Wri Synthesis Essays
What is synthesis?
When you synthesize two or more texts in an essay, you find connections between the texts. You create a dialogue of sorts between the texts, showing how they �speak� to each other.
What is the purpose of synthesis?
Synthesis is a common academic exercise. When you synthesize texts, you come to a new or deeper understanding of those texts and the ideas within them. When you look at the ideas in one text alone, you focus only on your interpretation of that particular author�s ideas. When you open your analysis up to two or more texts, you can see the ideas in a new light by looking at how multiple authors complement and/or contradict each other.
How is synthesis different from compare and contrast?
In some ways, these two activities are similar. But think of synthesis as going beyond compare and contrast; in general, it is a more complex intellectual task. Instead of looking at two separate things and finding similarities or differences, you focus on how these two things (texts, in this case) actually work together to create a deeper understanding of a theme or idea.
How should I organize a synthesis essay?
Because you want to show a strong connection between the texts and maintain that throughout your essay, I would encourage you to follow the general organizational pattern below. Notice that you�re going back and forth, from one text to the other, so that connection is always there.
I. Introduction (introduce theme, texts, thesis statement)
II. First point about the theme
A. Text #1�s perspective on/treatment of that theme
B. Text #2�s perspective on/treatment of that theme
III. Second point about the theme
A. Text #1�s perspective on/treatment of that theme
B. Text #2�s perspective on/treatment of that theme
- You may choose to focus on more than two points. The number of points you develop should be dictated by the content of your thesis, not by a formula. Because of this, the number of points in each essay will vary from student to student.
- Depending on the complexity of your suppor points, you may choose to write a paragraph that introduces the point in general, then follow with a separate paragraph for each text that develops the point.
- Again, depending on the complexity and number of your suppor points, you may choose to write a paragraph that introduces the point in general, then follow that with a paragraph (or paragraphs) that refer to both texts (in the same paragraph).
Sample synthesis thesis statements:
� In The Woman Warrior, Maxine Hong Kingston challenges the controlling image of the lotus blossom as introduced by Yen Le Esperitu in �Ideological Racism and Cultural Resistance.�
� George Orwell, in his classic essay, �Shoo an Elephant,� describes how, as a police officer for the British Imperial government, he acted against his own desires to salvage his pride. In �Just Walk On By� Brent Staples reveals how, as a black man, he has come to change his behavior because white people are uncomfortable with his presence. In critically examining these two essays together, it becomes clear that both Orwell and Staples understand that most behavior is motivated by concerns for how others view or judge us.
Urinary tract infections (UTIs) are a severe public health problem and are caused by a range of pathogens, but most commonly by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis and Staphylococcus saprophyticus. High recurrence rates and increasing antimicrobial resistance among uropathogens threaten to greatly increase the economic burden of these infections. In this Review, we discuss how basic science studies are elucidating the molecular details of the crosstalk that occurs at the host–pathogen interface, as well as the consequences of these interactions for the pathophysiology of UTIs. We also describe current efforts to translate this knowledge into new clinical treatments for UTIs.
Urinary tract infections (UTIs) are some of the most common bacterial infections, affecting 150 million people each year worldwide1. In 2007, in the United States alone, there were an estimated 10.5 million office visits for UTI symptoms (constituting 0.9% of all ambulatory visits) and 2–3 million emergency department visits2–4. Currently, the societal costs of these infections, including health care costs and time missed from work, are approximately US$3.5 billion per year in the United States alone. UTIs are a significant cause of morbidity in infant boys, older men and females of all ages. Serious sequelae include frequent recurrences, pyelonephritis with sepsis, renal damage in young children, pre-term birth and complications caused by frequent antimicrobial use, such as high-level antibiotic resistance and Clostridium difficile colitis.
Clinically, UTIs are categorized as uncomplicated or complicated. Uncomplicated UTIs typically affect individuals who are otherwise healthy and have no structural or neurological urinary tract abnormalities5,6; these infections are differentiated into lower UTIs (cystitis) and upper UTIs (pyelonephritis)5,7. Several risk factors are associated with cystitis, including female gender, a prior UTI, sexual activity, vaginal infection, diabetes, obesity and genetic susceptibility3,7. Complicated UTIs are defined as UTIs associated with factors that compromise the urinary tract or host defence, including urinary obstruction, urinary retention caused by neurological disease, immunosuppression, renal failure, renal transplantation, pregnancy and the presence of foreign bodies such as calculi, indwelling catheters or other drainage devices8,9. In the United States, 70–80% of complicated UTIs are attributable to indwelling catheters10, accounting for 1 million cases per year4. Catheter-associated UTIs (CAUTIs) are associated with increased morbidity and mortality, and are collectively the most common cause of secondary bloodstream infections. Risk factors for developing a CAUTI include prolonged catheterization, female gender, older age and diabetes11.
UTIs are caused by both Gram-negative and Gram-positive bacteria, as well as by certain fungi (FIG. 1). The most common causative agent for both uncomplicated and complicated UTIs is uropathogenic Escherichia coli (UPEC). For the agents involved in uncomplicated UTIs, UPEC is followed in prevalence by Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, group B Streptococcus (GBS), Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Candida spp.3,6,12,13 (FIG. 1). For complicated UTIs, the order of prevalence for causative agents, following UPEC as most common, is Enterococcus spp., K. pneumoniae, Candida spp., S. aureus, P. mirabilis, P. aeruginosa and GBS9,14–16 (FIG. 1).
Epidemiology of urinary tract infections
Patients suffering from a symptomatic UTI are commonly treated with antibiotics; these treatments can result in long-term alteration of the normal micro-biota of the vagina and gastrointestinal tract and in the development of multidrug-resistant microorganisms17. The availability of niches that are no longer filled by the altered microbiota can increase the risk of colonization with multidrug-resistant uropathogens. Importantly, the ‘golden era’ of antibiotics is waning, and the need for rationally designed and alternative treatments is therefore increasing. Recent studies have used RNA sequencing to directly analyse uropathogens from the urine of women experiencing symptomatic UTIs. These studies, together with basic science and improved animal models, have been crucial in enabling us to understand the molecular details of how uropathogens adhere, colonize and adapt to the nutritionally limited bladder environment; evade immune surveillance; and persist and disseminate in the urinary tract. These studies have therefore revealed key virulence factors that can be targeted to prevent and counteract the pathogenic mechanisms that are important in UTIs7,17,18. In this Review, we discuss the molecular mechanisms of pathogenesis during bladder and kidney infection, comparing and contrasting the virulence factors used by the major uropathogens UPEC, K. pneumoniae, P. mirabilis, E. faecalis and P. aeruginosa. Furthermore, we discuss current antibiotic treatments, antibiotic resistance mechanisms, new combination therapies and future therapeutic interventions that use vaccines and small molecules to target virulence factors.
Adherence and colonization
Adherence is a key event initiating each step in UTI pathogenesis. A UTI typically starts with periurethral contamination by a uropathogen residing in the gut, followed by colonization of the urethra and subsequent migration of the pathogen to the bladder, an event that requires appendages such as flagella and pili (FIG. 2). In the bladder, the consequences of complex host–pathogen interactions ultimately determine whether uropathogens are successful in colonization or eliminated.
Pathogenesis of urinary tract infections
Multiple bacterial adhesins recognize receptors on the bladder epithelium (also known as the uroepithelium) and mediate colonization (TABLE 1). Uropathogens such as UPEC survive by invading the bladder epithelium, producing toxins and proteases to release nutrients from the host cells, and synthesizing siderophores to obtain iron (FIG. 2; TABLE 1). By multiplying and overcoming host immune surveillance, the uropathogens can subsequently ascend to the kidneys, again attaching via adhesins or pili to colonize the renal epithelium and then producing tissue-damaging toxins (FIG. 2; TABLE 1). Consequently, the uropathogens are able to cross the tubular epithelial barrier to access the blood stream, initiating bacteraemia.
Virulence factors used by the main uropathogens
The uropathogens that cause uncomplicated UTIs, including UPEC, K. pneumoniae and S. saprophyticus, have the ability to bind directly to the bladder epithelium, which is composed of the umbrella cells (also known as superficial facet cells), intermediate cells and basal cells19 (TABLE 1). UPEC and K. pneumoniae bind to uroplakins, which are the major protein components of the umbrella cell apical membrane19 and which form a crystalline array protecting the mammalian bladder tissue from damaging agents in urine20. In addition to uroplakins, α3β1 integrins, which are expressed at the surface of uroepithelial cells, can also serve as receptors for UPEC21. By contrast, complicated UTIs are initiated when the bacteria bind to a urinary catheter, a kidney stone or a bladder stone, or when they are retained in the urinary tract by a physical obstruction. Some pathogens (for example, UPEC) can cause both uncomplicated and complicated UTIs. However, others such as P. mirabilis, P. aeruginosa and Enterococcus spp. predominantly cause complicated UTIs (FIG. 2). Subsequently, these uropathogens often form biofilms that are responsible for colonization and persistence22,23 (BOX 1).
Biofilms and morphological plasticity
Uropathogens use different mechanisms for survival in response to stresses in the bladder such as starvation and immune responses. By forming biofilms and undergoing morphological changes, uropathogens can persist and cause recurrent infections40,129,130.
Extracellular DNA (eDNA), exopolysaccharides called extracellular polymeric substances, pili, flagella and other adhesive fibres create a scaffold to form a multicellular bacterial community that is protected from immune responses, antimicrobial agents and other stresses40. The antimicrobial recalcitrance of uropathogens increases on biofilm maturation, as the biofilm provides a physical barrier to antibiotic entry. Therefore, understanding species-specific biofilm formation and dispersal mechanisms is crucial for the development of novel therapies that prevent colonization, such as biofilm inhibitors, anti-adhesive molecules and molecules that induce bacterial dispersion.
Uropathogenic Escherichia coli (UPEC) forms biofilm-like intracellular bacterial communities (IBCs) that protect their members from neutrophils, antibiotics and other stresses38 (FIG. 3). Type 1 pili, antigen 43 and adhesive surface fibres called curli induce biofilm formation by mediating interbacterial interactions and attachment to surfaces. Transcription of antigen 43 is regulated by oxidative stress regulator (OxyR; also known as hydrogen peroxide-inducible genes activator)131, whereas type 1 pilus and curli fibre genes are regulated by polymyxin-resistant protein B (PrmB; also known as BasS) on iron sensing3, leading to phosphorylation of polymyxin-resistant protein A (PmrA; also known as BasR) and quorum sensing regulator B (QseB)131. UPEC biofilm formation on catheters is dependent on type 1 pili35.
Virulence factors of uropathogenic Escherichia coli that contribute to urinary tract infections
Proteus mirabilis produces urease, which hydrolyses urea to carbon dioxide and ammonia. This increases the urine pH and generates calcium crystals and magnesium ammonium phosphate precipitates, which are incorporated into polysaccharide capsules, forming crystalline biofilms on the catheter (FIG. 4). The phosphotransferase regulator of swarming behaviour (RsbA) upregulates polysaccharide expression, represses swarming23 and enhances biofilm formation. Mannose-resistant Proteus-like (MR/P) pili intimately associate with the crystal layers, promoting biofilm formation. Oxygen limitation in the biofilm activates the expression of MR/P pili by inducing the recombinase MrpI to reorient the promoter of the pilus genes. Similarly, expression of the fimbrial operon regulator MrpJ leads to decreased motility, promoting biofilm formation53,132.
Mechanisms of pathogenesis during catheter-associated urinary tract infections
Pseudomonas aeruginosa has the ability to form biofilms on catheters and damaged bladder tissue82 through several mechanisms, including quorum sensing autoinducers that bind to the transcriptional regulators LasR (which regulates elastase (LasB) expression) and RhlR (which regulates the synthesis of rhamnolipids). Quorum sensing induces the production of eDNA, rhamnolipids, lectins, elastases and toxins. The amphiphilic rhamnolipids allow microcolony formation by changing the hydrophobicity of the P. aeruginosa surface133. Biofilm maturation is promoted by lectin adhesins, which are important for bacterial cell–cell interactions134. The production of alginates and extracellular polymeric substances is activated when cyclic di-GMP binds to the transcriptional regulators alginate biosynthesis 44 (Alg44) and pellicle formation regulator D (PelD)135. Small RNAs from the regulator of secondary metabolites (rsm) family, such as rsmZ and rsmY, regulate exopolysaccharide production by reducing the availability of RsmA, which is the transcriptional repressor for exopolysaccharide-encoding genes81,136,137.
Uropathogens also adopt morphological changes, such as filamentation, to circumvent the host immune system130,138. During IBC maturation, expression of suppressor of lon (SulA) inhibits FtsZ polymerization in a subpopulation of UPEC, blocking septation ring formation and cell division138. When the resulting filamentous bacterial cells emerge from epithelial cells, they are resistant to killing by neutrophils and can colonize other naive uroepithelial cells and re-enter the IBC cycle129,138 (FIG. 3). Alternatively, during colonization by P. mirabilis, the bacteria adopt a filamentous morphology as a result of the sensor activities of flagella on contact with a urinary catheter. Contact creates a torsional change in the outer membrane, and this is sensed by upregulator of the flagellar master operon (Umo) proteins, which induce the expression of flagella to produce the highly flagellated cells that are required for swarming during a UTI6,23,53,139 (FIG. 4).
Chaperone–usher pathway pili
Many uropathogens initiate a UTI using pili that mediate adhesion to host and environmental surfaces, facilitate invasion into the host tissues and promote interbacterial interactions to form biofilms24–27. For example, numerous Gram-negative pathogenic bacteria — including E. coli, Klebsiella spp., Proteus spp., Pseudomonas spp., Haemophilus spp., Salmonella spp. and Yersinia spp.16,27–29 — express a large, highly conserved family of adhesive fibres called chaperone–usher pathway (CUP) pili25,26. CUP pili are assembled by the chaperone–usher molecular machinery24,25 and are composed of pilin subunits with incomplete immunoglobulin-like folds that lack the typical carboxy-terminal seventh β-strand30,31. Briefly, in a process termed donor-strand complementation, a dedicated periplasmic chaperone ‘donates’ a β-strand to complete the immunoglobulin fold of the subunits, forming a complex with each subunit and ensuring their proper folding and stabilization. The chaperone–subunit complex is then targeted to the usher assembly protein in the outer membrane, where the usher selectively differentiates chaperone–subunit complexes and catalyses the ordered assembly of pili on the cell surface via a mechanism termed donor-strand exchange. During donor-strand exchange, the final folding of a subunit occurs as the donated β-strand of the chaperone is replaced by an amino-terminal extension on the next incoming subunit32. Importantly, understanding the most basic principles of molecular biology — such as how a protein folds into domains that serve as assembly modules for building large supramolecular structures, and how an outer-membrane macromolecular machine (the usher) assembles these structures from individual subunits, which are delivered as chaperone–subunit complexes and then transported in a regulated manner across a biological membrane — has led to the development of anti-virulence compounds that block CUP pilus assembly or function and that result in the dysregulation of virulence factors. These compounds have the potential for broad-spectrum activity against numerous Gram-negative bacteria (see below).
Uropathogenic Escherichia coli
Thirty-eight distinct CUP pilus operons have been identified in E. coli genomes, and a single UPEC strain can encode more than 12 different CUP pili25. However, the distribution of CUP operons is not uniform across different UPEC isolates; some operons are found ubiquitously in UPEC, whereas others are present in only a handful of strains. The multitude of CUP pili encoded by UPEC are tipped with different adhesins, some of which are known to mediate distinct tropisms in the lower and upper urinary tract by recognizing receptors with stereochemical specificity, notably in the bladder or kidney epithelium33.
Type 1 pili and pyelonephritis-associated (P) pili are the better characterized CUP pili. Type 1 pili are essential for colonization, invasion and persistence of UPEC in the mouse bladder34 (FIG. 3). Type 1 pili are tipped with the adhesin FimH7, which recognizes mannosylated uroplakins and α1β3 integrins with stereochemical specificity21,35 to initiate colonization and invasion into umbrella cells7,21. Type 1 pili binding to these cells triggers a signal transduction cascade that activates Rho GTPases, such as those from the Rac family, to cause actin rearrangement and internalization of UPEC by a zippering mechanism consisting of a plasma membrane sheathe that engulfs the bacterium36 (FIG. 3). Invasion allows UPEC to subvert certain host defences and become recalcitrant to antibiotic treatments. However, an innate defence expulsion mechanism defends the uroepithelium from UPEC invasion; this expulsion mechanism depends on Toll-like receptor 4 (TLR4) expression by uroepithelial cells. Lipopolysaccharide (LPS)-mediated activation of TLR4 stimulates adenylyl cyclase 3 (AC3) to produce cyclic AMP, which induces the exocytosis of vesicular UPEC into the apical plasma membrane of the umbrella cells37 (FIG. 3). Importantly, by escaping into the cytoplasm (through an unknown mechanism), UPEC can subvert the expulsion pathway and rapidly multiply, forming transient biofilm-like intracellular bacterial communities (IBCs)38,39 (BOX 1; FIG. 3). After their maturation, bacteria disperse from the IBC to invade other cells, where the IBC cycle is repeated38–40. IBC formation is a common mechanism for clinical UPEC isolates and has been observed in multiple mouse backgrounds and also in exfoliated uroepithelial cells in the urine of patients with acute UTIs but not in the cells in urine from healthy controls41,42. The process of invasion and IBC formation provides UPEC with the ability to survive stringent bottlenecks in the urinary tract, including TLR4-mediated expulsion, umbrella cell exfoliation, ascension to the kidneys, urination and inflammation7,43. UPEC also establishes quiescent intracellular reservoirs (QIRs) in underlying transitional cells, within membrane-bound compartments enmeshed in F-actin (FIG. 3). In contrast to the metabolically active IBCs, QIRs typically contain 4–10 non-replicating bacteria that can remain viable for months and can be re-activated to serve as seeds that initiate a recurrent UTI7. It has been proposed that during uroepithelial turnover, in which the underlying immature cells terminally differentiate into umbrella cells, the redistribution of actin and perhaps other associated signals might trigger UPEC revival from QIRs, releasing the bacteria back into the bladder lumen44.
Unlike the mannose-binding adhesin FimH of type 1 pili, the adhesin of P pili, PapG, binds globosides containing glycolipids that are present in the human kidneys33 (FIG. 3). In addition, PapG modulates the local secretory-antibody immune response by interacting with TLR4 to reduce polymeric immunoglobulin receptor (PlGR) expression, thus impairing immunoglobulin A transport through the lamina propria and epithelial cells to the kidney lumen45 (FIG. 3). By inhibiting immunoglobulin A transport into the urinary space, UPEC evades a key host protective mechanism, allowing the establishment of ascending infection45,46.
Importantly, the initial innate host response to UPEC colonization and invasion not only dictates the outcome of the original infection but is also crucial for determining host susceptibility to subsequent infections39. An increased susceptibility to recurrent UTIs can occur not because of a deficient host response to UPEC infection, as is commonly accepted, but rather as a result of an unrestrained lymphocyte-dependent innate inflammatory response to acute infection, leading to severe acute injury to the mucosal uroepithelium and potentiating subsequent infections39.
Similarly to UPEC, K. pneumoniae uses type 1 pili for biofilm formation and bladder colonization47 (TABLE 1). Interestingly, although the K. pneumoniae adhesin FimH is highly homologous to UPEC FimH, they have different binding specificities48. K. pneumoniae FimH-mediated biofilm formation is inhibited by heptyl mannose, as opposed to the methyl mannose-mediated inhibition of UPEC FimH. Moreover, K. pneumoniae FimH has a weaker adherence to the bladder than UPEC FimH, resulting in significantly lower K. pneumoniae titers in the mouse bladder and fewer IBCs than are seen for UPEC. Despite the relatively poor adhesive properties of K. pneumoniae FimH in the urinary tract, it remains an important virulence factor for K. pneumoniae during colonization, biofilm formation and persistence in both UTIs and CAUTIs48–50. In addition, K. pneumoniae encodes numerous other CUP pili, including type 3 pili, which also play an important part in colonization, biofilm formation and persistence during UTIs and in biofilm formation during CAUTIs35,51,52.
Following initial attachment, P. mirabilis produces mannose-resistant Proteus-like (MR/P) pili, which are CUP pili that facilitate biofilm formation and colonization of the bladder and kidneys, and are crucial for catheter-associated biofilm formation6,16,23,53 (BOX 1; FIG. 4). Other CUP pili encoded by P. mirabilis include P. mirabilis-like fimbriae (PMFs), which are important for bladder and kidney colonization53, and non-agglutinating fimbriae (NAFs), which are able to attach to uroepithelial cells in vitro53. However, the in vivo mechanistic roles of PMFs, NAFs and their receptors have not yet been established.
In addition to CUP pili, P. mirabilis encodes two autotransporters, TaaP (trimeric autoagglutinin autotransporter of Proteus) and AipA (adhesion and invasion mediated by the Proteus autotransporter), which are important for bladder and kidney infection, respectively53. AipA can adhere to human bladder and kidney cell lines in vitro but is only required for kidney infection (and not for bladder infection) in mice. Conversely, TaaP is required for bladder infection by P. mirabilis in mice. Importantly, both autotransporters bind to extracellular-matrix proteins in vitro: AipA preferentially binds to collagen I, and TaaP to laminin, which might provide an explanation for their different tissue tropisms.
Enterococci encode several adhesion factors, including the collagen adhesin Ace, enterococcal surface protein (Esp), enterococcal polysaccharide antigen (Epa), and endocarditis- and biofilm-associated (Ebp) pili54 (TABLE 1). Of these, Ebp pili contribute to CAUTIs54–56 and are required for persistence during infection55,56. Clinical studies have shown that mechanical stress induced by urinary catheterization produces histological and immunological changes in the bladder, resulting in a robust inflammatory response, exfoliation, oedema, and mucosal lesions of the uroepithelium and kidneys57,58. Importantly, a mouse model of CAUTI seems to recapitulate these immunological changes that are induced by urinary catheterization, exhibiting catheter-induced inflammation, severe uroepithelial damage, exfoliation and the onset of bladder wall oedema, which is exacerbated by increased catheterization time59. Urinary catheters provide a surface for E. faecalis attachment and biofilm formation, which promotes E. faecalis persistence in the bladder and further dissemination to the kidneys55 (FIG. 4). However, E. faecalis is unable to bind to catheter material in vitro and is unable to grow in urine60. This apparent paradox was resolved by the finding that urinary catheterization induces fibrinogen release into the bladder as part of the inflammatory response; this fibrinogen subsequently accumulates in the bladder and is deposited on the implanted catheter60 (FIGS 2,4). Following fibrinogen deposition, the Ebp pilus adhesin — EbpA, which contains an N-terminal fibrinogen-binding domain — mediates catheter colonization and biofilm formation during CAUTIs caused by E. faecalis60,61 (FIG. 4). Furthermore, E. faecalis can use fibrinogen for growth, enhancing biofilm formation on the catheter60 (FIG. 4). This resolution of the paradox has been recapitulated in vitro by the demonstration that E. faecalis attaches to fibrinogen-coated catheters and grows in urine supplemented with fibrinogen60.
Other virulence factors
The bladder environment is limited in nutrients; thus, in order to survive and grow within the urinary tract, uropathogens produce proteases and toxins that damage the host tissue to release nutrients, while also providing a niche for bacterial invasion and dissemination (TABLE 1).
Proteases and toxins
UPEC secretes high concentrations of α-haemolysin (HlyA), which oligomerizes and integrates in the cholesterol-rich microdomains in the host cell membrane in a Ca+-dependent manner62,63. This results in pore formation in the umbrella cells and promotes their lysis, which facilitates iron and nutrient acquisition by the bacteria (FIG. 3). HlyA also triggers exfoliation, exposing deeper layers of the uroepithelium for colonization and promoting bacterial spread to other hosts following cell expulsion in the urine62–65 (FIG. 3). Furthermore, HlyA is highly expressed in IBCs, suggesting that it is important during this stage of infection39,63,66.
UPEC also secretes cytotoxic necrotizing factor 1 (CNF1), which affects actin remodelling in the host cell through three small RHO GTPases: RAC1, RHOA and cell division control 42 (CDC42)67,68. CNF1 enters the host cell in endocytic vesicles, by binding to the receptor basal cell adhesion molecule (BCAM; also known as LU)69, and then constitutively activates RHO GTPases via deamination of a glutamine residue; this causes actin cytoskeletal rearrangements and membrane ruffling, leading to increased levels of bacterial internalization67,70. In addition, the activation of RAC1–GTP induces the host cell anti-apoptotic and pro-survival pathways (through the interaction of phosphoinositide 3-kinase (PI3K), AKT (also known as PKB) and nuclear factor-κB (NF-κB)); this prevents apoptosis of the colonized uroepithelium, thus facilitating UPEC survival and protecting the niche67,71 (FIG. 3).
P. mirabilis produce two toxins, haemolysin (HpmA) and Proteus toxic agglutinin (Pta), which are implicated in tissue damage and dissemination to the kidneys, initiating acute pyelonephritis16,72. HpmA is a Ca+-dependent pore-forming cytolysin that destabilizes the host cell by inserting itself into the cell membrane and causing a Na+ efflux16 (FIG. 4). By contrast, the surface-associated cytotoxic protease Pta is functional only in an alkaline pH, such as that induced by the activity of P. mirabilis urease73. In the proposed mode of action, Pta punctures the host cell membrane, causing leakage of the cytosol, osmotic stress and depolymerization of actin filaments; the structural integrity of the cell is therefore compromised, resulting in bladder and kidney damage53,73 (FIG. 4). Pta also induces bacterial cell–cell interaction via autoaggregation53,73.
P. aeruginosa produces elastases, exoenzyme S (ExoS) and haemolytic phospholipase C, all of which have been implicated in UTI initiation and dissemination, and subsequent pyelonephritis74,75 (TABLE 1). The GTPase activity of ExoS downregulates macrophage RAC1 function, interfering with lamellopodium formation and inducing membrane ruffle formation. The ADP-ribosyltransfease activity of ExoS targets RHO family proteins (RAS proteins and RalA), affecting cell adherence and morphology76. Elastase induces tissue destruction through its protease activity, releasing nutrients (including iron) for continued bacterial growth77. Phospholipase C is an α-toxin that hydrolyses phosphatidylcholine from the host cell membrane, compromising cell integrity and resulting in organ damage78–80. The expression of all of these virulence factors is regulated by the quorum sensing system81. Quorum sensing is activated at high cell density by the accumulation of small molecules called autoinducers. When a threshold level of autoinducers is reached, they bind to transcriptional activator proteins and activate the expression of virulence factors81,82 (BOX 1).
Urease is encoded by several uropathogens, including P. mirabilis53,83, S. saprophyticus84, K. pneumoniae85 and P. aureginosa86, and is important for colonization and persistence during P. mirabilis and S. saprophyticus UTIs83,84 (FIG. 4; TABLE 1). This enzyme catalyses the hydrolysis of urea to carbon dioxide and ammonia87, resulting in elevated urine pH and the production of calcium crystals (apatite) and magnesium ammonium phosphate ammoprecipitates (struvite) in urine and on catheters53 (FIG. 4). Importantly, the accumulation of ammonia becomes toxic for the uroepithelial cells, inducing direct tissue damage88. The P. mirabilis urease, one of the best studied ureases involved in UTIs, is a Ni2+-dependent metalloenzyme that is essential for colonization of the bladder and kidneys and promotes the formation of stones23,53,87. The P. mirabilis urease is induced by urea and is constitutively expressed during growth in urine89. This urease is highly active, hydrolysing urea several times faster than those produced by other species, such as Providencia stuartii, Providencia rettgeri, Proteus vulgaris and Morganella morganii90. The high activity level of the P. mirabilis enzyme induces rapid crystal formation, and these crystals become trapped within the polysaccharides produced by attached bacterial cells, forming crystalline biofilms on catheters23,89,91. The crystalline biofilms provide P. mirabilis with protection from the host immune system and antibiotics88 (BOX 1; FIG. 4). These structures also block urine drainage from the ureters, potentially resulting in reflux and promoting progression to pyelonephritis, septicaemia and shock53.
The bladder environment is limited in iron. Thus, to be able to grow in human urine, uropathogens utilize siderophore systems for iron (Fe3+) scavenging; these systems are composed of the siderophore assembly machinery, a siderophore responsible for binding iron and a membrane receptor that internalizes the iron bound to the siderophore92 (TABLE 1).
UPEC produces several siderophores93, of which two — aerobactin and yersiniabactin — are essential in the urinary tract93 (FIG. 3). Aerobactin is highly expressed, stable at low pH and displays higher levels of iron binding than enterobactin94,95. Yersiniabactin is important in bio-film formation in urine and has a protective role against intracellular killing by copper stress, as it sequesters host-derived copper96.
Numerous iron-scavenging siderophore systems are utilized by other uropathogens: K. pneumoniae produces enterobactin and aerobactin85; P. mirabilis uses proteobactin and yersiniabactin-related97; and P. aeruginosa produces pyochelin and pyoverdin86 (TABLE 1). Siderophore systems are important potential targets for vaccine development98 and for designing small molecules that interfere with their function.
Treatment of urinary tract infections
UTIs result in considerable economic and public health burdens and substantially affect the life quality of afflicted individuals17. Currently, antibiotics — such as trimethoprim sulfamethoxazole, ciprofloxacin and ampicillin — are the most commonly recommended therapeutics for UTIs4. However, increasing rates of antibiotic resistance and high recurrence rates threaten to greatly enhance the burden that these common infections place on society. Ideally, alternative therapies will be established that will be recalcitrant to the development of resistance. Many promising approaches are being developed, from leveraging what we have learned about the basic biology of UTI pathogenesis to specifically target virulence pathways. These antivirulence therapeutics should theoretically allow us to effectively neutralize, or ‘disarm’, the capacity of UTI pathogens to cause disease, without altering the gut commensal microbiota, because antivirulence therapeutics target processes that are critical for UTI pathogenesis but that are not required for the essential processes of growth and cell division (which are the targets of conventional antibiotics).
Below, we discuss the current challenges that have arisen from the emergence of multidrug-resistant bacterial strains and highlight the progress that is being made towards the development of antivirulence therapeutics for UTIs. We also discuss how an understanding of the evolution of bacterial resistance mechanisms and their spread is providing new approaches for the modification and improvement of current therapeutic options.
UTIs are becoming increasingly difficult to treat owing to the widespread emergence of an array of antibiotic resistance mechanisms3,4,15,99–102 (see Supplementary information S1 (table)). Of particular concern are members of the family Enterobacteriaceae, including E. coli and K. pneumoniae, which have both acquired plasmids encoding extended-spectrum β-lactamases (ESBLs). These plasmids rapidly spread resistance to third-generation cephalosporins as well as other antibiotics15,99–103 (BOX 2). Other Enterobacteriaceae family members produce the class C β-lactamases (AmpC enzymes) that are active against cephamycin in addition to third-generation cephalosporins, and are also resistant to β-lactamase inhibitors99–102. The expression of AmpC enzymes is also associated with carbapenem resistance in K. pneumoniae strains lacking a 42 kDa outer-membrane protein15,99–102 (BOX 2).
Multidrug-resistant uropathogenic organisms are becoming an expanding public health threat, as Enterobacteriaceae family members increasingly acquire extended-spectrum β-lactamases (ESBLs) such as cefotaximases (CTX-Ms) and oxacillinases (OXAs), AmpC-type β-lactamases and carbapenemases.
Originating in Klebsiella pneumoniae and Escherichia coli, ESBLs are now prevalent throughout the Enterobacteriaceae family, as frequent use of cephalosporins in the nosocomial setting and the carriage of ESBL-encoding genes on transferrable elements together create an ideal environment for the selection of antibiotic resistance99,102. ESBLs are plasmid-encoded or chromosomally encoded β-lactamases with broad activity against penicillins and cephalosporins. They function by splitting the amide bond of the β-lactam ring, thus inactivating β-lactam antibiotics102. Troublingly, ESBLs are encoded on plasmids that typically carry other resistance genes which provide activity against aminoglycosides, sulfonamides and quinolones, making the bacteria that acquire these plasmids multidrug resistant101,102.
The plasmids encoding the ESBLs CTX-Ms form a new plasmid phylum that is phylogenetically distinct from other plasmid-encoded β-lactamases. CTX-Ms are active against narrow-, broad- and extended-spectrum penicillins, classical and extended-spectrum cephalosporins, and monobactams99,102,103. Notably, they also confer high-level cefotaxime resistance99,103. CTX-Ms are the most prevalent β-lactamases in community-associated isolates and are typically encoded on plasmids with other resistance genes102. CTX-Ms efficiently hydrolyse the β-lactam ring via nucleophilic attack of a ring carbonyl carbon by a conserved serine in the β-lactamase, resulting in a ring-opened product that is inactive140.
OXAs are ESBLs that are typically encoded by plasmids and mediate resistance to ampicillin, cephalothin, oxacillin and cloxacillin by hydrolysing the β-lactam rings99,103. In addition, OXAs are characterized by their ability to resist the β-lactamase inhibitor clavulante103. To date, OXAs have been shown to be expressed only in Pseudomonas aeruginosa99,103.
The chromosomally encoded AmpC enzymes hydrolyse penicillins, third-generation and extended-spectrum cephalosporins, and cephamycins, and are resistant to β-lactamase inhibitors, including clavulanate99,102. AmpC expression is induced in response to β-lactams, cephamycin and cephalosporin exposure.
Carbapenemases are ESBLs that confer the ability to inactive carbapenems in addition to penicillins and extended-spectrum cephalosporins99,101,102. The two most clinically relevant carbapenemases, K. pneumoniae (serine) carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM-1), originated in K. pneumoniae and rapidly spread throughout the Enterobacteriaceae family, creating carbapenem-resistant Enterobacteriaceae (CRE)15,99,101,102. The broad activity of carbapenemases confers resistance against a wide range of extended-spectrum β-lactam antibiotics, particularly carbapenem.
Multidrug resistance is also common among enterococci, as they are naturally resistant to trimethoprim, clindamycin, cephalosporins and penicillins15,101,102. Recently, Enterococcus spp. have developed high-level resistance to glycopeptides, including vancomycin, which is considered to be one of the last lines of defence against multidrug-resistant organisms. Specifically, enterococci evolved resistance to glycopeptides through the expression of vancomycin and teicoplanin A-type resistance (van) genes that encode the penicillin-binding proteins (PBPs) VanA, VanB, VanD, VanE, VanG and VanL101,102. The mechanism of resistance for VanA, the most common PBP expressed by enterococci, is to replace the cell wall precursor D-alanine–D-alanine with D-alanine–D-lactose, effectively reducing the binding affinity of vancomycin104. The troubling trend towards a high prevalence of multi-drug-resistant uropathogens has spurred the development of alternative control measures and treatment options.
New antimicrobials that are resistant to inactivation by ESBLs are under development for use in combination with new classes of β-lactamase inhibitors, which target both β-lactamases and K. pneumoniae carbapenemases (KPCs)105–107. These combination therapies have been shown to be effective in vitro against carbapenem-resistant members of the family Enterobacteriaceae. Furthermore, clinical trials involving complicated UTIs revealed that ceftazidime, a third-generation cephalosporin that is active against Gram-positive and Gram-negative organisms, is effective against ESBL- and carbapenemase-producing Gram-negative bacteria when combined with the β-lactamase inhibitor avibactam105. Future studies are needed to test the efficacy of ceftazidime–avibactam against ESBL-, KPC- and AmpC-producing Gram-negative pathogens during infection, as the drug combination has the potential to be effective against a broad range of cephalosporin-resistant Enterobacteriaceae family members. Although these antibiotic–inhibitor combinations are promising, the development of resistance to β-lactamase inhibitors is not well characterized105. Moreover, the effectiveness of specific antibiotic–inhibitor therapies is dependent on the antimicrobial-resistance patterns encoded by each pathogen, as the expression of certain combinations of ESBLs and carbapenemases can provide resistance to an antibiotic–inhibitor therapy105–107. For example, the combination of BAL30072-BAL29880–clavulanate (two β-lactam antibiotics and a β-lactamase inhibitor) is effective against many carbapenem-resistant Enterobacteriaceae family members, but K. pneumoniae strains that typically produce KPCs and SHVs (another type of ESBL), or AmpC enzymes are resistant106. Therefore, it is crucial to know which antibiotic mechanisms are available to a specific uropathogen in order to determine an effective treatment.
Vaccines targeting bacterial adhesion
As adherence has a key role at nearly every step of UTI pathogenesis, one attractive strategy for the development of antivirulence therapies, including vaccines, has been to target CUP pili. As a general rule, vaccination with whole pili has been ineffective at generating an antibody response that can protect against UTIs. However, adhesin-based vaccines have been shown to be effective at blocking host–pathogen interactions, thus preventing the establishment of disease108–112. Experiments using mouse and cynomolgus monkey models of UTIs determined that immunization with PapD–PapG or FimC–FimH chaperone–adhesin complexes protected against UTIs108–112. The effectiveness of the FimC–FimH vaccine was shown to be due, in large part, to antibodies that block the function of FimH in bladder colonization110. Furthermore, the anti-FimH antibodies did not seem to alter the E. coli niche in the gut microbiota109. Modifications of this vaccine are currently under development, with the aim of inducing greater immune stimulation108,112. For example, one approach has been to fuse FimH to the flagellin FliC in order to induce a more substantial acute inflammatory response, which functions through TLR4 signalling via the MYD88 pathway112. A Phase I clinical trial began in January 2014 to evaluate the efficacy of a FimC–FimH vaccine using a synthetic analogue of monophosphoryl lipid A as the adjuvant.
In addition to the UPEC adhesins, adhesins from P. mirabilis and E. faecalis have also been used as vaccine targets60,113. In a mouse model of UTI, vaccination with the P. mirabilis MR/P pilus adhesin, MrpH, reduced bacterial burdens compared with those of unvaccinated controls, similar to the results observed with UPEC in the FimH vaccine trials110,113. Moreover, a vaccine strategy that is efficacious against E. faecalis CAUTIs is being developed based on vaccination with the Ebp pilus adhesin, EbpA. This strategy induced high antibody titers and reduced bacterial burdens in a mouse model of CAUTI60. In conclusion, adhesin-based vaccines represent a promising area for the development of therapeutics against uropathogens. Thus, understanding the molecular basis of host–pathogen interactions is crucial for vaccine development strategies.
Vaccines targeting bacterial toxins and proteases
The UPEC pore-forming toxin HlyA has also received attention as a potential vaccine target and was evaluated in a mouse model of pyelonephritis to assess protection against renal damage114,115. Vaccination with HlyA reduced the incidence of renal scaring compared with controls; however, it did not protect against UPEC colonization of the kidneys115. In addition, in a mouse model of UTI, vaccination with the P. mirabilis haemolysin, HpmA, did not provide protection against bacterial colonization116. However, vaccination with Pta, an alkaline protease with toxic effects towards epithelial cells, displayed promising results in a mouse model of UTI, protecting against upper UTI, although bacterial burdens in the bladder remained unaffected116. Thus, although haemolysins and proteases might provide effective vaccine targets for preventing upper UTIs, additional studies are needed to determine the effectiveness of these enzymes as targets for vaccines.
Vaccines targeting siderophores
Iron acquisition systems have shown great promise as targets for vaccine development because uropathogens require a source of iron during colonization and persistence. Furthermore, siderophore and haem acquisition systems have been shown to be upregulated during experimental infection, as well as in the urine of women with a UTI86,94,97,98. These parameters sparked vaccine development based on ferric yersiniabactin uptake receptor (FyuA), haem acquisition protein (Hma), iron uptake transport aerobactin receptor (IutA) and the siderophore receptor iron-responsive element A (IreA)98. Vaccination with FyuA and Hma protected mice against pyelonephritis98,117, whereas vaccination with IutA and IreA reduced bladder colonization in mice, confirming the importance of these proteins during infection98,117. Interestingly, the differential tissue-specific protection seen with these four proteins suggests that these systems have different roles or expression profiles in different niches, including the bladder or kidneys.
Vaccinations with other siderophore systems in mouse models of UTI, including the iron receptors FitA and ChuA98, were not protective against infection and were correlated, to a large extent, with lower antigen-specific humoral responses during experimental UTI. These studies suggest that effective siderophore-based vaccines function in part by preventing cognate siderophore uptake, as is the case with FyuA, Hma, IutA and IreA98,117, making this an exciting area of therapeutic development against UTIs.
Small molecules targeting urease
Several urease inhibitors have been developed as potential drugs for UTI treatment, with varying results89. Many of the early inhibitors were active against ureases from several different bacterial species, including Helicobacter pylori, P. mirabilis and S. saprophyticus, and many of these inhibitors showed great promise, as they had low binding and inhibitory concentrations. The best characterized urease inhibitor, acetohydroxamic acid (AHA), even had some success in treating UTIs caused by urease-producing organisms; this inhibitor works by preventing urine alkalization and was approved by the FDA in 1983 (REF. 89). However, many of these inhibitors had severe side effects related to toxicity. For example, AHA resulted in teratogenicity, as well as psychoneurological and musculo-integumentary effects. Subsequent studies showed that derivatives of AHA also had considerable inhibitory properties, but again, these compounds had mutagenic properties that made them undesirable therapeutics89