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At least 235 records · Page 13

Pseudomonas aeruginosa Adhesion and Biofilm Formation on Poly(l-lysine)-Tethered Hydrogels: Synergistic Effect of Substrate Stiffness and Positive Charge Density

Infections associated with antibacterial-resistant Pseudomonas aeruginosa (P. aeruginosa) are the major cause of morbidity and mortality of patients, presenting one of the greatest therapeutic challenges for treatment of community-acquired and nosocomial infections. To develop antimicrobial hydrogel coatings to control the adhesion and subsequent biofilm formation of P. aeruginosa, we have used photo-cross-linked poly(ethylene glycol) diacrylate (PEGDA) hydrogels with varied cross-linking densities and covalently grafted poly(l-lysine) (PLL) at different weight compositions (ϕ PLL ). Both surface stiffness and positive charge density of the hydrogels were efficiently tuned over a broad range to investigate their effects on two main strains of P. aeruginosa, PA01 and PA14. We found that both number and viability of attached cells were positively correlated with the hydrogel stiffness, leading to thicker and larger coverage of cell colonies at 72 h postseeding on the stiffer substrates. The dependence of both PA01 and PA14 strains on ϕ PLL , however, was nonmonotonic. Positive charges from dissociated amine groups in the grafted PLL chains significantly promoted initial adhesion and proliferation of both strains at low ϕ PLL and developed into the thickest biofilms on the stiffest hydrogels grafted with ϕ PLL of 1–2%. Nevertheless, on the softest hydrogels grafted with PLL at high ϕ PLL of 7–10%, the bacteria no longer attached or survived. These results not only improved our fundamental understanding of bacteria-material interactions but also provided a series of PLL-grafted PEGDA hydrogels with controlled stiffness and positive charge density as ideal surface coating materials to prevent bacterial infections.

bacteria↗

Effects of Concentration, Salinity and Temperature on the Conformations of Zwitterionic Poly(2-Vinylpyridine- N -Oxide) Chains in Semidilute Solutions Probed by Small-Angle X-Ray and Neutron Scattering

Polyzwitterions, composed of repeating units with equal numbers of anionic and cationic groups, have drawn considerable interest for applications ranging from antimicrobial coatings and antifouling membranes to low-friction interfaces, energy storage media, and actuators. A characteristic feature of many polyzwitterions is the anti-polyelectrolyte effect salt-induced chain expansion in aqueous solution a phenomenon often implicated in their functional behavior. In this study, we investigated the conformational behavior of poly(2-vinylpyridine-N-oxide) (P2VPNO) using smallangle X-ray and neutron scattering (SAXS and SANS). Small-angle X-ray scattering (SAXS) revealed that, in salt-free aqueous solution, P2VPNO adopts an expanded wormlike conformation stabilized by hydration. With increasing concentration, the chains contract due to screening of intrachain excluded-volume interactions, qualitatively consistent with de Gennes’ scaling predictions for neutral polymers. Small-angle neutron scattering (SANS) further demonstrated that P2VPNO exhibits the characteristic anti-polyelectrolyte response to added salt, as observed in many other polyzwitterions. At elevated temperatures, chain flexibility increases, leading to a shorter Kuhn length and reduced radius of gyration. Notably, however, salt-induced chain expansion persists, indicating that the anti-polyelectrolyte effect remains operative under elevated thermal conditions. These findings provide the first experimental evidence of scaling behavior in polyzwitterions, as well as the first observation of their altered antipolyelectrolyte response at elevated temperatures, offering new insights into the solution physics of this important class of polymers.

Conformation↗

Catalytic Strategy for Conversion of Triacetic Acid Lactone to Potassium Sorbate

Here this study shows a new route to produce potassium sorbate (KS) from triacetic acid lactone (TAL), which is a chemical platform that can be biologically synthesized from natural sources. Sorbic acid and its potassium salt (KS) are widely used as preservatives in foods and pharmaceuticals. Three steps are used to produce KS from TAL: 1) hydrogenation of TAL into 4-hydroxy- 6-methyltetrahydro-2-pyrone (HMP), 2) dehydration of HMP to parasorbic acid (PSA), and 3) ring-opening and hydrolysis of PSA to KS. TAL can be fully hydrogenated over Ni/SiO2 to give near quantitative yields of HMP. A three-step reaction kinetics model was developed for dehydration of HMP into PSA. This model was used to show that the highest PSA yield occurs at low temperatures. An experimental PSA yield of 84.2% with respect to TAL was obtained which agreed with the prediction of the reaction kinetics model. KOH was used as a coreactant for the ring-opening hydrolysis of PSA to produce >99.9% yield of KS from PSA. Tetrahydrofuran (THF) was used to purify the TAL derived-KS (TAL-KS). The TAL-KS had a KS purity of 95.5%. The overall yield of TAL-KS with respect to TAL was calculated to be 77.3%. TAL-KS produced in this study had similar antimicrobial activities as commercial KS.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Restricted Rotational Flexibility of the C5α-Methyl-Substituted Carbapenem NA-1-157 Leads to Potent Inhibition of the GES-5 Carbapenemase

Carbapenem antibiotics are used as a last-resort treatment for infections caused by multidrug-resistant bacteria. The wide spread of carbapenemases in Gram-negative bacteria has severely compromised the utility of these drugs and represents a serious public health threat. To combat carbapenemase-mediated resistance, new antimicrobials and inhibitors of these enzymes are urgently needed. Here, we describe the interaction of the atypically C5α-methyl-substituted carbapenem, NA-1-157, with the GES-5 carbapenemase. MICs of this compound against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii producing the enzyme were reduced 4–16-fold when compared to MICs of the commercial carbapenems, reaching clinically sensitive breakpoints. When NA-1-157 was combined with meropenem, a strong synergistic effect was observed. Kinetic and ESI-LC/MS studies demonstrated that NA-1-157 is a potent inhibitor of GES-5, with a high inactivation efficiency of (2.9 ± 0.9) × 105 M –1 s –1 . Acylation of GES-5 by NA-1-157 was biphasic, with the fast phase completing within seconds, and the slow phase taking several hours and likely proceeding through a reversible tetrahedral intermediate. Deacylation was extremely slow (k 3 = (2.4 ± 0.3) × 10 –7 s –1 ), resulting in a residence time of 48 ± 6 days. MD simulation of the GES-5-meropenem and GES-5-NA-1-157 acyl-enzyme complexes revealed that the C5α-methyl group in NA-1- 157 sterically restricts rotation of the 6α-hydroxyethyl group preventing ingress of the deacylating water into the vicinity of the scissile bond of the acyl-enzyme intermediate. Furthermore, these data demonstrate that NA-1-157 is a potent irreversible inhibitor of the GES-5 carbapenemase.

60 APPLIED LIFE SCIENCES↗

Electrochemical Synthesis of Zeolite Coatings with Controlled Crystal Polymorphism and Self-Regulating Growth

Zeolite coatings are studied as molecular sieves for membrane separation, membrane reactors, and chemical sensor applications. They are also studied as anticorrosive films for metals and alloys, antimicrobial and hydrophobic films for heating, ventilation, and air conditioning, and dielectrics for semiconductor applications. Zeolite coatings are synthesized by hydrothermal, ionothermal, and dry-gel conversion approaches, which require high process temperatures and lengthy times (ranging from hours to days). Here, we report the first zeolite coatings synthesized via electrochemical deposition on a cathodic electrode, with controlled crystal polymorphism achieved within subhourly duration. We demonstrate this approach by developing sodium zeolite (e.g., sodalite (SOD), NaA (LTA), and Linde Type N (LTN)) coatings on a titanium electrode and extending the synthesis method to porous stainless steel. The coating morphology and crystallinity depend on the temperature, time, and applied current. The coating thickness is independent of the applied current, showing the presence of a self-regulating mechanism to ensure a uniform coating thickness across the metal surface. The electrochemical zeolite growth mechanism was elucidated with high-resolution transmission electron microscopy, and applications of the resultant zeolite coatings for oil/water separation and ethanol/water pervaporation were exploited. Electrochemical synthesis represents a novel, simple, fast, and environmentally friendly approach to preparing zeolite coatings. It can potentially be generalized for developing zeolite materials with diverse framework structures, morphologies, and orientations for substrates with complicated geometries.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A small RNA that cooperatively senses two stacked metabolites in one pocket for gene control

Riboswitches are structured non-coding RNAs often located upstream of essential genes in bacterial messenger RNAs. Such RNAs regulate expression of downstream genes by recognizing a specific cellular effector. Although nearly 50 riboswitch classes are known, only a handful recognize multiple effectors. Here, we report the 2.60-Å resolution co-crystal structure of a class I type I preQ 1 -sensing riboswitch that reveals two effectors stacked atop one another in a single binding pocket. These effectors bind with positive cooperativity in vitro and both molecules are necessary for gene regulation in bacterial cells. Stacked effector recognition appears to be a hallmark of the largest subgroup of preQ 1 riboswitches, including those from pathogens such as Neisseria gonorrhoeae. We postulate that binding to stacked effectors arose in the RNA World to closely position two substrates for RNA-mediated catalysis. These findings expand known effector recognition capabilities of riboswitches and have implications for antimicrobial development.

59 BASIC BIOLOGICAL SCIENCES↗

PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

59 BASIC BIOLOGICAL SCIENCES↗

Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model

Bacteriophage therapy is a promising approach to address antimicrobial infections though questions remain regarding the impact of the immune response on clinical effectiveness. Here, we develop a mouse model to assess phage treatment using a cocktail of five phages from the Myoviridae and Siphoviridae families that target Vancomycin-Resistant Enterococcus gut colonization. Phage treatment significantly reduces fecal bacterial loads of Vancomycin-Resistant Enterococcus. We also characterize immune responses elicited following administration of the phage cocktail. While minimal innate responses are observed after phage administration, two rounds of treatment induces phage-specific neutralizing antibodies and accelerate phage clearance from tissues. Interestingly, the myophages in our cocktail induce a more robust neutralizing antibody response than the siphophages. This anti-phage immunity reduces the effectiveness of the phage cocktail in our murine model. Collectively, this study shows phage-specific immune responses may be an important consideration in the development of phage cocktails for therapeutic use.

60 APPLIED LIFE SCIENCES↗

Structural basis of undecaprenyl phosphate glycosylation leading to polymyxin resistance in Gram-negative bacteria

In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral membrane glycosyltransferase, attaches a formylated form of aminoarabinose to the lipid undecaprenyl phosphate, enabling its association with the bacterial inner membrane. Here, we present cryo-electron microscopy structures of ArnC from S. enterica in apo and nucleotide-bound conformations. These structures reveal a conformational transition that takes place upon binding of the partial donor substrate. Using coarse-grained and atomistic simulations, we provide insights into substrate coordination before and during catalysis, and we propose a catalytic mechanism that may operate on all similar metal-dependent polyprenyl phosphate glycosyltransferases. The reported structures provide a new target for drug design aiming to combat polymyxin resistance.

Ashraf, Khuram U. [Rutgers University, Newark, NJ ↗

A mycobacterial ABC transporter mediates the uptake of hydrophilic compounds

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen and the causative agent of tuberculosis. Although Mtb can synthesize vitamin B 12 (cobalamin) de novo, uptake of cobalamin has been linked to pathogenesis of tuberculosis. Mtb does not encode any characterized cobalamin transporter; however, the gene rv1819c was found to be essential for uptake of cobalamin. This result is difficult to reconcile with the original annotation of Rv1819c as a protein implicated in the transport of antimicrobial peptides such as bleomycin. In addition, uptake of cobalamin seems inconsistent with the amino acid sequence, which suggests that Rv1819c has a bacterial ATP-binding cassette (ABC)-exporter fold. In this paper, we present structures of Rv1819c, which reveal that the protein indeed contains the ABC-exporter fold, as well as a large water-filled cavity of about 7,700 Å, which enables the protein to transport the unrelated hydrophilic compounds bleomycin and cobalamin. On the basis of these structures, we propose that Rv1819c is a multi-solute transporter for hydrophilic molecules, analogous to the multidrug exporters of the ABC transporter family, which pump out structurally diverse hydrophobic compounds from cells.

59 BASIC BIOLOGICAL SCIENCES↗

Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence

Abstract cGAS is an evolutionarily conserved enzyme that has a pivotal role in immune defence against infection 1–3 . In vertebrate animals, cGAS is activated by DNA to produce cyclic GMP–AMP (cGAMP) 4,5 , which leads to the expression of antimicrobial genes 6,7 . In bacteria, cyclic dinucleotide (CDN)-based anti-phage signalling systems (CBASS) have been discovered 8–11 . These systems are composed of cGAS-like enzymes and various effector proteins that kill bacteria on phage infection, thereby stopping phage spread. Of the CBASS systems reported, approximately 39% contain Cap2 and Cap3 , which encode proteins with homology to ubiquitin conjugating (E1/E2) and deconjugating enzymes, respectively 8,12 . Although these proteins are required to prevent infection of some bacteriophages 8 , the mechanism by which the enzymatic activities exert an anti-phage effect is unknown. Here we show that Cap2 forms a thioester bond with the C-terminal glycine of cGAS and promotes conjugation of cGAS to target proteins in a process that resembles ubiquitin conjugation. The covalent conjugation of cGAS increases the production of cGAMP. Using a genetic screen, we found that the phage protein Vs.4 antagonized cGAS signalling by binding tightly to cGAMP (dissociation constant of approximately 30 nM) and sequestering it. A crystal structure of Vs.4 bound to cGAMP showed that Vs.4 formed a hexamer that was bound to three molecules of cGAMP. These results reveal a ubiquitin-like conjugation mechanism that regulates cGAS activity in bacteria and illustrates an arms race between bacteria and viruses through controlling CDN levels.

Science & Technology - Other Topics↗

Synthetic antibacterial minerals: harnessing a natural geochemical reaction to combat antibiotic resistance

The overuse of antibiotics in clinical and livestock settings is accelerating the selection of multidrug resistant bacterial pathogens. Antibiotic resistant bacteria result in increased mortality and financial strain on the health care and livestock industry. The development of new antibiotics has stalled, and novel strategies are needed as we enter the age of antibiotic resistance. Certain naturally occurring clays have been shown to have antimicrobial properties and kill antibiotic resistant bacteria. Harnessing the activity of compounds within these clays that harbor antibiotic properties offers new therapeutic opportunities for fighting the potentially devastating effects of the post antibiotic era. However, natural samples are highly heterogenous and exhibit variable antibacterial effectiveness, therefore synthesizing minerals of high purity with reproducible antibacterial activity is needed. Here we describe for the first time synthetic smectite clay minerals and Fe-sulfide microspheres that reproduce the geochemical antibacterial properties observed in natural occurring clays. We show that these mineral formulations are effective at killing the ESKAPE pathogens ( Enterococcus sp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter sp., Pseudomonas aeruginosa and Enterobacter sp.) by maintaining Fe 2+ solubility and reactive oxygen species (ROS) production while buffering solution pH, unlike the application of metals alone. Our results represent the first step in utilizing a geochemical process to treat antibiotic resistant topical or gastrointestinal infections in the age of antibiotic resistance.

58 GEOSCIENCES↗

Genotypic analyses of IncHI2 plasmids from enteric bacteria

Incompatibility (Inc) HI2 plasmids are large (typically > 200 kb), transmissible plasmids that encode antimicrobial resistance (AMR), heavy metal resistance (HMR) and disinfectants/biocide resistance (DBR). To better understand the distribution and diversity of resistance-encoding genes among IncHI2 plasmids, computational approaches were used to evaluate resistance and transfer-associated genes among the plasmids. Complete IncHI2 plasmid (N - 667) sequences were extracted from GenBank and analyzed using AMRFinderPlus, IntegronFinder and Plasmid Transfer Factor database. The most common IncHI2-carrying genera included Enterobacter (N = 209), Escherichia (N = 208), and Salmonella (N = 204). Resistance genes distribution was diverse, with plasmids from Escherichia and Salmonella showing general similarity in comparison to Enterobacter and other taxa, which grouped together. Plasmids from Enterobacter and other taxa had a higher prevalence of multiple mercury resistance genes and arsenic resistance gene, arsC, compared to Escherichia and Salmonella. For sulfonamide resistance, sul1 was more common among Enterobacter and other taxa, compared to sul2 and sul3 for Escherichia and Salmonella. Similar gene diversity trends were also observed for tetracyclines, quinolones, β-lactams, and colistin. Over 99% of plasmids carried at least 25 IncHI2-associated conjugal transfer genes. These findings highlight the diversity and dissemination potential for resistance across different enteric bacteria and value of computational-based approaches for the resistance-gene assessment.

59 BASIC BIOLOGICAL SCIENCES↗

Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography

Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B 6 -derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Predicting permeation of compounds across the outer membrane of P. aeruginosa using molecular descriptors

The ability Gram-negative pathogens have at adapting and protecting themselves against antibiotics has increasingly become a public health threat. Data-driven models identifying molecular properties that correlate with outer membrane (OM) permeation and growth inhibition while avoiding efflux could guide the discovery of novel classes of antibiotics. Here we evaluate 174 molecular descriptors in 1260 antimicrobial compounds and study their correlations with antibacterial activity in Gram-negative Pseudomonas aeruginosa. The descriptors are derived from traditional approaches quantifying the compounds’ intrinsic physicochemical properties, together with, bacterium-specific from ensemble docking of compounds targeting specific MexB binding pockets, and all-atom molecular dynamics simulations in different subregions of the OM model. Using these descriptors and the measured inhibitory concentrations, we design a statistical protocol to identify predictors of OM permeation/inhibition. We find consistent rules across most of our data highlighting the role of the interaction between the compounds and the OM. An implementation of the rules uncovered in our study is shown, and it demonstrates the accuracy of our approach in a set of previously unseen compounds. Our analysis sheds new light on the key properties drug candidates need to effectively permeate/inhibit P. aeruginosa, and opens the gate to similar data-driven studies in other Gram-negative pathogens.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Tunable, Bacterio-Instructive Scaffolds Made from Functional Graphenic Materials

The balance of bacterial populations in the human body is critical for human health. Researchers have aimed to control bacterial populations using antibiotic substrates. However, antibiotic materials that non-selectively kill bacteria can compromise health by eliminating beneficial bacteria, which leaves the body vulnerable to colonization by harmful pathogens. Due to their chemical tunablity and unique surface properties, graphene oxide (GO)-based materials – termed “functional graphenic materials” (FGMs) – have been previously designed to be antibacterial but have the capacity to actively adhere and instruct probiotics to maintain human health. Numerous studies have demonstrated that negatively and positively charged surfaces influence bacterial adhesion through electrostatic interactions with the negatively charged bacterial surface. We found that tuning the surface charge of FGMs provides an avenue to control bacterial attachment without compromising vitality. Using E. coli as a model organism for gram-negative bacteria, we demonstrate that negatively charged Claisen graphene (CG), a reduced and carboxylated FGM, is bacterio-repellent through electrostatic repulsion with the bacterial surface. Though positively charged poly-L-lysine (PLL) is antibacterial when free in solution by inserting into the bacterial cell wall, here, we found that covalent conjugation of PLL to CG (giving PLLn-G) masks the antimicrobial activity of PLL by restricting polypeptide mobility. This allows the immobilized positive charge of the PLLn-Gs to be leveraged for E. coli adhesion through electrostatic attraction. We identified the magnitude of positive charge of the PLLn-G conjugates, which is modulated by the length of the PLL peptide, as an important parameter to tune the balance between the opposing forces of bacterial adhesion and proliferation. We also tested adhesion of gram-positive B. subtilis to these FGMs and found that the effect of FGM charge is less pronounced. B. subtilis adheres nondiscriminatory to all FGMs, regardless of charge, but adhesion is scarce and localized. Overall, this work demonstrates that FGMs can be tuned to selectively control bacterial response, paving the way for future development of FGM-based biomaterials as bacterio-instructive scaffolds through careful design of FGM surface chemistry.

Eckhart, Karoline E.↗

Assessment of a host–guest interaction in a bilayer membrane model

Supramolecular interactions are well recognized and many of them have been extensively studied in chemistry. The formation of supramolecular complexes that rely on weak force interactions are less well studied in bilayer membranes. Herein, a supported bilayer membrane is used to probe the penetration of a complex between tetracycline and a macrocyclic polyether. In a number of bacterial systems, the presence of the macrocycle has been found to significantly enhance the potency of the antimicrobial in vitro. The crown·tetracycline complex has been characterized in solution, neutron reflectometry has probed complex penetration, and the phenomena have been modeled by computational methods.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗