Role of flavohemoglobin in combating nitrosative stress in uropathogenic Escherichia coliimplications for urinary tract infection. During the course of urinary tract infection (UTI) nitric oxide (NO) is generated as part of host response. The aim of this study was to investigate the significance of the NO-detoxifying enzymes flavohemoglobin (Hmp) and flavorubredoxin (Norv) in protection of uropathogenic Escherichia coli (UPEC) against nitrosative stress. Hmp (J96∆hmp) and norV (J96∆norV) knockout mutants of UPEC strain J96 were constructed using a single-gene deletion strategy. Bacterial tolerance and expression of hmp and norV in response to the NO-donor DETA/NO was evaluated in Luria broth and urine from healthy volunteers. Bacterial NO consumption and respiratory inhibition were assessed when exposed to NO. Expression of hmp and norV from E. coli originating from patients with UTI was evaluated using real-time PCR. The colonizing ability of J96 wild-type (wt) compared to an hmp-deficient mutant was assessed using a competition-based mouse UTI model. The viability of J96∆hmp and J96∆norV was significantly reduced compared to the wildtype strain after exposure to DETA/NO. The hmp expression in DETA/NO-exposed cultures was similar in J96wt and J96∆norV, while J96∆hmp showed an increased norV expression compared to J96wt. The NO consumption in J96∆hmp, but not in J96∆norV, was significantly impaired compared to J96wt. An up-regulation of hmp expression was found in E. coli isolated from all UTIpatients while norV expression increased in 50% of the patients. In the mouse UTI model, the hmp-mutant strain was significantly out-competed by the wild-type strain in the bladder and kidney. Hmp and NorV contribute to the protection of UPEC against NOmediated toxicity in vitro. Screening UPEC isolates from UTI patients revealed an increased hmp expression in all patients which confirms that hmp expression occurs in vivo in the infected human urinary tract. The ability to colonize the mouse urinary tract was impaired in the hmp-deficient mutant compared to the wild-type strain. NO-detoxification by Hmp is suggested to be an important characteristic for UPEC in protection against nitrosative stress and may be a virulence-facilitating factor.

Extracellular ATP is released from a variety of cells not only as a consequence of cell injury or cell death but also via nonlytic mechanisms (25). ATP has attracted increasing attention as a danger signal released during the initial phase of infection and inflammation in order to trigger innate immunity (18). In a healthy bladder, release of ATP from uroepithelial cells by stretch and distension mediates the sensation of bladder fullness by activation of P2X 3 receptors on suburothelial sensory nerves (5). Pathophysiological conditions cause enhanced bladder release of ATP as shown in patients diagnosed with interstitial cystitis (29,30,31). Although several studies have shown that pathology often results in augmented ATP release from the urothelium, there have not been any studies of ATP release from urothelium infected with bacteria. Patients with bacteriuria have increased urinary levels of ATP (21), but it is not known whether the bacteria, host, or both produce ATP during infection. In their function as extracellular mediators, nucleotides activate P2 nucleotide receptors that include the ionotrophic P2X receptors (P2X 1 to P2X 7 ) and G-proteincoupled P2Y receptors (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 to P2Y 14 ) (33). In the urinary tract, extracellular ATP may communicate through P2X and P2Y receptors with different cells, such as uroepithelial cells, inflammatory cells, nerves, and myofibroblasts (5). Evidence that P2 receptors, in particular P2Y receptors, may regulate host immunity and modulate phagocytosis, chemotaxis, and cytokine production is now accumulating (14, 16). The P2Y receptor subtypes P2Y 1 , P2Y 2 , and P2Y 4 are expressed throughout the cat bladder urothelium (7), but P2Y receptor expression has not been examined in the human urothelium.Innate immunity is the first line of defense in the urinary tract and triggered by uropathogenic bacteria through activation of Toll-like receptors (TLRs) located on the uroepithelial cell surface (32). Lipopolysaccharide (LPS) is an established ligand for Toll-like receptor 4 (TLR4), but associated coeffector proteins, such as CD14 and MD2, are needed for LPS-induced stimulation of TLR4 (4, 27). TLR4 and CD14 expression by the human urinary tract epithelium remains controversial (3,23,24), and in vitro studies show that the TLR4 receptor can be activated in a LPS-independent manner by P-fimbriated uropathogenic Escherichia coli (UPEC) (12). Once activated, the uroepithelial cells play a central role in the host proinflammatory response through production of chemotactic substances, such as interleukin 6 (IL-6) and IL-8. Several lines of evidence demonstrate that IL-8 is essential for neutrophil transmigration across the infected urothelium and that neutrophils are important for clearance of bacterial infections in the urinary tract (32).

Interleukin-8 and 6 release after purinergic stimulation in uroepithelial A498 cells is mediated through P2Y(2) receptor activation.

Background/Aims: Increased nitric oxide (NO) production or inducible form of NO synthase activity have been documented in patients suffering from urinary tract infection (UTI), but the role of NO in this infection is unclear. We investigated whether NO can affect the host response in human renal epithelial cells by modulating IL-6 production and mRNA expression. Methods: The human renal epithelial cell line A498 was infected with a uropathogenic Escherichia coli (UPEC) strain and/or the NO donor DETA/NO. The IL-6 production and mRNA expression were evaluated by ELISA and real-time RT-PCR. IL-6 mRNA stability was evaluated by analyzing mRNA degradation by real-time RT-PCR. Results: DETA/NO caused a significant (p < 0.05) increase in IL-6 production. Inhibitors of p38 MAPK and ERK1/2 signaling, but not JNK, were shown to significantly suppress DETA/NO-induced IL-6 production. UPEC-induced IL-6 production was further increased (by 73 ± 23%, p < 0.05) in the presence of DETA/NO. The IL-6 mRNA expression increased 2.1 ± 0.17-fold in response to DETA/NO, while the UPEC-evoked increase was pronounced (20 ± 4.5-fold). A synergistic effect of DETA/NO on UPEC-induced IL-6 expression was found (33 ± 7.2-fold increase).The IL-6 mRNA stability studies showed that DETA/NO partially attenuated UPEC-induced degradation of IL-6 mRNA. Conclusions: NO was found to stimulate IL-6 in renal epithelial cells through p38 MAPK and ERK1/2 signaling pathways and also to increase IL-6 mRNA stability in UPEC-infected cells. This study proposes a new role for NO in the host response during UTI by modulating the transcription and production of the cytokine IL-6.