Host-Pathogen Interactions and Host Defense Mechanisms
Keira Melican
Ferdinand X. Choong
Agneta Richter-Dahlfors
Urinary tract infections (UTIs) represent the most common urologic disease in the United States based on the Centers for Disease Control’s (CDC) statistics of visits to office-based physicians and hospital outpatient clinics. Women account for a majority of cases. The 2005/2006 Ambulatory Medical Care Utilization Estimates attributed an annual figure of approximately 8.1 million of all diagnosed patients with UTIs as the main ailment.1a Although no clear cost estimate is available currently, total expenditures in the year of 2000 alone was estimated to amount to US$3.5 billion. 2a
The human urinary tract is a normally sterile environment that presents invading bacteria with numerous challenges of a dynamic nature. These challenges include the mechanical stress of urine flow, various physical barriers such as the mucosal epithelium, and the attack of invading immune cells that form part of the host’s immune response. The dynamic nature of these challenges means that invading pathogens must rapidly adapt to their changing niche in order to enable colonization. Infections of the urinary tract occur when pathogens, often originating from fecal flora, enter the urethra. Although continuous cycles of urine production, storage, and voiding relentlessly expel invading organisms, pathogens are able to migrate to the bladder, where they may cause symptomatic cystitis or asymptomatic bacteriuria (ABU). 1 Pyelonephritis manifest when pathogens ascend further up to the kidney, colonizing the tubules of the nephrons.
Asymptomatic bacteriuria is defined as the presence of bacteria in the urinary tract, which do not cause any obvious clinical symptoms in the patient. ABU has been described as similar to a commensal state 2 where patients may carry up to 10 5 CFU per milliliter of urine without symptoms. ABU strains are genetically similar to those that cause symptomatic infections, but they notably tend to lack adhesion organelles.
Cystitis, or a lower UTI, occurs when the pathogens that have entered the urinary bladder attach themselves to cells of the bladder epithelial lining, where they start multiplying. A lower UTI often presents with clinical symptoms such as pain and urgency of urination. The urine of cystitis patients often appears cloudy due to the presence of bacteria, white blood cells (WBCs), and sloughed epithelial cells. 3 A urine examination and culture are essential for a diagnosis, and the infection is usually treated with antibiotics.
Further migration of bacteria up the ureters leads to an infection of the kidneys. 4 A bacterial infection of the kidney is medically termed pyelonephritis, indicating that the infection has reached the renal pelvis—the so-called pyelum —of the kidney ( nephros ). Upper UTI infections are more difficult to diagnose than cystitis. They show similar symptoms to a lower UTI but are often accompanied by a sudden increase in temperature and unilateral or bilateral flank pain. 5 Pyelonephritis is commonly defined as a tubulointerstitial disorder based on the pathologic picture observed in renal biopsies. This indicates that the tubules and interstitial tissue are most commonly involved. 6 In light of the greater level of inflammation, as compared to cystitis, pyelonephritis is considered a serious infection. 7 Gross pathology includes abscess formation in the renal parenchyma and edema, often leading to irreversible scar formation. Renal scar formation with fibrosis can contribute toward the development of renal insufficiency. 7
The normal kidney is considered relatively resistant to infection but abnormalities in the structure and function of the urinary tract can increase susceptibility. 8 Risk factors in children include voiding dysfunction and vesicoureteral reflux, whereas in adults, genetic susceptibilities and behavioral risk factors are most relevant. 9
An essential step in bacterial colonization and the initiation of a UTI is the bacterial binding to the urinary epithelium. However, the epithelia that line the urinary tract are far from uniform. The bladder is lined by a transitional stratified epithelium consisting of multiple layers, topped with facet or umbrella cells, and covered with apical plaques of hexagonal uroplakin. 10 The bladder epithelium, together with the transitional epithelium, covers the ureters and the renal pelvis and is also known as uroepithelium. The Bowman capsule (the epithelial structure that surrounds the glomerular capillary tufts) is lined with thin squamous epithelial cells. The tubular systems of the nephron all consist of a
single layer of epithelium, which expresses a unique structure and function depending on the tubule segment. 11,12,13,14 The proximal tubule consists of cuboidal/columnar epithelia covered with microvilli (˜150 per square micrometer of cell surface), which function to increase the surface area for tubule reabsorption. 12 Distal tubule cells lack microvilli and constitute a tight epithelium, displaying low endocytic capacity and low permeability to water. 13,14 This suggests that bacteria do not only meet very different epithelial linings on their way up the urinary tract, but also that they do so while being exposed to a continuously altered composition of filtrate/urine.
single layer of epithelium, which expresses a unique structure and function depending on the tubule segment. 11,12,13,14 The proximal tubule consists of cuboidal/columnar epithelia covered with microvilli (˜150 per square micrometer of cell surface), which function to increase the surface area for tubule reabsorption. 12 Distal tubule cells lack microvilli and constitute a tight epithelium, displaying low endocytic capacity and low permeability to water. 13,14 This suggests that bacteria do not only meet very different epithelial linings on their way up the urinary tract, but also that they do so while being exposed to a continuously altered composition of filtrate/urine.
Uropathogenic Escherichia coli (UPEC) are implicated as the causative agent in up to 80% of community-acquired UTIs, making it the leading urinary pathogen. 7 However, other gram-negative bacterial species are also associated with UTIs, including Klebsiella , Enterobacter, Pseudomonas, and Proteus mirabilis. 7 The latter accounts for more than 40% of UTIs in infant boys. Gram-positive bacteria implicated in UTIs include Staphylococcus strains, epidermidis , and aureus , as well as Enterococcus faecalis. 7 Using culturebased methods, a Canadian study of bacterial isolates in ambulatory patients with community-acquired UTIs revealed that 74.2% contained Escherichia coli, 6.2% contained Klebsiella pneumoniae , 5.3% contained Enterococcus , 2.8% contained Streptococcus agalactiae, 2% contained Proteus mirabilis , 1.4% contained Staphylococcus saprophyticus , 0.9% contained Viridans streptococci , 0.9% contained Klebsiella oxytoca , and 0.8% contained Pseudomonas aeruginosa. 15 Although the proportion of isolates vary depending on the region, the disease state, and the patient type, UPEC unanimously remains the major causative microbe for UTIs and, as such, has been used as the primary pathogen in a number of molecular studies of the UTI process. This chapter will accordingly focus on current knowledge gained from UPEC-induced UTIs.
VIRULENCE OF UROPATHOGENIC ESCHERICHIA COLI
Bacteria entering the urinary tract must rapidly adapt to their new environmental niche. To enable this adaptation, UPEC strains express specific genes that encode a class of proteins termed virulence factors. This name originates from the finding that these factors assist in the initiation and progression of the infection. UPEC strains have a larger genome and therefore contain more genes than their nonvirulent ABU or commensal E. coli counterparts. For example, the clinical isolate CFT073 has 590,209 more base pairs in its genome than the K-12 MG1655 strain. 16 Based on recent genetic approaches, it was proposed that UPEC, ABU, and commensal strains may have evolved from the same virulent ancestral parent, with the ABU and commensal strains having lost virulence factors. 17,18 Due to the relatively minor genetic variations between the UPEC, ABU, and a commensal gut E. coli , genetic mutations and differences in expression of certain genes may actually differentiate virulence potential rather than genomic content itself. 17
The extra genes that confer virulence are commonly located in specific regions of the chromosome, termed pathogenicity-associated islands (PAIs). 19,20 This unique subset of genomic islands has been acquired by horizontal gene transfer. 21 PAIs were originally identified by Hacker and colleagues 22 when they analyzed segments of chromosomal regions that encode multiple, distinct virulence-associated phenotypes in UPEC strain 536. 23 Further characterization has demonstrated that PAIs are present in a wide range of bacterial pathogens, that PAI segments range from 10 to 200 kb in size, and that they are rich in virulence and antibiotic resistance gene insertion sequences or other mobile genetic elements. PAIs are easily identifiable by their unique G+C content, 24 and their location near or within tRNA genes. One bacterium may possess multiple PAIs, 25,26 as exemplified by UPEC strain 536, which contains six wellcharacterized PAIs. 27,28,29
The chromosomes of E. coli appear highly diverse aside from a core genome that is highly homogeneous in G+C content. 24 A large proportion of this diversity arises from a variable pool of mobile genetic elements, conjugative plasmids, bacteriophages, transposons, insertion elements, as well as by the recombination of foreign DNA into host DNA. 24 This has been highlighted in comparative studies of the nonpathogenic E. coli K-12 lab strain MG1655, 28 the enterohemorrhagic O157:H7 strain EDL933, 30 and the UPEC strain CFT073. 16 They were shown to differ significantly in genome size, sharing only 39.2% of proteins in common. 16 The flexibility of bacterial genomes arising from mobile genetic elements may facilitate the timely emergence of new clones, 31,32 which provides new virulence and antibiotic resistance profiles.
However the genetics may look or may have evolved, it remains that pathogenic UPEC strains express proteins that are considered essential for virulence. These virulence factors characterize disease isolates. 33 Although the early definitions of virulence factors came from the basic epidemiology practice of comparing properties of fecal strains from healthy controls with urinary isolates from patients, 34 Falkow 35 introduced a new view in 1988, which he named the molecular Koch’s postulates for pathogenesis. These postulates include 36 :
The phenotype or property under investigation should be associated with the pathogenic members of a genus or pathogenic strains of a species.
Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence, or the gene(s) associated with the supposed virulence trait should be isolated by molecular methods. Specific inactivation or deletion of the gene(s) should lead to loss of function in the clone.
Reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity, or the replacement of the modified gene(s) for its allelic counterpart in the strain of origin should lead to a loss of function and a loss of pathogenicity or virulence. Restoration of pathogenicity should accompany the reintroduction of the wild-type gene(s).
Fifteen years later, Falkow 36 reviewed these postulates in light of the new advances in technology available to infection biologists. Here he described how the postulates should be considered as a “working hypothesis for the study of the genetic and molecular basis of pathogenicity” and not a ridged determination of virulence factors. Some virulence factors may play different roles in different model systems and, as technology advances, the definitions of what a virulence factor is may need to evolve. Some of these factors may also be considered as “fitness factors” (i.e., factors that enhance the growth and colonization of the bacteria but may not be absolutely essential for infection). Siderophores, which allow bacteria to sequester iron, have been annotated as fitness factors because their expression is advantageous but not essential to virulence. 37,38 Conversely, the acquisition of certain traits such as antibiotic resistance, which would appear advantageous for virulence, can have a negative effect on bacterial fitness. 39,40
UROPATHOGENIC ESCHERICHIA COLI ADHESION
The traditionally annotated UPEC virulence factors include adhesion factors, exotoxins, lipopolysaccharides, capsules, proteases, and iron acquisition systems. 41,42 Research on these factors has been carried out in vitro and forms the foundation for their current definition. Thus, the expression of certain adhesion factors is still defined by their in vitro agglutination abilities. 34,42 In UPEC, the best described virulence factors are involved in bacterial adhesion to the uroepithelium, and these proteinaceous structures are referred to as fimbriae or pili. 20 These organelles allow UPEC to bind to the epithelium and help bacteria to withstand the stress of filtrate and urine flow. UPEC express numerous different fimbriae including P, type 1, F1C, S, and Afa/Dr adhesins. 42 The great redundancy in fimbriae expression is further illustrated by the fact that one bacterium contains the genes for many different fimbriae. 43 The current understanding of the roles of the various fimbriae in UTIs is described in detail in the following paragraphs.
Bacteria have many tools aiding their rapid adaptation to changing microenvironments. They contain genes for numerous different fimbriae and it has been shown there is a redundancy between these fimbriae. 43 Phase variation means the bacteria can vary their fimbriae expression, thereby altering their nature of adhesion depending on the microenvironment. This common feature not only allows for rapid adaptation but also, at the same time, allows for the development of a heterogeneous bacterial population. 44 A genetic switch, the so-called fim switch, controls the phase variation of type 1 fimbriae expression. This invertible element contains the main promoter for the fimbrial structural subunits. 44 Negative cross-talk between type 1 and P fimbriae has been demonstrated, with PapB being shown to repress the FimB-promoted off-to-on inversion of the fim switch. 45 This means that UPEC express either Type 1 or P fimbriae but it is unlikely that they express both simultaneously. A cross-talk between P fimbriae expression and motility has also been reported, showing that the expression of P fimbriae also regulates the synthesis of flagellum, a protein-based extrusion that mediates bacterial mobility. The PapX protein, encoded at the end of the pheV associated pap gene cluster in UPEC strain CFT073, represses motility by binding to the flhD promoter, thereby repressing transcription of FlhD2 C2 , the master regulator of flagella. 46 These regulatory mechanisms highlight one mechanism by which bacteria can fine-tune their expression to adapt to the challenging microenvironments they encounter upon infection.
Type 1 Fimbriae
Type 1 fimbriae are attachment organelles produced by the vast majority of E. coli strains, both commensal and pathogenic. Initially visualized in 1950, 47 type 1 fimbriae are mannose sensitive adhesion organelles, which means their ability to agglutinate erythrocytes is inhibited by mannose. 48,49 This feature, found in the mid 1950s, is still used today to define type 1 fimbrial expression.
The type 1 fimbriae are made up of 500 to 3,000 repeating subunits of the FimA protein, which is formed into a 7-nm thick right-handed helical rod. At the outermost end of the rod is located a 3-nm thick distal tip that contains several copies of the adapter proteins FimG and FimF as well as the tip adhesin FimH. 50,51,52 Assembly of the rod-like type 1 fimbriae occurs via the chaperone-usher pathway, which represents a common assembly pathway for fimbriae in gram-negative bacteria. 52 The chaperone and usher proteins required for the formation of type 1 fimbriae are all encoded in the fim operon. 51 FimC is the periplasmic chaperone, which delivers bound subunits to the outer membrane usher protein FimD. From here, the subunits are incorporated into the growing fimbriae. 51
The ability of the tip adhesion FimH to bind mannosecontaining glycoproteins means that type 1 fimbriated bacteria can adhere to a wide range of human target cells. 53,54 The crystal structure for FimH was recently resolved. 55,56 Whereas intestinal E. coli express certain variants of the FimH adhesion, 57,58 UPEC express a FimH that has an increased affinity for terminal monomannose (M1) residues 59 and displays a 20-fold higher ability to bind uroepithelium. 57,58,60 The traditional view of type 1-mediated binding is that the mannose moiety is present on cells or structures associated with the mucosal lining, or alternatively, that mannose is bound to abiotic surfaces. Whereas the first situation refers
to bacterial colonization on the mucosal lining, the latter is implicated in bacterial biofilm formation in vitro. 61 Recently, a novel alternative was presented, demonstrating the importance of type 1 fimbriae in mediating interbacterial contact and biofilm formation in vivo, thus providing a means for bacteria to withstand the shear stress from the renal filtrate (see the following text for further details). 62
to bacterial colonization on the mucosal lining, the latter is implicated in bacterial biofilm formation in vitro. 61 Recently, a novel alternative was presented, demonstrating the importance of type 1 fimbriae in mediating interbacterial contact and biofilm formation in vivo, thus providing a means for bacteria to withstand the shear stress from the renal filtrate (see the following text for further details). 62
In UPEC strains, the role of type 1 fimbriae in cystitis has been extensively described. The uroplakins on the surface of bladder epithelium contain monomannose moieties to which FimH binds. 42,55 Therefore, uroplakins serve as anchoring sites allowing UPEC to gain a foothold in the bladder.63 Although the kidney lacks mannose moieties, a new hypothesis was recently proposed in which the FimH tip adhesions of type 1 fimbriae facilitate interbacterial binding and, in synergy with P fimbriae, thus enable tubule colonization.62 This broadens the role for type 1 fimbriae to infectious niches other than those with surface-bound mannose moieties.
The urinary tract represents a compartment continuously exposed to some degree of mechanical flow, primarily in the form of urine. Bacteria entering into this compartment will thus be exposed to shear stress generated by the flow of urine over the epithelial surface. Over recent years, it has become increasingly appreciated that the stress may affect bacterial adhesion. The UPEC FimH protein has been shown to display enhanced binding to mannosylated surfaces in vitro in the presence of shear stress. 64,65 This interaction is reported to operate via a force-enhanced allosteric catch-bond mechanism, functioning via a finger-traplike β sheet twisting mechanism. 64
In the initial report of FimH shear-dependent binding, it was shown that at a shear of 0.02 dynes per cubic centimeter, the binding strength of FimH was weak, whereas as the shear increased to 0.8 dynes per centimeter, this binding became stronger. 65 The same laboratory also showed that UPEC positively select for a FimH variant that maintains an attachment following a drop in shear, as compared to fecal or vaginal E. coli isolates. 66 This variation in the signal peptide of FimH, which results in expression of less, though longer, fimbriae, may be very relevant under the fluctuating conditions facing UPEC in vivo. The fact that certain bacterial adhesion events are enhanced by tensile force, as opposed to bacteria being washed away, is particularly relevant in an environment such as the urinary tract where bacteria must bind in the face of fluctuating filtrate flow. Thus far, FimH is the best described bacterial adhesin in terms of shear- enhanced adhesion, whereas binding of PapG, the tip adhesion of P fimbriae, has been shown to be shear-independent, being able to mediate binding even under relatively low shear. 67
Although the shear-mediated adhesion may assist type 1 adhesion to the bladder epithelium via mannose binding, it may also function during UPEC colonization of the renal tubule, albeit via a different mechanism. Within the nephron, FimH may mediate interbacterial binding and help prevent bacterial washout by renal filtrate. 62 Interestingly, FimH is present in all virotypes of E. coli, 60 and a role for FimH in interbacterial binding may explain a general function for these fimbriae in diverse perfused environmental niches.
Type 1 fimbriae have been found to fulfill molecular Koch’s postulates. Microarray studies of an in vivo mouse model show high levels of type 1 expression. 68 A mutant unable to make FimH is severely deficient in colonization of the urinary tract in a mouse UTI model, and complementation of the mutant has been shown to restore virulence. 69
P Fimbriae
P fimbriae were one of the first virulence factors associated with UPEC. In 1976, it was demonstrated that E. coli from patients with acute pyelonephritis adhered in greater numbers to uroepithelial cells in vitro than strains causing asymptomatic bacteriuria. 70 Their adherence was not inhibited by the prototypic type 1 inhibitor mannose, and further investigation led to the identification of P fimbriae. They were designated P because of their ability to agglutinate red blood cells (RBCs) of the P blood group when analyzed in vitro. 34,71 P fimbriae are encoded by the pap (pyelonephritis-associated pilus) operon, which consists of 11 genes located on chromosomal pathogenicity islands. Unlike the fim operon, the pap operon is selectively distributed in E. coli. 24,72 The morphology of this fimbriae is extremely similar to type 1 fimbriae. 73 P fimbriae are hetero-polymers consisting of a helical rod of PapA subunits with a tip consisting of the minor pilins PapE and PapF. The tip adhesion PapG mediates attachment to Galα1-4Gαlβ containing glycolipids, which are often found on the renal epithelium.73,74 PapG is known to have at least three allele variants: class I, class II, and class III. Class II is primarily linked to human pyelonephritis and class III is linked to cystitis. 71,75,76 Some strains, such as the prototypical UPEC strain CFT073, carries two pap gene clusters, both of which encode for the PapGII allele. 16,71
Although P fimbriae have long been considered an important virulence factor in UTIs, they do not fulfill the molecular Koch’s postulates. P fimbriae are expressed by a majority, but not all, clinical isolates. 34,77 Approximately 80% of UPEC strains express P fimbriae, 78 and a strong relationship exists between the severity of infection and the prevalence of P fimbriae. Indeed, clinical isolates lacking PapG adhesin were observed to cause comparatively less kidney damage than the PapG positive counterpart. Interestingly, despite the known role of P fimbriae in the adhesion colonization capability of strains in both the kidneys and bladder, it was found to be independent of PapG mediated adhesion. 79,80,81,82
Expression of P fimbriae is controlled by phase variation, and varies depending on environmental conditions. The reversible epigenetic switch that controls the initiation of pap operon transcription allows bacteria to fine-tune their P fimbriae expression to suit their changing environment in vivo.44,83,84
Recently, P fimbria were shown to facilitate the early stage of UPEC colonization of renal tubules. 62 Using high-resolution live animal imaging, it was shown that strains
expressing P fimbriae were able to bind and initiate colonization in the face of sheer stress from renal filtrate flow. It was also demonstrated that the P fimbriae act in synergy with type 1 fimbriae in a heterogeneous bacterial community to facilitate renal tubule colonization. P fimbriae were shown to mediate bacterial binding to the epithelium, whereas the type 1 fimbriae mediated interbacterial binding as the colony expanded into the tubule and away from the epithelium. 62 It is interesting to note that unlike type 1 fimbriae, only E. coli and not other gram-negative rods carry the genes for P fimbriae. 85
expressing P fimbriae were able to bind and initiate colonization in the face of sheer stress from renal filtrate flow. It was also demonstrated that the P fimbriae act in synergy with type 1 fimbriae in a heterogeneous bacterial community to facilitate renal tubule colonization. P fimbriae were shown to mediate bacterial binding to the epithelium, whereas the type 1 fimbriae mediated interbacterial binding as the colony expanded into the tubule and away from the epithelium. 62 It is interesting to note that unlike type 1 fimbriae, only E. coli and not other gram-negative rods carry the genes for P fimbriae. 85
Dr Adhesin
Aside from type 1 and P fimbriae, several other adhesins are implicated in mediating urinary tract infections, though their roles are not as established. The Dr adhesins family embraces fimbrial and afimbrial structures, which are found on the extracellular surface of E. coli, and have in common that they bind to Dr blood group antigens. 86 The Dr blood group antigen is a component of the decay-accelerating factor (DAF), a membrane protein that prevents host lysis by complement.87,88 This binding leads to the internalization of Dr+ E. coli into nonfusogenic intracellular vacuoles where bacteria are shielded from the host immune system. 89 Dr adhesin mediated binding of E. coli to the bladder epithelium has also been correlated with recurrent UTIs in young adults and with pyelonephritis in pregnant women. 90 Among the Dr adhesin family, only Dr fimbria possess the ability to bind both type IV collagen of basement membranes and DAF. 91 The latter is mediated via the subunit DraE. 92 When investigating the significance of DraE-type IV collagen binding, it was shown that disruption of this capability resulted in the inability of E. coli to cause a persistent kidney infection. 93
F1C Fimbriae and the S Fimbria Family
Although the role of type F1C fimbriae for UTIs has not been fully determined, epidemiologic data suggest this fimbriae to be more prevalent in pyelonephritis and cystitis strains than among fecal strains of E. coli. 94 Data suggest these fimbriae are expressed in vivo and provide bacteria the capacity to adhere to human distal tubular and collecting tubular epithelium, as well as the vascular endothelium on kidney tissue sections. 95,96 The two minor glycosphingolipids, galactosylceramide and globotriaosylceramide, have been identified as target tissue receptors for F1C fimbriae in rats, canines, and humans. Galactosylceramide is found throughout the urinary tract, with the exception of the urethra, whereas globotriaosylceramide is unique to the kidney. 95 The binding of F1C-fimbriated bacteria to renal epithelial cells in vitro was shown to induce similar levels of interleukin (IL)-8 production as compared to those levels produced by the adhesion of type 1- and P-fimbriated bacteria, thus supporting a role for F1C in pyelonephritis. 95
BACTERIAL TOXINS AND VIRULENCE FACTORS
α-Hemolysin
The 107 kDa lipoprotein α-hemolysin (Hly) is considered an important UPEC virulence factor, yet no more than 50% of pyelonephritogenic E. coli organisms express this toxin. The Hly operon is commonly located adjacent to genes encoding P fimbriae, 99,100 which may account for the two- to threefold higher probability of UPEC having hly genes over fecal strains. 101 Hly exerts concentration-dependent, biphasic activities on target cells. The traditional view focuses on Hlys cytotoxic effect. Hly is lytic for numerous cell types, including erythrocytes, polymorphonuclear leukocytes, monocytes, mast cells, basophils, and lymphocytes. 102,103 More recently, the sublytic concentration of Hly was shown to elicit Ca 2+ signaling in primary proximal tubule cells. 104 Via frequency-modulated activation of the transcription factor nuclear factor-kappa B (NF-κB), Hly activated proinflammatory signaling in epithelial cells. When analyzing a role for Hly in vivo, intravital imaging of the infection process within a nephron of a rat was applied. This showed that the same end result was achieved whether or not UPEC expressed Hly. However, the kinetics of the tissue response was severely influenced. 105
Cytotoxic Necrotizing Factor
CNF1 is a toxin contributing to UPECs invasion of the epithelium.106,107 The toxin induces the formation of stress fibers via the deamination-dependent activation of small, actin-regulatory GTPase proteins of the Rho family. 108,109 The gene encoding CNF1 is positioned adjacent to hemolysin,110,111 and coregulation of their expression is mediated by RfaH. 112,113 Although the role of CNF1 in vivo remains unclear, in vitro studies do suggest a role for the toxin in urinary tract disease. 114,115
Secreted Autotransporter Toxin
Among the array of toxins studied in UTI models, the 107kDa Sat protein is more frequently secreted from pyelonephritogenic E. coli strains than fecal isolates, suggesting a possible role of the toxin in pathogenesis. 116 Sat has serine protease activity and shows cytopathic effects (cytoplasmic vacuolization) on human bladder and kidney cell lines, and in the mouse kidney. 117 However, Sat is not required for kidney colonization. 117 Originally isolated from the prototypic UPEC strain CFT073, Sat was found to share homology with various virulence-related proteins from a range of E. coli pathotypes.118 Among these, Sat possess a high similarity to Pet and EspC, two SPATE (serine protease autotransporters of Enterobacteriaceae) proteins. 118
Siderophores
Mammals possess efficient systems such as the proteins transferrin and lactoferrin to efficiently scavenge free iron within the host. During an infection, the deprivation of free iron is used as a host defense mechanism as upregulation of iron acquisition and storage mechanisms are up-regulated. Low iron availability limits bacterial viability. To counteract this, bacteria produce low-molecular-weight chelators called siderophores (Greek sideros, iron; and phoros, bearing). Siderophores are secreted into the extracellular environment where they bind ferric iron (Fe 3+) and internalize it via receptor-mediated mechanisms. UPEC strains produce four distinct siderophore systems: enterobactin, salmochelin, yersiniabactin, and aerobactin. Among these, enterobactin is conserved in all isolates. 119,120 UPEC also expresses siderophore- associated receptors such as ireA 121 and iroN, 122 and other iron acquisition systems. 16,20 Strains with impaired iron acquisition capability were shown to have decreased fitness and virulence in mouse models. 123
The precise contribution of each iron uptake mechanism to bacterial virulence is presently unclear. However, a study of coincident urinary and rectal strains from patients with recurrent UTIs suggested UPEC infections are facilitated by yersiniabactin and salmochelin. 119 Some UPEC strains express siderophore receptors but not siderophore. These strains are hypothesized to take advantage of neighboring siderophore-expressing bacteria in a polymicrobial setting by competitively scavenging excreted iron-bound siderophores. 20,124,125
Lipopolysaccharide
The serotyping of E. coli strains is based on three determinants: the somatic antigen O, the capsular antigen K, and the flagella antigen H. 126 This system, developed by Kauffmann in 1940, has identified more than 50,000 different E. coli serotypes of various combinations of the 173 O, 80 K, and 56 H types, in addition to all nontypable strains. The association of O-antigen serogroups with UTIs is complex. Although studies have observed the presence of certain serogroups (O1, O2, O4, O6-8, O18, O25, and O75) to be more frequent in E. coli
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