Exotoxin Secretion Research Articles

Design, synthesis, anti-infective potency and mechanism study of novel Ru-based complexes containing substituted adamantane as antibacterial agents

Infections caused by Staphylococcus aureus (S. aureus) are increasing difficult to treat because this pathogen is easily resistant to antibiotics. However, the development of novel antibacterial agents with high antimicrobial activity and low frequency of resistance remains a huge challenge. Here, building on the coupling strategy, an adamantane moiety was linked to the membrane-active Ru-based structure and then developed three novel metalloantibiotics: [Ru(bpy)2(L)](PF6)2 (Ru1) (bpy = 2,2-bipyridine, L = amantadine modified ligand), [Ru(dmb)2(L)](PF6)2 (Ru2) (dmb = 4,4′-dimethyl-2,2′-bipyridine) and [Ru(dpa)2(L)](PF6)2 (Ru3), (dpa = 2,2′-dipyridylamine). Notably, complex Ru1 was identified to be the best candidate agent, showing greater efficacy against S. aureus than most of clinical antibiotics and low resistance frequencies. Mechanism studies demonstrated that Ru1 could not only increase the permeability of bacterial cell membrane and then caused the leakage of bacterial contents, but also promoted the production of reactive oxygen species (ROS) in bacteria. Importantly, complex Ru1 inhibited the biofilm formation, exotoxin secretion and increased the potency of some clinical used antibiotics. In addition, Ru1 showed low toxic in vivo and excellent anti-infective efficacy in two animal infection model. Thus, Ru-based metalloantibiotic bearing adamantane moiety are promising antibacterial agents, providing a certain research basis for the future antibiotics research.

Unraveling extracellular protein signatures to enhance live attenuated vaccine development through type II secretion system disruption in Vibriomimicus

Type II secretion systems (T2SS) are important molecular machines used by bacteria to transport a wide range of proteins across the outer membrane from the periplasm. Vibrio mimicus is an epidemic pathogen threats to both aquatic animals and human health. Our previous study demonstrates that T2SS deletion reduced virulence by 307.26 times in yellow catfish. However, the specific effects of T2SS-mediated extracellular protein secretion in V. mimicus, including its potential role in exotoxin secretion or other mechanisms, require further investigation. Through proteomics and phenotypic analyses, this study observed that the ΔT2SS strain exhibited significant self-aggregation and dynamic deficiency, with a notable negative correlation with subsequent biofilm formation. The proteomics analysis revealed 239 different abundances of extracellular proteins after T2SS deletion, including 19 proteins with higher abundance and 220 proteins with lower and even absent in the ΔT2SS strain. These extracellular proteins are involved in various pathways, such as metabolism, virulence factors expression, and enzymes. Among them, purine, pyruvate, and pyrimidine metabolism, and the Citrate cycle, were the primary pathways affected by T2SS. Our phenotypic analysis is consistent with these findings, suggesting that the decreased virulence of ΔT2SS strains is due to the effect of T2SS on these proteins, which negatively impacts growth, biofilm formation, auto-aggregation, and motility of V. mimicus. These results provide valuable insights for designing deletion targets for attenuated vaccines development against V. mimicus and expand our understanding of the biological functions of T2SS.

Discovery of inhibitors of Pseudomonas aeruginosa virulence through the search for natural-like compounds with a dual role as inducers and substrates of efflux pumps.

Multidrug efflux pumps are ancient elements encoded in every genome, from bacteria to humans. In bacteria, in addition to antibiotics, efflux pumps extrude a wide range of substrates, including quorum sensing signals, bacterial metabolites, or plant-produced compounds. This indicates that their original functions may differ from their recently acquired role in the extrusion of antibiotics during human infection. Concerning plant-produced compounds, some of them are substrates and inducers of the same efflux pump, suggesting a coordinated plant/bacteria coevolution. Herein we analyse the ability of 1243 compounds from a Natural Product-Like library to induce the expression of P. aeruginosa mexCD-oprJ or mexAB-oprM efflux pumps' encoding genes. We further characterized natural-like compounds that do not trigger antibiotic resistance in P. aeruginosa and that act as virulence inhibitors, choosing those that were not only inducers but substrates of the same efflux pump. Four compounds impair swarming motility, exotoxin secretion through the Type 3 Secretion System (T3SS) and the ability to kill Caenorhabditis elegans, which might be explained by the downregulation of genes encoding flagellum and T3SS. Our results emphasize the possibility of discovering new anti-virulence drugs by screening natural or natural-like libraries for compounds that behave as both, inducers and substrates of efflux pumps.

Proteomic profiling unveils citral modulating expression of IsaA, CodY and SaeS to inhibit biofilm and virulence in Methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the dangerous human pathogens and it is categorized as a high priority multi-drug resistant bacterium by WHO. Biofilm forming ability of MRSA is responsible for persistent infections and also difficult to eradicate using antibiotic therapy as biofilm is much more resistant to antibiotics. Thus, targeting biofilm and virulence has become an alternative approach to attenuate the pathogenicity of bacterium without affecting the growth. Hence, the present study was aimed at evaluation of antibiofilm potential of citral against MRSA and to decode the possible mode of action. Citral inhibited biofilm formation by MRSA without affecting growth at 100 μg/mL. Microscopic analyses evidenced that citral greatly hampered the surface adherence of MRSA. Effect of citral on cellular proteome of MRSA was studied using two-dimensional gel electrophoresis (2DGE) and differentially regulated proteins were identified using nano LC-MS/MS and MALDI-TOF/TOF analysis. Gene ontology and STRING analysis revealed that citral differentially regulated the proteins involved in pleotropic transcriptional repression (CodY), cell wall homeostasis (IsaA), regulation of exotoxin secretion (SaeS), cell adhesion, hemolysis, capsular polysaccharide biosynthesis and pathogenesis. Gene expression analysis and in vitro assays further validated the alteration in synthesis of slime, hemolysin, lipase, staphyloxanthin and oxidant susceptibility. Thus, the present study unveiled the multiple protein targeted antibiofilm potential of citral and portrays citral as a promising therapeutic agent to combat biofilm mediated MRSA infections with less possibility of resistance development.

Full Transcriptomic Response of Pseudomonas aeruginosa to an Inulin-Derived Fructooligosaccharide.

Pseudomonas aeruginosa is an ubiquitous gram-negative opportunistic human pathogen which is not considered part of the human commensal gut microbiota. However, depletion of the intestinal microbiota (Dysbiosis) following antibiotic treatment facilitates the colonization of the intestinal tract by Multidrug-Resistant P. aeruginosa. One possible strategy is based on the use of functional foods with prebiotic activity. The bifidogenic effect of the prebiotic inulin and its hydrolyzed form (fructooligosaccharide: FOS) is well established since they promote the growth of specific beneficial (probiotic) gut bacteria such as bifidobacteria. Previous studies of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 have shown that inulin and to a greater extent FOS reduce growth and biofilm formation, which was found to be due to a decrease in motility and exotoxin secretion. However, the transcriptional basis for these phenotypic alterations remains unclear. To address this question we conducted RNA-sequence analysis. Changes in the transcript level induced by inulin and FOS were similar, but a set of transcript levels were increased in response to inulin and reduced in the presence of FOS. In the presence of inulin or FOS, 260 and 217 transcript levels, respectively, were altered compared to the control to which no polysaccharide was added. Importantly, changes in transcript levels of 57 and 83 genes were found to be specific for either inulin or FOS, respectively, indicating that both compounds trigger different changes. Gene pathway analyses of differentially expressed genes (DEG) revealed a specific FOS-mediated reduction in transcript levels of genes that participate in several canonical pathways involved in metabolism and growth, motility, biofilm formation, β-lactamase resistance, and in the modulation of type III and VI secretion systems; results that have been partially verified by real time quantitative PCR measurements. Moreover, we have identified a genomic island formed by a cluster of 15 genes, encoding uncharacterized proteins, which were repressed in the presence of FOS. The analysis of isogenic mutants has shown that genes of this genomic island encode proteins involved in growth, biofilm formation and motility. These results indicate that FOS selectively modulates bacterial pathogenicity by interfering with different signaling pathways.

Near-Infrared Light-Enhanced Protease-Conjugated Gold Nanorods As A Photothermal Antimicrobial Agent For Elimination Of Exotoxin And Biofilms.

PurposeTreatment strategies to eliminate bacterial infections have long emphasized bacterial killing as a goal. However, bacteria secrete toxins that sustain chronic disease and dead cells release DNA that can promote the spread of antibiotic resistance even when viable cells are eradicated. Meanwhile, biofilms regulated by quorum-sensing system, protect bacteria and promote the development of antibiotic resistance. Thus, all of these factors underscore the need for novel antimicrobial therapeutic treatments as alternatives to traditional antibiotics. Here, a smart material was developed that incorporated gold nanorods and an adsorbed protease (protease-conjugated gold nanorods, PGs). When illuminated with near-infrared (NIR) light, PGs functioned to physically damage bacteria, prevent biofilm and exotoxin production, eliminate pre-existing biofilm and exotoxin, and inhibit bacterial quorum-sensing systems.MethodsPGs were incubated with suspensions of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria followed by exposure to 808-nm NIR laser irradiation. Bacterial viability was determined using a colony-forming unit assay followed by an exploration of cell-damage mechanisms using transmission electron microscopy, scanning electron microscopy, agarose gel electrophoresis, and SDS-PAGE. Quantification of biofilm mass was performed using crystal violet staining. A commercial enterotoxin ELISA kit was used to test inhibitory and degradative effects of PGs on secreted exotoxin.ResultsUse of the remote-controlled antibacterial system reduced surviving bacterial populations to 3.2% and 2.1% of untreated control numbers for E. coli and S. aureus, respectively, and inhibited biofilm formation and exotoxin secretion even in the absence of NIR radiation. However, enhanced degradation of existing biofilm and exotoxin was observed when PGs were used with NIR laser irradiation.ConclusionThis promising new strategy achieved both the reduction of viable microorganisms and elimination of biofilm and exotoxin. Thus, this strategy addresses the long-ignored issue of persistence of bacterial residues that perpetuate chronic illness in patients even after viable bacteria have been eradicated.

A stealth adhesion factor contributes to Vibrio vulnificus pathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation.

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the Δtad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the Δtad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The Δtad123 mutant cells showed attenuated host cell adhesion, decreased biofilm formation, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The Δtad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Complement depletion by treating with anti-C5 antibody rescued the viable count of Δtad123 in infected mouse bloodstream to the level comparable to wild type strain. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.

Tools of Aggregatibacter actinomycetemcomitans to Evade the Host Response.

Periodontitis is an infection-induced inflammatory disease that affects the tooth supporting tissues, i.e., bone and connective tissues. The initiation and progression of this disease depend on dysbiotic ecological changes in the oral microbiome, thereby affecting the severity of disease through multiple immune-inflammatory responses. Aggregatibacter actinomycetemcomitans is a facultative anaerobic Gram-negative bacterium associated with such cellular and molecular mechanisms associated with the pathogenesis of periodontitis. In the present review, we outline virulence mechanisms that help the bacterium to escape the host response. These properties include invasiveness, secretion of exotoxins, serum resistance, and release of outer membrane vesicles. Virulence properties of A. actinomycetemcomitans that can contribute to treatment resistance in the infected individuals and upon translocation to the circulation, also induce pathogenic mechanisms associated with several systemic diseases.

Repurposing the anticancer drug cisplatin with the aim of developing novel Pseudomonas aeruginosa infection control agents.

Antibiotic resistance threatens effective treatment of microbial infections globally. This situation has spurred the hunt for new antimicrobial compounds in both academia and the pharmaceutical industry. Here, we report how the widely used antitumor drug cisplatin may be repurposed as an effective antimicrobial against the nosocomial pathogen Pseudomonas aeruginosa. Cisplatin was found to effectively kill strains of P. aeruginosa. In such experiments, transcriptomic profiling showed upregulation of the recA gene, which is known to be important for DNA repair, implicating that cisplatin could interfere with DNA replication in P. aeruginosa. Cisplatin treatment significantly repressed the type III secretion system (T3SS), which is important for the secretion of exotoxins. Furthermore, cisplatin was also demonstrated to eradicate in vitro biofilms and in vivo biofilms in a murine keratitis model. This showed that cisplatin could be effectively used to eradicate biofilm infections which were otherwise difficult to be treated by conventional antibiotics. Although cisplatin is highly toxic for humans upon systemic exposure, a low toxicity was demonstrated with topical treatment. This indicated that higher-than-minimal inhibitory concentration (MIC) doses of cisplatin could be topically applied to treat persistent and recalcitrant P. aeruginosa infections.

Umbrella Sampling and X-ray Crystallographic Analysis Unveil an Arg-Asp Gate Facilitating Inhibitor Binding Inside Phosphopantetheine Adenylyltransferase Allosteric Cleft.

Phosphopantetheine adenylyltransferase (PPAT) is a rate-limiting enzyme essential for biosynthesis of coenzyme A (CoA), which in turn is responsible to regulate the secretion of exotoxins via type III secretion system in Pseudomonas aeruginosa, causing severe health concerns ranging from nosocomial infections to respiratory failure. Acetyl coenzyme A (AcCoA) is a newly reported inhibitor of PPAT, believed to regulate the cellular levels of CoA and thereby the pathogenesis. Very little is known so far regarding the mechanistic details of AcCoA binding inside PPAT-binding cleft. Herein, we have used extensive umbrella sampling simulations to decipher mechanistic insight into the inhibitor accommodation inside the binding cavity. We found that R90 and D94 residues act like a gate near the binding cavity to accommodate and stabilize the incoming ligand. Mutational models concerning these residues also show considerable difference in AcCoA-binding thermodynamics. To substantiate our findings, we have solved the first crystal structure of apo-PPAT from P. aeruginosa, which also found to agree with the simulation results. Collectively, these results describe the mechanistic details of accommodation of inhibitor molecule inside PPAT-binding cavity and also offer valuable insight into regulating cellular levels of CoA/AcCoA and thus controlling the pathogenicity.

Subinhibitory concentrations of punicalagin reduces expression of virulence-related exoproteins by Staphylococcus aureus.

Staphylococcus aureus produces a number of virulence factors. The major virulence factors exhibited by S aureus include various antigens, enzymes, cytotoxins and exotoxins (e.g. hemolysins, enterotoxins and toxic shock syndrome toxin). In this report, we show the influence of punicalagin on the secretion of exoprotein from S aureus by western blotting, tumor necrosis factor (TNF) release assay and quantitative RT-PCR. When added to S aureus cultures at an OD600 of 0.9, graded subinhibitory concentrations of punicalagin reduced the production of α-toxin, SEA and SEB in methicillin-resistant Staphylococcus aureus in a dose-dependent manner. Consistently, punicalagin reduced TNF-inducing activity by S aureus culture supernatants. Here, the transcriptional level of agr (accessory gene regulator) in S aureus was inhibited by punicalagin, suggesting that the reduced transcription may affect the secretion of exotoxins. These findings suggest that the expression of α-toxin and enterotoxins in S aureus is sensitive to the action of punicalagin, which may be an advantageous candidate in the treatment of toxigenic staphylococcal disease.

Draft Genome Sequence of Pseudomonas aeruginosa Strain WS394, a Multidrug-Resistant and Highly Cytotoxic Wound Isolate from Chronic Ulcus Cruris.

Pseudomonas aeruginosa is a frequent human pathogen that increasingly causes chronic infections of nonhealing wounds. Here we present the 6.8 Mb draft genome of strain WS394, a multidrug-resistant chronic ulcer isolate that exhibited outstanding high cell cytotoxicity despite defective secretion of exotoxin U, suggesting a habitat-dependent adaptation process.

Novel Antimicrobial Peptides That Inhibit Gram Positive Bacterial Exotoxin Synthesis

Gram-positive bacteria, such as Staphylococcus aureus, cause serious human illnesses through combinations of surface virulence factors and secretion of exotoxins. Our prior studies using the protein synthesis inhibitor clindamycin and signal transduction inhibitors glycerol monolaurate and α-globin and β-globin chains of hemoglobin indicate that their abilities to inhibit exotoxin production by S. aureus are separable from abilities to inhibit growth of the organism. Additionally, our previous studies suggest that inhibition of exotoxin production, in absence of ability to kill S. aureus and normal flora lactobacilli, will prevent colonization by pathogenic S. aureus, while not interfering with lactobacilli colonization. These disparate activities may be important in development of novel anti-infective agents that do not alter normal flora. We initiated studies to explore the exotoxin-synthesis-inhibition activity of hemoglobin peptides further to develop potential agents to prevent S. aureus infections. We tested synthesized α-globin chain peptides, synthetic variants of α-globin chain peptides, and two human defensins for ability to inhibit exotoxin production without significantly inhibiting S. aureus growth. All of these peptides were weakly or not inhibitory to bacterial growth. However, the peptides were inhibitory to exotoxin production with increasing activity dependent on increasing numbers of positively-charged amino acids. Additionally, the peptides could be immobilized on agarose beads or have amino acid sequences scrambled and still retain exotoxin-synthesis-inhibition. The peptides are not toxic to human vaginal epithelial cells and do not inhibit growth of normal flora L. crispatus. These peptides may interfere with plasma membrane signal transduction in S. aureus due to their positive charges.

Reprint of “GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC)”

Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures. Here, we apply a solution-phase electrophoretic platform (GELFrEE) followed by MS to characterize the secreted proteome of a wild type and mutant strain of EPEC (ΔsepD), exhibiting enhanced secretion of effector proteins. Through peptide-level analysis, a total of 363 and 155 proteins were identified from the wild type and mutant strains, respectively. Semi-quantitative analysis of the MS data reveals the effector proteins EspB, EspC, and EspD appear in a relatively greater abundance from wild type EPEC, while two major virulence factors in EPEC, Tir and NleA appear in greater abundance from the secreted proteome of the mutant strain. Additionally, intact proteins may further be characterized following GELFrEE with MS to improve throughput of analysis. This study demonstrates the application of GELFrEE-MS to differentiate wild type and mutant strains of EPEC. This platform is therefore a powerful tool to study exotoxin secretion from pathogenic bacteria.

Salmonella entericadelivers its genotoxin through outer membrane vesicles secreted from infected cells

Cytolethal-distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram-negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella-containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71-CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71-CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71-CDT also secreted OMVs-like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin-loaded OMVs by bystander cells was dependent on dynamin-1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria.

GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures. Here, we apply a solution-phase electrophoretic platform (GELFrEE) followed by MS to characterize the secreted proteome of a wild type and mutant strain of EPEC (ΔsepD), exhibiting enhanced secretion of effector proteins. Through peptide-level analysis, a total of 363 and 155 proteins were identified from the wild type and mutant strains, respectively. Semi-quantitative analysis of the MS data reveals the effector proteins EspB, EspC, and EspD appear in a relatively greater abundance from wild type EPEC, while two major virulence factors in EPEC, Tir and NleA appear in greater abundance from the secreted proteome of the mutant strain. Additionally, intact proteins may further be characterized following GELFrEE with MS to improve throughput of analysis. This study demonstrates the application of GELFrEE-MS to differentiate wild type and mutant strains of EPEC. This platform is therefore a powerful tool to study exotoxin secretion from pathogenic bacteria.

Relative contribution of three main virulence factors in Pseudomonas aeruginosa pneumonia*

The pathogenesis and the outcome of Pseudomonas aeruginosa ventilator-acquired pneumonia depend on the virulence factors displayed by the bacteria as well as the host response. Thus, quorum sensing, lipopolysaccharide, and type 3 secretion system have each individually been shown to be important virulence systems in laboratory reference strains. However, the relative contribution of these three factors to the in vivo pathogenicity of clinically relevant strains has never been studied. We analyzed the virulence of 56 nonclonal Pseudomonas aeruginosa strains isolated from critically ill patients with ventilator-acquired pneumonia. To avoid the variation of human immune response, we used a murine model of pneumonia. The aim was to determine which virulence factor was the most important. Research laboratory of a university. Male adult BALB/c mice. In vitro, the phenotype of each strain was established as to the expression of quorum sensing-regulated factors (elastase and pyocyanin), type 3 secretion system exotoxin secretion (Exotoxin U, S and/or T, or "nonsecreting"), and lipopolysaccharide O-antigen serotype. Strain pathogenicity was evaluated in vivo in a mouse model of acute pneumonia through lung injury assessment by measuring alveolar-capillary barrier permeability to proteins, lung wet/dry weight ratio, and bacterial dissemination. Associations were then sought between virulence system phenotypes and levels of lung injury. In univariate analysis, elastase production, O11 serotype, and type 3 secretion system exotoxin secretion were associated with increased lung injury and exotoxin U was linked to an increase risk of bacteremia. In multivariate analysis, we observed that type 3 secretion system exotoxin secretion and to a lesser degree elastase production were associated with increased lung injury. In a murine model of pneumonia, our data suggest that type 3 secretion system and elastase are the most important virulence factors in clinically relevant P. aeruginosa strains.

Exotoxin Secretion: Getting Out to Find the Way In

During infection, most pathogenic bacteria deliver proteins to the host cell cytoplasm to manipulate host behavior. In this issue of Cell Host & Microbe, Spanò and colleagues describe a system where a bacterium produces an exotoxin while inside the host cell. Only after this exotoxin is transported to the mammalian cell surface and secreted into the extracellular milieu can it intoxicate the infected cell or noninfected distant cells.

The Type III Pseudomonal Exotoxin U Activates the c-Jun NH2-Terminal Kinase Pathway and Increases Human Epithelial Interleukin-8 Production

Microbial interactions with host cell signaling pathways are key determinants of the host cell response to infection. Many toxins secreted by bacterial type III secretion systems either stimulate or inhibit the host inflammatory response. We investigated the role of type III secreted toxins of the lung pathogen Pseudomonas aeruginosa in the inflammatory response of human respiratory epithelial cells to infection. Using bacteria with specific gene deletions, we found that interleukin-8 production by these cells was almost entirely dependent on bacterial type III secretion of exotoxin U (ExoU), a phospholipase, although other bacterial factors are involved. ExoU activated the c-Jun NH(2)-terminal kinase pathway, stimulating the phosphorylation and activation of mitogen-activated kinase kinase 4, c-Jun NH(2)-terminal kinase, and c-Jun. This in turn increased levels of transcriptionally competent activator protein-1. Although this pathway was dependent on the lipase activity of ExoU, it was independent of cell death. Activation of mitogen-activated kinase signaling by ExoU in this fashion is a novel mechanism by which a bacterial product can initiate a host inflammatory response, and it may result in increased epithelial permeability and bacterial spread.

Chronic Sinusitis with Nasal Polyps: Staphylococcal Exotoxin Immunoglobulin E and Cellular Inflammation

The etiology of chronic sinusitis with nasal polyposis (CS/NP) remains enigmatic. Frequently, Staphylococcus aureus is present in the nose of CS/NP patients, although the significance is unclear. Recent reports have suggested the hypothesis that these bacteria may secrete exotoxins triggering the inflammatory mucosal changes seen in CS/NP. This mechanism of immunopathology has been established in other diseases associated with Staphylococcus colonization and exotoxin secretion such as atopic dermatitis. In atopic dermatitis, the exotoxins incite a local superantigen response in which clonal T-cell activation and massive cytokine release occur in the affected skin. Second, these exotoxins can act as traditional allergens, stimulating a typical immunoglobulin E (IgE) response in the serum, which has been correlated with disease severity. This study is designed to begin the assessment of the hypothesis that a similar mechanism takes place in CS/NP. Serum was drawn from patients with CS/NP undergoing endoscopic sinus surgery as well as 13 atopic and nonatopic control subjects without sinusitis. IgE levels to S. aureus exotoxins A (SEA), SE exotoxins B (SEB), and toxic shock syndrome toxin 1 were measured using enzyme-linked immunosorbent assay. Tissue eosinophilia and the presence of lymphocytes on hemotoxylin and eosin-stained sections of polyps were scored by a blinded pathologist and correlated to presence of toxin IgE in the serum. Staphylococcal exotoxin (SE)-specific IgE was found in the serum of 5/10 (50%) of the patients with CS/NP. In contrast, 0/13 control patients had IgE to the exotoxins (p = 0.031). Polyp eosinophil, lymphocyte, and mononuclear cell counts were compared in IgE exotoxin-positive and -negative subjects. A trend toward increased eosinophil counts in patients with SE IgE (SE IgE+) was present, but not statistically significant. These results indicate that a high percentage of CS/NP patients show a systemic IgE response to S. aureus exotoxins in comparison with controls without CS/NP. Although these results are consistent with the actions of Staphylococcus toxins in other diseases, additional work is necessary to establish a local superantigen response in the nasal mucosa of CS/NP patients.