Uropathogenic Escherichia Coli Pathogenesis Research Articles

Oral supplementation of trans-cinnamaldehyde reduces uropathogenic Escherichia coli colonization in a mouse model.

In this study, we have demonstrated that oral supplementation of trans-cinnamaldehyde (TC) reduced uropathogenic Escherichia coli (UPEC)-associated lower urinary tract infection (UTI) in mice. Specifically, in-feed supplementation of TC significantly decreased UPEC populations in the urethra and bladder, thereby reducing the infectious load. These findings are particularly significant given the increase in incidence and prevalence of antibiotic-resistant UTIs. Our study offers new insights into the potential use of natural antimicrobials including TC, the active ingredient in cinnamon, as a nonantibiotic-based natural dietary intervention in the prophylaxis of lower UTIs.

Purine Biosynthesis Metabolically Constrains Intracellular Survival of Uropathogenic Escherichia coli.

The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement.

Genotypic Characterization of Virulence Factors in Escherichia coli Isolated from Patients with Acute Cystitis, Pyelonephritis and Asymptomatic Bacteriuria.

Urinary Tract Infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most common infections worldwide. It is well-documented that the pathogenesis of UPEC is mediated by the production of a wide variety of Virulence Factors (VFs). Thus, detection of these VFs and evaluation of their association with different clinical types of UTIs could help to understand the role of these factors in pathogenesis of UPEC isolates. To investigate the genotypic characteristics of UPEC isolates and to examine the relationship between VFs and different clinical symptoms of UTI. In this cross-sectional study conducted at Pasteur Institute of Iran, a total of 156 UPEC isolated from outpatients and inpatients (symptomatic and asymptomatic UTI patients) visiting general and private hospitals in Tehran, Iran between March 2014 and February 2015 were included. Among them, 49 patients experienced at least one episode of recurrent UTI. A Polymerase Chain Reaction (PCR) assay was developed to detect the presence of different VFs in the isolates. Moreover, Pulsed-Field Gel Electrophoresis (PFGE) was used to characterize clonal relationships among UPEC isolates. The prevalence of virulence genes ranged from 0% for cdtB to 100% for fimH. The papEF, hlyA and aer genes were found to be significantly more frequent in UPEC isolated from patients with pyelonephritis, while the afa gene, the only indicator of recurrent UTIs, was more prevalent in UPEC isolated from patients with cystitis. In the present study, 34 PFGE clonal groups were found in the UPEC genome. Our findings showed that from outpatients and patients with pyelonephritis, isolates were more virulent than those isolated from inpatients and cystitis patients, respectively. PFGE displayed a large diversity in the UPEC isolates that could be considered as an evolutionary strategy in the survival of the bacteria.

The yin-yang driving urinary tract infection and how proteomics can enhance research, diagnostics, and treatment.

Bacterial urinary tract infections (UTIs) afflict millions of people worldwide both in the community and the hospital setting. The onset, duration, and severity of infection depend on the characteristics of the invading pathogen (yin), as well as the immune response elicited by the infected individual (yang). Uropathogenic Escherichia coli (UPEC) account for the majority of UTIs, and extensive investigations by many scientific groups have elucidated an elaborate pathogenic UPEC life cycle, involving the occupation of extracellular and intracellular niches and the expression of an arsenal of virulence factors that facilitate niche occupation. This review will summarize the current knowledge on UPEC pathogenesis; the host immune responses elicited to combat infection; and it will describe proteomics approaches used to understand UPEC pathogenesis, as well as drive diagnostics and treatment options. Finally, new strategies are highlighted that could be applied toward furthering our knowledge regarding host-bacterial interactions during UTI.

Role of uropathogenic Escherichia coli outer membrane protein T in pathogenesis of urinary tract infection.

OmpT is one of the members of the outer membrane protein family that has been identified as a virulence factor in most of the uropathogenic Escherichia coli (UPEC). However, the exact role of OmpT in the urinary tract infections (UTIs) remains unclear. To determine the role of OmpT in the pathogenesis of UPEC, an isogenic deletion mutant of ompT (COTD) was constructed by the λ Red recombination. Human bladder epithelial cell line 5637(HBEC 5637) was used to evaluate the ability of bacterial adhesion/invasion. A murine model of UTI was established to study the formation of intracellular bacterial communities (IBCs) in the process of UTIs. The cytokines were also examined during the pathogenesis. The results showed that the COTD strain was deficient in bacterial adhesion and invasion as well as in IBC formation compare to the parent strain. ELISA quantification analysis of cytokines showed that the levels of TNF-α, IL-6 and IL-8 in the serum, bladder and kidney tissues of the mice infected with COTD were lower than that of the CFT073 group. In summary, these results suggest that OmpT plays a multifaceted role in pathogenesis of UTI, including increased bacterial adhesiveness/invasiveness, formation of IBCs and upregulated proinflammatory cytokines.

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5. A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria. CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS). Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity. A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria. CP/CPPS is primarily characterized by the presence of tactile allodynia. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Vaccination against urinary tract infections caused by <i>E. coli</i>

Uropathogenic Escherichia coli (UPEC) causes uncomplicated urinary tract infection (UTI) depicts a prevalent and potentially uncompromising infectious disease. In this analysis, we explained the functions of an immunoproteomics concept to vaccine development that has been successfully employed to recognize vaccine targets in other pathogenic bacteria. Pyelonephritis strains E. coli CFT073 are used for outer membrane isolation mimics urinary tract environment in which iron limitation, osmotic stress, human urine, and exposure to uroepithelial cells are included. During experiments of UTI, the antigens that induce the humoral immune response is to identified, two-dimensional gel electrophoresis are employed for the isolation of outer membrane protein and probed using pooled antisera from 20 CBA/J mice chronically infected with E. coli CFT073. 23 total outer membrane antigens, in which a unique iron compound receptor is included, are reacted with antisera and were identified by mass spectrometry. These antigens comprises of proteins with known functions in UPEC pathogenesis such as, ChuA, IroN, IreA, Iha, IutA, and FliC. These all information and data elaborate that these factors are associated with virulence during UTI are directed by antibody response. We also represents that the genes encoding ChuA, IroN, hypothetical protein c2482, and IutA are significantly more prevalent among UPEC strains than among fecal-commensal E. coli isolates. Therefore we concluded that, the outer membrane antigens are identified in this study are conserved, could be reflective part for the UTI vaccine generated to induce protective immunity against UPEC infections.

Combinatorial Small-Molecule Therapy Prevents Uropathogenic Escherichia coli Catheter-Associated Urinary Tract Infections in Mice

Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial urinary tract infections (UTIs) and pose significant clinical challenges. These infections are polymicrobial in nature and are often associated with multidrug-resistant pathogens, including uropathogenic Escherichia coli (UPEC). Urinary catheterization elicits major histological and immunological alterations in the bladder that can favor microbial colonization and dissemination in the urinary tract. We report that these biological perturbations impact UPEC pathogenesis and that bacterial reservoirs established during a previous UPEC infection, in which bacteriuria had resolved, can serve as a nidus for subsequent urinary catheter colonization. Mannosides, small molecule inhibitors of the type 1 pilus adhesin, FimH, provided significant protection against UPEC CAUTI by preventing bacterial invasion and shifting the UPEC niche primarily to the extracellular milieu and on the foreign body. By doing so, mannosides potentiated the action of trimethoprim-sulfamethoxazole in the prevention and treatment of CAUTI. In this study, we provide novel insights into UPEC pathogenesis in the context of urinary catheterization, and demonstrate the efficacy of novel therapies that target critical mechanisms for this infection. Thus, we establish a proof-of-principle for the development of mannosides to prevent and eventually treat these infections in the face of rising antibiotic-resistant uropathogens.

Involvement of mismatch repair in the reciprocal control of motility and adherence of uropathogenic Escherichia coli.

Type 1 fimbriae and flagella, two surface organelles critical for colonization of the urinary tract by uropathogenic Escherichia coli (UPEC), mediate opposing virulence objectives. Type 1 fimbriae facilitate adhesion to mucosal cells and promote bacterial persistence in the urinary tract, while flagella propel bacteria through urine and along mucous layers during ascension to the upper urinary tract. Using a transposon screen of the E. coli CFT073 fim locked-ON (L-ON) mutant, a construct that constitutively expresses type 1 fimbriae and represses motility, we identified six mutants that exhibited a partial restoration of motility. Among these six mutated genes was mutS, which encodes a component of the methyl-directed mismatch repair (MMR) system. When complemented with mutS in trans, motility was again repressed. To determine whether the MMR system, in general, is involved in this reciprocal control, we characterized the effects of gene deletions of other MMR components on UPEC motility. Isogenic deletions of mutS, mutH, and mutL were constructed in both wild-type CFT073 and fim L-ON backgrounds. All MMR mutants showed an increase in motility in the wild-type background, and ΔmutH and ΔmutS mutations increased motility in the fim L-ON background. Cochallenge of the wild-type strain with an MMR-defective strain showed a subtle but significant competitive advantage in the bladder and spleen for the MMR mutant using the murine model of ascending urinary tract infection after 48 h. Our findings demonstrate that the MMR system generally affects the reciprocal regulation of motility and adherence and thus could contribute to UPEC pathogenesis during urinary tract infections.

Regulation offimgenes in uropathogenicEscherichia coli

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections in women, causing significant morbidity and mortality in this population. Adherence to host epithelial cells is a pivotal step in the pathogenesis of UPEC. One of the most important virulence factors involved in mediating this attachment is the type 1 pilus (type 1 fimbria) encoded by a set of fim genes arranged in an operon. The expression of type 1 pili is controlled by a phenomenon known as phase variation, which reversibly switches between the expression of type 1 pili (Phase-ON) and loss of expression (Phase-OFF). Phase-ON cells have the promoter for the fimA structural gene on an invertible DNA element called fimS, which lines up to allow transcription, whereas transcription of the structural gene is silenced in Phase-OFF cells. The orientation of the fimS invertible element is controlled by two site-specific recombinases, FimB and FimE. Environmental conditions cause transcriptional and post-transcriptional changes in UPEC cells that affect the level of regulatory proteins, which in turn play vital roles in modulating this phase switching ability. The role of fim gene regulation in UPEC pathogenesis will be discussed.

Virulence Plasmid Harbored by UropathogenicEscherichia coliFunctions in Acute Stages of Pathogenesis

Urinary tract infections (UTIs), the majority of which are caused by uropathogenic Escherichia coli (UPEC), afflict nearly 60% of women within their lifetimes. Studies in mice and humans have revealed that UPEC strains undergo a complex pathogenesis cycle that involves both the formation of intracellular bacterial communities (IBC) and the colonization of extracellular niches. Despite the commonality of the UPEC pathogenesis cycle, no specific urovirulence genetic profile has been determined; this is likely due to the fluid nature of the UPEC genome as the result of horizontal gene transfer and numerous genes of unknown function. UTI89 has a large extrachromosomal element termed pUTI89 with many characteristics of UPEC pathogenicity islands and that likely arose due to horizontal gene transfer. The pUTI89 plasmid has characteristics of both F plasmids and other known virulence plasmids. We sought to determine whether pUTI89 is important for virulence. Both in vitro and in vivo assays were used to examine the function of pUTI89 using plasmid-cured UTI89. No differences were observed between UTI89 and plasmid-cured UTI89 based on growth, type 1 pilus expression, or biofilm formation. However, in a mouse model of UTI, a significant decrease in bacterial invasion, CFU and IBC formation of the pUTI89-cured strain was observed at early time points postinfection compared to the wild type. Through directed deletions of specific operons on pUTI89, the cjr operon was partially implicated in this observed defect. Our findings implicate pUTI89 in the early aspects of infection.

LeuX tRNA‐dependent and ‐independent mechanisms of Escherichia coli pathogenesis in acute cystitis

Uropathogenic Escherichia coli (UPEC) contain multiple horizontally acquired pathogenicity-associated islands (PAI) implicated in the pathogenesis of urinary tract infection. In a murine model of cystitis, type 1 pili-mediated bladder epithelial invasion and intracellular proliferation are key events associated with UPEC virulence. In this study, we examined the mechanisms by which a conserved PAI contributes to UPEC pathogenesis in acute cystitis. In the human UPEC strain UTI89, spontaneous excision of PAI II(UTI89) disrupts the adjacent leuX tRNA locus. Loss of wild-type leuX-encoded tRNA(5)(Leu) significantly delayed, but did not eliminate, FimB recombinase-mediated phase variation of type 1 pili. FimX, an additional FimB-like, leuX-independent recombinase, was also found to mediate type 1 pili phase variation. However, whereas FimX activity is relatively slow in vitro, it is rapid in vivo as a non-piliated strain lacking the other fim recombinases rapidly expressed type 1 pili upon experimental infection. Finally, we found that disruption of leuX, but not loss of PAI II(UTI89) genes, reduced bladder epithelial invasion and intracellular proliferation, independent of type 1 piliation. These findings indicate that the predominant mechanism for preservation of PAI II(UTI89) during the establishment of acute cystitis is maintenance of wild-type leuX, and not PAI II(UTI89) gene content.

Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney

P fimbria, a mannose-resistant adhesin of uropathogenic Escherichia coli (UPEC), has been shown to be associated with acute pyelonephritis. The pap gene cluster encodes the proteins required for P-fimbrial biogenesis, including papG, which encodes the tip adhesin. The three most studied PapG molecular variants, which are shown to bind distinct isoreceptors, are PapGI, -II, and -III. PapGII preferentially binds globoside, or GbO4, a glycolipid isoreceptor of the human kidney. Studies using different animal models of ascending urinary tract infection (UTI) have demonstrated a variable role for P fimbriae, and specifically PapGII-mediated adherence, in renal colonization. The disparities in the results obtained from those studies are likely to be attributed to the differences in animal models and UPEC strains utilized. One explanation that is discussed in detail is the contribution of multiple fimbriae of UPEC that potentially mediate adherence to the mammalian kidney. Overall, P fimbriae appear to play some role in mediating adherence to uroepithelial cells in vivo and establishing an inflammatory response during renal colonization, thus contributing to kidney damage during acute pyelonephritis. To verify that P fimbriae contribute to the pathogenesis of UPEC during ascending UTI (and in particular acute pyelonephritis), future studies should be conducted to satisfy fully all three tenets of the molecular Koch's postulates, including complementation of a mutated allele.

Uropathogenic Escherichia coli Outer Membrane Antigens Expressed during Urinary Tract Infection

Uncomplicated urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) represents a prevalent and potentially severe infectious disease. In this study, we describe the application of an immunoproteomics approach to vaccine development that has been used successfully to identify vaccine targets in other pathogenic bacteria. Outer membranes were isolated from pyelonephritis strain E. coli CFT073 cultured under conditions that mimic the urinary tract environment, including iron limitation, osmotic stress, human urine, and exposure to uroepithelial cells. To identify antigens that elicit a humoral response during experimental UTI, outer membrane proteins were separated by two-dimensional gel electrophoresis and probed using pooled antisera from 20 CBA/J mice chronically infected with E. coli CFT073. In total, 23 outer membrane antigens, including a novel iron compound receptor, reacted with the antisera and were identified by mass spectrometry. These antigens also included proteins with known roles in UPEC pathogenesis, such as ChuA, IroN, IreA, Iha, IutA, and FliC. These data demonstrate that an antibody response is directed against these virulence-associated factors during UTI. We also show that the genes encoding ChuA, IroN, hypothetical protein c2482, and IutA are significantly more prevalent (P < 0.01) among UPEC strains than among fecal-commensal E. coli isolates. Thus, we suggest that the conserved outer membrane antigens identified in this study could be rational candidates for a UTI vaccine designed to elicit protective immunity against UPEC infection.

The solution structure of PapGII from uropathogenic Escherichia coli and its recognition of glycolipid receptors.

Uropathogenic Escherichia coli (UPEC) is the primary cause of symptomatic urinary tract infection. The P-pili, a bacterial surface organelle, mediates the bacterial host--cell adhesion. The PapG adhesin has generated much interest in recent years, not only because of its clinical value, i.e. in the prevention of microbial adherence, but also because of its ability to promote virulence. Using multidimensional nuclear magnetic resonance (NMR) and deuteration we have determined the solution structure of the adhesin domain from PapGII (PapGII-198). The novel structure of PapGII-198 is composed of a large elongated jellyroll motif. Despite an automated search of the structural database failing to reveal any similar proteins, PapGII adhesin shares some structural similarities with FimH. Furthermore, interpretation of NMR-titration data has enabled us to identify the putative binding site for the globoseries of oligosaccharides. This work provides insight into UPEC pathogenesis as well as aiding the development of preventative therapies and the guidance of future mutagenesis programmes.