Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli.

The Toll/IL-1 receptor (TIR) domains are crucial signaling modules during innate immune responses involving the Toll-like receptors (TLRs) and IL-1 receptor (IL-1R). Myeloid differential factor 88 (MyD88) is a central TIR domain-containing adapter molecule responsible for nearly all TLR-mediated signaling and is targeted by a TIR domain-containing protein C (TcpC) from virulent uropathogenic Escherichia coli, a common human pathogen. The mechanism of such molecular antagonism has remained elusive. We present the crystal structure of the MyD88 TIR domain with distinct loop conformations that underscore the functional specialization of the adapter, receptor, and microbial TIR domains. Our structural analyses shed light on the genetic mutations at these loops as well as the Poc site. We demonstrate that TcpC directly associates with MyD88 and TLR4 through its predicted DD and BB loops to impair the TLR-induced cytokine induction. Furthermore, NMR titration experiments identify the unique CD, DE, and EE loops from MyD88 at the TcpC-interacting surface, suggesting that TcpC specifically engages these MyD88 structural elements for immune suppression. These findings thus provide a molecular basis for the subversion of TLR signaling by the uropathogenic E. coli virulence factor TcpC and furnish a framework for the design of novel therapeutic agents that modulate immune activation.

Guanine modification of inhibitory oligonucleotides potentiates their suppressive function.

Inhibitory TLR7 and/or TLR9 oligonucleotides (inhibitory oligonucleotide [INH-ODN]) are characterized by a phosphorothioate backbone and a CC(T)XXX3₋5GGG motif, respectively. INH-ODN 2088 is a prototypic member of this class of INH-ODN and acts as a TLR7 and TLR9 antagonist. It contains a G quadruple that leads to higher order structures by the formation of G tetrads. These structures are unfavorable for the prediction of their pharmacological and immunological behavior. We show in this study that modification of Gs within the G quadruple by 7-deaza-guanine or 7-deaza-2'-O-methyl-guanine avoids higher order structures and improves their inhibitory potential. Whereas TLR9-induced TNF-α secretion of bone marrow-derived macrophages and conventional dendritic cells was equally inhibited by INH-ODN 2088 and G-modified INH-ODNs such as INH-ODN 24888, TLR7-induced TNF-α release and TLR7- and TLR9-induced IL-12p40 release were significantly more impaired by G-modified INH-ODNs. Similarly, the IL-6 release of B cells from wild-type and autoimmune MRL/Mp-lpr/lpr mice was more efficiently impaired by G-modified INH-ODNs. Surprisingly, INH-ODN 2088 stimulated B cells to proliferate when used in higher doses. Finally, in vivo, in wild-type and autoimmune MRL/Mp-lpr/lpr mice, G-modified INH-ODN 24888 was significantly more efficient than unmodified INH-ODN 2088. In summary, G modification allows the development of INH-ODNs with superior inhibitory potency for inflammatory diseases with high medical need such as systemic lupus erythematosus.

Risk factors for increased antimicrobial resistance: a retrospective analysis of 309 acute cholangitis episodes.

OBJECTIVES: To assess the risk factors for increased antimicrobial resistance among Enterobacteriaceae representing the most common biliary pathogens. METHODS: A retrospective analysis was conducted of 276 patients with acute cholangitis treated at a German tertiary centre between April 1996 and May 2009. The resistance patterns among Enterobacteriaceae isolated from blood/bile cultures were compared and related to age, sex, the genesis of the cholangitis and the type and number of previous interventional procedures [percutaneous transhepatic cholangiography (PTC)/endoscopic retrograde cholangiography (ERC)]. Univariate and multivariate generalized estimation equation models were used to compute ORs with corresponding 95% CIs for the binomial outcomes. RESULTS: According to the univariate analysis, patients undergoing stent therapy had a smaller proportion of Enterobacteriaceae with susceptibility to quinolones (ofloxacin/ciprofloxacin) (184/239 versus 205/221; P < 0.001) and to ceftriaxone (208/239 versus 209/222; P = 0.014). Logistic regression analysis revealed that the odds for acquiring ceftriaxone-resistant Enterobacteriaceae were 4-fold higher than in patients who had not undergone stent therapy (P = 0.039). Furthermore, an increased number of interventional procedures (PTC/ERC) was associated with lower susceptibility. The odds for susceptibility to ampicillin, ampicillin/sulbactam, ceftriaxone, quinolones and co-trimoxazole decreased by 2%, 2%, 4%, 6% and 3%, respectively, per interventional procedure. Age, sex and type of interventional procedure displayed no significant relationship to the development of antimicrobial resistance. CONCLUSIONS: Stent therapy was found to be a risk factor for increased antimicrobial resistance in patients with acute cholangitis, particularly those who had undergone numerous interventional procedures prior to the onset of the cholangitis.

A Comparative Analysis of the Mechanism of Toll-Like Receptor-Disruption by TIR-Containing Protein C from Uropathogenic Escherichia coli.

The TIR-containing protein C (TcpC) of uropathogenic Escherichia coli strains is a powerful virulence factor by impairing the signaling cascade of Toll-like receptors (TLRs). Several other bacterial pathogens like Salmonella, Yersinia, Staphylococcus aureus but also non-pathogens express similar proteins. We discuss here the pathogenic potential of TcpC and its interaction with TLRs and TLR-adapter proteins on the molecular level and compare its activity with the activity of other bacterial TIR-containing proteins. Finally, we analyze and compare the structure of bacterial TIR-domains with the TIR-domains of TLRs and TLR-adapters.

Uropathogenic Escherichia coli strain CFT073 disrupts NLRP3 inflammasome activation.

Successful bacterial pathogens produce an array of virulence factors that allow subversion of the immune system and persistence within the host. For example, uropathogenic Escherichia coli strains, such as CFT073, express Toll/IL-1 receptor-containing (TIR-containing) protein C (TcpC), which impairs TLR signaling, thereby suppressing innate immunity in the urinary tract and enhancing persistence in the kidneys. Here, we have reported that TcpC also reduces secretion of IL-1ß by directly interacting with the NACHT leucin-rich repeat PYD protein 3 (NLRP3) inflammasome, which is crucial for recognition of pathogens within the cytosol. At a low MOI, IL-1ß secretion was minimal in CFT073-infected macrophages; however, IL-1ß release was markedly increased in macrophages infected with CFT073 lacking tcpC. Induction of IL-1ß secretion by CFT073 and tcpC-deficient CFT073 required the NLRP3 inflammasome. TcpC attenuated activation of the NLRP3 inflammasome by binding both NLRP3 and caspase-1 and thereby preventing processing and activation of caspase-1. Moreover, in a murine urinary tract infection model, CFT073 infection rapidly induced expression of the NLRP3 inflammasome in the bladder mucosa; however, the presence of TcpC in WT CFT073 reduced IL-1ß levels in the urine of infected mice. Together, these findings illustrate how uropathogenic E. coli use the multifunctional virulence factor TcpC to attenuate innate immune responses in the urinary tract.

Guanine-modified inhibitory oligonucleotides efficiently impair TLR7- and TLR9-mediated immune responses of human immune cells.

Activation of TLR7 and TLR9 by endogenous RNA- or DNA-containing ligands, respectively, is thought to contribute to the complicated pathophysiology of systemic lupus erythematosus (SLE). These ligands induce the release of type-I interferons by plasmacytoid dendritic cells and autoreactive antibodies by B-cells, both responses being key events in perpetuating SLE. We recently described the development of inhibitory oligonucleotides (INH-ODN), which are characterized by a phosphorothioate backbone, a CC(T)XXX3-5GGG motif and a chemical modification of the G-quartet to avoid the formation of higher order structures via intermolecular G-tetrads. These INH-ODNs were equally or significantly more efficient to impair TLR7- and TLR9-stimulated murine B-cells, macrophages, conventional and plasmacytoid dendritic cells than the parent INH-ODN 2088, which lacks G-modification. Here, we evaluate the inhibitory/therapeutic potential of our set of G-modified INH-ODN on human immune cells. We report the novel finding that G-modified INH-ODNs efficiently inhibited the release of IFN-α by PBMC stimulated either with the TLR7-ligand oligoribonucleotide (ORN) 22075 or the TLR9-ligand CpG-ODN 2216. G-modification of INH-ODNs significantly improved inhibition of IL-6 release by PBMCs and purified human B-cells stimulated with the TLR7-ligand imiquimod or the TLR9-ligand CpG-ODN 2006. Furthermore, inhibition of B-cell activation analyzed by expression of activation markers and intracellular ATP content was significantly improved by G-modification. As observed with murine B-cells, high concentrations of INH-ODN 2088 but not of G-modified INH-ODNs stimulated IL-6 secretion by PBMCs in the absence of TLR-ligands thus limiting its blocking efficacy. In summary, G-modification of INH-ODNs improved their ability to impair TLR7- and TLR9-mediated signaling in those human immune cells which are considered as crucial in the pathophysiology of SLE.

Microbial colonization of pancreatic duct stents: a prospective analysis.

OBJECTIVE: The aim of the study was to analyze the microbial colonization rate as well as the spectrum and number of microorganisms in relation to the indwelling time of pancreatic stents. METHODS: Forty pancreatic stents were prepared according to a standardized protocol and subsequently sonicated to optimize bacterial release from the biofilm on the stents. RESULTS: Two hundred forty-six microorganisms were identified. Thirty-nine of 40 stents were colonized with microorganisms. Aerobic gram-positive microorganisms (106/246 [43%]) accounted for the greatest proportion. The predominant microorganisms were Streptococcus species (46/246 [19%]), which were isolated from 27 (68%) of 40 stents. Stents with a short indwelling time (3-13 days) were mainly colonized with aerobic gram-positive bacteria (82%) and Candida species (63%). In contrast, anaerobes (P < 0.01, 69% vs 18%) and aerobic gram-negative microorganisms (P < 0.01, 93% vs 45%) such as Enterobacteriaceae (P < 0.01, 86% vs 27%) were significantly more present on stents with a long indwelling time (29-93 days), compared with stents with a short indwelling time. CONCLUSIONS: Microbial analysis of pancreatic duct stents revealed a very high colonization rate. Furthermore, the spectrum and number of microorganisms altered with the indwelling time of the stent. However, clinical relevance of our findings remains unclear.

Granzyme A produces bioactive IL-1ß through a nonapoptotic inflammasome-independent pathway.

Bacterial components are recognized by the immune system through activation of the inflammasome, eventually causing processing of the proinflammatory cytokine interleukin-1? (IL-1?), a pleiotropic cytokine and one of the most important mediators of inflammation, through the protease caspase-1. Synthesis of the precursor protein and processing into its bioactive form are tightly regulated, given that disturbed control of IL-1? release can cause severe autoinflammatory diseases or contribute to cancer development. We show that the bacterial Pasteurella multocida toxin (PMT) triggers Il1b gene transcription in macrophages independently of Toll-like receptor signaling through RhoA/Rho-kinase-mediated NF-?? activation. Furthermore, PMT mediates signal transducer and activator of transcription (STAT) protein-controlled granzyme A (a serine protease) expression in macrophages. The exocytosed granzyme A enters target cells and mediates IL-1? maturation independently of caspase-1 and without inducing cytotoxicity. These findings show that macrophages can induce an IL-1?-initiated immune response independently of inflammasome activity.

Spectrum of pathogens in acute cholangitis in patients with and without biliary endoprosthesis.

BACKGROUND: Knowledge of bacterial spectrum for acute cholangitis is essential for adequate empiric antibiotic treatment. Main focus of the study was to analyse the spectrum of pathogens in acute cholangitis with and without biliary endoprosthesis. METHODS: Retrospective cohort study of 1024 patients with acute cholangitis treated at a German tertiary center. RESULTS: 447 cholangitis episodes with positive bile and/or blood cultures obtained from 388 patients were studied. In total, 1088 pathogen were isolated. The predominant strains were Enterococcus species (25%), followed by Escherichia coli (18%) and Klebsiella species (14%). Bacteraemia was mainly caused by E. coli (91/282; 32%) and Enterococcus species (550/282; 18%). The incidences of Enterococcus species [121(74%) vs. 89(60%); p = 0.011] and non-fermenters [41(25%) vs. 16(11%); p = 0.001] were significantly higher in cholangitis episodes with biliary endoprosthesis compared to cholangitis episodes without biliary endoprosthesis. In particular, more Pseudomonas aeruginosa [27(16%) vs. 12(8%); p = 0.027] and Enterococcus faecium [59(36%) vs. 34(23%); p = 0.013] were isolated from patients with a biliary endoprosthesis. CONCLUSIONS: Unlike cholangitis without stent, the presence of biliary endoprosthesis in patients with cholangitis can serve as a surrogate indicator of nosocomial pathogens and therefore should be considered, when selecting empiric antimicrobial therapy.