains. While all eight NRPS, PKS, and hybrid NRPS-PKS enzymes and eight of the nine accessory proteins encoded by this locus are required for cytotoxicity, the precise role of each remains unknown (265). Soluble unidentified toxins from an O6:K13:H1 uropathogenic strain were also found that induced apoptosis in a renal tubule cell line (43). Moreover, the complete genome sequences of CFT073 and other UPEC strains have revealed additional putative toxins whose functions have yet to be identified.
The importance of capsule and lipopolysaccharide O side chains in UPEC pathogenesis has been controversial. Russo et al. found no difference in the ability of a wild-type O4:K54:H5 blood isolate and an isogenic mutant unable to synthesize K54 capsule to cause UTIs in a mouse model (308, 315). Also, there is no experimental evidence to support the hypothesis that having a particular O antigen type per se increases the pathogenic potential for an E. coli strain. However, an isogenic mutant of an O4:K54:H5 strain that was no longer able to synthesize the O4 antigen was significantly impaired in its ability to colonize the mouse bladder, kidney, and urine than was the wild-type strain from which it was derived (308). This suggests that the inability to produce any O antigen may be detrimental to E. coli in the urinary tract. Schilling et al. also studied the role of LPS in the pathogenesis of UPEC (324). These authors found that UPEC LPS is required for invasion of the bladder epithelial cells, which leads to production of IL-6, both in cultured bladder epithelial cells and in mice.
LPS and capsular carbohydrates seem to play a role in the ability of certain strains of E. coli to grow in the presence of human serum. Cross et al. demonstrated that certain O antigens confer resistance to human serum, but, for some of these O antigens, the presence of the capsule was also required (49). By using a defined LPS mutant and an acapsular mutant of UPEC O75:K5, Burns and Hall showed that LPS was more important for serum resistance than the capsule and that the length of the O antigen is crucial for exerting such effect (38). Capsules are also known to play a role in resistance to phagocytosis in the absence of anticapsular opsonizing antibody (135). Indeed, both O and K antigens of UPEC O75:K5 were found to play roles in resistance to phagocytosis (39).
In a large-scale screen of signature-tagged transposon mutants of UPEC strain CFT073 in a murine model of UTI, several survival-attenuated mutants were found to have insertions in genes involved in the biogenesis of various extracellular polysaccharides (19). Three mutants that were deficient in colonization of the murine urinary tract had disruptions in loci linked to capsule production. Each of these mutants did not make detectable capsule when grown in vivo. Intriguingly, these capsule biosynthetic genes were found to be highly upregulated during growth in urine or in the mouse urinary tract compared with growth in laboratory medium (336). Furthermore, a mutant with a deletion of the gene cluster encoding the cluster II enzymes involved in synthesizing the K2 capsular antigen was outcompeted in the murine urinary tract by the wild-type CFT073 strain, but not when complemented with the genes that had been deleted (E. L. Buckles, X. Wang, M. C. Lane, C. V. Lockatell, D. E. Johnson, D. A. Rasko, H. L. Mobley, and M. S. Donnenberg, unpublished work). This mutant, but not the wild-type strain, was highly sensitive to killing in the presence of human serum, a phenotype that was also reversed by complementation. However, the mutant did not differ from wild type in sensitivity to killing by human neutrophils. Thus, the K2 capsule, but not the K54 capsule, has been confirmed to be a urovirulence factor. These results suggest that the roles of capsule and LPS may differ depending on the particular serotype studied or on other characteristics of specific strains.
Iron, an essential cofactor for enzymes found in all organisms, is sequestered in the human body by a variety of iron-binding proteins; and, therefore, bacteria that colonize host tissues must have systems for procuring this element. It has long been appreciated that the ability to produce aerobactin, an iron-chelating hydroxamate siderophore, is more common among strains of E. coli isolated from the urine of patients with UTIs than among control strains isolated from the feces of healthy individuals (40, 157). Definitive genetic evidence of the role of iron acquisition in the pathogenesis of E. coli UTI was obtained by Torres and colleagues who isolated a spontaneous tonB mutant of a virulent UPEC strain. TonB is an inner membrane protein required for the function of all outer membrane ferric iron receptors in E. coli. These investigators found that the tonB mutant was significantly attenuated in its ability infect the kidney in a murine model of ascending UTI and could complement this defect by restoring tonB on a plasmid (358). To determine which of the several iron uptake systems was involved, they constructed mutants with defects in aerobactin-, enterobactin-, and heme-mediated iron uptake. They found that a strain with mutations in both the aerobactin and enterobactin systems was slightly deficient in kidney colonization, but that strains with either system alone still intact were not (they did not complement these mutants). Furthermore, using the more sensitive coinfection assay, they found that the wild-type strain could outcompete either a mutant with a defective aerobactin receptor or a mutant with a defective heme receptor when inoculated together in a mixed infection. Thus, they suggest that multiple iron uptake systems work in concert to allow growth of UPEC strains in the urinary tract.
Further evidence for the role of multiple iron acquisition systems in UPEC virulence is found in recent studies identifying additional iron uptake genes. The genome of UPEC strain CFT073 encodes at least 10 characterized ferric uptake systems, and several putative transporters (376) (Table 3). Similar to the original findings of Torres et al. (358), mutants deficient in the uptake of salmochelin, enterobactin, heme, and other siderophores are outcompeted by wild-type UPEC strains in coinfection experiments (112, 155, 310, 313). However, no single iron uptake locus has been found to be necessary for UPEC colonization of the urinary tract, suggesting that significant functional redundancy exists among these systems, and further highlighting the importance of iron acquisition for the survival of UPEC in vivo.
Flagellum-mediated motility has long been hypothesized to play a role in the pathogenesis of UTIs caused by UPEC. Flagella have also been implicated in the virulence of other pathogenic E. coli, by inducing IL-8 expression and Toll-like receptor 5 activation, and contributing to the adhesion of enteropathogenic E. coli to epithelial cells in vitro (61, 95). Moreover, flagella play a major role in the virulence of other uropathogens, such as Proteus mirabilis (250).
Flagella are complex organelles that protrude from the exterior of the bacterial outer membrane to mediate directed motility and chemotaxis (for details, see Chapter 10 of the last print edition of Escherichia coli and Salmonella: Cellular and Molecular Biology). The bacterial flagellum consists of a basal body, hook, and filament. These flagellar components are essentially constructed in an “inside-out” fashion with the basal body embedded in the membrane prior to the addition of the hook and filament (118). Genes for flagellum synthesis form an ordered and highly regulated cascade of three classes (45, 169, 191, 192, 208, 230). Class 1 genes include flhDC that encode the transcription factor necessary for transcription of the class 2 genes, which encode the basal body and hook of the flagellum, in addition to FliA (σ28) and FlgM (anti-σ28 factor) (169). FliA is the sigma factor that has been shown to be necessary and specific for transcription of the class 3 flagellar genes (207, 277). Intracellular FlgM inhibits FliA activity to ensure that the basal body and hook of the flagella are assembled before the class 3 genes are transcribed (136). The class 3 genes encode hook-associated proteins and the filament of the flagellum (FliC), as well as proteins necessary for motility and chemotaxis (such as MotA, MotB, CheW, and CheY) (169). Potential advantages of flagella-mediated motility during urinary tract colonization include the ability to disseminate to new sites of the urinary tract to obtain nutrients as well as to escape host immune responses.
To evaluate their contribution to virulence, six separate flagellar mutations were constructed in UPEC strain CFT073, including fliA and fliC mutants that do not produce flagella; a flgM mutant that has reduced motility, a motAB mutant that is nonmotile, and cheW and cheY mutants that are motile but nonchemotactic. While all of these mutants colonized the urinary tract during independent challenge, each was outcompeted by the wild-type strain to various degrees at specific time points during cochallenge (214). These results suggest that, while flagella and flagella-mediated motility/chemotaxis may not be absolutely required for virulence, these traits contribute to the fitness of UPEC, and therefore significantly enhance the pathogenesis of UTI caused by UPEC. In a study of intracellular invasion and dissemination of a uropathogenic strain UTI89 in the murine model of UTI, the use of flhDC and fliC fusions with gfp demonstrated poor expression in the intracellular bacterial communities of the bladder (381). These data are consistent with the poor flagellar gene expression observed for planktonic bacteria isolated from infected mice (336). Nevertheless, in this model, the wild-type strain outcompeted a fliC mutant in a competition experiment, supporting the concept that motility contributes to the fitness of UPEC in the urinary tract. Recently, by use of a lux bioluminescent construct under the control of the fliC promoter, whole-animal biophotonic imaging demonstrated that expression of flagella coincides with the ascension of UPEC to the kidneys (212). Furthermore, when a fliC mutant was inoculated into the murine urethra, it was unable to colonize the kidneys, establishing that flagella are required for an ascending infection (212).
Chemotaxis is a behavior that bacteria use to sense and respond to external chemical signals. In E. coli, four methyl-accepting chemotaxis protein receptors are necessary for chemotaxis to amino acids (Tar and Tsr), saccharides (Trg), and dipeptides (Tap) (120, 193, 232, 334). Interestingly, while tar and tsr were present or functional in 100% and 98% of all motile UPEC isolates (n = 34), respectively, trg and tap were significantly less prevalent or functional among uropathogenic isolates than fecal-commensal and diarrheagenic E. coli isolates (trg and tap, 6% and 6% uropathogenic [n = 160]; 50% and 60% fecal-commensal [n = 10]; and 88% and 88% diarrheagenic strains [n = 8]) (213). These data suggested that uropathogenic E. coli have evolved under different selective pressures than the intestinal E. coli. Alternatively, the differences may reflect different niches in the gut. In any event, retention of the two amino acid methyl-accepting chemotaxis protein receptors emphasizes the likely importance of chemotaxis to amino acids rather than saccharides by uropathogenic E. coli.
Flushing by urine is thought to be a primary defense mechanism of the host against microbial colonization of the urinary system. In addition, the low pH, high osmolality, and high concentration of urea in urine can be inhibiting factors for bacterial growth (181). UPEC strains, however, are able to grow in urine and this may contribute to the pathogenesis of UTIs (100). Given the high osmolarity of urine, a number of investigators have studied osmoregulatory proteins in UTI isolates. In one such study it was reported that a pyelonephritis isolate had approximately 3-fold higher capacity to transport proline betaine than did E. coli K-12. Furthermore, deletion of the proP locus encoding the proline betaine transporter resulted in a 100-fold decrease in the ability of the pyelonephritis isolate to colonize the bladder, but the mutation had no effect on kidney colonization (50). In pyelonephritis strain CFT073, however, deletion of both proP and proU, a glycine betaine transporter, did not affect either its growth in urine or its ability to colonize the murine urinary tract. Additional putative osmoprotectant systems present in this strain are thought to confer this phenotypic difference. Thus, further work is needed to define the roles of proP and proU in the context of these putative osmoregulatory systems (51).
In a screen for mutants of a prototrophic pyelonephritis isolate that are deficient for growth in urine, Hull and Hall found that guanine, arginine, and glutamine auxotrophs were severely defective for growth in urine, and that growth in urine was reduced in serine, proline, leucine, phenylalanine, and methionine auxotrophs (139). In contrast, in an in vivo screen of 20 uncharacterized auxotrophic mutants generated by signature-tagged mutagenesis, only a pyrimidine auxotroph was slightly attenuated for survival in mice (39), suggesting that perhaps the close association of the organisms with the urinary tract epithelium provides sufficient nutrients for survival of many UPEC auxotrophic mutants. In another study, Russo et al. (312) screened mutants of an E. coli blood isolate for diminished growth in urine or for induction of lacZ expression in urine. They found that mutations in guaA involved in purine synthesis caused the organism to be sensitive to urine. They also found that the argC locus was induced in urine and that argC mutants exhibited reduced growth in urine. The guaA mutant was significantly impaired in its ability to colonize the urine, bladder, and kidneys of mice, while the argC mutant was impaired in its ability to colonize only the kidneys (312).
A screen of ~2,000 signature-tagged mutants of UPEC strain CFT073 in a murine model of UTI revealed new uropathogenic genes in addition to the ones described previously (19). Among these genes was phoU, the last gene in the pst-phoU operon, which encodes a phosphate transport system and negatively regulates the Pho regulon (350). While it is not absolutely required for urinary tract colonization, phoU contributes to the fitness of UPEC in vivo, because a phoU mutant was significantly outcompeted by wild-type CFT073 in a murine competition model of UTI. This defect was reversed upon complementation (37). However, since the Pho regulon regulates many other genes, the effect of phoU gene mutation on bacterial survival may have been indirect.
A differential fluorescence induction approach was used to identify promoters in strain CFT073 that are active during murine peritonitis, but not active in vitro. Among these in vivo induced genes was degS, an inner membrane protease required for sigma E activity. An isogenic degS mutant was attenuated in both peritoneal and UTI infection models, a defect that could be restored by complementation (295). Further studies identified several genes in the sigma E regulon that contribute to the fitness of CFT073 during UTI (296).
Usp and microcins.
The gene encoding uropathogenic-specific protein (Usp) has been found significantly more often in uropathogenic strains than in fecal strains in humans (23, 175, 261) as well as companion animals (206), and there is supporting evidence that the protein enhances virulence and resides on a PAI (384). DNA sequence homology suggests that Usp is a bacteriocin (285, 331) and an adjacent orf encodes an immunity protein. The Usp amino acid sequence predicts an endonuclease domain at its C terminus (285).
Additionally, genes encoding microcins and their immunity proteins are found in the genomes of sequenced UPEC strains CFT073, 536, and UTI89. In commensal E. coli, microcin production was shown to be Fur-regulated, and a microcin-producing strain, but not its isogenic microcin mutant (ΔmchFEDC), outcompeted a sensitive strain in broth culture supplemented with iron chelator (286). Microcin uptake is thought to be mediated by catecholate siderophore receptors, as a fepA cir fiu mutant was resistant to microcin killing. Since UPEC strains typically encode a number of siderophore receptors, microcins may play a role in interstrain competition in the iron-depleted urinary tract.
Putative virulence factors.
Advances in genomic and functional genomic studies have facilitated the discovery of new virulence determinants of the uropathogens. Several investigators have used a subtractive hybridization strategy to reveal a number of genes on the chromosome of the prototypic UPEC strains CFT073 and 536, which are absent from the genome of E. coli K-12, thus representing potential virulence-associated genes (20, 149, 345). Genes found by this subtractive procedure include those encoding LPS, capsule biosynthesis, a siderophore receptor, other iron utilization genes, genes involved in the cleavage of the glycosidic linkages, bacteriophage-encoded toxin genes, and insertion sequences. In addition, 19 novel DNA fragments were found that were specific for strain CFT073.
In a more directed approach, pathogenicity islands were identified by examining sequences of strain CFT073 that flanked the two functional copies of the pap operon. The first was a 60-kb stretch of DNA that contained 44 ORFs, including genes encoding P fimbriae, hemolysin, and an iron acquisition system (107, 177). Nineteen ORFs showed no homology to E. coli K-12 sequences. A second PAI was identified that carries >72 kb with 89 ORFs including pap genes (294). DNA probes isolated from both of these PAI hybridized significantly more frequently with DNA from pyelonephritis and cystitis isolates than fecal strains (107).
Indeed, many of the genes identified by these genomic approaches were found to be unknown genes, some of which having a G + C content different from E. coli suggesting an origin in a foreign species. The presence of these genes was confirmed by the determination of the complete genomes of E. coli CFT073, 536, and UTI89. Based on annotation, homologs of known virulence genes have been identified. These genes can then be individually mutated and tested for virulence in an appropriate animal model of infection. Obviously, this may be a monumental task because the uropathogenic strains contain as much as a megabase of extra DNA located within pathogenicity islands and smaller chromosomal insertions.
The finding that certain phenotypic traits are more commonly expressed by strains isolated from the urine of patients with UTIs than from the feces of healthy individuals suggests that these factors may be involved in the pathogenesis of UTIs. However, these factors may merely be genetically linked to true virulence determinants and irrelevant to UTIs. One of the best-accepted methods to distinguish between these alternatives is to create a strain that has a specific mutation in a gene encoding a putative virulence factor and test this strain, along with the wild-type strain from which it was derived and the mutant strain complemented with the wild-type copy of the gene, in a suitable model of infection. Factors that are proven to play a role in infection by fulfilling these so-called Molecular Koch's (Falkow's) Postulates are considered to be bona fide virulence factors (77). To date, eight true virulence factors, type 1 fimbriae, Dr fimbriae, TonB, hemolysin, CNF-1, K2 capsule, PhoU, and DegS, have been confirmed for UPEC (Table 4).
As discussed previously, a nonsaturating signature-tagged transposon screen of 2,049 transposon mutants of strain CFT073 in the CBA murine model of ascending UTI led to the identification of 19 survival-defective mutants (19). Notably, type 1 fimbrial genes were hit repeatedly. Other genes included those involved in biosynthesis of extracellular polysaccharides including group I capsule, group II capsule, and enterobacterial common antigen, genes involved in metabolic pathways, as well as some genes of unknown function. Five of these genes were absent in an E. coli K-12 strain.
To further the understanding of the pathogenesis of urinary tract infection, a uropathogenic E. coli strain CFT073-specific DNA microarray that includes each ORF was used to analyze the transcriptome of CFT073 cells isolated directly from the urine of infected CBA/J mice (336). The in vivo expression profiles were compared with E. coli CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and LPS synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes such as pap and foc/sfa, and genes involved in motility and chemotaxis were downregulated in vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen- and iron-limiting, of high osmolarity, and of moderate oxygenation. This study represented the first assessment of any E. coli pathotype's transcriptome in vivo (336).
DNA microarrays were also used to describe the transcriptome of asymptomatic bacteriuria (ABU) strain 83972 (Table 2) in the human urinary tract (305). ABU strains, which effectively colonize the bladder without clinical symptoms, are thought to have evolved from pathogenic E. coli (186). Expression profiles of ABU strain 83972 isolated from three patients indicated significant upregulation of genes involved in iron acquisition, nitric oxide protection, and sugar, amino acid, and nitrate metabolism compared with growth in rich medium (305). Unlike the transcriptome of the pathogenic strain CFT073, no known ABU fimbriae or attachment genes were upregulated in patients (305, 336). Combined with the finding that the major fimbrial genes fim and pap encode nonfunctional proteins in ABU strain 83972 (186), this suggests that the ability to attach to host tissues represents a critical step in uropathogenesis.
Strain CFT073 carries a single circular chromosome of 5,231,438 bp representing 5,533 protein coding regions, but no plasmids. There are 89 tRNA genes and 22 rRNA genes organized in seven operons. The G + C content is 50.47%. The CFT073 genome is 590,209 bp longer than MG1655 and similar in size to enterohemorrhagic E. coli strain EDL933. However, surprisingly, all three strains share only 39.2% of their ORFs, and 247 CFT073-specific DNA segments >50 bp were found inserted into a conserved backbone sequence of 3.92 Mb. Sixty unique segments >4.0 kb encode known or potential virulence genes. CFT073-specific islands total 1.303 Mb and encode 2,004 genes. While this strain lacks a type III secretion sequence (for an exception reporting a UPEC strain with a TTSS, see reference 248), it does encode 10 chaperone-usher-assembled fimbriae (including two complete pap operons), two type IV pili, five or more iron acquisition systems, hemolysin, and seven autotransporters (including Sat, Pic, and Tsh).
The complete genome sequences of two additional UPEC strains, pyelonephritis isolate 536 (O6:K15:H31) and UTI89, a strain isolated from a patient with acute cystitis, have been described recently (35, 44). Although its genome is 292 kb smaller than that of CFT073, E. coli 536 shares 89% of its ORFs with CFT073 (35). Similarly, the UTI89 chromosome is 165 kb smaller than that of CFT073, but this strain also carries a 114-kb plasmid. In addition to plasmid replication and conjugation factors, the pUTI89 plasmid encodes a putative iron uptake system and putative toxin (44). Like CFT073, both 536 and UTI89 have a G + C content of approximately 50.5%. UTI89 and 536 encode chaperone-usher fimbriae, iron acquisition systems, adhesins, and autotransporters similar to those of E. coli CFT073. Notable exceptions include the presence of only one pap operon in these strains, several key differences in PAIs (Fig. 1), and the presence of genes encoding Pix fimbriae, enterotoxigenic E. coli-like adhesins, and a second hemolysin determinant in 536. While there is clearly much similarity between these three sequenced UPEC isolates, a number of strain-specific genes exist and we are still far from understanding the genetic profile of a uropathogen.
The urothelial layer of the bladder represents one of the first lines of defense against pathogens. This transitional epithelium, which can readily expand and contract, is composed of basal cells, intermediate cells, and superficial umbrella cells (reviewed in reference 13). Coated apically by hexagonal arrays of semicrystalline membrane proteins termed uroplakins (387, 388), terminally differentiated umbrella cells form a barrier between the bladder lumen and underlying epithelium. When damaged, these cells slough off and are eliminated with the voiding of urine, providing a means to eliminate adherent bacteria.
In addition to these physical defense mechanisms, a number of secreted factors function to protect the urinary tract from infection. Tamm Horsfall protein (THP), the most abundant protein in mammalian urine, is a glycoprotein synthesized in the renal loop of Henle (332). Characterized by high-mannose oligosaccharides (330), THP has a number of functions in the urinary tract, including defense against uropathogens. THP binds to type 1 fimbriated E. coli, an interaction that can be inhibited by D-mannose (251, 282). This binding subsequently blocks the ability of type 1 fimbriae to bind the uroepithelial receptors, uroplakin Ia and Ib (282). Consequently, THP appears to play an important role in bladder immunity, as THP−/− knockout mice inoculated with type 1 fimbriated E. coli were impaired in their ability to control bladder colonization compared with THP-sufficient controls (22, 249). However, no difference in bladder (249) or kidney (22) colonization was observed when knockout mice were inoculated with P-fimbriated E. coli.
Cathelicidin, an antimicrobial peptide expressed by circulating neutrophils, is also produced by the urothelium in response to contact with bacteria (47). When infected with E. coli CFT073, cathelicidin-deficient mice had higher bladder and kidney colonization levels than wild-type mice and showed more severe signs of infection. Pyelonephritis isolates had an approximately 2-fold higher cathelicidin MIC than did cystitis isolates, suggesting that more severe infections are associated with higher resistance to this antimicrobial peptide.
One of the first events after UPEC entry into the bladder is attachment to the urothelium via type 1 fimbriae. This attachment triggers exfoliation of the superficial layer, presumably as a host defense mechanism to eliminate bacteria from the bladder. Observations indicate that the underlying cells then differentiate rapidly to restore the superficial umbrella layer (90, 256). The molecular response of bladder cells to infection has been examined by use of a host microarray, supporting assays, and isogenic wild-type and FimH-deficient strains (259). Interaction with type 1 fimbriated E. coli suppresses transforming growth factor-β signaling to promote subsequent differentiation of basal/intermediate cells. Regulators of proliferation (such as epidermal growth factor) and epithelial differentiation, proapoptotic factors, cell tight junction components, and host defenses (including inducible nitric oxide synthase [iNOS] and mediators of proinflammatory responses) are triggered by UPEC. These events appear to depend at least in part on host interactions with type 1 fimbriae, as a FimH-deficient strain elicited a much weaker response (259).
Like other mucosal surfaces, innate immune defenses in the urinary tract play crucial roles in preventing infection in this exposed site. Reflecting this significance, most immune research in the field to date has focused on the innate response to UPEC. Toll-like receptor signaling, proinflammatory cytokines, and phagocytic cells are the effectors involved in bacterial clearance from the urinary tract.
Toll-like receptors (TLRs), which recognize conserved pathogen-associated molecules, are critical in facilitating the innate immune response to UTI. In humans, TLR4, which recognizes and responds to LPS, is expressed along the bladder, ureter, and renal epithelium (316). While the CD14 coreceptor was detected in several bladder epithelial cell lines (322), it was not detected in human urinary tract biopsy samples (316). Mice lacking TLR4 (117) or the flagellin-responsive TLR5 (4) are each more susceptible to UTI and maintain higher bacterial burdens than wild-type controls. An in vivo invasion assay similarly showed that E. coli invaded bladder epithelial cells in tlr4−/− mice at 10-fold greater levels than those of wild-type mice (343). TLR4 expressed on both epithelial and hematopoietic cells are required for these responses (321). In addition to the classical NF-κB pathway, TLR4 signaling on bladder epithelial cells may also use cAMP levels to induce rapid responses to LPS (344). Recently, a novel receptor, TLR11, that mediates recognition of UPEC (391) and a parasite-derived profilin-like protein (386) was identified in mice. However, because humans do not express this receptor, the role of TLR11 in innate resistance to UTI remains unclear (391).
While common bacterial products like LPS and flagellin indeed trigger an innate immune response in the urothelium, evidence for the role of type 1 and P fimbriae in the induction of inflammation is increasing. In human tissues, type 1 or P-fimbriated E. coli induced greater IL-8 and IL-6 production than did isogenic nonfimbriate strains (316). The precise mechanism of this activation is debated, however. Some investigators report that optimal response to LPS and type 1 fimbriated E. coli by uroepithelial cells requires CD14 (322). However, other studies show that both type 1 and P fimbriae can activate TLR4 signaling and cytokine production in LPS-hyporesponsive epithelial cells with severely reduced or absent CD14 expression (89, 125). Recently, the type 1 fimbrial adhesion FimH was identified as a novel TLR4 ligand (254), signaling through MyD88 to induce antimicrobial responses, including interferon production (15).
Signaling through TLRs and other receptors initiates a local proinflammatory response in the urothelium. Cytokines are important mediators of this response and serve as markers for innate immune activation. In epithelial cell lines, E. coli stimulated the expression and secretion of both IL-6 and IL-8 (121, 124). Elevated levels of these cytokines were also detected in the urine of patients with UTIs (123, 190). Indeed, human volunteers who were challenged with E. coli via a urethral catheter secreted urinary, but not serum IL-6 (122). Furthermore, in vitro challenge of human urothelium tissue indicated that IL-1β, IL-6, and IL-8 were rapidly induced within 2 h of challenge, while IL-4 and interferon-γ were not (316). However, another report suggested that interferon-γ may play a role in UTI immunity, because interferon-γ-deficient mice were more susceptible to E. coli bladder infection than wild-type controls (163).
Pyuria, or the presence of neutrophils in the urine, is a clinical hallmark of UTI. An acute proinflammatory response, including neutrophil infiltration, has long been appreciated as a key innate response to these infections. Elevated levels of IL-8, a potent neutrophil chemoattractant, were detected in the urine of nearly all patients with acute UTIs (190). In a mouse model, granulocyte colony-stimulating factor, a proinflammatory cytokine involved in neutrophil maturation, was induced in infected bladders and shown to be important for neutrophil infiltration into the bladder (145). Macrophage inflammatory protein-2, a murine IL-8 orthologue, is required for this migration across the urothelium into the bladder lumen (115). Neutrophils are indeed necessary for UTI resolution, because either preventing their migration (96) or depleting them with antibodies (117) resulted in increased bacterial burden in urinary tract tissues of infected mice compared with controls.
One mechanism of cell-mediated innate immune killing is via the production of toxic nitric oxide by iNOS. While iNOS expression was detected in mouse bladders and kidneys after infection, UPEC only weakly induced iNOS in human renal epithelial cells in culture (292, 293). Although UPEC strains appear to be more resistant to nitric oxide cytotoxicity in vitro than nonpathogenic E. coli (352), an iNOS-deficient mouse strain was as susceptible to UTI as wild-type animals were (163, 291). Furthermore, although tumor necrosis factor-α- and iNOS-producing dendritic cells are recruited to the murine bladder rapidly following experimental infection, knockout mice deficient for these cells did not display altered UTI susceptibility (73). Thus, it appears that iNOS plays, at most, a modest role in UTI clearance.
While innate immune defenses are clearly necessary components of the host response to UTI, adaptive immunity is also induced upon UPEC infection. Humoral responses appear to be particularly important. Indeed, secretory immunoglobulin A (IgA) that can block E. coli binding to urothelial cells was detected in the urine of patients with UTI, with higher levels observed in patients with pyelonephritis than in those with lower UTIs (351, 360). B- and T-cell-deficient severe combined immunodeficient mice are significantly more susceptible to bladder and kidney infection. Nude mice, however, which have T-cell defects, are as resistant as wild-type animals, suggesting that humoral T-cell-independent mechanisms are important for accelerated UTI clearance (134). Gamma delta T cells in these animals may contribute to clearance, though, because T-cell receptor δ-chain knockout mice were found to be more susceptible to E. coli UTI at 3 and 14 days postinfection (163). Furthermore, using an OVA-expressing UPEC strain as a model, Thumbikat and colleagues showed that adaptive responses contribute to UTI clearance, as they observed reduced bladder colonization after reinfection (357). By 2 weeks postinfection, increased antigen-specific serum IgG and IgM were detected in infected mice, and splenic T cells expressed the activation marker CD69. Finally, naïve mice could be partially protected from bladder colonization by the transfer of splenocytes or sera from infected mice (357). Further study of the adaptive immune response to UTI will be especially critical to refine our understanding and treatment of recurrent infections and to develop vaccines.
Despite the fact that the complete nucleotide sequences of three uropathogenic strains are completed, we do not fully appreciate the role of many ORFs in the development of UTI. Nevertheless, much prior research allows us to construct a model of pathogenesis (Fig. 4). Infection probably begins with the colonization of the bowel with a uropathogenic strain in addition to the commensal flora. This strain, which by virtue of factors encoded within pathogenicity islands and other numerous small insertions, is capable of infecting an immunocompetent host, colonizes the periurethral area, and ascends the urethra to the bladder. Little is understood about these early steps in UTI pathogenesis, but it is clear that certain risk factors (such as the use of spermicidal gel in the case of periurethral colonization and sexual activity in the case of introduction into the bladder) facilitate these processes. Between 4 and 24 h, the new environment in the bladder selects for the expression of type 1 fimbriae, which clearly play a critical role early in the development of a UTI. Type 1 fimbriated E. coli attach to mannose moieties of the uroplakin receptors that coat transitional epithelial cells. Attachment triggers apoptosis and exfoliation; invasion of bladder epithelium has also been observed, and it is argued that invaded epithelial cells may act as a reservoir for recurrent infection.
In cystitis strains, type 1 fimbriae are continually expressed and the infection is confined to the bladder. In pyelonephritis strains, the invertible element that controls type 1 fimbriae expression may turn to the off position and type 1 fimbriae expression ceases. One could argue that this releases the E. coli strain from bladder epithelial cell receptors and allows the organism to ascend the ureters to the kidneys. Indeed, flagella expression is upregulated when type 1 fimbriae are phase-off (not expressed) as bacteria ascend the ureters. In the kidney, the bacteria can attach via P fimbriae to digalactoside receptors expressed on the kidney epithelium triggering an inflammatory response. At this stage, hemolysin could damage epithelium and, together with other bacterial products including LPS, an acute inflammatory response recruits PMNs to the site. Secretion of Sat, a vacuolating cytotoxin, damages glomeruli and is cytopathic for surrounding epithelium. In some cases the barrier provided by the one-cell-thick proximal tubules can be breached and bacteria can penetrate the endothelial cell to enter the bloodstream, leading to bacteremia and in some cases, sepsis.
Early studies demonstrated that active or passive immunization against capsule polysaccharide or LPS O antigen could provide protection from UTI (167, 168, 347). Even oral vaccination with live E. coli induced specific anti-K13 and anti-O6 antibodies (239). Similarly, when chemically cross-linked to diphtheria toxoid and administered to naive mice, K13 capsule, one of the most common capsules among UTI-causing E. coli, provided renal protection and stimulated specific humoral and cellular responses (203). However, recent studies have suggested that the presence of capsule and O antigens may obstruct the development of protective immunity, as mucosal vaccination with a capsule- and O-antigen-negative ExPEC strain generated a greater humoral response than immunization with the wild-type strain (309).
Because of their abundance on the bacterial cell surface and role in UTI pathogenesis, fimbriae have been attractive targets for subunit vaccines against UPEC infection. Although most UPEC strains are capable of expressing more than 10 types of fimbriae, most research has focused on the two most prominent forms, type 1 and P fimbriae. Several studies have proposed FimH, the type 1 fimbrial adhesin that mediates mannose-specific binding to host cells, as a potential UTI vaccine candidate. A FimH subunit vaccine composed of either the N-terminal portion of the protein or full-length FimH conjugated to its chaperone, FimC, was shown to induce a specific antibody response in immunized C3H/J mice. Additionally, vaccinated animals showed a 99% reduction in bladder colonization upon challenge compared with controls (217). This vaccine was further shown to protect cynomolgus monkeys from bacteriuria and inflammation and demonstrated a correlation between protection and the presence of serum anti-FimH IgG (216).
Although the precise mechanism of protection by FimH vaccination is unknown, it has been suggested that inhibition of bacterial binding to host epithelial cells plays a role in fimbrial vaccine function (98). The minimum region of FimH required for binding has been mapped to the N-terminal 100 amino acids, a domain conserved among uropathogenic enterobacteria. Antibodies to this region were shown to block binding to mouse bladder epithelial cells, further supporting its function as a binding domain (355). In addition, sera from FimCH-vaccinated mice blocked binding of E. coli clinical isolates that expressed type 1 fimbriae to bladder epithelial cells by 94% (215). As additional support, a FimH mutant strain was not blocked by anti-FimH antibodies and retained its ability to bind to human uroepithelial cells (241).
A serum IgG response was also detected in C3H/HeJ (LPS nonresponder) mice immunized with purified whole Dr fimbriae. These sera blocked bacterial binding to bladder and kidney cells. Vaccinated mice also displayed decreased mortality over naïve animals, with 2% mortality compared with 20% of unvaccinated mice. However, a urine antibody response was not observed, and urinary tract colonization levels were not statistically different from unimmunized controls (98).
While a number of studies provide support for the use of UPEC adhesins as vaccine targets, other evidence suggests that these fimbriae-based vaccines may not be effective. Mutant strains lacking fimH or papG colonized the bladders of patients with spinal cord injury, showing that these proteins are not required for colonization of the human neurogenic bladder and suggesting that vaccines targeting adhesion factors may not block bacterial adherence in all patients (137). Similarly, when no differences in bacteriuria or peripheral leukocyte levels were observed between PapDG-vaccinated primates and controls, the authors suggested that other adhesins may be compensating for the loss of P-fimbriae function, thus allowing bladder colonization (302). Indeed, when type 1 and P-fimbrial genes are inactivated in strain CFT073, F1C fimbriae are upregulated to compensate (337). Antigenic diversity of adhesins also presents a major problem of vaccination with whole pili, potentially resulting in protection against only a small number of closely related strains (98). In addition, phase variation of fimbria expression may affect immune responses, as proposed when a patient infected with a P-fimbriae-negative E. coli strain was found to have a cell-mediated response to P fimbriae (176).
While much effort has been given to developing a fimbriae-based vaccine for UTI, other targets in UPEC have also been investigated. For example, a combination vaccine including whole P fimbriae and purified α-hemolysin (HlyA) protected infected mice from renal colonization and injury (271). The hlyA gene is highly conserved among hemolytic E. coli strains and exhibits only local variation, suggesting that, as a vaccine, it would have broad cross-reactivity against many infecting strains. Indeed, an anti-HlyA monoclonal antibody was found that bound 94% of clinical isolates (274). Additionally, immunization with purified, denatured IroN, an outer membrane siderophore receptor, increased specific serum, saliva, and urine IgG titers in mice. It also prevented renal, but not bladder, colonization in infected animals, leading the authors to propose it as a complement to cystitis vaccine targets such as FimH (314).
In 1959, Ed Kass, acknowledged as the founder of modern UTI research, stated, “It is apparent from the observations of many workers…that individual bacterial strains differ with respect to capacity of produce manifest disease of the urinary tract” (178, 392). Uropathogenic E. coli represent a heterogeneous group of isolates, restricted to a small number of O serogroups, with different phenotypes. Although many UTI isolates appear to be clonal, most likely belonging to the B2 phylogenetic group, there is clearly not a single phenotypic profile that causes UTI. Therefore, there may be several subclasses of uropathogenic E. coli. Indeed, by examining virulence factor patterns among a collection of E. coli isolates, Marrs and colleagues found that several virulence signatures are preferentially associated with uropathogenicity and suggest that there may be distinct UPEC pathotypes (237).
While no single pathogenicity profile can define UPEC, we can make some generalizations about this group of strains. Specific adhesins including P, S, Dr, and type 1 fimbriae appear to aid in colonization of the urinary tract. UPEC often produce toxins, including hemolysin, CNF-1, and an autotransported protease, Sat, which likely contribute to tissue damage and lead to a host inflammatory response. Multiple iron acquisition systems provide both redundancy and specificity of iron sources. These and other virulence genes are frequently colocalized on pathogenicity islands, large blocks of genes not found in the chromosome of fecal strains, although smaller genomic insertions are extremely common.
When weighing the contributions of various virulence determinants to construct a model for UPEC pathogenesis, it is important to consider that UTI is not a mechanism of spread. That is, it is unlikely that E. coli successfully infecting the urinary tract are transmitted to new hosts directly via urine. Thus, it is also unlikely that the urinary tract represents an essential step in the lifecycle of these strains. More probable is the model that certain E. coli strains, by virtue of their unique genetic composition, are able to take advantage of the distinct urinary tract niche, possibly avoiding competition with the intestinal microbiota. These considerations, along with analysis of the E. coli CFT073, UTI89, and 536 genomes and efforts to identify novel virulence genes should advance the field significantly and allow for the development of a comprehensive model of pathogenesis for uropathogenic E. coli.
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