Formation Of Intracellular Bacterial Communities Research Articles

Nur77 protects the bladder urothelium from intracellular bacterial infection

Intracellular infections by Gram-negative bacteria are a significant global health threat. The nuclear receptor Nur77 (also called TR3, NGFI-B, or NR4A1) was recently shown to sense cytosolic bacterial lipopolysaccharide (LPS). However, the potential role for Nur77 in controlling intracellular bacterial infection has not been examined. Here we show that Nur77 protects against intracellular infection in the bladder by uropathogenic Escherichia coli (UPEC), the leading cause of urinary tract infections (UTI). Nur77 deficiency in mice promotes the formation of UPEC intracellular bacterial communities (IBCs) in the cells lining the bladder lumen, leading to persistent infection in bladder tissue. Conversely, treatment with a small-molecule Nur77 agonist, cytosporone B, inhibits invasion and enhances the expulsion of UPEC from human urothelial cells in vitro, and significantly reduces UPEC IBC formation and bladder infection in mice. Our findings reveal a new role for Nur77 in control of bacterial infection and suggest that pharmacologic agonism of Nur77 function may represent a promising antibiotic-sparing therapeutic approach for UTI.

Modeling airway persistent infection of Moraxella catarrhalis and nontypeable Haemophilus influenzae by using human in vitro models.

Non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) are two common respiratory tract pathogens often associated with acute exacerbations in Chronic Obstructive Pulmonary Disease (COPD) as well as with otitis media (OM) in children. Although there is evidence that these pathogens can adopt persistence mechanisms such as biofilm formation, the precise means through which they contribute to disease severity and chronicity remains incompletely understood, posing challenges for their effective eradication. The identification of potential vaccine candidates frequently entails the characterization of the host-pathogen interplay in vitro even though this approach is limited by the fact that conventional models do not permit long term bacterial infections. In the present work, by using air-liquid-interface (ALI) human airway in vitro models, we aimed to recreate COPD-related persistent bacterial infections. In particular, we explored an alternative use of the ALI system consisting in the assembly of an inverted epithelium grown on the basal part of a transwell membrane with the aim to enable the functionality of natural defense mechanisms such as mucociliary clearance and cellular extrusion that are usually hampered during conventional ALI infection experiments. The inversion of the epithelium did not affect tissue differentiation and considerably delayed NTHi or Mcat infection progression, allowing one to monitor host-pathogen interactions for up to three weeks. Notably, the use of these models, coupled with confocal and transmission electron microscopy, revealed unique features associated with NTHi and Mcat infection, highlighting persistence strategies including the formation of intracellular bacterial communities (IBCs) and surface-associated biofilm-like structures. Overall, this study demonstrates the possibility to perform long term host-pathogen investigations in vitro with the aim to define persistence mechanisms adopted by respiratory pathogens and individuate potential new vaccine targets.

Nucleoside-diphosphate kinase of uropathogenic Escherichia coli inhibits caspase-1-dependent pyroptosis facilitating urinary tract infection

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infection (UTI). UPEC invades bladder epithelial cells (BECs) via fusiform vesicles, escapes into the cytosol, and establishes biofilm-like intracellular bacterial communities (IBCs). Nucleoside-diphosphate kinase (NDK) is secreted by pathogenic bacteria to enhance virulence. However, whether NDK is involved in UPEC pathogenesis remains unclear. Here, we find that the lack of ndk impairs the colonization of UPEC CFT073 in mouse bladders and kidneys owing to the impaired ability of UPEC to form IBCs. Furthermore, we demonstrate that NDK inhibits caspase-1-dependent pyroptosis by consuming extracellular ATP, preventing superficial BEC exfoliation, and promoting IBC formation. UPEC utilizes the reactive oxygen species (ROS) sensor OxyR to indirectly activate the regulator integration host factor, which then directly activates ndk expression in response to intracellular ROS. Here, we reveal a signaling transduction pathway that UPEC employs to inhibit superficial BEC exfoliation, thus facilitating acute UTI.

A Bird's-Eye View of the Pathophysiologic Role of the Human Urobiota in Health and Disease: Can We Modulate It?

For a long time, urine has been considered sterile in physiological conditions, thanks to the particular structure of the urinary tract and the production of uromodulin or Tamm-Horsfall protein (THP) by it. More recently, thanks to the development and use of new technologies, i.e., next-generation sequencing and expanded urine culture, the identification of a microbial community in the urine, the so-called urobiota, became possible. Major phyla detected in the urine are represented by Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Particularly, the female urobiota is largely represented by Lactobacillus spp., which are very active against urinary pathogenic Escherichia (E.) coli (UPEC) strains via the generation of lactic acid and hydrogen peroxide. Gut dysbiosis accounts for recurrent urinary tract infections (UTIs), so-called gut-bladder axis syndrome with the formation of intracellular bacterial communities in the course of acute cystitis. However, other chronic urinary tract infections are caused by bacterial strains of intestinal derivation. Monomicrobial and polymicrobial infections account for the outcome of acute and chronic UTIs, even including prostatitis and chronic pelvic pain. E. coli isolates have been shown to be more invasive and resistant to antibiotics. Probiotics, fecal microbial transplantation, phage therapy, antimicrobial peptides, and immune-mediated therapies, even including vaccines for the treatment of UTIs, will be described.

A human urothelial microtissue model reveals shared colonization and survival strategies between uropathogens and commensals.

Urinary tract infection is among the most common infections worldwide, typically studied in animals and cell lines with limited uropathogenic strains. Here, we assessed diverse bacterial species in a human urothelial microtissue model exhibiting full stratification, differentiation, innate epithelial responses, and urine tolerance. Several uropathogens invaded intracellularly, but also commensal Escherichia coli, suggesting that invasion is a shared survival strategy, not solely a virulence hallmark. The E. coli adhesin FimH was required for intracellular bacterial community formation, but not for invasion. Other shared lifestyles included filamentation (Gram-negatives), chaining (Gram-positives), and hijacking of exfoliating cells, while biofilm-like aggregates were formed mainly with Pseudomonas and Proteus. Urothelial cells expelled invasive bacteria in Rab-/LC3-decorated structures, while highly cytotoxic/invasive uropathogens, but not commensals, disrupted host barrier function and strongly induced exfoliation and cytokine production. Overall, this work highlights diverse species-/strain-specific infection strategies and corresponding host responses in a human urothelial microenvironment, providing insights at the microtissue, cell, and molecular level.

Bladder epithelial cell phosphate transporter inhibition protects mice against uropathogenic Escherichia coli infection.

Urinary tract infections are predominantly caused by uropathogenic Escherichia coli (UPEC). UPEC infects bladder epithelial cells (BECs) via fusiform vesicles, escapes into the cytosol to evade exocytosis, and establishes intracellular bacterial communities (IBCs) for the next round of infection. The UPEC vesicle escape mechanism remains unclear. Here we show that UPEC senses host immune responses and initiates escape by upregulating a key phospholipase. The UPEC phospholipase PldA disrupts the vesicle membrane, and pldA expression is activated by phosphate reduction in vesicles. The host phosphate transporter PIT1 is located on the fusiform vesicle membrane, transporting phosphate into the cytosol. UPEC infection upregulates PIT1 via nuclear factor κB (NF-κB), resulting in phosphate reduction. Silencing PIT1 blocks UPEC vesicle escape in BECs, inhibits IBC formation in mouse bladders, and protects mice from UPEC infection. Our results shed light on pathogenic bacteria responding to intracellular phosphate shortage and tackling host defense and provide insights for development of new therapeutic agents to treat UPEC infection.

Dynamic persistence of UPEC intracellular bacterial communities in a human bladder-chip model of urinary tract infection.

Uropathogenic Escherichia coli (UPEC) proliferate within superficial bladder umbrella cells to form intracellular bacterial communities (IBCs) during early stages of urinary tract infections. However, the dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. We develop a human bladder-chip model wherein umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and bladder filling and voiding mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel to sites of infection leads to swarm and neutrophil extracellular trap formation but does not prevent IBC formation. Subsequently, we tracked bacterial growth dynamics in individual IBCs through two cycles of antibiotic administration interspersed with recovery periods which revealed that the elimination of bacteria within IBCs by the antibiotic was delayed, and in some instances, did not occur at all. During the recovery period, rapid proliferation in a significant fraction of IBCs reseeded new foci of infection through bacterial shedding and host cell exfoliation. These insights reinforce a dynamic role for IBCs as harbors of bacterial persistence, with significant consequences for non-compliance with antibiotic regimens.

Early invasion of the bladder wall by solitary bacteria protects UPEC from antibiotics and neutrophil swarms in an organoid model.

Recurrence of uropathogenic Escherichia coli (UPEC) infections has been attributed to reactivation of quiescent intracellular reservoirs (QIRs) in deep layers of the bladder wall. QIRs are thought to arise late during infection following dispersal of bacteria from intracellular bacterial communities (IBCs) in superficial umbrella cells. Here, we track the formation of QIR-like bacteria in a bladder organoid model that recapitulates the stratified uroepithelium within a volume suitable for high-resolution live-cell imaging. Bacteria injected into the organoid lumen enter umbrella-like cells and proliferate to form IBC-like bodies. In parallel, single bacteria penetrate deeper layers of the organoid wall, where they localize within or between uroepithelial cells. These "solitary" bacteria evade killing by antibiotics and neutrophils and are morphologically distinct from bacteria in IBCs. We conclude that bacteria with QIR-like properties may arise at early stages of infection, independent of IBC formation and rupture.

Transcriptomic analysis reveals that the small protein MgtS contributes to the virulence of uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the most common pathogen causing urinary tract infections (UTIs). The pathogenesis of UPEC relies on the formation of intracellular bacterial communities (IBCs) after invading bladder epithelial cells (BECs). In this study, the gene expression profiles of UPEC after invading BECs were comprehensively analyzed using RNA sequencing to reveal potential virulence-related genes. The small protein MgtS, which is transcriptionally upregulated in BECs, was further investigated. It was found that MgtS contributed positively to UPEC invasion of BECs and colonization in murine bladders. A two-component regulatory system, PhoPQ was confirmed as a direct activator of mgtS expression in BECs, and magnesium limitation is proposed as a host cue for the activation. This study provides the first comprehensive analysis of the transcriptome profile of UPEC during its intra-BECs life, revealing a new virulence-associated gene and its regulatory mechanism.

Testosterone regulates the intracellular bacterial community formation of uropathogenic Escherichia coli in prostate cells via STAT3

BackgroundUPEC can internalize clonally in prostate to form biofilm-like intracellular bacterial communities (IBCs) for recurrent or chronic infection. We previously indicated that the exposure of prostate cells to testosterone could suppress UPEC invasion and their persistent survival within cells by effectively inhibiting the JAK/STAT1 signaling pathway. However, the regulatory mechanism by which testosterone affects UPEC-induced prostatitis via STAT3, another latent transcription factor signaling pathway is still unclear. The present study aimed to clarify the role of STAT3 in the process of UPEC-induced inflammation and colonization in prostate epithelial cells. MethodsThe effects of testosterone-mediated inhibition were compared between the prostatitis by different UPEC strains (CFT073 and J96) through the specific GFP-UPEC-infected prostate cell model. Fluorescence microscopy was used for UPEC IBCs detection and quantifying, and Flow cytometry, RT-PCR and western blotting were used for analyzing related gene and protein expressions. Pretreatment of JAK and STAT3 inhibitors were also applied to verify the regulation of transduction pathway in testosterone-mediated anti-UPEC infection. ResultsThis study revealed that testosterone effectively suppresses UPEC infection and IBC formation in prostate cells through the JAK/STAT3 pathway. The results show that CFT073 and J96 UPEC infection rates and colony numbers were dose-dependently reduced in RWPE-1 cells pretreated with 5 and 20 μg/mL testosterone at 0 and 24 h post-infection. Further, testosterone reduced the amounts of UPEC infecting and surviving within the prostate cells, as well as suppressed the size of IBCs formed. We demonstrated that pretreating testosterone effectively inhibited UPEC infection along with dose-dependent suppression of STAT3 and the phosphorylated-STAT3 expression in prostate cells, especially in 24 h J96 UPEC infected groups. The STAT inhibitor, SOCS3 also up-regulated at the same time. In addition, we pretreated the JAK1 or STAT3 inhibitor with testosterone to block the signaling transduction before CFT073 and J96 UPEC infection, and found the significant restoring in both the sizes of IBCs and bacterial numbers in RWPE-1 cells. Therefore, our results suggest that the suppression of STAT3 by testosterone treatment attenuate UPEC growing within IBCs and interfere with their infection to prostate cells. ConclusionsOverall, our study demonstrates that testosterone suppresses the initial infection of prostate epithelial cells by UPEC and reduces the survival of UPEC within IBCs after infection. These results indicate a critical role for STAT3 in facilitating UPEC infection and persistence, and its participation in driving testosterone-suppressive responses in prostate epithelial cells. In conclusion, this study suggests that testosterone may be beneficial in treating clinically recurrent UPEC infections and, thus, the persistent recurrence of prostatic inflammation.

Sitafloxacin has a potent activity for eradication of extended spectrum β-lactamase-producing fluoroquinolone-resistant Escherichia coli forming intracellular bacterial communities in uroepithelial cells

IntroductionEradication of asymptomatic bacteriuria (ASB) before urological procedures is important to reduce the risk for infectious complications after surgery. However, the appropriate regimen for antimicrobial treatment has not been fully determined. We experienced continuous (over 10 months) isolation of extended spectrum β-lactamase (ESBL)-producing fluoroquinolone-resistant Escherichia coli from urine of an asymptomatic patient. The four isolates obtained (SMESC1 to 4) were international high-risk clones of O25b:H4-ST131-H30R, and originated from one strain, as revealed by the whole genome sequences. Although the patient received meropenem (MEPM) and fosfomycin (FOM), to which the strains were susceptible before the urological procedures, they could not be eradicated. MethodsTo explore the reason for the continuous isolation even after MEPM and FOM administration, antimicrobial killing of adherent and/or intracellular bacterial communities (IBC) formed by coculture of the E. coli cells and T24 bladder epithelial cells were examined. ResultsFOM and levofloxacin did not decrease viable E. coli cells compared with gentamicin. MEPM partly decreased them, and sitafloxacin (STFX) decreased them most potently. These observations indicate that E. coli can survive in the urinary tract under antimicrobial administration, and some antimicrobials such as FOM and MEPM cannot eradicate E. coli in uroepithelial cells. Adhesion on urinary epithelial cells and/or IBC formation might result in continuous isolation from the urinary tract and recurrence of ASB and urinary tract infections. ConclusionsThe present study suggests that STFX is a promising optional agent for the eradication of ESBL-producing fluoroquinolone-resistant E. coli in the urinary tract before urological procedures.

The Tol-Pal System of Uropathogenic Escherichia coli Is Responsible for Optimal Internalization Into and Aggregation Within Bladder Epithelial Cells, Colonization of the Urinary Tract of Mice, and Bacterial Motility.

Urinary tracts infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is a common infectious disease. With the shortage of new antimicrobial agents, the increase in UPEC resistance to commonly used drugs, such as fluoroquinolones and β-lactams including carbapenems is a critical issue. UPEC invades urinary tract cells, where it aggregates, and subsequently, forms biofilm-like multicellular colonies termed intracellular bacterial communities (IBCs). This process allows the bacteria to establish infections and so may be a good potential target for new drugs to treat infections. Here, we show that deletion of the tolB gene, encoding a protein of the Tol-Pal system that was originally characterized as a protein complex for colicin uptake and maintenance of the outer membrane, decreases the level of bacterial internalization into and aggregation within cultured bladder epithelial cells and also inhibits the colonization of mice urinary tracts. The tolB mutant also exhibited defective motility because of impaired flagellum syntheses. The fliC and motA mutants, which are non-motile strains, also exhibited lower levels of bacterial internalization and aggregation than their wild-type parent. Additional deletion of tolB in the fliC mutant did not further decrease these, suggesting that the attenuated virulence of the tolB mutant is a result of defective motility. The tolA, tolQ, tolR, and pal mutants that lack other members of the Tol-Pal system also exhibited lower levels of motility and aggregation within bladder epithelial cells compared to their wild-type parent. These combined results suggest another role of the Tol-Pal system, i.e., that it is responsible for optimal internalization, aggregation followed by IBC formation within urinary tract cells, and bacterial motility.

Intraepithelial neutrophils in mammary, urinary and gall bladder infections

Neutrophil mobilization is a crucial response to protect the host against invading microorganisms. Neutrophil recruitment and removal have to be tightly regulated to prevent uncontrolled inflammation and excessive release of their toxic content causing tissue damage and subsequent organ dysfunctions. We show here the presence of live and apoptotic neutrophils in the cytoplasm of inflamed mammary, urinary and gall bladder epithelial cells following infection with E. coli and Salmonella bacteria. The entry process commenced with adherence of transmigrated neutrophils to the apical membrane of inflamed epithelial cells. Next, nuclear rearrangement and elongation associated with extensive actin polymerization enabled neutrophils to crawl and invaginate the apical membrane into cytoplasmic double membrane compartments. Scission of the invaginated cell membrane from the entry point and loss of these surrounding membranes released intracellular neutrophils into the cytoplasm where they undergone apoptotic death. The co-occurrence of this observation with bacterial invasion and formation of intracellular bacterial communities (IBCs) might link entry of infected neutrophils to the formation of IBCs and chronic carriage in E. coli mastitis and cystitis and Salmonella cholecystitis.

Urinary tract infections, immunity, and vaccination.

Urinary tract infections (UTI) are considered one of the main causes of morbidity worldwide, and uropathogenic Escherichia coli (UPEC) is the etiological agent associated with these infections. The high morbidity produced by the UTI and the limitation of antibiotic treatments promotes the search for new alternatives against these infections. The knowledge that has been generated regarding the immune response in the urinary tract is important for the development of effective strategies in the UTI prevention, treatment, and control. Molecular biology and bioinformatic tools have allowed the construction of fusion proteins as biomolecules for the development of a viable vaccine against UTI. The fimbrial adhesins (FimH, CsgA, and PapG) of UPEC are virulence factors that contribute to the adhesion, invasion, and formation of intracellular bacterial communities. The generation of recombinant proteins from fimbrial adhesins as a single molecule is obtained by fusion technology. A few in vivo and in vitro studies have shown that fusion proteins provide an efficient immune response and protection against UTI produced by UPEC. Intranasal immunization of immunogenic molecules has generated a response in the urinary tract mucosa compared with other routes of immunization. The objective of this review was to propose a vaccine designed against UTI caused by UPEC, describing the general scenario of the infection, the mechanism of pathogenicity of bacteria, and the immune response of the host.

MP23-03 IL-6/STAT3 SIGNALING IS A HOST DETERMINANT OF UPEC VIRULENCE VIA ALTERED BACTERIAL INVASION AND INTRACELLULAR BACTERIAL COMMUNITY FORMATION

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder II1 Apr 2018MP23-03 IL-6/STAT3 SIGNALING IS A HOST DETERMINANT OF UPEC VIRULENCE VIA ALTERED BACTERIAL INVASION AND INTRACELLULAR BACTERIAL COMMUNITY FORMATION Christina Ching, Sudipti Gupta, and Brian Becknell Christina ChingChristina Ching More articles by this author , Sudipti GuptaSudipti Gupta More articles by this author , and Brian BecknellBrian Becknell More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.735AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our understanding of bacterial virulence in urinary tract infection (UTI) is primarily limited to identification of bacteria-specific determinants. Host mediators are less well known. IL-6 is a cytokine that is induced during UTI and results in subsequent Stat3 phosphorylation, but without readily apparent functional significance. We sought to evaluate the impact of IL-6/Stat3 signaling on host susceptibility to UTI via urothelial bacterial invasion and intracellular bacterial community formation. METHODS We performed in vivo and in vitro studies in order to evaluate the impact of IL-6 and Stat3 in bacterial invasion. We infected 6 week old female wild type (WT) C57BL/6J, IL-6 knock out (KO), or tamoxifen-inducible Stat3 conditional KO mice transurethrally with UPEC. We evaluated bladder and urine bacterial burden at varying time points of sacrifice by serial plating of tissue homogenates on LB agar and IBC formation via beta-galactosidase staining. We similarly evaluated the impact of recombinant IL-6 rescue in IL-6 KO mice on IBC formation and antimicrobial formation. We evaluated the impact of recombinant IL-6 incubation and Stat3 inhibition on intracellular bacterial invasion with gentamicin protection assay in serum starved 5637 urothelial cells. Results were evaluated by Mann-Whitney U test with p <0.05 being significant. RESULTS We observed an early transient (6 hours post inoculation - hpi) increase in bladder bacterial burden in our IL-6 KO mice compared to the WT controls, with a subsequent and persistent increase in urine bacterial burden starting at 24 hpi. This matched an initial significant difference in bladder IBC number in the IL-6 KO vs. WT that resolved by 24 hpi and was confirmed in our Stat3 CKO mice. Recombinant intravesical IL-6 rescued this phenotype in the IL-6 KO mouse and also significantly increased antimicrobial peptide expression. Similarly, we found that there was a significant decrease in bacterial invasion in vitro when 5637 cells were preincubated with IL-6. Stat3 inhibition resulted in a reversal of this phenotype with an increase in bacterial invasion on par with if not above unstimulated cells. CONCLUSIONS IL-6 plays an important role in host susceptibility to UTI as a mediator of bacterial invasion and IBC formation, potentially via antimicrobial peptide formation. Given IBC formation represents a significant bottleneck in UPEC survival during cystitis, these findings could suggest a role of IL-6 in UTI persistence and chronicity. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e282 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Christina Ching More articles by this author Sudipti Gupta More articles by this author Brian Becknell More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Interleukin-6/Stat3 signaling has an essential role in the host antimicrobial response to urinary tract infection

The signaling networks regulating antimicrobial activity during urinary tract infection (UTI) are incompletely understood. Interleukin-6 (IL-6) levels increase with UTI severity, but the specific contributions of IL-6 to host immunity against bacterial uropathogens are unknown. To clarify this we tested whether IL-6 activates the Stat3 transcription factor, to drive a program of antimicrobial peptide gene expression in infected urothelium during UTI. Transurethral inoculation of uropathogenic Escherichia coli led to IL-6 secretion, urothelial Stat3 phosphorylation, and activation of antimicrobial peptide transcription, in a Toll-like receptor 4-dependent manner in a murine model of cystitis. Recombinant IL-6 elicited Stat3 phosphorylation in primary urothelial cells invitro, and systemic IL-6 administration promoted urothelial Stat3 phosphorylation and antimicrobial peptide expression invivo. IL-6 deficiency led to decreased urothelial Stat3 phosphorylation and antimicrobial peptide mRNA expression following UTI, a finding mirrored by conditional Stat3 deletion. Deficiency in IL-6 or Stat3 was associated with increased formation of intracellular bacterial communities, and exogenous IL-6 reversed this phenotype in IL-6 knockout mice. Moreover, chronic IL-6 depletion led to increased renal bacterial burden and severe pyelonephritis in C3H/HeOuJ mice. Thus, IL-6/Stat3 signaling drives a transcriptional program of antimicrobial gene expression in infected urothelium, with key roles in limiting epithelial invasion and ascending infection.

The Catabolite Repressor Protein-Cyclic AMP Complex Regulates csgD and Biofilm Formation in Uropathogenic Escherichia coli.

The extracellular matrix protects Escherichia coli from immune cells, oxidative stress, predation, and other environmental stresses. Production of the E. coli extracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenic E. coli (UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms through csgD The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion of cyaA resulted in reduced extracellular matrix production and biofilm formation. The catabolite repressor protein (CRP) positively regulated csgD transcription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaA and Δcrp did not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within the csgD-csgB intergenic region, and purified CRP could gel shift the csgD-csgB intergenic region. Additionally, we found that CRP binded upstream of kpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influence E. coli biofilms through transcriptional regulation of csgD IMPORTANCE The catabolite repressor protein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on the Escherichia coli chromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874-5893, 2004, https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibits E. coli biofilm formation, and ΔcyaA and Δcrp mutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406-3410, 2002, https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms through csgD Additionally, we propose that cAMP may work as a signaling compound for uropathogenic E. coli (UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation.

Persistence of uropathogenic Escherichia Coli in the bladders of female patients with sterile urine after antibiotic therapies.

This study aimed to provide evidence of persistent uropathogenic Escherichia coli (UPEC) in female patients with recurrent urinary tract infection (UTI) after antibiotic therapy. We collected biopsies of the bladder, and clean-catch urine samples from 32 women who had episodes of recurrent UTI and were given antibiotic therapy. Urine samples and biopsies were analyzed by conventional bacteriological techniques. Phylogenetic group and 16 virulence factors (VFs) of UPEC were determined using polymerase chain reaction (PCR). The infection capability of UPEC was confirmed in a mouse model. Immunofluorescence and electron microscopy were used to detect intracellular bacterial communities (IBCs) in the mouse model. The results showed that all urine specimens were detected sterile. E. coli was found in 6 of 32 biopsies (18.75%), and was identified to be UPEC by PCR. Different VFs associated with the formation of IBCs were identified in all six UPEC isolates. Each UPEC isolate was capable of forming IBCs within the bladder epithelial cells of mice. In conclusion, UPEC with distinctive pathological traits and the capability of IBC formation was first found in the bladders of women after antibiotic therapy, suggesting that the IBC pathogenic pathway may occur in humans and it plays an important role in UTI recurrence.

Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). This bacterium adheres to and invades the host cells in the bladder, where it forms biofilm-like polymicrobial structures termed intracellular bacterial communities (IBCs) that protect UPEC from antimicrobial agents and the host immune systems. Using genetic screening, we found that deletion of the fur gene, which encodes an iron-binding transcriptional repressor for iron uptake systems, elevated the expression of type I fimbriae and motility when UPEC was grown under iron-rich conditions, and it led to an increased number of UPEC cells adhering to and internalized in bladder epithelial cells. Consequently, the IBC colonies that the fur mutant formed in host cells were denser and larger than those formed by the wild-type parent strain. Fur is inactivated under iron-restricted conditions. When iron was depleted from the bacterial cultures, wild-type UPEC adhesion, invasion, and motility increased, similar to the case with the fur mutant. The purified Fur protein bound to regions upstream of fimA and flhD, which encode type I fimbriae and an activator of flagellar expression that contributes to motility, respectively. These results suggest that Fur is a repressor of fimA and flhD and that its repression is abolished under iron-depleted conditions. Based on our in vitro experiments, we conclude that UPEC adhesion, invasion, IBC formation, and motility are suppressed by Fur under iron-rich conditions but derepressed under iron-restricted conditions, such as in patients with UTIs.

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.