Formation Of Intracellular Bacterial Communities Research Articles

Detection of intracellular bacterial communities in a child withEscherichia colirecurrent urinary tract infections

The formation of intracellular bacterial communities (IBC) has been proposed as a new pathogenic model for urinary tract infections. Scarce reports describe this phenomenon in humans. We describe the presence of IBC in uroepithelial cells of a child with recurrent urinary infections. Urine specimen was collected from a child with Escherichia coli UTI and analyzed by light and confocal laser scanning microscopy (CLSM). The capability of this strain to produce intracellular infection in bladder tissue was confirmed in mice models. Escherichia coli phylogenetic group, presence of virulence factors genes, and its multiple locus sequence type were determined. CLSM showed large collections of morphologically coccoid and rod bacteria in eukaryotic cells cytoplasm, even seemingly protruding from the cells. Escherichia coli EC7U, ST3626, harbored type 1, P, and S/F1C fimbriae and K1 capsule genes. In this report, we confirm the presence of IBC in children with UTI, as it has been described before in women.

Transposon Mutagenesis Identifies Uropathogenic Escherichia coli Biofilm Factors

Uropathogenic Escherichia coli (UPEC), which accounts for 85% of urinary tract infections (UTI), assembles biofilms in diverse environments, including the host. Besides forming biofilms on biotic surfaces and catheters, UPEC has evolved an intracellular pathogenic cascade that culminates in the formation of biofilm-like intracellular bacterial communities (IBCs) within bladder epithelial cells. Rapid bacterial replication during IBC formation augments a build-up in bacterial numbers and persistence within the host. Relatively little is known about factors mediating UPEC biofilm formation and how these overlap with IBC formation. To address this gap, we screened a UPEC transposon mutant library in three in vitro biofilm conditions: Luria broth (LB)-polyvinyl chloride (PVC), YESCA (yeast extract-Casamino Acids)-PVC, and YESCA-pellicle that are dependent on type 1 pili (LB) and curli (YESCA), respectively. Flagella are important in all three conditions. Mutants were identified that had biofilm defects in all three conditions but had no significant effects on the expression of type 1 pili, curli, or flagella. Thus, this approach uncovered a comprehensive inventory of novel effectors and regulators that are involved in UPEC biofilm formation under multiple conditions. A subset of these mutants was found to be dramatically attenuated and unable to form IBCs in a murine model of UTI. Collectively, this study expands our insights into UPEC multicellular behavior that may provide insights into IBC formation and virulence.

914 IN VIVO ROLE OF RAB27B IN UROTHELIAL ADHESION AND INVASION BY UROPATHOGENIC E.COLI

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Kidney & Bladder1 Apr 2012914 IN VIVO ROLE OF RAB27B IN UROTHELIAL ADHESION AND INVASION BY UROPATHOGENIC E.COLI Duane Hickling, Yan Liu, Victor Nitti, Herbert Lepor, Tung-Tien Sun, Gert Kreibich, and Xue-Ru Wu Duane HicklingDuane Hickling New York, NY More articles by this author , Yan LiuYan Liu New York, NY More articles by this author , Victor NittiVictor Nitti New York, NY More articles by this author , Herbert LeporHerbert Lepor New York, NY More articles by this author , Tung-Tien SunTung-Tien Sun New York, NY More articles by this author , Gert KreibichGert Kreibich New York, NY More articles by this author , and Xue-Ru WuXue-Ru Wu New York, NM More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1010AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Uropathogenic E. coli (UPEC) can adhere to and invade urothelial cells. This process is critical to the formation of intracellular bacterial communities (IBCs) and quiescent intracellular reservoirs (QIRs), potentially important sources of recurrent urinary tract infection (UTI). Uroplakin Ia (UPIa) is the putative urothelial receptor for UPEC binding but the molecular bases underlying invasion remain poorly understood. Rab27b belongs to a family of membrane-associated GTPases that coordinate host cell signal transduction, cytoskeletal organization, and vesicular traffic. Rab27b is highly expressed in the urothelium and Bishop and colleagues recently showed that UPECs exist in urothelial cytoplasmic vesicles positive for Rab27b and UPIa. However, in vivo evidence is still lacking as to what exact roles Rab27b plays in the invasion of UPEC into the urothelial cell. METHODS Ascending UTI experiments were performed using Rab 27b knockout (KO) mice, lacking urothelial Rab27b expression, and wild-type (WT) control mice. 6-9 wk old females were inoculated, transurethrally, with a UPEC cystitis strain UTI89. Mice (5/genotype/time point) were sacrificed at 1, 12, 32 h post-infection and subject to antibody staining and Western blotting to identify and quantify UPEC, uroplakins and extent of apoptosis. RESULTS Rab27b KO mice had significantly fewer UPECs attached to the urothelial surface at 1 h post-inoculation when compared to WT mice. At 12 h post-inoculation, the superficial urothelial cells of WT mice harbored a large number of IBCs whereas Rab27b KO urothelial cells were almost devoid of IBCs. This trend held at 32 h post-inoculation, as there were considerably fewer QIRs in Rab27b KO versus WT mice. Additionally, urothelial cells of the Rab27b KO mice expressed significantly lower level of UPIa and sustained less apoptosis in response to UPEC. CONCLUSIONS Our data provide the first in vivo evidence implicating Rab27b as an important player in UTI pathogenesis. Rab27b affects the expression of urothelial receptors for UPECs and potentially alters UPEC invasion into and/or exit from the urothelium. Because intracellular vesicular trafficking mediated by molecules like Rab27b is affected in various human genetic diseases, it will be of interest to determine if patients with these conditions are more prone or resistant to UTI. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e372-e373 Peer Review Report Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Duane Hickling New York, NY More articles by this author Yan Liu New York, NY More articles by this author Victor Nitti New York, NY More articles by this author Herbert Lepor New York, NY More articles by this author Tung-Tien Sun New York, NY More articles by this author Gert Kreibich New York, NY More articles by this author Xue-Ru Wu New York, NM More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Host–pathogen interactions in urinary tract infection

The urinary tract is a common site of bacterial infections; nearly half of all women experience at least one urinary tract infection (UTI) during their lifetime. These infections are classified based on the condition of the host. Uncomplicated infections affect otherwise healthy individuals and are most commonly caused by uropathogenic Escherichia coli, whereas complicated infections affect patients with underlying difficulties, such as a urinary tract abnormality or catheterization, and are commonly caused by species such as Proteus mirabilis. Virulence and fitness factors produced by both pathogens include fimbriae, toxins, flagella, iron acquisition systems, and proteins that function in immune evasion. Additional factors that contribute to infection include the formation of intracellular bacterial communities by E. coli and the production of urease by P. mirabilis, which can result in urinary stone formation. Innate immune responses are induced or mediated by pattern recognition receptors, antimicrobial peptides, and neutrophils. The adaptive immune response to UTI is less well understood. Host factors TLR4 and CXCR1 are implicated in disease outcome and susceptibility, respectively. Low levels of TLR4 are associated with asymptomatic bacteriuria while low levels of CXCR1 are associated with increased incidence of acute pyelonephritis. Current research is focused on the identification of additional virulence factors and therapeutic or prophylactic targets that might be used in the generation of vaccines against both uropathogens.

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.

Polysaccharide Capsule and Sialic Acid-Mediated Regulation Promote Biofilm-Like Intracellular Bacterial Communities during Cystitis

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4(+) and TLR4(-) mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.

Positive selection identifies an in vivo role for FimH during urinary tract infection in addition to mannose binding

FimH, the type 1 pilus adhesin of uropathogenic Escherichia coli (UPEC), contains a receptor-binding domain with an acidic binding pocket specific for mannose. The fim operon, and thus type 1 pilus production, is under transcriptional control via phase variation of an invertible promoter element. FimH is critical during urinary tract infection for mediating colonization and invasion of the bladder epithelium and establishment of intracellular bacterial communities (IBCs). In silico analysis of FimH gene sequences from 279 E. coli strains identified specific amino acids evolving under positive selection outside of its mannose-binding pocket. Mutating two of these residues (A27V/V163A) had no effect on phase variation, pilus assembly, or mannose binding in vitro. However, compared to wild-type, this double mutant strain exhibited a 10,000-fold reduction in mouse bladder colonization 24 h after inoculation and was unable to form IBCs even though it bound normally to mannosylated receptors in the urothelium. In contrast, the single A62S mutation altered phase variation, reducing the proportion of piliated cells, reduced mannose binding 8-fold, and decreased bladder colonization 30-fold in vivo compared to wild-type. A phase-locked ON A62S mutant restored virulence to wild-type levels even though in vitro mannose binding remained impaired. Thus, positive selection analysis of FimH has separated mannose binding from in vivo fitness, suggesting that IBC formation is critical for successful infection of the mammalian bladder, providing support for more general use of in silico positive selection analysis to define the molecular underpinnings of bacterial pathogenesis.

QseC‐mediated dephosphorylation of QseB is required for expression of genes associated with virulence in uropathogenic Escherichia coli

Bacteria sense environmental cues and regulate gene expression accordingly so as to persist in diverse niches. QseC is a membrane sensor kinase shown in enterohemorrhagic Escherichia coli to respond to host and bacterial signals by phosphorylating the QseB response regulator at residue D51, resulting in QseB activation and presumably upregulation of virulence genes. We studied QseBC in uropathogenic E. coli (UPEC). UPEC establish infection by colonizing and invading bladder cells. After invasion, UPEC can escape into the cytoplasm where they can form intracellular bacterial communities. Deletion of qseC significantly attenuated intracellular bacterial community formation and virulence, whereas paradoxically qseB deletion did not impact pathogenesis. We found that QseB upregulates its own expression in the qseC mutant, arguing that it is activated even in the absence of QseC. However, expression of QseB, but not a QseB_D51A mutant, in the absence of QseC resulted in downregulation of type 1 pili, curli and flagella. We observed similar phenotypes with enterohemorrhagic E. coli, showing that this is not a UPEC-specific phenomenon. Target gene expression is restored when QseC is present. We discovered that QseC has phosphatase activity required for QseB dephosphorylation. Thus, the QseC phosphatase capacity is critical for modulating QseB activity and subsequent gene expression.

Urothelial Cultures Support Intracellular Bacterial Community Formation by UropathogenicEscherichia coli

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.

Functional Genomic Studies of Uropathogenic Escherichia coli and Host Urothelial Cells when Intracellular Bacterial Communities Are Assembled

Uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infection in women, colonizes the gut as well as the genitourinary tract. Studies of mice inoculated with UTI89, a sequenced isolate, have revealed a complex life cycle that includes formation of intracellular bacterial communities (IBCs) in bladder urothelial cells. To understand how UPEC adapts to life in IBCs, we have used GeneChips and/or quantitative reverse transcriptase PCR to study UTI89 recovered from the distal gut of gnotobiotic mice and from IBCs harvested by laser capture microdissection from the bladder urothelium of infected C3H/HeJ female mice. Host responses were characterized in laser capture microdissected urothelial cells that do or do not contain IBCs. The results reveal components of ferric iron acquisition systems in UTI89 that are expressed at significantly higher levels in IBCs compared with the intestine, including the hemin receptor chuA (1,390 +/- 188-fold). Localized urothelial responses to IBCs help oppose bacterial salvage of host cell iron (e.g. up-regulation of Tfrc (transferrin receptor) and Lcn2 (lipocalin 2)), facilitate glucose import (e.g. Hk2 (hexokinase 2)), and maintain epithelial structural integrity (e.g. Ivl (involucrin) and Sbsn (suprabasin)). DeltachuA mutants produce significantly smaller IBCs compared with wild type UTI89. This difference was not observed in strains lacking sitA (ABC-type iron/manganese transporter subunit), iroN (salmochelin receptor), hlyA (alpha-hemolysin), or entF (enterobactin synthetase subunit). Together, these studies indicate that heme- and siderophore-associated iron play key roles in IBC development and provide a series of microbial and host biomarkers for comparing UPEC strains isolated from humans.

Filamentation by Escherichia coli subverts innate defenses during urinary tract infection.

To establish disease, an infecting organism must overcome a vast array of host defenses. During cystitis, uropathogenic Escherichia coli (UPEC) subvert innate defenses by invading superficial umbrella cells and rapidly increasing in numbers to form intracellular bacterial communities (IBCs). In the late stages of the IBC pathway, filamentous and bacillary UPEC detach from the biofilm-like IBC, fluxing out of this safe haven to colonize the surrounding epithelium and initiate subsequent generations of IBCs, and eventually they establish a quiescent intracellular reservoir. Filamentous UPEC are not observed during acute infection in mice lacking functional Toll-like receptor 4 (TLR4), suggesting that the filamentous phenotype arises in response to host innate immunity. We investigated SulA, a cell division inhibitor associated with the SOS response, to gain insight into the role of filamentous UPEC in pathogenesis. A transcriptional reporter from P(sulA) revealed spatial and temporal differences in expression within IBCs, and it was active in the majority of filamentous UPEC. Although UTI89 and UTI89 DeltasulA both formed first-generation IBCs equally well, UTI89 DeltasulA was sharply attenuated in formation of second-generation IBCs and establishment of the quiescent intracellular reservoir. The virulence of UTI89 DeltasulA was restored in TLR4-deficient mice, suggesting that filamentation facilitates the transition to additional rounds of IBC formation by subverting innate immune responses. These findings demonstrate that transient SulA-mediated inhibition of cell division is essential for UPEC virulence in the murine model of cystitis.

Escherichia coli from Urine of Female Patients with Urinary Tract Infections Is Competent for Intracellular Bacterial Community Formation

Nearly 50% of women experience at least one urinary tract infection (UTI) in their lifetime. Studies with mice have revealed that uropathogenic Escherichia coli (UPEC) isolates invade superficial umbrella cells that line the bladder, allowing them to find a safe haven and subvert clearance by innate host responses. Rapid intracellular replication results in the formation of distinctive intracellular bacterial communities (IBCs). In this study, we evaluated whether UPEC strains cultured from the urine of women and classified as causing acute cystitis, recurrent cystitis, asymptomatic bacteriuria, or pyelonephritis could progress through the IBC cascade in a well-characterized mouse model of cystitis. Of 18 UPEC isolates collected from women, 15 formed IBCs. Variations in the size, number, and kinetics of IBC formation were observed with strains isolated from women with different clinical syndromes. Two of the three isolates that did not form IBCs when inoculated alone were able to do so when coinoculated with an isolate that was capable of generating IBCs. The mixed infections dramatically altered the behavior of the coinfecting bacteria relative to their behavior in a single infection. The study also showed that mice with five different genetic backgrounds can support IBC formation. Although UPEC isolates differ genetically in their virulence factors, the majority of UPEC isolates from different types of UTI proceed through the IBC pathway, confirming the generality of IBCs in UTI pathogenesis in mice.

Host Subversion by Formation of Intracellular Bacterial Communities in the Urinary Tract

No AccessJournal of UrologyUrological Survey: Infection and Inflammation of Genitourinary Tract1 Nov 2005Host Subversion by Formation of Intracellular Bacterial Communities in the Urinary Tract Richard E. BergerM.D. Richard E. BergerRichard E. Berger More articles by this author View All Author Informationhttps://doi.org/10.1097/01.ju.0000183182.90538.3aAboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "Host Subversion by Formation of Intracellular Bacterial Communities in the Urinary Tract." The Journal of Urology, 174(5), p. 1843 © 2005 by American Urological Association, Inc.FiguresReferencesRelatedDetailsCited ByAtala A (2018) Re: Selective Depletion of Uropathogenic E. coli from the Gut by a FimH AntagonistJournal of Urology, VOL. 199, NO. 1, (30-31), Online publication date: 1-Jan-2018. Volume 174Issue 5November 2005Page: 1843 Advertisement Copyright & Permissions© 2005 by American Urological Association, Inc.MetricsAuthor Information Richard E. Berger More articles by this author Expand All Advertisement PDF DownloadLoading ...

Uropathogenic Escherichia coli Flagella Aid in Efficient Urinary Tract Colonization

In the murine model of urinary tract infections (UTI), cystitis by uropathogenic Escherichia coli (UPEC) occurs through an intimate relationship with the bladder superficial umbrella cell entailing cycles of adherence, invasion, intracellular bacterial community (IBC) formation, and dispersal (fluxing) from the intracellular environment. IBC dispersal is a key step that results in the spread of bacteria over the epithelial surface to initiate additional rounds of IBC formation. We investigated the role of flagella in mediating adherence and motility during UTI, hypothesizing that the dispersion of the IBC would be incomplete in the absence of motility, thus interrupting the IBC pathway and attenuating the infection. Using gfp reporter fusions, the expression of the flagellar class I flhDC and class III fliC genes was monitored to track key points of regulation throughout the pathogenic cascade. In vitro, growth under conditions promoting motility resulted in the robust expression of both fusions. In contrast, only the class I fusion produced significant expression throughout early stages of IBC development including the dispersion stage. Thus, unlike in vitro modeling of motility, the regulatory cascade appeared incomplete in vivo. Throughout IBC formation, nonmotile DeltafliC mutants achieved the same number of IBCs as the wild-type (wt) strain, demonstrating that flagella are neither essential nor required for first- or second-generation IBC formation. However, in competition experiments between wt and DeltafliC strains, the wt was shown to have a fitness advantage in persisting throughout the urinary tract for 2 weeks, demonstrating a subtle but measurable role for flagella in virulence.

Host subversion by formation of intracellular bacterial communities in the urinary tract

Urinary tract infections pose a serious health threat with respect to antibiotic resistance and high recurrence rates. While the host robustly responds to bacterial infiltration into the bladder, uropathogenic Escherichia coli can survive the onslaught to persist for months after initially infecting. To accomplish this feat, uropathogenic E. coli forms intracellular bacterial communities, with many biofilm-like properties, within the bladder epithelium. These communities may allow bacteria to subvert host defenses and form a persistent reservoir in the bladder.