Urothelial Cultures Support Intracellular Bacterial Community Formation by Uropathogenic Escherichia coli

ABSTRACT Uropathogenic Escherichia coli (UPEC) causes most community-acquired and nosocomial urinary tract infections (UTI). In a mouse model of UTI, UPEC invades superficial bladder cells and proliferates rapidly, forming biofilm-like structures called intracellular bacterial communities (IBCs). Using a gentamicin protection assay and fluorescence microscopy, we developed an in vitro model for studying UPEC proliferation within immortalized human urothelial cells. By pharmacologic manipulation of urothelial cells with the cholesterol-sequestering drug filipin, numbers of intracellular UPEC CFU increased 8 h and 24 h postinfection relative to untreated cultures. Enhanced UPEC intracellular proliferation required that the urothelial cells, but not the bacteria, be filipin treated prior to infection. However, neither UPEC frequency of invasion nor early intracellular trafficking events to a Lamp1-positive compartment were modulated by filipin. Upon inspection by fluorescence microscopy, cultures with enhanced UPEC intracellular proliferation exhibited large, dense bacterial aggregates within cells that resembled IBCs but were contained with Lamp1-positive vacuoles. While an isogenic fimH mutant was capable of forming these IBC-like structures, the mutant formed significantly fewer than wild-type UPEC. Similar to IBCs, expression of E. coli iron acquisition systems was upregulated by intracellular UPEC. Expression of other putative virulence factors, including hlyA, cnf1, fliC, kpsD, and the biofilm adhesin yfaL also increased, while expression of fimA decreased and that of flu did not change. These results indicate that UPEC differentially regulates virulence factors in the intracellular environment. Thus, immortalized urothelial cultures that recapitulate IBC formation in vitro represent a novel system for the molecular and biochemical characterization of the UPEC intracellular life cycle.

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A Conserved PapB Family Member, TosR, Regulates Expression of the Uropathogenic Escherichia coli RTX Nonfimbrial Adhesin TosA while Conserved LuxR Family Members TosE and TosF Suppress Motility

Infection and Immunity ◽

10.1128/iai.01608-14 ◽

2014 ◽

Vol 82 (9) ◽

pp. 3644-3656 ◽

Cited By ~ 6

Author(s):

Michael D. Engstrom ◽

Christopher J. Alteri ◽

Harry L. T. Mobley

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Virulence Factors ◽

Upper Urinary Tract ◽

Host Cells ◽

Promoter Regions ◽

Content Type ◽

Tract Infections

ABSTRACTA heterogeneous subset of extraintestinal pathogenicEscherichia coli(ExPEC) strains, referred to as uropathogenicE. coli(UPEC), causes most uncomplicated urinary tract infections. However, no core set of virulence factors exists among UPEC strains. Instead, the focus of the analysis of urovirulence has shifted to studying broad classes of virulence factors and the interactions between them. For example, the RTX nonfimbrial adhesin TosA mediates adherence to host cells derived from the upper urinary tract. The associatedtosoperon is well expressedin vivobut poorly expressedin vitroand encodes TosCBD, a predicted type 1 secretion system. TosR and TosEF are PapB and LuxR family transcription factors, respectively; however, no role has been assigned to these potential regulators. Thus, the focus of this study was to determine how TosR and TosEF regulatetosAand affect the reciprocal expression of adhesins and flagella. Among a collection of sequenced UPEC strains, 32% (101/317) were found to encode TosA, and nearly all strains (91% [92/101]) simultaneously carried the putative regulatory genes. Deletion oftosRalleviatestosArepression. Thetospromoter was localized upstream oftosRusing transcriptional fusions of putative promoter regions withlacZ. TosR binds to this region, affecting a gel shift. A 100-bp fragment 220 to 319 bp upstream oftosRinhibits binding, suggesting localization of the TosR binding site. TosEF, on the other hand, downmodulate motility when overexpressed by preventing the expression offliC, encoding flagellin. Deletion oftosEFincreased motility. Thus, we present an additional example of the reciprocal control of adherence and motility.

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The Salmochelin Siderophore Receptor IroN Contributes to Invasion of Urothelial Cells by Extraintestinal Pathogenic Escherichia coli In Vitro

Infection and Immunity ◽

10.1128/iai.00656-06 ◽

2007 ◽

Vol 75 (6) ◽

pp. 3183-3187 ◽

Cited By ~ 35

Author(s):

Friederike Feldmann ◽

Liisa Johanna Sorsa ◽

Kirsten Hildinger ◽

Sören Schubert

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Iron Uptake ◽

Dual Role ◽

Urothelial Cells ◽

Pathogenic Escherichia Coli ◽

Tract Infections

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) strains possess several siderophore-dependent iron uptake systems. In this study we demonstrated that the salmochelin siderophore receptor IroN is involved in the invasion of urothelial cells by ExPEC in vitro. Thus, IroN may play a dual role in the establishment of urinary tract infections, displaying an iron uptake receptor as well as an internalization factor.

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A bacterial "shield and sword": A previously uncharacterized two-component system protects uropathogenic Escherichia coli from host-derived oxidative insults and promotes hemolysin-mediated host cell pyroptosis

10.1101/2021.04.19.440293 ◽

2021 ◽

Author(s):

Hongwei Gu ◽

Xuwang Cai ◽

Xinyang Zhang ◽

Jie Luo ◽

Xiaoyang Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Virulence Factors ◽

Bacterial Pathogen ◽

Methionine Sulfoxide ◽

Uropathogenic Escherichia Coli ◽

Methionine Sulfoxide Reductase ◽

Renal Epithelial Cell ◽

Two Component ◽

Tract Infections

Uropathogenic Escherichia coli (UPEC) deploys an array of virulence factors to successfully establish urinary tract infections. Coordinated expression of these various virulence factors is critical for UPEC's overall fitness in the host. Two-component signaling systems (TCSs) are a major mechanism by which bacteria sense environmental cues and initiate adaptive responses. Here, we report a previously uncharacterized TCS encoded on a pathogenicity island in UPEC that directly activates the expression of a putative methionine sulfoxide reductase system (C3566/C3567) and a pore-forming hemolysin in response to host-derived hydrogen peroxide (H2O2) exposure. The TCS increases UPEC resistance to H2O2 in vitro and survival in macrophages in tissue culture via C3566/C3567. Additionally, the TCS mediates hemolysin-induced renal epithelial cell and macrophage death via a pyroptosis pathway. Taken together, our data suggest a paradigm in which this signal transduction system coordinates both bacterial pathogen defensive and offensive traits in the presence of host-derived signals.

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Identification ofIn Vivo-Induced Antigens Including an RTX Family Exoprotein Required for Uropathogenic Escherichia coli Virulence

Infection and Immunity ◽

10.1128/iai.00110-11 ◽

2011 ◽

Vol 79 (6) ◽

pp. 2335-2344 ◽

Cited By ~ 35

Author(s):

Patrick D. Vigil ◽

Christopher J. Alteri ◽

Harry L. T. Mobley

Keyword(s):

Escherichia Coli ◽

Iron Acquisition ◽

Expression Library ◽

Upec Strain ◽

Infected Female ◽

Content Type ◽

Strain Cft073 ◽

Tract Infections

ABSTRACTUncomplicated urinary tract infections (UTI) are caused most commonly by uropathogenicEscherichia coli(UPEC). Whole-genome screening approaches, including transcriptomic, proteomic, and signature-tagged mutagenesis, have shown that UPEC highly expresses or requires genes for translational machinery, capsule, lipopolysaccharide, type 1 fimbriae, and iron acquisition systems during UTI. To identify additional genes expressed by UPEC during UTI, an immunoscreening approach termedin vivo-induced antigen technology (IVIAT) was employed to identify antigens produced during experimental infection that are not produced duringin vitroculture. An inducible protein expression library, constructed from genomic DNA isolated from UPEC strain CFT073, was screened using exhaustively adsorbed pooled sera from 20 chronically infected female CBA/J mice. Using this approach, we identified 93 antigens induced by UPECin vivo. A representative subset of these genes was tested by quantitative PCR for expression by CFT073in vivoand during growth in human urine or LB mediumin vitro;proWX,narJI,lolA,lolD,tosA(upxA), c2432,katG,ydhX,kpsS, andyddQwere poorly expressedin vitrobut highly expressedin vivo. Of these,tosA, a gene encoding a predicted repeat-in-toxin family member, was expressed exclusively during UTI. Deletion oftosAin UPEC strain CFT073 resulted in significant attenuation in bladder and kidney infections during ascending UTI. By screening forin vivo-induced antigens, we identified a novel UPEC virulence factor and additional proteins that could be useful as potential vaccine targets.

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Quinolones Induce Partial or Total Loss of Pathogenicity Islands in Uropathogenic Escherichia coli by SOS-Dependent or -Independent Pathways, Respectively

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.2.649-653.2006 ◽

2006 ◽

Vol 50 (2) ◽

pp. 649-653 ◽

Cited By ~ 60

Author(s):

S. M. Soto ◽

M. T. Jimenez de Anta ◽

J. Vila

Keyword(s):

Escherichia Coli ◽

Virulence Factors ◽

Reca Protein ◽

Total Loss ◽

Pathogenicity Islands ◽

E Coli ◽

Cytotoxic Necrotizing Factor ◽

Tract Infections ◽

Cytotoxic Necrotizing Factor 1

ABSTRACT Escherichia coli is the most common microorganism causing urinary tract infections. Quinolone-resistant E. coli strains have fewer virulence factors than quinolone-susceptible strains. Several urovirulence genes are located in pathogenicity islands (PAIs). We investigated the capacity of quinolones to induce loss of virulence factors such as hemolysin, cytotoxic necrotizing factor 1, P fimbriae, and autotransporter Sat included in PAIs in three uropathogenic E. coli strains. In a multistep selection, all strains lost hemolytic capacity at between 1 and 4 passages when they were incubated with subinhibitory concentrations of ciprofloxacin, showing a partial or total loss of the PAI containing the hly (hemolysin) and cnf-1 (cytotoxic necrotizing factor 1) genes. RecA− mutants were obtained from the two E. coli strains with partial or total loss of the PAI. The inactivation of the RecA protein affected only the partial loss of the PAI induced by quinolones. No spontaneous loss of PAIs was observed on incubation in the absence of quinolones in either the wild-type or mutant E. coli strains. Quinolones induce partial or total loss of PAIs in vitro in uropathogenic E. coli by SOS-dependent or -independent pathways, respectively.

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Escherichia coli virulence factors iutA, an iron acquisition factor, and ibeA, an invasion factor, are related to severity of bacteremic acute biliary tract infections

10.21203/rs.3.rs-73526/v1 ◽

2020 ◽

Author(s):

Mahoko Ikeda ◽

Tatsuya Kobayashi ◽

Fumie Fujimoto ◽

Yuta Okada ◽

Yoshimi Higurashi ◽

...

Keyword(s):

Escherichia Coli ◽

Tract Infection ◽

Virulence Factors ◽

Biliary Tract ◽

Iron Acquisition ◽

Care Hospital ◽

Biliary Tract Infection ◽

E Coli ◽

Severe Group ◽

Tract Infections

Abstract BackgroundAlthough Escherichia coli is the most frequently isolated organism in acute biliary tract infections with bacteremia, data regarding its virulence are limited.ResultsBacteremic acute biliary tract infection cases in a retrospective study were collected from 2013 to 2015 at a tertiary care hospital in Japan. Factors related to the severity of infection were investigated, including patient background, phylogenetic typing, and virulence factors of E. coli, such as adhesion, invasion, toxins, and iron acquisition. In total, 72 E. coli strains were identified in 71 cases, most of which primarily belonged to the phylogenetic B2 group (68%). The presence of IutA (77.3% in the non-severe group, 46.4% in the severe group, P = 0.011) and ibeA (9.1% in the non-severe group, and 35.7% in the severe group, P = 0.012) were significantly associated with severity of infection, whereas patient characteristics did not relate to the severity of infection.ConclusionsWe showed that bacteremic E. coli strains from acute biliary tract infections belonged to a virulent (B2) group. The severity of biliary tract infection depended on iutA and ibeA.

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Iron Acquisition of Urinary Tract Infection Escherichia coli Involves Pathogenicity in Caenorhabditis elegans

Microorganisms ◽

10.3390/microorganisms9020310 ◽

2021 ◽

Vol 9 (2) ◽

pp. 310

Author(s):

Masayuki Hashimoto ◽

Yi-Fen Ma ◽

Sin-Tian Wang ◽

Chang-Shi Chen ◽

Ching-Hao Teng

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Caenorhabditis Elegans ◽

Large Scale ◽

Iron Acquisition ◽

Infection Model ◽

High Throughput Analysis ◽

E Coli ◽

C Elegans ◽

Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major bacterial pathogen that causes urinary tract infections (UTIs). The mouse is an available UTI model for studying the pathogenicity; however, Caenorhabditis elegans represents as an alternative surrogate host with the capacity for high-throughput analysis. Then, we established a simple assay for a UPEC infection model with C. elegans for large-scale screening. A total of 133 clinically isolated E. coli strains, which included UTI-associated and fecal isolates, were applied to demonstrate the simple pathogenicity assay. From the screening, several virulence factors (VFs) involved with iron acquisition (chuA, fyuA, and irp2) were significantly associated with high pathogenicity. We then evaluated whether the VFs in UPEC were involved in the pathogenicity. Mutants of E. coli UTI89 with defective iron acquisition systems were applied to a solid killing assay with C. elegans. As a result, the survival rate of C. elegans fed with the mutants significantly increased compared to when fed with the parent strain. The results demonstrated, the simple assay with C. elegans was useful as a UPEC infectious model. To our knowledge, this is the first report of the involvement of iron acquisition in the pathogenicity of UPEC in a C. elegans model.

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The prevalence of the iutA and ibeA genes in Escherichia coli isolates from severe and non-severe patients with bacteremic acute biliary tract infection is significantly different

Gut Pathogens ◽

10.1186/s13099-021-00429-1 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Mahoko Ikeda ◽

Tatsuya Kobayashi ◽

Fumie Fujimoto ◽

Yuta Okada ◽

Yoshimi Higurashi ◽

...

Keyword(s):

Escherichia Coli ◽

Tract Infection ◽

Biliary Tract ◽

Iron Acquisition ◽

Tertiary Care ◽

Care Hospital ◽

Biliary Tract Infection ◽

E Coli ◽

Severe Group ◽

Tract Infections

Abstract Background Although Escherichia coli is the most frequently isolated microorganism in acute biliary tract infections with bacteremia, data regarding its virulence are limited. Results Information on cases of bacteremia in acute biliary tract infection in a retrospective study was collected from 2013 to 2015 at a tertiary care hospital in Japan. Factors related to the severity of infection were investigated, including patient background, phylogenetic typing, and virulence factors of E. coli, such as adhesion, invasion, toxins, and iron acquisition. In total, 72 E. coli strains were identified in 71 cases, most of which primarily belonged to the B2 phylogroup (68.1%). The presence of the iutA gene (77.3% in the non-severe group, 46.4% in the severe group, P = 0.011) and the ibeA gene (9.1% in the non-severe group, and 35.7% in the severe group, P = 0.012) was significantly associated with the severity of infection. Among the patient characteristics, diabetes mellitus with organ involvement and alkaline phosphatase were different in the severe and non-severe groups. Conclusions We showed that bacteremic E. coli strains from acute biliary tract infections belonged to the virulent (B2) phylogroup. The prevalence of the iutA and ibeA genes between the two groups of bacteremia severity was significantly different.

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New Aspects of RpoE in Uropathogenic Proteus mirabilis

Infection and Immunity ◽

10.1128/iai.02232-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 966-977 ◽

Cited By ~ 10

Author(s):

Ming-Che Liu ◽

Kuan-Ting Kuo ◽

Hsiung-Fei Chien ◽

Yi-Lin Tsai ◽

Shwu-Jen Liaw

Keyword(s):

Urinary Tract ◽

Virulence Factors ◽

Stress Responses ◽

Proteus Mirabilis ◽

Immune Cell ◽

Polymyxin B ◽

Urothelial Cells ◽

Cationic Antimicrobial Peptide ◽

Content Type ◽

Tract Infections

Proteus mirabilisis a common human pathogen causing recurrent or persistent urinary tract infections (UTIs). The underlying mechanisms forP. mirabilisto establish UTIs are not fully elucidated. In this study, we showed that loss of the sigma factor E (RpoE), mediating extracytoplasmic stress responses, decreased fimbria expression, survival in macrophages, cell invasion, and colonization in mice but increased the interleukin-8 (IL-8) expression of urothelial cells and swarming motility. This is the first study to demonstrate that RpoE modulated expression of MR/P fimbriae by regulatingmrpI, a gene encoding a recombinase controlling the orientation of MR/P fimbria promoter. By real-time reverse transcription-PCR, we found that the IL-8 mRNA amount of urothelial cells was induced significantly by lipopolysaccharides extracted fromrpoEmutant but not from the wild type. These RpoE-associated virulence factors should be coordinately expressed to enhance the fitness ofP. mirabilisin the host, including the avoidance of immune attacks. Accordingly,rpoEmutant-infected mice displayed more immune cell infiltration in bladders and kidneys during early stages of infection, and therpoEmutant had a dramatically impaired ability of colonization. Moreover, it is noteworthy that urea (the major component in urine) and polymyxin B (a cationic antimicrobial peptide) can induce expression ofrpoEby the reporter assay, suggesting that RpoE might be activated in the urinary tract. Altogether, our results indicate that RpoE is important in sensing environmental cues of the urinary tract and subsequently triggering the expression of virulence factors, which are associated with the fitness ofP. mirabilis, to build up a UTI.

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Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2343-2351.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2343-2351 ◽

Cited By ~ 91

Author(s):

Patricia Komp Lindgren ◽

Linda L. Marcusson ◽

Dorthe Sandvang ◽

Niels Frimodt-Møller ◽

Diarmaid Hughes

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Infection Model ◽

Resistance Mutations ◽

Topoisomerase Iv ◽

E Coli ◽

Tract Infections ◽

Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

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