Discovery of cyanophycin dipeptide hydrolase enzymes suggests widespread utility of the natural biopolymer cyanophycin.

Cyanophycin is a bacterial polymer mainly used for nitrogen storage. It is composed of a peptide backbone of L-aspartate residues with L-arginines attached to their side chains through isopeptide bonds. Cyanophycin is degraded in two steps: Cyanophycinase cleaves the polymer into ß-Asp-Arg dipeptides, which are hydrolyzed into free Asp and Arg by enzymes possessing isoaspartyl dipeptide hydrolase activity. Two unrelated enzymes with this activity, isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA) have been shown to degrade ß-Asp-Arg dipeptides, but bacteria which encode cyanophycin-metabolizing genes can lack iaaA and iadA genes. In this study, we investigate a previously uncharacterized enzyme whose gene can cluster with cyanophycin-metabolizing genes. This enzyme, which we name cyanophycin dipeptide hydrolase (CphZ), is specific for dipeptides derived from cyanophycin degradation. Accordingly, a co-complex structure of CphZ and ß-Asp-Arg shows that CphZ, unlike IadA or IaaA, recognizes all portions of its ß-Asp-Arg substrate. Bioinformatic analyses showed that CphZ is found in very many proteobacteria and is homologous to an uncharacterized protein encoded in the "arginine/ornithine transport" (aot) operon of many pseudomonas species, including Pseudomonas aeruginosa. In vitro assays show that AotO is indeed a CphZ, and in cellulo growth experiments show that this enzyme and the aot operon allow P. aeruginosa to take up and use ß-Asp-Arg as a sole carbon and nitrogen source. Together the results establish the novel, highly specific enzyme subfamily of CphZs, suggesting that cyanophycin is potentially used by a much wider range of bacteria than previously appreciated.

Defining the functional properties of cyclopropane fatty acid synthase from Pseudomonas aeruginosa PAO1.

Cyclopropane fatty acid synthases (CFAS) catalyze the conversion of unsaturated fatty acids to cyclopropane fatty acids (CFAs) within bacterial membranes. This modification alters the biophysical properties of membranes and has been correlated with virulence in several human pathogens. Despite the central role played by CFAS enzymes in regulating bacterial stress responses, the mechanistic properties of the CFAS enzyme family and the consequences of CFA biosynthesis remain largely uncharacterized in most bacteria. We report the first characterization of the CFAS enzyme from Pseudomonas aeruginosa (PA), an opportunistic human pathogen with complex membrane biology that is frequently associated with antimicrobial resistance and high tolerance to various external stressors. We demonstrate that CFAs are produced by a single enzyme in PA and that cfas gene expression is upregulated during the transition to stationary phase and in response to oxidative stress. Analysis of PA lipid extracts reveal a massive increase in CFA production as PA cells enter stationary phase and help define the optimal membrane composition for in vitro assays. The purified PA-CFAS enzyme forms a stable homodimer and preferentially modifies phosphatidylglycerol lipid substrates and membranes with a higher content of unsaturated acyl chains. Bioinformatic analysis across bacterial phyla shows highly divergent amino acid sequences within the lipid-binding domain of CFAS enzymes, perhaps suggesting distinct membrane-binding properties among different orthologs. This work lays an important foundation for further characterization of CFAS in P. aeruginosa and for examining the functional differences between CFAS enzymes from different bacteria.

LasR-deficient Pseudomonas aeruginosa variants increase airway epithelial mICAM-1 expression and enhance neutrophilic lung inflammation.

Pseudomonas aeruginosa causes chronic airway infections, a major determinant of lung inflammation and damage in cystic fibrosis (CF). Loss-of-function lasR mutants commonly arise during chronic CF infections, are associated with accelerated lung function decline in CF patients and induce exaggerated neutrophilic inflammation in model systems. In this study, we investigated how lasR mutants modulate airway epithelial membrane bound ICAM-1 (mICAM-1), a surface adhesion molecule, and determined its impact on neutrophilic inflammation in vitro and in vivo. We demonstrated that LasR-deficient strains induce increased mICAM-1 levels in airway epithelial cells compared to wild-type strains, an effect attributable to the loss of mICAM-1 degradation by LasR-regulated proteases and associated with enhanced neutrophil adhesion. In a subacute airway infection model, we also observed that lasR mutant-infected mice displayed greater airway epithelial ICAM-1 expression and increased neutrophilic pulmonary inflammation. Our findings provide new insights into the intricate interplay between lasR mutants, LasR-regulated proteases and airway epithelial ICAM-1 expression, and reveal a new mechanism involved in the exaggerated inflammatory response induced by lasR mutants.

Cranberry-Derived Proanthocyanidins Potentiate ß-Lactam Antibiotics against Resistant Bacteria.

The emergence and spread of extended-spectrum ß-lactamases (ESBLs), metallo-ß-lactamases (MBLs), or variant low-affinity penicillin-binding proteins (PBPs) pose a major threat to our ability to treat bacterial infection using ß-lactam antibiotics. Although combinations of ß-lactamase inhibitors with ß-lactam agents have been clinically successful, there are no MBL inhibitors in current therapeutic use. Furthermore, recent clinical use of new-generation cephalosporins targeting PBP2a, an altered PBP, has led to the emergence of resistance to these antimicrobial agents. Previous work shows that natural polyphenols such as cranberry-extracted proanthocyanidins (cPAC) can potentiate non-ß-lactam antibiotics against Gram-negative bacteria. This study extends beyond previous work by investigating the in vitro effect of cPAC in overcoming ESBL-, MBL-, and PBP2a-mediated ß-lactam resistance. The results show that cPAC exhibit variable potentiation of different ß-lactams against ß-lactam-resistant Enterobacteriaceae clinical isolates as well as ESBL- and MBL-producing E. coli We also discovered that cPAC have broad-spectrum inhibitory properties in vitro on the activity of different classes of ß-lactamases, including CTX-M3 ESBL and IMP-1 MBL. Furthermore, we observe that cPAC selectively potentiate oxacillin and carbenicillin against methicillin-resistant but not methicillin-sensitive staphylococci, suggesting that cPAC also interfere with PBP2a-mediated resistance. This study motivates the need for future work to identify the most bioactive compounds in cPAC and to evaluate their antibiotic-potentiating efficacy in vivoIMPORTANCE The emergence of ß-lactam-resistant Enterobacteriaceae and staphylococci compromises the effectiveness of ß-lactam-based therapy. By acquisition of ESBLs, MBLs, or PBPs, it is highly likely that bacteria may become completely resistant to the most effective ß-lactam agents in the near future. In this study, we described a natural extract rich in proanthocyanidins which exerts adjuvant properties by interfering with two different resistance mechanisms. By their broad-spectrum inhibitory ability, cranberry-extracted proanthocyanidins could have the potential to enhance the effectiveness of existing ß-lactam agents.

Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase Pseudomonas aeruginosa.

Metabolically quiescent bacteria represent a large proportion of those in natural and host environments, and they are often refractory to antibiotic treatment. Such drug tolerance is also observed in the laboratory during stationary phase, when bacteria face stress and starvation-induced growth arrest. Tolerance requires (p)ppGpp signaling, which mediates the stress and starvation stringent response (SR), but the downstream effectors that confer tolerance are unclear. We previously demonstrated that the SR is linked to increased antioxidant defenses in Pseudomonas aeruginosa We now demonstrate that superoxide dismutase (SOD) activity is a key factor in SR-mediated multidrug tolerance in stationary-phase P. aeruginosa Inactivation of the SR leads to loss of SOD activity and decreased multidrug tolerance during stationary phase. Genetic or chemical complementation of SOD activity of the ΔrelA spoT mutant (ΔSR) is sufficient to restore antibiotic tolerance to WT levels. Remarkably, we observe high membrane permeability and increased drug internalization upon ablation of SOD activity. Combined, our results highlight an unprecedented mode of SR-mediated multidrug tolerance in stationary-phase P. aeruginosa and suggest that inhibition of SOD activity may potentiate current antibiotics.

Hydrophilic Mechano-Bactericidal Nanopillars Require External Forces to Rapidly Kill Bacteria.

Nanopillars have been shown to mechanically damage bacteria, suggesting a promising strategy for future antibacterial surfaces. However, the mechanisms underlying this phenomena remain unclear, which ultimately limits translational potential toward real-world applications. Using real-time and end-point analysis techniques, we demonstrate that in contrast to initial expectations, bacteria on multiple hydrophilic "mechano-bactericidal" surfaces remained viable unless exposed to a moving air-liquid interface, which caused considerable cell death. Reasoning that normal forces arising from surface tension may underlie this mechano-bactericidal activity, we developed computational and experimental models to estimate, manipulate, and recreate the impact of these forces. Our experiments together demonstrate that a critical level of external force acting on cells attached to nanopillar surfaces can rapidly deform and rupture bacteria. These studies provide fundamental physical insight into how nanopillar surfaces can serve as effective antibacterial materials and suggest use-conditions under which such nanotechnology approaches may provide practical value.

Pressure-driven changes to spontaneous flow in active nematic liquid crystals.

We consider the effects of a pressure gradient on the spontaneous flow of an active nematic liquid crystal in a channel, subject to planar anchoring and no-slip conditions on the boundaries of the channel. We employ a model based on the Ericksen-Leslie theory of nematics, with an additional active stress accounting for the activity of the fluid. By directly solving the flow equation, we consider an asymptotic solution for the director angle equation for large activity parameter values and predict the possible values of the director angle in the bulk of the channel. Through a numerical solution of the full nonlinear equations, we examine the effects of pressure on the branches of stable and unstable equilibria, some of which are disconnected from the no-flow state. In the absence of a pressure gradient, solutions are either symmetric or antisymmetric about the channel midpoint; these symmetries are changed by the pressure gradient. Considering the activity-pressure state space allows us to predict qualitatively the extent of each solution type and to show, for large enough pressure gradients, that a branch of non-trivial director angle solutions exists for all activity values.

Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

Chronic Pseudomonas aeruginosa lung infections are associated with progressive epithelial damage and lung function decline. In addition to its role in tissue injury, the persistent presence of P. aeruginosa-secreted products may also affect epithelial repair ability, raising the need for new antivirulence therapies. The purpose of our study was to better understand the outcomes of P. aeruginosa exoproducts exposure on airway epithelial repair processes to identify a strategy to counteract their deleterious effect. We found that P. aeruginosa exoproducts significantly decreased wound healing, migration, and proliferation rates, and impaired the ability of directional migration of primary non-cystic fibrosis (CF) human airway epithelial cells. Impact of exoproducts was inhibited after mutations in P. aeruginosa genes that encoded for the quorum-sensing (QS) transcriptional regulator, LasR, and the elastase, LasB, whereas impact was restored by LasB induction in ΔlasR mutants. P. aeruginosa purified elastase also induced a significant decrease in non-CF epithelial repair, whereas protease inhibition with phosphoramidon prevented the effect of P. aeruginosa exoproducts. Furthermore, treatment of P. aeruginosa cultures with 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a QS inhibitor, abrogated the negative impact of P. aeruginosa exoproducts on airway epithelial repair. Finally, we confirmed our findings in human airway epithelial cells from patients with CF, a disease featuring P. aeruginosa chronic respiratory infection. These data demonstrate that secreted proteases under the control of the LasR QS system impair airway epithelial repair and that QS inhibitors could be of benefit to counteract the deleterious effect of P. aeruginosa in infected patients.-Ruffin, M., Bilodeau, C., Maillé, É., LaFayette, S. L., McKay, G. A., Trinh, N. T. N., Beaudoin, T., Desrosiers, M.-Y., Rousseau, S., Nguyen, D., Brochiero, E. Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

The stringent response modulates 4-hydroxy-2-alkylquinoline biosynthesis and quorum-sensing hierarchy in Pseudomonas aeruginosa.

As a ubiquitous environmental organism and an important human pathogen, Pseudomonas aeruginosa readily adapts and responds to a wide range of conditions and habitats. The intricate regulatory networks that link quorum sensing and other global regulators allow P. aeruginosa to coordinate its gene expression and cell signaling in response to different growth conditions and stressors. Upon nutrient transitions and starvation, as well as other environmental stresses, the stringent response is activated, mediated by the signal (p)ppGpp. P. aeruginosa produces a family of molecules called HAQ (4-hydroxy-2-alkylquinolines), some of which exhibit antibacterial and quorum-sensing signaling functions and regulate virulence genes. In this study, we report that (p)ppGpp negatively regulates HAQ biosynthesis: in a (p)ppGpp-null (ΔSR) mutant, HHQ (4-hydroxyl-2-heptylquinoline) and PQS (3,4-dihydroxy-2-heptylquinoline) levels are increased due to upregulated pqsA and pqsR expression and reduced repression by the rhl system. We also found that (p)ppGpp is required for full expression of both rhl and las AHL (acyl-homoserine lactone) quorum-sensing systems, since the ΔSR mutant has reduced rhlI, rhlR, lasI, and lasR expression, butanoyl-homoserine lactone (C4-HSL) and 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C12-HSL) levels, and rhamnolipid and elastase production. Furthermore, (p)ppGpp significantly modulates the AHL and PQS quorum-sensing hierarchy, as the las system no longer has a dominant effect on HAQ biosynthesis when the stringent response is inactivated.

Titanium-Dioxide-Nanoparticle-Embedded Polyelectrolyte Multilayer as an Osteoconductive and Antimicrobial Surface Coating.

Bioactive surface coatings have retained the attention of researchers and physicians due to their versatility and range of applications in orthopedics, particularly in infection prevention. Antibacterial metal nanoparticles (mNPs) are a promising therapeutic, with vast application opportunities on orthopedic implants. The current research aimed to construct a polyelectrolyte multilayer on a highly porous titanium implant using alternating thin film coatings of chitosan and alginate via the layer-by-layer (LbL) self-assembly technique, along with the incorporation of silver nanoparticles (AgNPs) or titanium dioxide nanoparticles (TiO2NPs), for antibacterial and osteoconductive activity. These mNPs were characterized for their physicochemical properties using quartz crystal microgravimetry with a dissipation system, nanoparticle tracking analysis, scanning electron microscopy, and atomic force microscopy. Their cytotoxicity and osteogenic differentiation capabilities were assessed using AlamarBlue and alkaline phosphatase (ALP) activity assays, respectively. The antibiofilm efficacy of the mNPs was tested against Staphylococcus aureus. The LbL polyelectrolyte coating was successfully applied to the porous titanium substrate. A dose-dependent relationship between nanoparticle concentration and ALP as well as antibacterial effects was observed. TiO2NP samples were also less cytotoxic than their AgNP counterparts, although similarly antimicrobial. Together, these data serve as a proof-of-concept for a novel coating approach for orthopedic implants with antimicrobial and osteoconductive properties.