Pseudomonas aeruginosa-Derived DnaJ Induces the Expression of IL-1ß by Engaging the Interplay of p38 and ERK Signaling Pathways in Macrophages.

As members of pathogen-associated molecular patterns, bacterial heat shock proteins (HSPs) are widely recognized for their role in initiating innate immune responses. This study aimed to examine the impact of DnaJ, a homolog of HSP40 derived from Pseudomonas aeruginosa (P. aeruginosa), on the regulation of IL-1ß expression in macrophages. We demonstrated that DnaJ modulates macrophages to secrete IL-1ß by activating NF-κB and MAPK signaling pathways. Specifically, ERK was identified as a positive mediator for IL-1ß expression, while p38 acted as a negative mediator. These results suggest that the reciprocal actions of these two crucial MAPKs play a vital role in controlling IL-1ß expression. Additionally, the reciprocal actions of MAPKs were found to regulate the activation of inflammasome-related molecules, including vimentin, NLRP3, caspase-1, and GSDMD. Furthermore, our investigation explored the involvement of CD91/CD40 in ERK signaling-mediated IL-1ß production from DnaJ-treated macrophages. These findings emphasize the importance of understanding the signaling mechanisms underlying IL-1ß induction and suggest the potential utility of DnaJ as an adjuvant for stimulating inflammasome activation.

MvaT binds to the PexsC promoter to repress the type III secretion system in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a variety of acute and chronic infections. Its type III secretion system (T3SS) plays a critical role in pathogenesis during acute infection. ExsA is a master regulator that activates the expression of all T3SS genes. Transcription of exsA is driven by two distinct promoters, its own promoter PexsA and its operon promoter PexsC. Here, in combination with a DNA pull-down assay and mass spectrometric analysis, we found that a histone-like nucleoid-structuring (H-NS) family protein MvaT can bind to the PexsC promoter. Using EMSA and reporter assays, we further found that MvaT directly binds to the PexsC promoter to repress the expression of T3SS genes. The repression of MvaT on PexsC is independent of ExsA, with MvaT binding to the -429 to -380 bp region relative to the transcription start site of the exsC gene. The presented work further reveals the complex regulatory network of the T3SS in P. aeruginosa.

DnaJ-induced miRNA-146a negatively regulates the expression of IL-8 in macrophages.

As a member of the damage-associated molecular patterns, heat shock proteins (HSPs) are widely recognized for their role in initiating innate immune responses. These highly conserved proteins are expressed ubiquitously in both prokaryotes and eukaryotes. In this study, our aim was to investigate how DnaJ, a HSP40 homolog derived from Pseudomonas aeruginosa (P. aeruginosa), influences the regulation of IL-8 expression in macrophages. Treatment with DnaJ served as a stimulus, inducing a more robust expression of IL-8 compared to other HSP homologs, including DnaK, GroEL, and HtpG. This effect was achieved through the activation of the NF-κB signaling pathway. Interestingly, DnaJ treatment also significantly increased the expression of microRNA-146a (miR-146a), which appears to play a role in modulating the expression of innate defense genes. As a consequence, pre-treatment with DnaJ led to a reduction in the extent of IL-8 induction in response to P. aeruginosa treatment. Notably, this reduction was counteracted by transfection of a miR-146a inhibitor, highlighting the involvement of miR-146a in P. aeruginosa-mediated induction of IL-8 expression. Therefore, this study uncovers the role of DnaJ in triggering the expression of miR-146a, which, in turn, modulates the excessive expression of IL-8 induced by P. aeruginosa infection.

MvfR Controls Tolerance to Polymyxin B by Regulating rfaD in Pseudomonas aeruginosa.

Polymyxins are currently the last-resort antibiotics for the treatment of multidrug-resistant Gram-negative bacterial infections. To expand the understanding of the intrinsic resistance mechanism against polymyxins, a laboratory strain of Pseudomonas aeruginosa PAO1 was subjected to serial passage in the presence of sublethal doses of polymyxin B over a period of 30 days. By whole-genome sequencing of successively isolated polymyxin B-resistant isolates, we identified a frameshift mutation (L183fs) in the mvfR gene that further increased polymyxin resistance in the pmrB mutant background. A ΔmvfR mutation alone showed higher tolerance to polymyxin B due to altered lipopolysaccharide (LPS) on the surface of bacterial cells, which decreases its outer membrane permeability. In the ΔmvfR mutant, polymyxin B treatment caused the upregulation of rfaD, the gene involved in LPS core oligosaccharide synthesis, which is responsible for polymyxin tolerance. To the best of our knowledge, this is the first report of mvfR mutation conferring polymyxin resistance in P. aeruginosa via increased integrity of bacterial outer membrane. IMPORTANCE Antibiotic resistance imposes a considerable challenge for the treatment of P. aeruginosa infections. Polymyxins are the last-resort antibiotics for the treatment of multidrug-resistant P. aeruginosa infections. Understanding the development and mechanisms of bacterial resistance to polymyxins may provide clues for the development of new or improved therapeutic strategies effective against P. aeruginosa. In this study, using an in vitro evolution assay in combination with whole-genome sequencing, we demonstrated that MvfR controls tolerance to polymyxin B by regulating the rfaD gene in P. aeruginosa. Our results reveal a novel mechanism employed by P. aeruginosa in the defense against polymyxin antibiotics.

Delivery of spike-RBD by bacterial type three secretion system for SARS-CoV-2 vaccine development.

COVID-19 pandemic continues to spread throughout the world with an urgent demand for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a bacterial vector COVID-19 vaccine (aPA-RBD) that carries the gene for the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Live-attenuated strains of Pseudomonas aeruginosa (aPA) were constructed which express the recombinant RBD and effectively deliver RBD protein into various antigen presenting cells through bacterial type 3 secretion system (T3SS) in vitro. In mice, two-dose of intranasal aPA-RBD vaccinations elicited the development of RBD-specific serum IgG and IgM. Importantly, the sera from the immunized mice were able to neutralize host cell infections by SARS-CoV-2 pseudovirus as well as the authentic virus variants potently. T-cell responses of immunized mice were assessed by enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) assays. aPA-RBD vaccinations can elicit RBD-specific CD4+and CD8+T cell responses. T3SS-based RBD intracellular delivery heightens the efficiency of antigen presentation and enables the aPA-RBD vaccine to elicit CD8+T cell response. Thus, aPA vector has the potential as an inexpensive, readily manufactured, and respiratory tract vaccination route vaccine platform for other pathogens.

High-Level Expression of Cell-Surface Signaling System Hxu Enhances Pseudomonas aeruginosa Bloodstream Infection.

Bloodstream infections (BSIs) caused by Pseudomonas aeruginosa are associated with a high mortality rate in the clinic. However, the fitness mechanisms responsible for the evolution of virulence factors that facilitate the dissemination of P. aeruginosa to the bloodstream are poorly understood. In this study, a transcriptomic analysis of the BSI-associated P. aeruginosa clinical isolates showed a high-level expression of cell-surface signaling (CSS) system Hxu. Whole-genome sequencing and comparative genomics of these isolates showed that a mutation in rnfE gene was responsible for the elevated expression of the Hxu-CSS pathway. Most importantly, deletion of the hxuIRA gene cluster in a laboratory strain PAO1 reduced its BSI capability while overexpression of the HxuIRA pathway promoted BSI in a murine sepsis model. We further demonstrated that multiple components in the blood plasma, including heme, hemoglobin, the heme-scavenging proteins haptoglobin, and hemopexin, as well as the iron-delivery protein transferrin, could activate the Hxu system. Together, these studies suggested that the Hxu-CSS system was an important signal transduction pathway contributing to the adaptive pathogenesis of P. aeruginosa in BSI.

Weigela florida inhibits the expression of inflammatory mediators induced by Pseudomonas aeruginosa and Staphylococcus aureus infection.

Inflammatory responses involve the action of inflammatory mediators that are necessary for the clearance of invading bacterial pathogens. However, excessive production of inflammatory mediators can damage tissues, thereby impairing bacterial clearance. Here, we examined the effects of Weigela florida on the expression of inflammatory cytokines induced by Pseudomonas aeruginosa or Staphylococcus aureus infection in macrophages. The results showed that pre-treatment with W. florida markedly downregulated the bacterial infection-mediated expression of cytokines. Additionally, post-treatment also triggered anti-inflammatory effects in cells infected with S. aureus to a greater extent than in those infected with P. aeruginosa. Bacterial infection activated inflammation-associated AKT (Thr308 and Ser473)/NF-κB and MAPK (p38, JNK, and ERK) signaling pathways, whereas W. florida treatment typically inhibited the phosphorylation of AKT/NF-κB and p38/JNK, supporting the anti-inflammatory effects of W. florida. The present results suggest that W. florida decreases the infection-mediated expression of inflammatory mediators by inhibiting the AKT/NF-κB and MAPK signaling pathways, implying that it may have potential use as an inhibitory agent of excessive inflammatory responses.

Acetylation of CspC Controls the Las Quorum-Sensing System through Translational Regulation of rsaL in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous pathogenic bacterium that can adapt to a variety environments. The ability to effectively sense and respond to host local nutrients is critical for the infection of P. aeruginosa. However, the mechanisms employed by the bacterium to respond to nutrients remain to be explored. CspA family proteins are RNA binding proteins that are involved in gene regulation. We previously demonstrated that the P. aeruginosa CspA family protein CspC regulates the type III secretion system in response to temperature shift. In this study, we found that CspC regulates the quorum-sensing (QS) systems by repressing the translation of a QS negative regulatory gene, rsaL. Through RNA immunoprecipitation coupled with real-time quantitative reverse transcription-PCR (RIP-qRT-PCR) and electrophoretic mobility shift assays (EMSAs), we found that CspC binds to the 5' untranslated region of the rsaL mRNA. Unlike glucose, itaconate (a metabolite generated by macrophages during infection) reduces the acetylation of CspC, which increases the affinity between CspC and the rsaL mRNA, leading to upregulation of the QS systems. Our results revealed a novel regulatory mechanism of the QS systems in response to a host-generated metabolite. IMPORTANCE Bacterial infectious diseases impose a severe threat to human health. The ability to orchestrate virulence determinant in response to the host environment is critical for the pathogenesis of bacterial pathogens. Pseudomonas aeruginosa is a leading pathogen that causes various infections in humans. In P. aeruginosa, the quorum-sensing (QS) systems play an important role in regulating the production of virulence factors. In this study, we find that a small RNA binding protein, CspC, regulates the QS systems by repressing the expression of a QS negative regulator. We further demonstrate that CspC is acetylated in response to a host-derived metabolite, itaconate, which alters the function of CspC in regulating the QS system. The importance of this work is in elucidation of a novel regulatory pathway that regulates virulence determinants in P. aeruginosa in response to a host signal.

DnaJ-induced TLR7 mediates an increase in interferons through the TLR4-engaged AKT/NF-κB and JNK signaling pathways in macrophages.

Toll-like receptor 7 (TLR7) signaling plays pivotal roles in innate immunity by sensing viral single-stranded RNA thereby triggering inflammatory signaling cascades and eliciting protective antiviral responses. In this study, we found that TLR7 expression is highly induced in response to Pseudomonas aeruginosa (P. aeruginosa) infection in a dose- and time-dependent manner. P. aeruginosa-derived DnaJ, a homolog of HSP40, was identified as a related inducing agent for TLR7 expression, and expression of DnaJ was stimulated when host cells were infected with P. aeruginosa. Interestingly, DnaJ was not involved in mediating an increase in the expression levels of TLR3 and TLR8, other well-known antiviral receptors. The induction of TLR7 in response to DnaJ was mediated by the activation of the AKT (Thr308 and Ser473)/NF-κB and p38/JNK MAPKs signaling pathways, consequently transmitting related signals for the expression of interferons (IFNs). Of note, these antiviral responses were regulated, at least in part, by TLR4, which senses the presence of DnaJ and then promotes downstream activation of the AKT (Ser473)/NF-κB and JNK signaling cascades. Taken together, these results suggest that P. aeruginosa-derived DnaJ is sufficient to promote an increase in TLR7 expression in the TLR4-engaged AKT/NF-κB and JNK signaling pathways, thereby promoting an increased antiviral response through the elevated expression of IFNs.

ECF Sigma Factor HxuI Is Critical for In Vivo Fitness of Pseudomonas aeruginosa during Infection.

The opportunistic pathogen Pseudomonas aeruginosa often adapts to its host environment and causes recurrent nosocomial infections. The extracytoplasmic function (ECF) sigma factor enables bacteria to alter their gene expression in response to host environmental stimuli. Here, we report an ECF sigma factor, HxuI, which is rapidly induced once P. aeruginosa encounters the host. Host stresses such as iron limitation, oxidative stress, low oxygen, and nitric oxide induce the expression of hxuI. By combining RNA-seq and promoter-lacZ reporter fusion analysis, we reveal that HxuI can activate the expression of diverse metabolic and virulence pathways which are critical to P. aeruginosa infections, including iron acquisition, denitrification, pyocyanin synthesis, and bacteriocin production. Most importantly, overexpression of the hxuI in the laboratory strain PAO1 promotes its colonization in both murine lung and subcutaneous infections. Together, our findings show that HxuI, a key player in host stress-response, controls the in vivo adaptability and virulence of P. aeruginosa during infection. IMPORTANCE P. aeruginosa has a strong ability to adapt to diverse environments, making it capable of causing recurrent and multisite infections in clinics. Understanding host adaptive mechanisms plays an important guiding role in the development of new anti-infective agents. Here, we demonstrate that an ECFσ factor of P. aeruginosa response to the host-inflicted stresses, which promotes the bacterial in vivo fitness and pathogenicity. Furthermore, our findings may help explain the emergence of highly transmissible strains of P. aeruginosa and the acute exacerbations during chronic infections.

Pseudomonas aeruginosa DnaK Stimulates the Production of Pentraxin 3 via TLR4-Dependent NF-κB and ERK Signaling Pathways.

Microbe-derived factors trigger innate immune responses through the production of inflammatory mediators, including pentraxin 3 (PTX3). PTX3 is a soluble pattern recognition molecule that stimulates the clearance of clinically important bacterial pathogens such as Pseudomonas aeruginosa. However, the P. aeruginosa factors responsible for the production of PTX3 have not been elucidated. In this study, we found that P. aeruginosa DnaK, a homolog of heat shock protein 70, induced PTX3 production. Induction was mediated by intracellular signals transmitted through the Toll-like receptor 4 (TLR4) signaling pathway. Following receptor engagement, the stimulatory signals were relayed initially through the nuclear factor kappa B (NF-κB) signaling pathway and subsequently by extracellular signal-regulated kinases (ERK), which are mitogen-activated protein kinases. However, ERK activation was negatively controlled by NF-κB, implying the existence of negative crosstalk between the NF-κB and the ERK pathways. These data suggest that P. aeruginosa DnaK acts as a pathogen-associated molecular pattern to trigger modulation of host defense responses via production of PTX3.

The Anti-Tumor Effects of Oenothera odorata Extract Are Mediated by Inhibition of Glycolysis and Cellular Respiration in Cancer Cells.

Cancer is caused by uncontrolled cell division and is a leading cause of mortality worldwide. Oenothera odorata (O. odorata) extract is used in herbal medicine to inhibit inflammation, but its potential anti-tumor properties have not been fully evaluated. Here, we demonstrated that O. odorata extract inhibits the proliferation of lung adenocarcinoma and melanoma cell lines In Vitro, and also inhibits the growth of melanoma cells In Vivo. After partitioning the extract with n-hexane, chloroform, ethyl acetate, and n-butanol, it was found that the butanol-soluble (OOB) and water-soluble (OOW) fractions of O. odorata extract are effective at inhibiting tumor cell growth In Vivo although OOW is more effective than OOB. Interestingly, these fractions did not inhibit the growth of non-cancerous cells. The anti-proliferative effects of the OOW fraction were found to be mediated by inhibition of glycolysis and cellular respiration. UPLC of both fractions showed two major common peaks, which were predicted to be hydrolyzable tannin-related compounds. Taken together, these data suggest that O. odorata extract has anti-tumor properties, and the molecular mechanism involves metabolic alterations and inhibition of cell proliferation. O. odorata extract therefore holds promise as a novel natural product for the treatment of cancer.

HSP70-Homolog DnaK of Pseudomonas aeruginosa Increases the Production of IL-27 through Expression of EBI3 via TLR4-Dependent NF-κB and TLR4-Independent Akt Signaling.

IL-27, a heterodimeric cytokine composed of the p28 subunit and Epstein-Barr virus-induced gene 3 (EBI3), acts as a potent immunosuppressant and thus limits pathogenic inflammatory responses. IL-27 is upregulated upon Pseudomonas aeruginosa infection in septic mice, increasing susceptibility to the infection and decreasing clearance of the pathogen. However, it remains unclear which P. aeruginosa-derived molecules promote production of IL-27. In this study, we explored the mechanism by which P. aeruginosa DnaK, a heat shock protein 70-like protein, induces EBI3 expression, thereby promoting production of IL-27. Upregulation of EBI3 expression did not lead to an increase in IL-35, which consists of the p35 subunit and EBI3. The IL-27 production in response to DnaK was biologically active, as reflected by stimulation of IL-10 production. DnaK-mediated expression of EBI3 was driven by two distinct signaling pathways, NF-κB and Akt. However, NF-κB is linked to TLR4-associated signaling pathways, whereas Akt is not. Taken together, our results reveal that P. aeruginosa DnaK potently upregulates EBI3 expression, which in turn drives production of the prominent anti-inflammatory cytokine IL-27, as a consequence of TLR4-dependent activation of NF-κB and TLR4-independent activation of the Akt signaling pathway.

The Pseudomonas aeruginosa HSP90-like protein HtpG regulates IL-8 expression through NF-κB/p38 MAPK and CYLD signaling triggered by TLR4 and CD91.

Pulmonary infection activates acute inflammatory responses by recruiting neutrophils to the infection site; this recruitment is promoted by interleukin-8 (IL-8). However, IL-8 production in response to Pseudomonas aeruginosa HtpG (PA1596), a homolog of heat shock protein 90, has yet not been characterized in detail. htpG expression in P. aeruginosa strain was elevated upon infection of host cells, and HtpG was released into bacterial culture supernatant. Treatment of dTHP-1 macrophages with recombinant HtpG (rHtpG) increased production of IL-8 in a dose- and time-dependent manner, and this effect was abolished by inhibition of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) p38 signaling. By contrast, the rHtpG-mediated production of IL-8 was increased by suppression of cylindromatosis (CYLD), suggesting that CYLD is a negative regulator of this pathway. The upregulation of expression was coordinated by signals transmitting through toll-like receptor 4 (TLR4) with the aid of CD91. Together, these observations suggest that P. aeruginosa HtpG activates NF-κB, CYLD, and p38 MAPK in a TLR4-and CD91-dependent manner, leading to stimulation of IL-8 production in macrophages.

Negative regulation of interleukin 1ß expression in response to DnaK from Pseudomonas aeruginosa via the PI3K/PDK1/FoxO1 pathways.

Interleukin (IL)-1ß is crucial for a wide range of inflammatory responses. Previously, we reported that IL-1ß is produced in response to Pseudomonas aeruginosa-derived DnaK via NF-κB and JNK pathways; however, the signaling pathways that counter the process to maintain IL-1ß homeostasis are unknown. Here, we show that DnaK-mediated expression of IL1ß is increased markedly in macrophages upon blockade of PI3K/PDK1. This was verified by measuring released IL-1ß protein. The negative effect of PI3K on IL-1ß production was dependent on suppression of both NF-κB and JNK activation. Intriguingly, PDK1 (an underlying mediator of PI3K) acted as an upstream regulator for the activation of NF-κB, but downregulated JNK activation. Furthermore, production of IL-1ß and activation of JNK were triggered by inhibition of phosphorylated FoxO1; phosphorylation of FoxO1 was controlled by PDK1 signaling in response to DnaK. Thus, IL-1ß production is modulated by P. aeruginosa-derived DnaK via cross-talk between JNK and PI3K/PDK1/FoxO1 pathways.

Maackiain, a compound derived from Sophora flavescens, increases IL-1ß production by amplifying nigericin-mediated inflammasome activation.

Sophora flavescens is used as a traditional herbal medicine to modulate inflammatory responses. However, little is known about the impact of (-)-maackiain, a compound derived from S. flavescens, on the activation of inflammasome/caspase-1, a key factor in interleukin-1ß (IL-1ß) processing. Here, we report that (-)-maackiain potently amplified caspase-1 cleavage in macrophages in response to nigericin (Nig). In macrophages primed with either lipopolysaccharide or monophosphoryl lipid A, Nig-mediated caspase-1 cleavage was also markedly promoted by (-)-maackiain. Notably, (-)-maackiain induced the production of vimentin, an essential mediator for the activation of the NOD-, LRR-, and pyrin domain-containing protein 3 inflammasome, thereby contributing to promotion of the formation of the inflammasome complex to activate caspase-1. Taken together, our data suggest that (-)-maackiain exerts an immunostimulatory effect by promoting IL-1ß production via activation of the inflammasome/caspase-1 pathway. Thus, the potent inflammasome-activating effect of (-)-maackiain may be clinically useful as an acute immune-stimulating agent.

The Pseudomonas aeruginosa HSP70-like protein DnaK induces IL-1ß expression via TLR4-dependent activation of the NF-κB and JNK signaling pathways.

IL-1ß expression is increased in response to P. aeruginosa infection, but the responsible proteins have not been clearly elucidated. Here, we demonstrate for the first time that IL-1ß expression is induced in response to the heat shock protein 70-like protein DnaK. Treatment with recombinant DnaK (rDnaK) increased IL-1ß expression in a dose- and time-dependent manner, and the release of mature IL-1ß in response to rDnaK was detected to an extent similar to that stimulated by the well-known agonists, lipopolysaccharide and nigericin. rDnaK-mediated IL-1ß expression was driven by the NF-κB signaling pathway. In addition, expression was controlled by the JNK signaling pathway, although these two signaling cascades act independently upon rDnaK stimulation. Finally, rDnaK-induced IL-1ß expression was initiated via the action of TLR4. Taken together, the data reveal that P. aeruginosa-derived DnaK induces expression of IL-1ß via TLR4-dependent activation of the NF-κB and JNK signaling pathways.

Decreased Expression of Sphingosine-1-Phosphate Receptor 1 in the Blood Leukocyte of Rheumatoid Arthritis Patients.

Sphingosine-1-phosphate (S1P) plays an important role in trafficking leukocytes and developing immune disorders including autoimmunity. In the synovium of rheumatoid arthritis (RA) patients, increased expression of S1P was reported, and the interaction between S1P and S1P receptor 1 (S1P1) has been suggested to regulate the expression of inflammatory genes and the proliferation of synovial cells. In this study, we investigated the level of S1P1 mRNA expression in the blood leukocytes of RA patients. In contrast to the previous reports, the expression level of this gene was not correlated to their clinical scores, disease durations and ages. However, S1P1 was transcribed at a significantly lower level in the circulating leukocytes of RA patients when compared to age-, and sex-matched healthy controls. Since these data may suggest the participation of S1P1, further studies are needed to determine the role of this receptor in the pathogenesis of RA.

Pik3ca is required for mouse uterine gland development and pregnancy.

The PI3K/AKT signaling pathway plays a critical role in the maintenance of equilibrium between cell survival and apoptosis. The Pik3ca gene is mutated in a range of human cancers. It has been found to be oncogenic, and mutations lead to constitutive activation of the PI3K/AKT pathway. The expression patterns of PIK3CA proteins in the uterus of mice during early pregnancy indicate that it may play a role in the regulation of glandular epithelial cells, which is required to support uterine receptivity. To further investigate the role of Pik3ca in uterine function, Pik3ca was conditionally ablated only in the PGR-positive cells (Pgrcre/+Pik3caf/f; Pik3cad/d). A defect of uterine gland development and decidualization led to subfertility observed in Pik3cad/d mice. Pik3cad/d mice showed significantly decreased uterine weight compared to Pik3caf/f mice. Interestingly, a significant decrease of gland numbers were detected in Pik3cad/d mice compared to control mice. In addition, we found a decrease of Foxa2 expression, which is a known uterine gland marker in Pik3cad/d mice. Furthermore, the excessive proliferation of endometrial epithelial cells was observed in Pik3cad/d mice. Our studies suggest that Pik3ca has a critical role in uterine gland development and female fertility.

T3SS effector ExoY reduces inflammasome-related responses by suppressing bacterial motility and delaying activation of NF-κB and caspase-1.

Type III-secreted effectors are essential for modulating host immune responses during the pathogenesis of Pseudomonas aeruginosa infections. Little is known about the impact of one of the effectors, ExoY, on inflammasome activation, which results in IL-1ß production and pyroptotic cell death. In this study, we found that transcriptional expression of Il-1ß was induced to a lesser extent in response to an exoY-harboring strain than to a deleted mutant. This suppressive effect of ExoY was verified by complementation assay as well as by direct translocation of exoY into host cells. In addition to the production of IL-1ß, pyroptotic cell death was also diminished in response to an exoY-harboring strain. These inflammasome responses were mediated by the adenylate cyclase activity of ExoY, which plays a role in delaying the activation of NF-κB and caspase-1, a key component of inflammasome-mediated responses. Moreover, the negative effects of ExoY on these responses were in part conferred by the suppression of bacterial motility, which could reduce the degree of bacterial contact with cells. Together, these results demonstrate that the adenylate cyclase activity of P. aeruginosa ExoY can reduce inflammasome-related responses by influencing both the host and the bacterium itself by delaying the activation of inflammatory pathways and suppressing bacterial motility.