Targeting the Pseudomonas aeruginosa Virulence Factor Phospholipase C With Engineered Liposomes.

Engineered liposomes composed of the naturally occurring lipids sphingomyelin (Sm) and cholesterol (Ch) have been demonstrated to efficiently neutralize toxins secreted by Gram-positive bacteria such as Streptococcus pneumoniae and Staphylococcus aureus. Here, we hypothesized that liposomes are capable of neutralizing cytolytic virulence factors secreted by the Gram-negative pathogen Pseudomonas aeruginosa. We used the highly virulent cystic fibrosis P. aeruginosa Liverpool Epidemic Strain LESB58 and showed that sphingomyelin (Sm) and a combination of sphingomyelin with cholesterol (Ch:Sm; 66 mol/% Ch and 34 mol/% Sm) liposomes reduced lysis of human bronchial and red blood cells upon challenge with the Pseudomonas secretome. Mass spectrometry of liposome-sequestered Pseudomonas proteins identified the virulence-promoting hemolytic phospholipase C (PlcH) as having been neutralized. Pseudomonas aeruginosa supernatants incubated with liposomes demonstrated reduced PlcH activity as assessed by the p-nitrophenylphosphorylcholine (NPPC) assay. Testing the in vivo efficacy of the liposomes in a murine cutaneous abscess model revealed that Sm and Ch:Sm, as single dose treatments, attenuated abscesses by >30%, demonstrating a similar effect to that of a mutant lacking plcH in this infection model. Thus, sphingomyelin-containing liposome therapy offers an interesting approach to treat and reduce virulence of complex infections caused by P. aeruginosa and potentially other Gram-negative pathogens expressing PlcH.

Selection of M2+ -Independent RNA-Cleaving DNAzymes with Side-Chains Mimicking Arginine and Lysine.

Sequence-specific cleavage of RNA by nucleic acid catalysts in the absence of a divalent metal cation (M2+ ) has remained an important goal in biomimicry with potential therapeutic applications. Given the lack of functional group diversity in canonical nucleotides, modified nucleotides with amino acid-like side chains were used to enhance self-cleavage rates at a single embedded ribonucleoside site. Previous works relied on three functional groups: an amine, a guanidine and an imidazole ensconced on three different nucleosides. However, to date, few studies have systematically addressed the necessity of all three modifications, as the value of any single modified nucleoside is contextualized at the outset of selection. Herein, we report on the use of only two modified dNTPs, excluding an imidazole, i. e. 5-(3-guanidinoallyl)-2'-dUTP (dUga TP) and 5-aminoallyl-2'-dCTP (dCaa TP), to select in-vitro self-cleaving DNAzymes that cleave in the absence of M2+ in a pH-independent fashion. Cleavage shows biphasic kinetics with rate constants that are significantly higher than in unmodified DNAzymes and compare favorably to certain DNAzymes involving an imidazole.

Surfing motility is a complex adaptation dependent on the stringent stress response in Pseudomonas aeruginosa LESB58.

Cystic fibrosis (CF) is a genetic disease that affects mucin-producing body organs such as the lungs. Characteristic of CF is the production of thick, viscous mucus, containing the glycoprotein mucin, that can lead to progressive airway obstruction. Recently, we demonstrated that the presence of mucin induced a rapid surface adaptation in motile bacteria termed surfing motility, which data presented here indicates is very different from swarming motility. Pseudomonas aeruginosa, the main colonizing pathogen in CF, employs several stress coping mechanisms to survive the highly viscous environment of the CF lung. We used motility-based assays and RNA-Seq to study the stringent stress response in the hypervirulent CF isolate LESB58 (Liverpool Epidemic Strain). Motility experiments revealed that an LESB58 stringent response mutant (ΔrelAΔspoT) was unable to surf. Transcriptional profiling of ΔrelAΔspoT mutant cells from surfing agar plates, when compared to wild-type cells from the surfing edge, revealed 2,584 dysregulated genes. Gene Ontology and KEGG enrichment analysis revealed effects of the stringent response on amino acid, nucleic acid and fatty acid metabolism, TCA cycle and glycolysis, type VI secretion, as well as chemotaxis, cell communication, iron transport, nitrogen metabolic processes and cyclic-di-GMP signalling. Screening of the ordered PA14 transposon library revealed 224 mutants unable to surf and very limited overlap with genes required for swarming. Mutants affecting surfing included two downstream effector genes of the stringent stress response, the copper regulator cueR and the quinolone synthase pqsH. Both the cueR and pqsH cloned genes complemented the surfing deficiency of ΔrelAΔspoT. Our study revealed insights into stringent stress dependency in LESB58 and showed that surfing motility is stringently-controlled via the expression of cueR and pqsH. Downstream factors of the stringent stress response are important to investigate in order to fully understand its ability to colonize and persist in the CF lung.

Liposomal Therapy Attenuates Dermonecrosis Induced by Community-Associated Methicillin-Resistant Staphylococcus aureus by Targeting α-Type Phenol-Soluble Modulins and α-Hemolysin.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), typified by the pulse-field type USA300, is an emerging endemic pathogen that is spreading rapidly among healthy people. CA-MRSA causes skin and soft tissue infections, life-threatening necrotizing pneumonia and sepsis, and is remarkably resistant to many antibiotics. Here we show that engineered liposomes composed of naturally occurring sphingomyelin were able to sequester cytolytic toxins secreted by USA300 and prevent necrosis of human erythrocytes, peripheral blood mononuclear cells and bronchial epithelial cells. Mass spectrometric analysis revealed the capture by liposomes of phenol-soluble modulins, α-hemolysin and other toxins. Sphingomyelin liposomes prevented hemolysis induced by pure phenol-soluble modulin-α3, one of the main cytolytic components in the USA300 secretome. In contrast, sphingomyelin liposomes harboring a high cholesterol content (66 mol/%) were unable to protect human cells from phenol-soluble modulin-α3-induced lysis, however these liposomes efficiently sequestered the potent staphylococcal toxin α-hemolysin. In a murine cutaneous abscess model, a single dose of either type of liposomes was sufficient to significantly decrease tissue dermonecrosis. Our results provide further insights into the promising potential of tailored liposomal therapy in the battle against infectious diseases.