The Role of NLRP3 in Regulation of Antimicrobial Peptides and Estrogen Signaling in UPEC-Infected Bladder Epithelial Cells.

The NLRP3 inflammasome, estrogen and antimicrobial peptides have all been found to have a vital role in the protection of the bladder urothelium. However, the interdependence between these protective factors during a bladder infection is currently unknown. Our aim was to investigate the role of NLRP3 in the regulation of antimicrobial peptides and estrogen signaling in bladder epithelial cells during a UPEC infection. Human bladder epithelial cells and CRISPR/Cas9-generated NLRP3-deficient cells were stimulated with the UPEC strain CFT073 and estradiol. The gene and protein expression were evaluated with microarray, qRT-PCR, western blot and ELISA. Microarray results showed that the expression of most antimicrobial peptides was reduced in CFT073-infected NLRP3-deficient cells compared to Cas9 control cells. Conditioned medium from NLRP3-deficient cells also lost the ability to suppress CFT073 growth. Moreover, NLRP3-deficient cells had lower basal release of Beta-defensin-1, Beta-defensin-2 and RNase7. The ability of estradiol to induce an increased expression of antimicrobial peptides was also abrogated in NLRP3-deficient cells. The decreased antimicrobial peptide expression might be linked to the observed reduced expression and activity of estradiol receptor beta in NLRP3-deficient cells. This study suggests that NLRP3 may regulate the release and expression of antimicrobial peptides and affect estrogen signaling in bladder epithelial cells.

TMAO Suppresses Megalin Expression and Albumin Uptake in Human Proximal Tubular Cells Via PI3K and ERK Signaling.

Trimethylamine-N-oxide (TMAO) is a uremic toxin, which has been associated with chronic kidney disease (CKD). Renal tubular epithelial cells play a central role in the pathophysiology of CKD. Megalin is an albumin-binding surface receptor on tubular epithelial cells, which is indispensable for urine protein reabsorption. To date, no studies have investigated the effect of TMAO on megalin expression and the functional properties of human tubular epithelial cells. The aim of this study was first to identify the functional effect of TMAO on human renal proximal tubular cells and second, to unravel the effects of TMAO on megalin-cubilin receptor expression. We found through global gene expression analysis that TMAO was associated with kidney disease. The microarray analysis also showed that megalin expression was suppressed by TMAO, which was also validated at the gene and protein level. High glucose and TMAO was shown to downregulate megalin expression and albumin uptake similarly. We also found that TMAO suppressed megalin expression via PI3K and ERK signaling. Furthermore, we showed that candesartan, dapagliflozin and enalaprilat counteracted the suppressive effect of TMAO on megalin expression. Our results may further help us unravel the role of TMAO in CKD development and to identify new therapeutic targets to counteract TMAOs effects.

The role of caspase-1, caspase-4 and NLRP3 in regulating the host cell response evoked by uropathogenic Escherichia coli.

The inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 have been emphasised to be essential in the host cell response during urinary tract infection (UTI) by regulating IL-1ß release. Our aim was to investigate how the inflammasome-associated proteins regulate the cell response of bladder epithelial cells during infection with uropathogenic Escherichia coli (UPEC). Human bladder epithelial cells (5637) and CRISPR/Cas9 generated caspase-1, caspase-4 and NLRP3 knockdown cells were stimulated with the UPEC strain CFT073. Using Olink proteomics and real time RT-PCR, we showed that caspase-1, caspase-4 and NLRP3 are vital for the expression of many inflammatory genes and proteins from bladder epithelial cells. When investigating the effect of inflammasome-associated proteins on neutrophils, we found that conditioned medium from UPEC-infected caspase-4 knockdown cells significantly increased phagocytosis of CFT073 and significantly decreased ROS production from neutrophils. In contrast, conditioned medium from UPEC-infected NLRP3 knockdown cells significantly decreased the phagocytosis of CFT073 and significantly increased the ROS production from neutrophils. In conclusion, we showed that the inflammasome-associated proteins contribute to the host cell response during UPEC infection.

Transcriptional alterations in bladder epithelial cells in response to infection with different morphological states of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) may undergo a cyclic cascade of morphological alterations that are believed to enhance the potential of UPEC to evade host responses and re-infect host cell. However, knowledge on the pathogenic potential and host activation properties of UPEC during the morphological switch is limited. Microarray analysis was performed on mRNA isolated from human bladder epithelial cells (HBEP) after exposure to three different morphological states of UPEC (normal coliform, filamentous form and reverted form). Cells stimulated with filamentous bacteria showed the lowest number of significant gene alterations, although the number of enriched gene ontology classes was high suggesting diverse effects on many different classes of host genes. The normal coliform was in general superior in stimulating transcriptional activity in HBEP cells compared to the filamentous and reverted form. Top-scored gene entities activated by all three morphological states included IL17C, TNFAIP6, TNF, IL20, CXCL2, CXCL3, IL6 and CXCL8. The number of significantly changed canonical pathways was lower in HBEP cells stimulated with the reverted form (32 pathways), than in cells stimulated with the coliform (83 pathways) or filamentous bacteria (138 pathways). A host cell invasion assay showed that filamentous bacteria were unable to invade bladder cells, and that the number of intracellular bacteria was markedly lower in cells infected with the reverted form compared to the coliform. In conclusion, the morphological state of UPEC has major impact on the host bladder response both when evaluating the number and the identity of altered host genes and pathways.

The Fibrotic Effects of TMAO on Human Renal Fibroblasts Is Mediated by NLRP3, Caspase-1 and the PERK/Akt/mTOR Pathway.

Trimethylamine N-oxide (TMAO), a product of gut microbiota metabolism, has previously been shown to be implicated in chronic kidney disease. A high TMAO-containing diet has been found to cause tubulointerstitial renal fibrosis in mice. However, today there are no data linking specific molecular pathways with the effect of TMAO on human renal fibrosis. The aim of this study was to investigate the fibrotic effects of TMAO on renal fibroblasts and to elucidate the molecular pathways involved. We found that TMAO promoted renal fibroblast activation and fibroblast proliferation via the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 signaling. We also found that TMAO increased the total collagen production from renal fibroblasts via the PERK/Akt/mTOR pathway. However, TMAO did not induce fibronectin or TGF-ß1 release from renal fibroblasts. We have unraveled that the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 mediates TMAO's fibrotic effect on human renal fibroblasts. Our results can pave the way for future research to further clarify the molecular mechanism behind TMAO's effects and to identify novel therapeutic targets in the context of chronic kidney disease.

Activation of NLRP3 by uropathogenic Escherichia coli is associated with IL-1ß release and regulation of antimicrobial properties in human neutrophils.

The NLRP3 inflammasome and IL-1ß have recently been linked to the severity of uropathogenic Escherichia coli (UPEC)-mediated urinary tract infection (UTI). However, not much is known about the contribution of NLRP3 to the antimicrobial properties of neutrophils and the release of IL-1ß during UPEC infection. The purpose of this study was to elucidate the mechanisms behind UPEC-induced IL-1ß release from human neutrophils, and to investigate the contribution of the NLRP3 inflammasome in neutrophil-mediated inhibition of UPEC growth. We found that the UPEC strain CFT073 increased the expression of NLRP3 and increased caspase-1 activation and IL-1ß release from human neutrophils. The IL-1ß release was mediated by the NLRP3 inflammasome and by serine proteases in an NF-κB-and cathepsin B-dependent manner. The UPEC virulence factors α-hemolysin, type-1 fimbriae and p-fimbriae were all shown to contribute to UPEC mediated IL-1ß release from neutrophils. Furthermore, inhibition of caspase-1 and NLRP3 activation increased neutrophil ROS-production, phagocytosis and the ability of neutrophils to suppress UPEC growth. In conclusion, this study demonstrates that UPEC can induce NLRP3 and serine protease-dependent release of IL-1ß from human neutrophils and that NLRP3 and caspase-1 can regulate the antimicrobial activity of human neutrophils against UPEC.

IL-1RA is part of the inflammasome-regulated immune response in bladder epithelial cells and influences colonization of uropathogenic E. coli.

The NLRP3 inflammasome, IL-1ß release and pyroptosis (cell lysis) have recently been proposed to be essential for the progression of urinary tract infection (UTI) and elimination of intracellular bacterial niches. However, the effects of IL-1R antagonist (IL-1RA) on immune responses during UTI, except for its ability to disrupt IL-1ß signalling, are not well understood. The aim of this study was to investigate the role of IL-1RA in UPEC colonization of bladder epithelial cells and the subsequent host inflammatory response. Human bladder epithelial cells (5637) and CRISPR/Cas9 generated NLRP3 and caspase-1 knockdown cells and IL-1RA knockout cells were stimulated with the UPEC isolate CFT073. The results showed that the UPEC virulence factor α-hemolysin is essential for IL-1RA release, and that the inflammasome-associated proteins caspase-1 and NLRP3 affect the release of IL-1RA. IL-1RA deficient cells showed a reduced adherence and invasion by CFT073 compared to wild-type cells, suggesting that IL-1RA may oppose mechanisms that protects against bacterial colonization. A targeted protein analysis of inflammation-related proteins showed that the basal expression of 23 proteins and the UPEC-induced expression of 10 proteins were significantly altered in IL-1RA deficient bladder epithelial cells compared to Cas9 control cells. This suggests that IL-1RA has a broad effect on the inflammatory response in bladder epithelial cells.

Host-Derived Nitric Oxide and Its Antibacterial Effects in the Urinary Tract.

Urinary tract infection (UTI) is one of the most common bacterial infections in humans, and the majority are caused by uropathogenic Escherichia coli (UPEC). The rising antibiotic resistance among UPEC and the frequent failure of antibiotics to effectively treat recurrent UTI and catheter-associated UTI motivate research on alternative ways of managing UTI. Abundant evidence indicates that the toxic radical nitric oxide (NO), formed by activation of the inducible nitric oxide synthase, plays an important role in host defence to bacterial infections, including UTI. The major source of NO production during UTI is from inflammatory cells, especially neutrophils, and from the uroepithelial cells that are known to orchestrate the innate immune response during UTI. NO and reactive nitrogen species have a wide range of antibacterial targets, including DNA, heme proteins, iron-sulfur clusters, and protein thiol groups. However, UPEC have acquired a variety of defence mechanisms for protection against NO, such as the NO-detoxifying enzyme flavohemoglobin and the NO-tolerant cytochrome bd-I respiratory oxidase. The cytotoxicity of NO-derived intermediates is nonspecific and may be detrimental to host cells, and a balanced NO production is crucial to maintain the tissue integrity of the urinary tract. In this review, we will give an overview of how NO production from host cells in the urinary tract is activated and regulated, the effect of NO on UPEC growth and colonization, and the ability of UPEC to protect themselves against NO. We also discuss the attempts that have been made to develop NO-based therapeutics for UTI treatment.

Activation of the NLRP3 Inflammasome Pathway by Uropathogenic Escherichia coli Is Virulence Factor-Dependent and Influences Colonization of Bladder Epithelial Cells.

The NLRP3 inflammasome and IL-1ß release have recently been suggested to be important for the progression of urinary tract infection (UTI). However, much is still unknown regarding the interaction of UPEC and the NLRP3 inflammasome. The purpose of this study was to elucidate what virulence factors uropathogenic Escherichia coli (UPEC) use to modulate NLRP3 inflammasome activation and subsequent IL-1ß release and the role of NLRP3 for UPEC colonization of bladder epithelial cells. The bladder epithelial cell line 5637, CRISPR/Cas9 generated NLRP3, caspase-1 and mesotrypsin deficient cell lines and transformed primary bladder epithelial cells (HBLAK) were stimulated with UPEC isolates and the non-pathogenic MG1655 strain. We found that the UPEC strain CFT073, but not MG1655, induced an increased caspase-1 activity and IL-1ß release from bladder epithelial cells. The increase was shown to be mediated by α-hemolysin activation of the NLRP3 inflammasome in an NF-κB-independent manner. The effect of α-hemolysin on IL-1ß release was biphasic, initially suppressive, later inductive. Furthermore, the phase-locked type-1-fimbrial ON variant of CFT073 inhibited caspase-1 activation and IL-1ß release. In addition, the ability of CFT073 to adhere to and invade NLRP3 deficient cells was significantly reduced compare to wild-type cells. The reduced colonization of NLRP3-deficient cells was type-1 fimbriae dependent. In conclusion, we found that the NLRP3 inflammasome was important for type-1 fimbriae-dependent colonization of bladder epithelial cells and that both type-1 fimbriae and α-hemolysin can modulate the activity of the NLRP3 inflammasome.

Transcriptional Alterations of Virulence-Associated Genes in Extended Spectrum Beta-Lactamase (ESBL)-Producing Uropathogenic Escherichia coli during Morphologic Transitions Induced by Ineffective Antibiotics.

It is known that an ineffective antibiotic treatment can induce morphological shifts in uropathogenic Escherichia coli (UPEC) but the virulence properties during these shifts remain to be studied. The present study examines changes in global gene expression patterns and in virulence factor-associated genes in an extended spectrum beta-lactamase (ESBL)-producing UPEC (ESBL019) during the morphologic transitions induced by an ineffective antibiotic and in the presence of human primary bladder epithelial cells. Microarray results showed that the different morphological states of ESBL019 had significant transcriptional alterations of a large number of genes (Transition; 7%, Filamentation; 32%, and Reverted 19% of the entities on the array). All three morphological states of ESBL019 were associated with a decreased energy metabolism, altered iron acquisition systems and altered adhesion expression. In addition, genes associated with LPS synthesis and bacterial motility was also altered in all the morphological states. Furthermore, the transition state induced a significantly higher release of TNF-α from bladder epithelial cells compared to all other morphologies, while the reverted state was unable to induce TNF-α release. Our findings show that the morphological shifts induced by ineffective antibiotics are associated with significant transcriptional virulence alterations in ESBL-producing UPEC, which may affect survival and persistence in the urinary tract.

Global gene expression profiling and antibiotic susceptibility after repeated exposure to the carbon monoxide-releasing molecule-2 (CORM-2) in multidrug-resistant ESBL-producing uropathogenic Escherichia coli.

Treatment of urinary tract infections is today a challenge due to the increasing prevalence of multidrug-resistant ESBL-producing uropathogenic Escherichia coli (UPEC). There is an urgent need for new treatment strategies for multidrug-resistant UPEC and preferably with targets that have low potential for development of resistance. Carbon monoxide-releasing molecules (CORMs) are novel and potent antibacterial agents. The present study examines the transcriptomic targets of CORM-2 in a multidrug-resistant ESBL-producing UPEC isolate in response to a single exposure to CORM-2 and after repeated exposure to CORM-2. The bacterial viability and minimal inhibitory concentration (MIC) were also examined after repeated exposure to CORM-2. Microarray analysis revealed that a wide range of processes were affected by CORM-2, including a general trend of down-regulation in energy metabolism and biosynthesis pathways and up-regulation of the SOS response and DNA repair. Several genes involved in virulence (ibpB), antibiotic resistance (marAB, mdtABC) and biofilm formation (bhsA, yfgF) were up-regulated, while some genes involved in virulence (kpsC, fepCEG, entABE), antibiotic resistance (evgA) and biofilm formation (artIP) were down-regulated. Repeated exposure to CORM-2 did not alter the gene expression patterns, the growth inhibitory response to CORM-2 or the MIC values for CORM-2, cefotaxime, ciprofloxacin and trimethoprim. This study identifies several enriched gene ontologies, modified pathways and single genes that are targeted by CORM-2 in a multidrug-resistant UPEC isolate. Repeated exposure to CORM-2 did not change the gene expression patterns or fold changes and the susceptibility to CORM-2 remained after repeated exposure.

Carbon monoxide releasing molecule-2 (CORM-2) inhibits growth of multidrug-resistant uropathogenic Escherichia coli in biofilm and following host cell colonization.

BACKGROUND: Increased resistance to antimicrobial agents is a characteristic of many bacteria growing in biofilms on for example indwelling urinary catheters or in intracellular bacterial reservoirs. Biofilm-related infections caused by multidrug-resistant bacteria, such as extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, are a major challenge. The aim of this study was to investigate if a carbon monoxide-releasing molecule (CORM-2) has antibacterial effects against ESBL-producing uropathogenic E. coli (UPEC) in the biofilm mode of growth and following colonization of host bladder epithelial cells. RESULTS: The effect of CORM-2 was examined on bacteria grown within an established biofilm (biofilm formed for 24 h on plastic surface) by a live/dead viability staining assay. CORM-2 (500 µM) exposure for 24 h killed approximately 60 % of the ESBL-producing UPEC isolate. A non-ESBL-producing UPEC isolate and the E. coli K-12 strain TG1 were also sensitive to CORM-2 exposure when grown in biofilms. The antibacterial effect of CORM-2 on planktonic bacteria was reduced and delayed in the stationary growth phase compared to the exponential growth phase. In human bladder epithelial cell colonization experiments, CORM-2 exposure for 4 h significantly reduced the bacterial counts of an ESBL-producing UPEC isolate. CONCLUSION: This study shows that CORM-2 has antibacterial properties against multidrug-resistant UPEC under biofilm-like conditions and following host cell colonization, which motivate further studies of its therapeutic potential.

Ceftibuten-induced filamentation of extended spectrum beta lactamase (ESBL)-producing uropathogenic Escherichia coli alters host cell responses during an in vitro infection.

Inadequate and delayed antibiotic treatment of extended spectrum beta-lactamase (ESBL)-producing isolates have been associated with increased mortality of affected patients. The purpose of this study was to compare the host response of human renal epithelial cells and polymorphonuclear leucocyte (PMN) cells when infected by ESBL-producing uropathogenic Escherichia coli (UPEC) isolates in the presence or absence of ineffective antibiotics. The renal epithelial cell line A498 and PMN cells were stimulated with ESBL-producing UPEC isolates in the presence or absence of three different antibiotics (trimetoprim, ceftibuten and ciprofloxacin). Host cell responses were evaluated as release of cytokines (IL-6, IL-8), reactive oxygen species (ROS), ATP and endotoxins. Bacterial morphology and PMN phagocytosis were evaluated by microscopy. In the presence of ceftibuten, 2 out of 3 examined ESBL-isolates changed their morphology into a filamentous form. The presence of ceftibuten enhanced IL-6, IL-8 and ROS-production from host cells, but only from cells stimulated by the filamentous isolates. The bacterial supernatant and not the filamentous bacteria per se was responsible for the increased release of IL-6, IL-8 and ROS. Increased endotoxin and ATP levels were found in the bacterial supernatants from filamentous isolates. Apyrase decreased IL-6 secretion from A498 cells and polymyxin B abolished the increased ROS-production from PMN cells. PMN were able to inhibit the bacterial growth of some ESBL-isolates in the presence of ceftibuten. In conclusion, antibiotic-induced filamentation of ESBL-producing UPEC isolates and the associated release of ATP and endotoxins can alter the host cell response in the urinary tract.

IL-8 and global gene expression analysis define a key role of ATP in renal epithelial cell responses induced by uropathogenic bacteria.

The recent recognition of receptor-mediated ATP signalling as a pathway of epithelial pro-inflammatory cytokine release challenges the ubiquitous role of the TLR4 pathway during urinary tract infection. The aim of this study was to compare cellular responses of renal epithelial cells infected with uropathogenic Escherichia coli (UPEC) strain IA2 to stimulation with ATP-γ-S. A498 cells were infected or stimulated in the presence or absence of apyrase, that degrades extracellular ATP, or after siRNA-mediated knockdown of ATP-responding P2Y2 receptors. Cellular IL-8 release and global gene expression were analysed. Both IA2 and A498 cells per se released ATP, which increased during infection. IA2 and ATP-γ-S caused a ∼5-fold increase in cellular release of IL-8 and stimulations performed in the presence of apyrase or after siRNA knockdown of P2Y2 receptors resulted in attenuation of IA2-mediated IL-8 release. Microarray results show that both IA2 and ATP-γ-S induced marked changes in gene expression of renal cells. Thirty-six genes were in common between both stimuli, and many of these are key genes belonging to classical response pathways of bacterial infection. Functional analysis shows that 88 biological function-annotated cellular pathways were identical between IA2 and ATP-γ-S stimuli. Results show that UPEC-induced release of IL-8 is dependent on P2Y2 signalling and that cellular responses elicited by UPEC and ATP-γ-S have many identical features. This indicates that renal epithelial responses elicited by bacteria could be mediated by bacteria- or host-derived ATP, thus defining a key role of ATP during infection.

Characterization of CTX-M-producing Escherichia coli by repetitive sequence-based PCR and real-time PCR-based replicon typing of CTX-M-15 plasmids.

The emergence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is a major global concern. CTX-M is the dominating ESBL type worldwide, and CTX-M-15 is the most widespread CTX-M type. The dissemination of CTX-M appears to be in part due to global spread of the Escherichia coli clone O25b-ST131. However, the gene-encoding CTX-M is mainly located on mobile genetic elements, such as plasmids, that also promote the horizontal dissemination of the CTX-M genes. In this study, 152 CTX-M-producing E. coli isolated in 1999-2008 in Örebro County, Sweden, were typed using a commercial repetitive sequence-based PCR (the DiversiLab system), and the prevalence of ST131 was investigated by pabB PCR. Real-time PCR-based plasmid replicon typing was performed on 82 CTX-M-15-producing E. coli isolates. In general, the CTX-M-producing E. coli population was genetically diverse; however, ST131 was highly prevalent (27%), and the dominating clone in our area. The blaCTX -M-15 gene was mainly located on IncF plasmids (69%), but a relatively high proportion of IncI1 plasmids (29%) were also detected among E. coli with diverse rep-PCR patterns, indicating that horizontal transmission of IncI1 plasmids carrying blaCTX -M-15 may have occurred between different E. coli strains.

The antibacterial effect of nitric oxide against ESBL-producing uropathogenic E. coli is improved by combination with miconazole and polymyxin B nonapeptide.

BACKGROUND: Nitric oxide (NO) is produced as part of the host immune response to bacterial infections, including urinary tract infections. The enzyme flavohemoglobin, coded by the hmp gene, is involved in protecting bacterial cells from the toxic effects of NO and represents a potentially interesting target for development of novel treatment concepts against resistant uropathogenic bacteria. The aim of the present study was to investigate if the in vitro antibacterial effects of NO can be enhanced by pharmacological modulation of the enzyme flavohemoglobin. RESULTS: Four clinical isolates of multidrug-resistant extended-spectrum ß-lactamase (ESBL)-producing uropathogenic E. coli were included in the study. It was shown that the NO-donor substance DETA/NO, but not inactivated DETA/NO, caused an initial growth inhibition with regrowth noted after 8 h of exposure. An hmp-deficient strain showed a prolonged growth inhibition in response to DETA/NO compared to the wild type. The imidazole antibiotic miconazole, that has been shown to inhibit bacterial flavohemoglobin activity, prolonged the DETA/NO-evoked growth inhibition. When miconazole was combined with polymyxin B nonapeptide (PMBN), in order to increase the bacterial wall permeability, DETA/NO caused a prolonged bacteriostatic response that lasted for up to 24 h. CONCLUSION: An NO-donor in combination with miconazole and PMBN showed enhanced antimicrobial effects and proved effective against multidrug-resistant ESBL-producing uropathogenic E. coli.

Multiresistant uropathogenic extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli are susceptible to the carbon monoxide releasing molecule-2 (CORM-2).

Carbon monoxide (CO) releasing molecules (CO-RMs) have been shown to inhibit growth of commensal Escherichia coli (E. coli). In the present study we examined the effect of CORM-2 on uropathogenic E. coli (UPEC) that produces extended-spectrum ß-lactamase (ESBL). Viability experiments showed that CORM-2 inhibited the growth of several different ESBL-producing UPEC isolates and that 500 µM CORM-2 had a bactericidal effect within 4 h. The bactericidal effect of CORM-2 was significantly more pronounced than the effect of the antibiotic nitrofurantoin. CORM-2 demonstrated a low level of cytotoxicity in eukaryotic cells (human bladder epithelial cell line 5637) at the concentrations and time-points where the antibacterial effect was obtained. Real-time RT-PCR studies of different virulence genes showed that the expression of capsule group II kpsMT II and serum resistance traT was reduced and that some genes encoding iron acquisition systems were altered by CORM-2. Our results demonstrate that CORM-2 has a fast bactericidal effect against multiresistant ESBL-producing UPEC isolates, and also identify some putative UPEC virulence factors as targets for CORM-2. CO-RMs may be candidate drugs for further studies in the field of finding new therapeutic approaches for treatment of uropathogenic ESBLproducing E. coli.

Comparison of host response mechanisms evoked by extended spectrum beta lactamase (ESBL)--and non-ESBL-producing uropathogenic E. coli.

BACKGROUND: Infections caused by extended spectrum beta-lactamases (ESBL)-producing bacteria have been emerging worldwide and the majority of ESBL-producing E. coli strains are isolated from patients with urinary tracts infections. The purpose of this study was to compare the host-response mechanisms in human polymorphonucleated leukocytes (PMN) and renal epithelial cells when stimulated by ESBL- or non-ESBL-producing uropathogenic E. coli (UPEC) isolates. The host-pathogen interaction of these ESBL-producing strains in the urinary tract is not well studied. RESULTS: The ability of ESBL strains to evoke ROS-production from PMN cells was significantly higher than that of the non-ESBL strains. The growth of ESBL strains was slightly suppressed in the presence of PMN compared to non-ESBL strains after 30 min and 2h, but the opposite was observed after 5 and 6h. The number of migrating PMN was significantly higher in response to ESBL strains compared to non-ESBL strains. Stimulation of A498 cells with ESBL strains elicited lower production of IL-6 and IL-8 compared to non-ESBL strains. CONCLUSION: Significant differences in host-response mechanisms were identified when host cells were stimulated by ESBL- or non-ESBL producing strains. The obtained results on the early interactions of ESBL-producing strains with the host immune system may provide valuable information for management of these infections.

Expression of suppressor of cytokine signalling 3 (SOCS3) in human bladder epithelial cells infected with uropathogenic Escherichia coli.

Suppressor of cytokine signalling (SOCS) proteins inhibit pro-inflammatory signalling mediated by Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) pathways. To evade the immune response some pathogens appear to modify the host SOCS proteins. Uropathogenic Escherichia coli (UPEC) are able to subvert the host response evoked by bladder epithelial cells, but the mechanisms are not fully understood. The objective of this study was to investigate whether UPEC can modify the host SOCS and STAT3 response. Real time RT-PCR studies demonstrated an increased SOCS1 and SOCS3 expression in the isolated human bladder epithelial cell lines (RT-4 and 5637) in response to cytokines. UPEC strain IA2 increased SOCS3, but not SOCS1, mRNA levels with a peak at 6 h after infection. The increase of SOCS3 was confirmed at the protein level by Western blotting. The UPEC strain IA2 caused a time-dependent decrease in the phosphorylation of STAT3. This study demonstrates that UPEC are able to affect SOCS3 and STAT3 signalling in human uroepithelial cells. The finding that UPEC are able to induce mediators involved in suppression of host cytokine signalling may help to elucidate how UPEC may circumvent the host response during urinary tract infection.