Co-regulation of β-lactam resistance, alginate production and quorum sensing in Pseudomonas aeruginosa

Development of β-lactam resistance, production of alginate and modulation of virulence factor expression that alters host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. In this study, we propose that a co-regulatory network exists between these mechanisms. We compared the promoter activities of ampR, algT/U, lasR, lasI, rhlR, rhlI and lasA genes, representing the β-lactam antibiotic resistance master regulatory gene, the alginate switch operon, the las and rhl quorum-sensing (QS) genes, and the LasA staphylolytic protease, respectively. Four isogenic P. aeruginosa strains, the prototypic Alg− PAO1, Alg− PAOampR, the mucoid Alg+ PAOmucA22 (Alg+ PDO300) and Alg+ PAOmucA22ampR (Alg+ PDOampR) were used. We found that in the presence of AmpR regulator and β-lactam antibiotic, the extracytoplasmic function sigma factor AlgT/U positively regulated PampR, whereas AmpR negatively regulated PalgT/U. On the basis of this finding we suggest the presence of a negative feedback loop to limit algT/U expression. In addition, the functional AlgT/U caused a significant decrease in the expression of QS genes, whereas loss of ampR only resulted in increased PlasI and PlasR transcription. The upregulation of the las QS system is likely to be responsible for the increased lasA promoter and the LasA protease activities in Alg− PAOampR and Alg+ PDOampR. The enhanced expression of virulence factors in the ampR strains correlated with a higher rate of Caenorhabditis elegans paralysis. Hence, this study shows that the loss of ampR results in increased virulence, and is indicative of the existence of a co-regulatory network between β-lactam resistance, alginate production, QS and virulence factor production, with AmpR playing a central role

Bacillus anthracis edema factor substrate specificity: evidence for new modes of action

Since the isolation of Bacillus anthracis exotoxins in the 1960s, the detrimental activity of edema factor (EF) was considered as adenylyl cyclase activity only. Yet the catalytic site of EF was recently shown to accomplish cyclization of cytidine 5'-triphosphate, uridine 5'-triphosphate and inosine 5'-triphosphate, in addition to adenosine 5'-triphosphate. This review discusses the broad EF substrate specificity and possible implications of intracellular accumulation of cyclic cytidine 3':5'-monophosphate, cyclic uridine 3':5'-monophosphate and cyclic inosine 3':5'-monophosphate on cellular functions vital for host defense. In particular, cAMP-independent mechanisms of action of EF on host cell signaling via protein kinase A, protein kinase G, phosphodiesterases and CNG channels are discussed

Anthrax Lethal Toxin Suppresses Murine Cardiomyocyte Contractile Function and Intracellular Ca2+ Handling via a NADPH Oxidase-Dependent Mechanism

OBJECTIVES: Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca(2+) properties. METHODS: Murine cardiomyocyte contractile function and intracellular Ca(2+) handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR(90)), intracellular Ca(2+) rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca(2+) decay rate. Stress signaling and Ca(2+) regulatory proteins were assessed using Western blot analysis. RESULTS: In vitro exposure to a lethal toxin (0.05-50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca(2+) properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca(2+) decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca(2+) responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca(2+) regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure. CONCLUSIONS: Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca(2+) through a NADPH oxidase-dependent mechanism

Anthrax Toxins Induce Shock in Rats by Depressed Cardiac Ventricular Function

Anthrax infections are frequently associated with severe and often irreversible hypotensive shock. The isolated toxic proteins of Bacillus anthracis produce a non-cytokine-mediated hypotension in rats by unknown mechanisms. These observations suggest the anthrax toxins have direct cardiovascular effects. Here, we characterize these effects. As a first step, we administered systemically anthrax lethal toxin (LeTx) and edema toxin (EdTx) to cohorts of three to twelve rats at different doses and determined the time of onset, degree of hypotension and mortality. We measured serum concentrations of the protective antigen (PA) toxin component at various time points after infusion. Peak serum levels of PA were in the µg/mL range with half-lives of 10–20 minutes. With doses that produced hypotension with delayed lethality, we then gave bolus intravenous infusions of toxins to groups of four to six instrumented rats and continuously monitored blood pressure by telemetry. Finally, the same doses used in the telemetry experiments were given to additional groups of four rats, and echocardiography was performed pretreatment and one, two, three and twenty-four hours post-treatment. LeTx and EdTx each produced hypotension. We observed a doubling of the velocity of propagation and 20% increases in left ventricular diastolic and systolic areas in LeTx-treated rats, but not in EdTx-treated rats. EdTx-but not LeTx-treated rats showed a significant increase in heart rate. These results indicate that LeTx reduced left ventricular systolic function and EdTx reduced preload. Uptake of toxins occurs readily into tissues with biological effects occurring within minutes to hours of serum toxin concentrations in the µg/mL range. LeTx and EdTx yield an irreversible shock with subsequent death. These findings should provide a basis for the rational design of drug interventions to reduce the dismal prognosis of systemic anthrax infections

Identification of Genes in the σ22 Regulon of Pseudomonas aeruginosa Required for Cell Envelope Homeostasis in Either the Planktonic or the Sessile Mode of Growth

The Pseudomonas aeruginosa extracytoplasmic functioning (ECF) sigma factor σ22 is encoded by algT/algU and is inhibited by anti-sigma factor MucA. σ22 was originally discovered for its essential role in the expression of the exopolysaccharide alginate by mucoid strains associated with chronic pulmonary infection. However, σ22 is now known to also have a large regulon associated with the response to cell wall stress. Our recent transcriptome analysis identified 293 open reading frames (ORFs) in the σ22 stress stimulon that include genes for outer envelope biogenesis and remodeling, although most of the genes have undefined functions. To better understand the σ22-dependent stress response, mutants affected in 27 genes of the σ22 stimulon were examined and expression was studied with lacZ fusions. Mutants constructed in the 27 genes showed no major change in response to cell wall-acting antibiotics or growth at elevated temperatures nor in alginate production. The mutants were examined for their effects on the expression of the σ22-dependent promoter of the alginate biosynthetic operon (PalgD) as a measure of σ22 derepression from MucA. By testing PalgD expression under both planktonic and sessile growth conditions, 11 genes were found to play a role in the stress response that activates σ22. Some mutations caused an increase or a decrease in the response to cell wall stress. Interestingly, mutations in 7 of the 11 genes caused constitutive PalgD expression under nonstressed conditions and thus showed that these genes are involved in maintaining envelope homeostasis. Mutations in PA0062 and PA1324 showed constitutive PalgD expression during both the planktonic and the sessile modes of growth. However, the PA5178 mutation caused constitutive PalgD expression only during planktonic growth. In contrast, mutations in PA2717, PA0567, PA3040, and PA0920 caused constitutive PalgD expression only in the sessile/biofilm mode of growth. This provides evidence that the σ22 stimulon for cell envelope homeostasis overlaps with biofilm control mechanisms

Emergence of Anthrax Edema Toxin as a Master Manipulator of Macrophage and B Cell Functions

Anthrax edema toxin (ET), a powerful adenylyl cyclase, is an important virulence factor of Bacillus anthracis. Until recently, only a modest amount of research was performed to understand the role this toxin plays in the organism’s immune evasion strategy. A new wave of studies have begun to elucidate the effects this toxin has on a variety of host cells. While efforts have been made to illuminate the effect ET has on cells of the adaptive immune system, such as T cells, the greatest focus has been on cells of the innate immune system, particularly the macrophage. Here we discuss the immunoevasive activities that ET exerts on macrophages, as well as new research on the effects of this toxin on B cells

Salmonella enterica biofilm-mediated dispersal by nitric oxide donors in association with cellulose nanocrystal hydrogels

Protected by extracellular polymers, microbes within biofilms are significantly more resistant to disinfectants. Current research has been instrumental in identifying nitric oxide donors and hydrogels as potential disinfectant additives. Nitric oxide (NO) donors are considered a very promising molecule as biofilm dispersal agents and hydrogels have recently attracted a lot of interest due to their biocompatible properties and ability to form stable thin films. When the NO donor MAHMA NONOate was dissolved in phosphate saline buffer, it was able to reduce the biomass of well-established biofilms up to 15% for at least 24 h of contact time. Encapsulation of MAHMA NONOate and molsidomine within a hydrogel composed of cellulose nanocrystals (CNC) has shown a synergistic effect in dispersing well-established biofilms: after 2 h of exposure, moderate but significant dispersion was measured. After 6 h of exposure, the number of cells transitioning from the biofilm to the planktonic state was up to 0.6 log higher when compared with non-treated biofilms. To further explore the transport processes of NO donors within hydrogels, we measured the nitric oxide flux from gels, at 25°C for a composite of 0.1 µM MAHMA NONOate–CNC. Nitric oxide diffuses up to 500 µm from the hydrogel surface, with flux decreasing according to Fick’s law. 60% of NO was released from the hydrogel composite during the first 23 min. These data suggest that the combined treatments with nitric oxide donor and hydrogels may allow for new sustainable cleaning strategies

Cytoskeleton as an Emerging Target of Anthrax Toxins

Bacillus anthracis, the agent of anthrax, has gained virulence through its exotoxins produced by vegetative bacilli and is composed of three components forming lethal toxin (LT) and edema toxin (ET). So far, little is known about the effects of these toxins on the eukaryotic cytoskeleton. Here, we provide an overview on the general effects of toxin upon the cytoskeleton architecture. Thus, we shall discuss how anthrax toxins interact with their receptors and may disrupt the interface between extracellular matrix and the cytoskeleton. We then analyze what toxin molecular effects on cytoskeleton have been described, before discussing how the cytoskeleton may help the pathogen to corrupt general cell processes such as phagocytosis or vascular integrity

Systematic analysis of the ability of Nitric Oxide donors to dislodge biofilms formed by Salmonella enterica and Escherichia coli O157:H7

Biofilms in the industrial environment could be problematic. Encased in extracellular polymeric substances, pathogens within biofilms are significantly more resistant to chlorine and other disinfectants. Recent studies suggest that compounds capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of sessile bacteria and provide a foundation for novel approaches to controlling biofilms formed by some microorganisms. In this work, we compared the ability of five nitric oxide donors (molsidomine, MAHMA NONOate, diethylamine NONOate, diethylamine NONOate diethylammonium salt, spermine NONOate) to dislodge biofilms formed by non-typhoidal Salmonella enterica and pathogenic E. coli on plastic and stainless steel surfaces at different temperatures. All five nitric oxide donors induced significant (35-80%) dispersal of biofilms, however, the degree of dispersal and the optimal dispersal conditions varied. MAHMA NONOate and molsidomine were strong dispersants of the Salmonella biofilms formed on polystyrene. Importantly, molsidomine induced dispersal of up to 50% of the pre-formed Salmonella biofilm at 4 degrees C, suggesting that it could be effective even under refrigerated conditions. Biofilms formed by E. coli O157:H7 were also significantly dispersed. Nitric oxide donor molecules were highly active within 6 hours of application. To better understand mode of action of these compounds, we identified Salmonella genomic region recA-hydN, deletion of which led to an insensitivity to the nitric oxide donors

DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription

<p>Abstract</p> <p>Background</p> <p>The clinical course of cystic fibrosis (CF) is characterized by recurrent pulmonary infections and chronic inflammation. We have recently shown that decreased methylation of the toll-like receptor-2 (TLR2) promoter leads to an apparent CF-related up-regulation of TLR2. This up-regulation could be responsible, in part, for the CF-associated enhanced proinflammatory responses to various bacterial products in epithelial cells. However, the molecular mechanisms underlying DNA hypomethylation-dependent enhancement of TLR2 expression in CF cells remain unknown.</p> <p>Results</p> <p>The present study indicates that there is a specific CpG region (CpG#18-20), adjacent to the SP1 binding site that is significantly hypomethylated in several CF epithelial cell lines. These CpGs encompass a minimal promoter region required for basal TLR2 expression, and suggests that CpG#18-20 methylation regulates TLR2 expression in epithelial cells. Furthermore, reporter gene analysis indicated that the SP1 binding site is involved in the methylation-dependent regulation of the TLR2 promoter. Inhibition of SP1 with mithramycin A decreased TLR2 expression in both CF and 5-azacytidine-treated non-CF epithelial cells. Moreover, even though SP1 binding was not affected by CpG methylation, SP1-dependent transcription was abolished by CpG methylation.</p> <p>Conclusion</p> <p>This report implicates SP1 as a critical component of DNA demethylation-dependent up-regulation of TLR2 expression in CF epithelial cells.</p