Pseudomonas aeruginosa ExoY infection of pulmonary microvascular endothelial cells releases cyclic nucleotides into the extracellular compartment.

Type three secretion system (TTSS) competent Pseudomonas aeruginosa expressing soluble promiscuous cyclase, ExoY, generates cyclic nucleotides in pulmonary microvascular endothelial cells (PMVECs). Within cells, cyclic nucleotide signals are highly compartmentalized, but these second messengers are also released into the extracellular space. While agonist-stimulation of endogenous adenylyl cyclase (AC) or the presence of ExoY increases cyclic nucleotides, the proportion of the signal that is in the intracellular versus extracellular compartments is unresolved. Further, it is unclear whether P. aeruginosa primary infection or treatment with sterile media supernatants derived from a primary infection, alters beta-adrenergic agonist-induced elevations in cAMP in PMVECs. Herein, we determine that PMVECs release cAMP into the extracellular space constitutively, following beta-adrenergic stimulation of endogenous AC, and following infection with P. aeruginosa expressing ExoY. Surprisingly, in PMVECs, only a small proportion of cGMP is detected within the cell at baseline or following P. aeruginosa ExoY-infection with a larger proportion of total cGMP being detected extracellularly. Thus, the ability of lung endothelium to generate cyclic nucleotides may be underestimated by examining intracellular cyclic nucleotides alone, since a large portion is delivered into the extracellular compartment. In addition, P. aeruginosa infection or treatment with sterile media supernatants from a primary infection suppress the beta-adrenergic cAMP response, which is further attenuated by the expression of functional ExoY. These findings reveal an overabundance of extracellular cyclic nucleotides following infection with ExoY expressing TTSS competent P. aeruginosa.

Bacterial Nucleotidyl Cyclases Activated by Calmodulin or Actin in Host Cells: Enzyme Specificities and Cytotoxicity Mechanisms Identified to Date.

Many pathogens manipulate host cell cAMP signaling pathways to promote their survival and proliferation. Bacterial Exoenzyme Y (ExoY) toxins belong to a family of invasive, structurally-related bacterial nucleotidyl cyclases (NC). Inactive in bacteria, they use proteins that are uniquely and abundantly present in eukaryotic cells to become potent, unregulated NC enzymes in host cells. Other well-known members of the family include Bacillus anthracis Edema Factor (EF) and Bordetella pertussis CyaA. Once bound to their eukaryotic protein cofactor, they can catalyze supra-physiological levels of various cyclic nucleotide monophosphates in infected cells. Originally identified in Pseudomonas aeruginosa, ExoY-related NC toxins appear now to be more widely distributed among various γ- and ß-proteobacteria. ExoY-like toxins represent atypical, poorly characterized members within the NC toxin family. While the NC catalytic domains of EF and CyaA toxins use both calmodulin as cofactor, their counterparts in ExoY-like members from pathogens of the genus Pseudomonas or Vibrio use actin as a potent cofactor, in either its monomeric or polymerized form. This is an original subversion of actin for cytoskeleton-targeting toxins. Here, we review recent advances on the different members of the NC toxin family to highlight their common and distinct functional characteristics at the molecular, cytotoxic and enzymatic levels, and important aspects that need further characterizations.

Pseudomonas aeruginosa exoenzyme Y directly bundles actin filaments.

Pseudomonas aeruginosa uses a type III secretion system (T3SS) to inject cytotoxic effector proteins into host cells. The promiscuous nucleotidyl cyclase, exoenzyme Y (ExoY), is one of the most common effectors found in clinical P. aeruginosa isolates. Recent studies have revealed that the nucleotidyl cyclase activity of ExoY is stimulated by actin filaments (F-actin) and that ExoY alters actin cytoskeleton dynamics in vitro, via an unknown mechanism. The actin cytoskeleton plays an important role in numerous key biological processes and is targeted by many pathogens to gain competitive advantages. We utilized total internal reflection fluorescence microscopy, bulk actin assays, and EM to investigate how ExoY impacts actin dynamics. We found that ExoY can directly bundle actin filaments with high affinity, comparable with eukaryotic F-actin-bundling proteins, such as fimbrin. Of note, ExoY enzymatic activity was not required for F-actin bundling. Bundling is known to require multiple actin-binding sites, yet small-angle X-ray scattering experiments revealed that ExoY is a monomer in solution, and previous data suggested that ExoY possesses only one actin-binding site. We therefore hypothesized that ExoY oligomerizes in response to F-actin binding and have used the ExoY structure to construct a dimer-based structural model for the ExoY-F-actin complex. Subsequent mutational analyses suggested that the ExoY oligomerization interface plays a crucial role in mediating F-actin bundling. Our results indicate that ExoY represents a new class of actin-binding proteins that modulate the actin cytoskeleton both directly, via F-actin bundling, and indirectly, via actin-activated nucleotidyl cyclase activity.

Salt Stress Enhances Early Symbiotic Gene Expression in Medicago truncatula and Induces a Stress-Specific Set of Rhizobium-Responsive Genes.

Salt stress is a major agricultural concern inhibiting not only plant growth but also the symbiotic association between legume roots and the soil bacteria rhizobia. This symbiotic association is initiated by a molecular dialogue between the two partners, leading to the activation of a signaling cascade in the legume host and, ultimately, the formation of nitrogen-fixing root nodules. Here, we show that a moderate salt stress increases the responsiveness of early symbiotic genes in Medicago truncatula to its symbiotic partner, Sinorhizobium meliloti while, conversely, inoculation with S. meliloti counteracts salt-regulated gene expression, restoring one-third to control levels. Our analysis of early nodulin 11 (ENOD11) shows that salt-induced expression is dynamic, Nod-factor dependent, and requires the ionic but not the osmotic component of salt. We demonstrate that salt stimulation of rhizobium-induced gene expression requires NSP2, which functions as a node to integrate the abiotic and biotic signals. In addition, our work reveals that inoculation with S. meliloti succinoglycan mutants also hyperinduces ENOD11 expression in the presence or absence of salt, suggesting a possible link between rhizobial exopolysaccharide and the plant response to salt stress. Finally, we identify an accessory set of genes that are induced by rhizobium only under conditions of salt stress and have not been previously identified as being nodulation-related genes. Our data suggest that interplay of core nodulation genes with different accessory sets, specific for different abiotic conditions, functions to establish the symbiosis. Together, our findings reveal a complex and dynamic interaction between plant, microbe, and environment.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Virulent Pseudomonas aeruginosa infection converts antimicrobial amyloids into cytotoxic prions.

Pseudomonas aeruginosa infection elicits the production of cytotoxic amyloids from lung endothelium, yet molecular mechanisms of host-pathogen interaction that underlie the amyloid production are not well understood. We examined the importance of type III secretion system (T3SS) effectors in the production of cytotoxic amyloids. P aeruginosa possessing a functional T3SS and effectors induced the production and release of cytotoxic amyloids from lung endothelium, including beta amyloid, and tau. T3SS effector intoxication was sufficient to generate cytotoxic amyloid release, yet intoxication with exoenzyme Y (ExoY) alone or together with exoenzymes S and T (ExoS/T/Y) generated the most virulent amyloids. Infection with lab and clinical strains engendered cytotoxic amyloids that were capable of being propagated in endothelial cell culture and passed to naïve cells, indicative of a prion strain. Conversely, T3SS-incompetent P aeruginosa infection produced non-cytotoxic amyloids with antimicrobial properties. These findings provide evidence that (1) endothelial intoxication with ExoY is sufficient to elicit self-propagating amyloid cytotoxins during infection, (2) pulmonary endothelium contributes to innate immunity by generating antimicrobial amyloids in response to bacterial infection, and (3) ExoY contributes to the virulence arsenal of P aeruginosa through the subversion of endothelial amyloid host-defense to promote a lung endothelial-derived cytotoxic proteinopathy.

α-Tocopherol Attenuates the Severity of Pseudomonas aeruginosa-induced Pneumonia.

Pseudomonas aeruginosa is a lethal pathogen that causes high mortality and morbidity in immunocompromised and critically ill patients. The type III secretion system (T3SS) of P. aeruginosa mediates many of the adverse effects of infection with this pathogen, including increased lung permeability in a Toll-like receptor 4/RhoA/PAI-1 (plasminogen activator inhibitor-1)-dependent manner. α-Tocopherol has antiinflammatory properties that may make it a useful adjunct in treatment of this moribund infection. We measured transendothelial and transepithelial resistance, RhoA and PAI-1 activation, stress fiber formation, P. aeruginosa T3SS exoenzyme (ExoY) intoxication into host cells, and survival in a murine model of pneumonia in the presence of P. aeruginosa and pretreatment with α-tocopherol. We found that α-tocopherol alleviated P. aeruginosa-mediated alveolar endothelial and epithelial paracellular permeability by inhibiting RhoA, in part, via PAI-1 activation, and increased survival in a mouse model of P. aeruginosa pneumonia. Furthermore, we found that α-tocopherol decreased the activation of RhoA and PAI-1 by blocking the injection of T3SS exoenzymes into alveolar epithelial cells. P. aeruginosa is becoming increasingly antibiotic resistant. We provide evidence that α-tocopherol could be a useful therapeutic agent for individuals who are susceptible to infection with P. aeruginosa, such as those who are immunocompromised or critically ill.

The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin.

Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 3'5'-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa is stimulated by the filamentous form of actin (F-actin). Utilizing yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays, and confocal microscopy, we demonstrate that the last nine amino acids of the C terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY that had been fused to a carrier protein bind to F-actin and that synthetic peptides corresponding to the extreme ExoY C terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants.

Bacterial Nucleotidyl Cyclase Inhibits the Host Innate Immune Response by Suppressing TAK1 Activation.

Exoenzyme Y (ExoY) is a type III secretion system effector found in 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY is a soluble nucleotidyl cyclase that increases the cytoplasmic levels of nucleoside 3',5'-cyclic monophosphates (cNMPs) to mediate endothelial Tau phosphorylation and permeability, its functional role in the innate immune response is still poorly understood. Transforming growth factor ß-activated kinase 1 (TAK1) is critical for mediating Toll-like receptor (TLR) signaling and subsequent activation of NF-κB and AP-1, which are transcriptional activators of innate immunity. Here, we report that ExoY inhibits proinflammatory cytokine production through suppressing the activation of TAK1 as well as downstream NF-κB and mitogen-activated protein (MAP) kinases. Mice infected with ExoY-deficient P. aeruginosa had higher levels of tumor necrosis factor (TNF) and interleukin-6 (IL-6), more neutrophil recruitment, and a lower bacterial load in lung tissue than mice infected with wild-type P. aeruginosa Taken together, our findings identify a previously unknown mechanism by which P. aeruginosa ExoY inhibits the host innate immune response.

cCMP and cUMP occur in vivo.

Mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. It is unknown whether these tentative new second messenger molecules occur in vivo. We used high performance liquid chromatography quadrupole tandem mass spectrometry to quantitate nucleoside 3',5'-cyclic monophosphates. cCMP was detected in all organs studied, most notably pancreas, spleen and the female reproductive system. cUMP was not detected in organs, probably due to the intrinsically low sensitivity of mass spectrometry to detect this molecule and organ matrix effects. Intratracheal infection of mice with recombinant Pseudomonas aeruginosa harboring the nucleotidyl cyclase toxin ExoY massively increased cUMP in lung. The identity of cCMP and cUMP in organs was confirmed by high performance liquid chromatography quadrupole time of flight mass spectrometry. cUMP also appeared in serum, urine and faeces following infection. Taken together, this report unequivocally shows for the first time that cCMP and cUMP occur in vivo.

Computational approach to attenuate virulence of Pseudomonas aeruginosa through bioinspired silver nanoparticles.

In this study we aim to investigate the computational docking approach of biofabricated silver nanoparticles against P. aeruginosa virulent exoenzymes, such as ExoS and ExoY. Therefore, the synthesis and characterization of biofabricated silver nanoparticles using Piper betle leaves (Pb-AgNPs) were carried out. The surface topology and functional group attachment on the surface of Pb-AgNPs were analyzed using UV-visible spectroscopy, Scanning Electron Microscopy, Fourier Transformed Infrared Spectroscopy (FTIR), and X-Ray Diffraction. The FTIR analysis revealed that the synthesized silver nanoparticles were capped with P. betle phytochemicals importantly Eugenol and Hydroxychavicol. These are the major bioactive compounds present in P. betle leaves; therefore, computational docking of Eugenol-conjugated AgNPs (PbEu-AgNPs) and Hydroxychavicol-conjugated AgNPs (PbHy-AgNPs) against ExoS and ExoY was performed. The active residues of PbEu-AgNPs and PbHy-AgNPs interacted with the active site of ExoS and ExoY exoenzymes. Biofabricated AgNP-mediated inhibition of these virulent exoenzymes blocked the adverse effect of P. aeruginosa on the host cell. The computational analysis provides new approach into the design of biofabricated AgNPs as promising anti-infective nanomedicine agents. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03367-0.

Prevalence of ExoY Activity in Pseudomonas aeruginosa Reference Panel Strains and Impact on Cytotoxicity in Epithelial Cells.

ExoY is among the effectors that are injected by the type III secretion system (T3SS) of Pseudomonas aeruginosa into host cells. Inside eukaryotic cells, ExoY interacts with F-actin, which stimulates its potent nucleotidyl cyclase activity to produce cyclic nucleotide monophosphates (cNMPs). ExoY has broad substrate specificity with GTP as a preferential substrate in vitro. How ExoY contributes to the virulence of P. aeruginosa remains largely unknown. Here, we examined the prevalence of active ExoY among strains from the international P. aeruginosa reference panel, a collection of strains that includes environmental and clinical isolates, commonly used laboratory strains, and sequential clonal isolates from cystic fibrosis (CF) patients and thus represents the large diversity of this bacterial species. The ability to secrete active ExoY was determined by measuring the F-actin stimulated guanylate cyclase (GC) activity in bacterial culture supernatants. We found an overall ExoY activity prevalence of about 60% among the 40 examined strains with no significant difference between CF and non-CF isolates. In parallel, we used cellular infection models of human lung epithelial cells to compare the cytotoxic effects of isogenic reference strains expressing active ExoY or lacking the exoY gene. We found that P. aeruginosa strains lacking ExoY were in fact more cytotoxic to the epithelial cells than those secreting active ExoY. This suggests that under certain conditions, ExoY might partly alleviate the cytotoxic effects of other virulence factors of P. aeruginosa.

Pseudomonas aeruginosa Type III Secretion System Virulotypes and Their Association with Clinical Features of Cystic Fibrosis Patients.

PURPOSE: Pseudomonas aeruginosa appears as the main pathogen in cystic fibrosis (CF) involved in recurrent pneumonia and pulmonary exacerbations. The type III secretion system (T3SS) is one of its main determinants of virulence and is associated with poor clinical progression and increased mortality. This study determined the relationship of clinical features of patients with CF and P. aeruginosa T3SS virulotypes. MATERIALS AND METHODS: From January 2018 to March 2019, P. aeruginosa were isolated from sputum and/or oropharyngeal swabs. T3SS markers (exoS, exoU, exoT and exoY) were detected by PCR. Clinical severity according to Shwachman-Kulckycki score and spirometry data were associated with T3SS virulotypes. RESULTS: A total of 49 patients had positive cultures for P. aeruginosa. T3SS virulence-related markers were detected as follows: exoS 97.9% (n=48), exoU 63.2% (n=31), exoT 95.9% (n=47) and exoY 97.9% (n=48). The prevalence of exoS+/exoU+ virulotype was higher than previously reported in CF settings, being detected in 61.2% of the evaluated isolates, present in 70% of intermittent infections and with a significantly higher frequency in cases of exacerbations. The presence of exoU in chronic infection was not associated with poor clinical results. In chronic infections, the exoS+/exoU- virulotype prevailed (77.8%) and was associated to worse clinical results according to the Shwachman-Kulckycki score and spirometric. CONCLUSION: Our findings revealed a high prevalence of the atypical exoS+/exoU+ virulotype among P. aeruginosa isolates from patients with CF, which was associated with intermittent infection and early clinical alterations, while the exoS+/exoU- virulotype was associated with chronic infection and worse clinical results. Finally, the presented data highlight the relevance of T3SS virulence markers in the clinical progression and disease severity in CF patients.

The Pseudomonas aeruginosa ExoY phenotype of high-copy-number recombinants is not detectable in natural isolates.

The nucleotidyl cyclase ExoY is an effector protein of the type III secretion system of Pseudomonas aeruginosa We compared the cyclic nucleotide production and lung disease phenotypes caused by the ExoY-overexpressing strain PA103ΔexoUexoT::Tc pUCPexoY, its vector control strain PA103ΔexoUexoT::Tc pUCP18, its loss-of-function control PA103ΔexoUexoT::Tc pUCPexoY K81M and natural ExoY-positive and ExoY-negative isolates in a murine acute airway infection model. Only the P. aeruginosa carrier of the exoY-plasmid produced high levels of cUMP and caused the most severe course of infection. The pathology ascribed to ExoY from studies using the high-copy-number plasmid on mammalian cells in vitro and in vivo was not observed with natural P. aeruginosa isolates. This indicates that the role of ExoY during infection with real-life P. aeruginosa still needs to be resolved.

ExoY, an actin-activated nucleotidyl cyclase toxin from P. aeruginosa: A minireview.

ExoY is one of four well-characterized Pseudomonas aeruginosa type 3 secretion system (T3SS) effectors. It is a nucleotidyl cyclase toxin that is inactive inside the bacteria, but becomes potently activated once it is delivered into the eukaryotic target cells. Recently, filamentous actin was identified as the eukaryotic cofactor that stimulates specifically ExoY enzymatic activity by several orders of magnitude. In this review, we discuss recent advances in understanding the biochemistry of nucleotidyl cyclase activity of ExoY and its regulation by interaction with filamentous actin.

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.