Immune recognition of Pseudomonas aeruginosa mediated by the IPAF/NLRC4 inflammasome

Pseudomonas aeruginosa is a Gram-negative bacterium that causes opportunistic infections in immunocompromised individuals. P. aeruginosa employs a type III secretion system to inject effector molecules into the cytoplasm of the host cell. This interaction with the host cell leads to inflammatory responses that eventually result in cell death. We show that infection of macrophages with P. aeruginosa results in activation of caspase-1 in an IPAF-dependent, but flagellin-independent, manner. Macrophages deficient in IPAF or caspase-1 were markedly resistant to P. aeruginosa–induced cell death and release of the proinflammatory cytokine interleukin (IL)-1β. A subset of P. aeruginosa isolates express the effector molecule exoenzyme U (ExoU), which we demonstrate is capable of inhibiting caspase-1–driven proinflammatory cytokine production. This study shows a key role for IPAF and capase-1 in innate immune responses to the pathogen P. aeruginosa, and also demonstrates that virulent ExoU-expressing strains of P. aeruginosa can circumvent this innate immune response

SAP Regulates TH2 Differentiation and PKC-θ-Mediated Activation of NF-κB1

AbstractXLP is caused by mutations affecting SAP, an adaptor that recruits Fyn to SLAM family receptors. SAP-deficient mice recapitulate features of XLP, including increased T cell activation and decreased humoral responses post-infection. SAP-deficient T cells also show increased TCR-induced IFN-γ and decreased TH2 cytokine production. We demonstrate that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-γ production. SAP-deficient cells respond normally to polarizing cytokines, yet show impaired TCR-mediated induction of GATA-3 and IL-4. Examination of TCR signaling revealed normal Ca2+ mobilization and ERK activation in SAP-deficient cells, but decreased PKC-θ recruitment, Bcl-10 phosphorylation, IκB-α degradation, and nuclear NF-κB1/p50 levels. Similar defects were observed in Fyn-deficient cells. SLAM engagement amplified PKC-θ recruitment in wt but not SAP- or Fyn-deficient cells, arguing that a SAP/Fyn-mediated pathway enhances PKC-θ/NF-κB1 activation and suggesting a role for this pathway in TH2 regulation

In Vivo Discrimination of Type 3 Secretion System-Positive and -Negative Pseudomonas aeruginosa via a Caspase-1-Dependent Pathway▿

Microbe-associated molecular patterns are recognized by Toll-like receptors of the innate immune system. This recognition enables a rapid response to potential pathogens but does not clearly provide a way for the innate immune system to discriminate between virulent and avirulent microbes. We find that pulmonary infection of mice with type 3 translocation-competent Pseudomonas aeruginosa triggers a rapid inflammatory response, while infection with isogenic translocation-deficient mutants does not. Discrimination between translocon-positive and -negative bacteria requires caspase-1 activity in bone marrow-derived cells and interleukin-1 receptor signaling. Thus, the activation of caspase-1 by bacteria expressing type 3 secretion systems allows for rapid recognition of bacteria expressing conserved functions associated with virulence

Molecular typing and growth curves of serially isolated strains of <i>B. petrii.</i>.

<p>(A) The DiversiLab Non fermentor typing kit was used for rep-PCR typing of <i>B. petrii</i> using DNA from clinical and reference strains. Amplicons were detected with the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA) and data analyzed with the DiversiLab software (version 3.3). Results generated include a dendrogram (left) and virtual gel images (right). (B) Genomic DNA was digested with the restriction endonuclease <i>XbaI</i> and separated by PFGE with a CHEF Mapper system. Asterisks indicate band differences among the patient strains. Ladder: Lambda DNA Ladder 48.5 KB–1 MB kb plugs (Lonza). (C) Growth curves were performed on LB broth at 37°C and growth assessed by colony-forming units (CFU) performed with serially diluted aliquots plated on SBA plates. Graph shows mean and SEM from three experiments. <i>B. petrii</i> 1–5: strains of <i>B. petrii</i> serially isolated from our patient; BAA-461: type strain of <i>B. petrii</i> (ATCC BAA-461); 13363: first described clinical strain of <i>B. petrii</i> (NCTC 13363).</p

Serum susceptibility of <i>B. petrii</i> 1 and <i>B. petrii</i> 3.

<p>Bacterial colonies grown on SBA were resuspended in 1% proteose peptone - phosphate-buffered saline (PP-PBS) to a concentration of 1×10⁷ CFU/ml. A 100-µl aliquot was then combined with an equal volume of 10% normal human serum (NHS) diluted in 1% PP-PBS. A 1% PP-PBS (0% serum) solution and heat-inactivated (56°C for 1 hour) normal human serum (HI-NHS) served as controls. Samples were incubated for 2 hours at 37°C with shaking. After incubation, samples were serially diluted, plated onto sheep blood agar plates, and grown 24–48 h at 37°C to determine the number of CFU and calculate % of survival.</p

Immunoblots with patient’s serum against her own and reference strains.

<p>An aliquot containing 10 µg of protein from the soluble (A) and insoluble (B) fractions obtained from <i>B. petrii</i> strains was electrophoresed on 12% SDS–PAGE, and then transferred to PVDF membrane. The membrane was incubated with the patient serum (1∶5000 dilution) obtained ∼2 months after isolation of <i>B. petrii</i> 3 and then horseradish peroxidase conjugated sheep anti-human IgG (1∶10000). The blots were developed using the enhanced chemiluminescence kit. <i>B. petrii</i> 4, <i>B. petrii</i> 4b, <i>B. petrii</i> 4c and <i>B. petrii</i> 4d refer to four different colonies obtained from the primary isolation plate of <i>B. petrii</i> 4. Fig. 2B had a shorter exposure time than Fig. 2A, so bands could be better visualized. If using similar exposure times, the intensity of the bands in Fig. 2B is 48% higher than in Fig. 2A (as determined by densitometric analysis).</p