Pseudomonas aeruginosa is an opportunistic Gram-negative human pathogen that is responsible for a broad range of infections in individuals with a variety of predisposing conditions. After infection, P. aeruginosa induces a marked inflammatory response in the host. However the mechanisms involved in bacterium recognition and induction of immune responses are poorly understood. Here we report that the Nod-like receptor family member Ipaf is required for optimal bacterial clearance in an in vivo model of P. aeruginosa lung infection. Further analysis showed that bacterial flagellin was essential for caspase-1 and IL-1b and this activity depended on Ipaf and the adaptor ASC but not TLR5. Notably, P. aeruginosa induced macrophage cell death and this event relied on flagellin and Ipaf but not on ASC. Analysis of Pseudomonas mutants revealed that different amino acid residues of flagellin were critical for sensing by Ipaf and TLR5. Finally, activation of caspase-1 and IL-1b secretion by P. aeruginosa required a functional type III secretion system, but not the effector molecules ExoS, ExoT and ExoY. These results provide new insight into the interaction of P. aeruginosa with host macrophages and suggest that distinct regions of flagellin are sensed by Ipaf and TLR5. Introduction IL-1b plays an important role in the induction of immune responses and in the development of inflammatory disease, fever and septic shock [1]. In response to proinflammatory stimuli including pathogenic bacteria, the IL-1b precursor is induced in monocytes and macrophages and processed into the biologically active IL-1b molecule by caspase-1 [2][3][4][5]. The protease caspase-1 is expressed in monocytes/macrophages as an inactive zymogen that is activated by self cleavage in large multi-protein complexes named 'inflammasomes' [6].The mechanism responsible for activation of caspase-1 in response to microbial stimuli has remained poorly understood. Recent studies have revealed members of the Nod-like receptor (NLR) family as critical components of the inflammasomes by linking microbial sensing to caspase-1 activation [7,8]. For example, Ipaf, an NLR family member and the adaptor ASC have been implicated in activation of caspase-1 in response to Salmonella and Legionella through the cytosolic sensing of flagellin [7,9,10]. Notably, flagellin is also recognized by TLR5 [11]. However, it is unclear whether Ipaf and TLR5 sense identical or distinct regions of flagellin. Similarly, Cryopyrin/Nalp3 is critical for caspase-1 activation and secretion of IL-1b and IL-18 in response to microbial RNA, synthetic purine-like compounds and endogenous urate crystals [12][13][14]. In addition, Cryopyrin regulates caspase-1 activation triggered by exogenous ATP or pore-forming toxins in macrophages stimulated with several TLR agonists [15,16]. Pseudomonas aeruginosa is a flagellated opportunistic Gram-negative human pathogen that is responsible for a broad range of infections in individuals with a variety of predisposing conditions including cystic fibrosis, immu...

Despite evidence that hyperactivity of the vasodeleterious axis (ACE/angiotensin II (Ang II)/AT1 receptor) of the renin-angiotensin system (RAS) is associated with the pathogenesis of diabetic retinopathy (DR) use of the inhibitors of this axis has met with limited success in the control of this pathophysiology. We investigated the hypothesis that enhancing the local activity of the recently established protective axis of the RAS, ACE2/Ang-(1-7), using adeno-associated virus (AAV)-mediated gene delivery of ACE2 or Ang-(1-7) would confer protection against diabetes-induced retinopathy. Genes expressing ACE2 and Ang-(1-7) were cloned in AAV vector. The effects of ocular AAV-ACE2/Ang-(1-7) gene transfer on DR in diabetic eNOS(-/-) mice and Sprague-Dawley (SD) rats were examined. Diabetes was associated with approximately tenfold and greater than threefold increases in the ratios of ACE/ACE2 and AT1R/Mas mRNA levels in the retina respectively. Intraocular administration of AAV-ACE2/Ang-(1-7) resulted in significant reduction in diabetes-induced retinal vascular leakage, acellular capillaries, infiltrating inflammatory cells and oxidative damage in both diabetic mice and rats. Our results demonstrate that DR is associated with impaired balance of retinal RAS. Increased expression of ACE2/Ang-(1-7) overcomes this imbalance and confers protection against DR. Thus, strategies enhancing the protective ACE2/Ang-(1-7) axis of RAS in the eye could serve as a novel therapeutic target for DR.

Type a flagellins from two strains of Pseudomonas aeruginosa, strains PAK and JJ692, were found to be glycosylated with unique glycan structures. In both cases, two sites of O-linked glycosylation were identified on each monomer, and these sites were localized to the central, surface-exposed domain of the monomer in the assembled filament. The PAK flagellin was modified with a heterogeneous glycan comprising up to 11 monosaccharide units that were O linked through a rhamnose residue to the protein backbone. The flagellin of JJ692 was less complex and had a single rhamnose substitution at each site. The role of the glycosylation island gene cluster in the production of each of these glycosyl moieties was investigated. These studies revealed that the orfA and orfN genes were required for attachment of the heterologous glycan and the proximal rhamnose residue, respectively.Protein glycosylation in prokaryotic organisms is now a wellestablished process, particularly in cell surface-associated or secreted molecules (5). Very recently, a number of examples of glycosylated surface proteins of bacterial pathogens have been described, including surface proteins of Streptococcus sanguis and Mycobacterium tuberculosis (11,14), the TIB adhesin of Escherichia coli (TIB) (4, 26), flagellum and periplasmic proteins of Campylobacter (12,40,43,44), flagellum proteins of Helicobacter pylori (35), an outer membrane protein of Chlamydia (24), and pilus proteins of Neisseria species (37) and Pseudomonas aeruginosa (8). However, information about the structures of the linked glycans found on prokaryotic glycoproteins, the mechanistic basis of the process, and the biological role of the process in bacterial pathogenesis is still limited.P. aeruginosa is an opportunistic pathogen which causes lifethreatening infections in immunocompromised individuals and burn wound victims and chronic infections in patients with cystic fibrosis (6). This organism produces a number of virulence factors, including toxins, secreted proteins, surface carbohydrates (mucoid exopolysaccharide and lipopolysaccharide [LPS]), and pili (18,27). A more recent addition to the list of putative virulence factors is the single, polar, nonsheathed flagellum of this organism, which traditionally is considered a motility organ but whose chief constituent, flagellin, is now known to be a potent stimulator of the inflammatory response via Toll-like receptor 5 (19). The flagellin protein can be classified in one of two major types, type a or type b, based on molecular weight and reactivity with specific antisera (1, 25). The type a flagellin appears to have two major subtypes of proteins, designated subtypes A1 and A2 (3). Type a flagellins have been shown to be heterologous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and to have molecular masses ranging from 45 to 52 kDa, while type b flagellins appear to have conserved sequences and a molecular mass of 53 kDa. The central domain of type a flagellin is described as hypervariable, and this is be...

Acute lung infection due to Pseudomonas aeruginosa is an increasingly serious problem that results in high mortality especially in the compromised host. In this study, we set out to ascertain what components of the TLR system are most important for innate immunity to this microorganism. We previously demonstrated that TLR2,4−/− mice were not hypersusceptible to infection by a wild-type P. aeruginosa strain. However, we now find that mice lacking both TLR2 and TLR4 (TLR2,4−/− mice) are hypersusceptible to infection following challenge with a P. aeruginosa mutant devoid of flagellin production. We demonstrate that this hypersusceptibilty is largely due to a lack of innate defense by the host that fails to control bacterial replication in the lung. Further evidence that a response to flagellin is a key factor in the failure of TLR2,4−/− mice to control the infection with the mutant strain was obtained by demonstrating that the intrapulmonary administration of flagellin over a 18 h period following infection, saved 100% of TLR2,4−/− mice from death. We conclude that the interactions of either TLR4 with LPS or TLR5 with flagellin can effectively defend the lung from P. aeruginosa infection and the absence of a response by both results in hypersusceptibility to this infection.

The Toll-like receptor 5 (TLR5) binding site has been predicted to be in the N terminus of the flagellin molecule. In order to better define the interaction between the N-terminal amino acids of Pseudomonas aeruginosa flagellin and TLR5, site-specific mutations were generated between residues 88 and 97 of P. aeruginosa PAK flagellin as well as outside of this region. The mutant flagellins were expressed in Escherichia coli BL21(plysS), purified by affinity chromatography, and passed through a polymyxin B column to remove contaminating lipopolysaccharide (LPS). Their ability to stimulate interleukin-8 (IL-8) release from A549 cells was examined. The cloned mutated genes were used to complement a PAK fliC mutant in order to test for effects on motility and on IL-8 release by purified flagellar preparations. All the mutations, single or double, in the predicted TLR5 binding region reduced IL-8 signaling to less than 95% of the wild-type flagellin levels, but the single mutation outside the binding region had no effect. Changes made at two amino acid sites resulted in loss/reduction of motility; however, changes made at single sites, i.e., Q83A, L88A, R90A, M91A, L94A, and Q97A, had no effect on motility. The mutated genes encoding two of the motile but poorly signaling flagellins had no compensatory mutations to allow motility. Thus, while it is speculated that pathogen-associated molecular patterns (PAMPs) have evolved in locations that are essential to maintain function, it appears that there is tolerance for at least single amino acid changes in the PAMP of P. aeruginosa flagellin. The purpose of flagellin glycosylation in P. aeruginosa is unknown. In order to examine its role, if any, in signaling an inflammatory response, we used whole flagella from the motile chromosomal mutant strains PAKrfbC and PAO1rfbC, which are defective in flagellin glycosylation. IL-8 release from A549 cells stimulated with nonglycosylated flagellar preparations (having less then 1 picogram of LPS/g) was significantly reduced compared to their respective wild-type flagellar preparations, indicating a role of flagellar glycosylation in the proinflammatory action of Pseudomonas flagellin. The basis of the latter activity is unknown, since the glycosylation sites are found in the D3 domain of flagellins and the TLR5 binding site is located in the D1 domain. Thus, P. aeruginosa flagellin has evolved additional flagellar signaling mechanisms over that described for Salmonella flagellin.