Chimeric Ligands of Pili and Lectin A Inhibit Tolerance, Persistence, and Virulence Factors of Pseudomonas aeruginosa over a Wide Range of Phenotypes.

Bacteria readily form resilient phenotypes to counter environmental and antibiotic stresses. Here, we demonstrate a class of small molecules that inhibit a wide range of Pseudomonas aeruginosa phenotypes and enable antibiotics to kill previously tolerant bacteria, preventing the transition of tolerant bacteria into a persistent population. We identified two proteins, type IV pili and lectin LecA, as receptors for our molecules by methods including a new label-free assay based on bacterial motility sensing the chemicals in the environment, the chemical inhibition of bacteriophage adsorption on pili appendages of bacteria, and fluorescence polarization. Structure-activity relationship studies reveal a molecule that inhibits only pili appendage and a class of chimeric ligands that inhibit both LecA and pili. Important structural elements of the ligand are identified for each protein. This selective ligand binding identifies the phenotypes each protein receptor controls. Inhibiting LecA results in reducing biofilm formation, eliminating small colony variants, and is correlated with killing previously tolerant bacteria. Inhibiting pili appendages impedes swarming and twitching motilities and pyocyanin and elastase production. Because these phenotypes are controlled by a broad range of signaling pathways, this approach simultaneously controls the multiple signaling mechanisms preventing bacteria to elude antibiotic treatments.

Chemical control over Asialo-GM1: A dual ligand for pili and Lectin A that activates swarming motility and facilitates adherence of Pseudomonas aeruginosa.

Glycolipid, ganglio-N-tetraosylceramide (asialo-GM1), on the mammalian cells are known to be recognized by type IV pili of Pseudomonas aeruginosa. In this work, we show that asialo-GM1 can also be recognized by Lectin A (LecA), another adhesin protein of the P. aeruginosa, by a fluorescent polarization assay, a label-free bacterial motility enabled binding assay, and bacterial mutant studies. On hydrated semi-solid gel surfaces, asialo-GM1 enables swarming and twitching motilities, while on solid surfaces facilitates the bacterial adherence of P. aeruginosa. These results indicate that asialo-GM1 can modulate bioactivities, adherence, and motilities, that are controlled by opposite signaling pathways. We demonstrate that when a solution of pilin monomers or LecA proteins are spread on hydrated gel surfaces, the asialo-GM1 mediated swarming motility is inhibited. Treatment of artificial liposomes containing asialo-GM1 as a component of lipid bilayer with pilin monomers or LecA proteins caused transient leakage of encapsulated dye from liposomes. These results suggest that pili and LecA proteins not only bind to asialo-GM1 but can also cause asialo-GM1 mediated leakage. We also show that both pili and LecA mutants of P. aeruginosa adhere to asialo-GM1 coated solid surfaces, and that a class of synthetic ligands for pili and LecA inhibits both pili and LecA-mediated adherence of P. aeruginosa on asialo-GM1-coated surfaces.

Exploration of Ligand-receptor Binding and Mechanisms for Alginate Reduction and Phenotype Reversion by Mucoid Pseudomonas aeruginosa.

Bacteria in general can develop a wide range of phenotypes under different conditions and external stresses. The phenotypes that reside in biofilms, overproduce exopolymers, and show increased motility often exhibit drug tolerance and drug persistence. In this work, we describe a class of small molecules that delay and inhibit the overproduction of alginate by a non-swarming mucoid Pseudomonas aeruginosa. Among these molecules, selected benzophenone-derived alkyl disaccharides cause the mucoid bacteria to swarm on hydrated soft agar gel and revert the mucoid to a nonmucoid phenotype. The sessile (biofilm) and motile (swarming) phenotypes are controlled by opposing signaling pathways with high and low intracellular levels of bis-(3',5')-cyclic diguanosine monophosphate (cdG), respectively. As our molecules control several of these phenotypes, we explored a protein receptor, pilin of the pili appendages, that is consistent with controlling these bioactivities and signaling pathways. To test this binding hypothesis, we developed a bacterial motility-enabled binding assay that uses the interfacial properties of hydrated gels and bacterial motility to conduct label-free ligand-receptor binding studies. The structure-activity correlation and receptor identification reveal a plausible mechanism for reverting mucoid to nonmucoid phenotypes by binding pili appendages with ligands capable of sequestering and neutralizing reactive oxygen species.

Experimentally Validated QSAR Model for Surface pK a Prediction of Heterolipids Having Potential as Delivery Materials for Nucleic Acid Therapeutics.

The application of lipid-based drug delivery technologies for bioavailability enhancement of drugs has led to many successful products in the market for clinical use. Recent studies on amine-containing heterolipid-based synthetic vectors for delivery of siRNA have witnessed the United States Food and Drug Administration (USFDA) approval of the first siRNA drug in the year 2018. The studies on various synthetic lipids investigated for delivery of such nucleic acid therapeutics have revealed that the surface pK a of the constructed nanoparticles plays an important role. The nanoparticles showing pK a values within the range of 6-7 have performed very well. The development of high-performing lipid vectors with structural diversity and falling within the desired surface pK a is by no means trivial and requires tedious trial and error efforts; therefore, a practical solution is called for. Herein, an attempt to is made provide a solution by predicting the statistically significant pK a through a predictive quantitative structure-activity relationship (QSAR) model. The QSAR model has been constructed using a series of 56 amine-containing heterolipids having measured pK a values as a data set and employing a partial least-squares regression coupled with stepwise (SW-PLSR) forward algorithm technique. The model was tested using statistical parameters such as r 2, q 2, and pred_r 2, and the model equation explains 97.2% (r 2 = 0.972) of the total variance in the training set and it has an internal (q 2) and an external (pred_r 2) predictive ability of ∼83 and ∼63%, respectively. The model was validated by synthesizing a series of designed heterolipids and comparing measured surface pK a values of their nanoparticle assembly using a 2-(p-toluidino)-6-napthalenesulfonic acid (TNS) assay. Predicted and measured surface pK a values of the synthesized heterolipids were in good agreement with a correlation coefficient of 93.3%, demonstrating the effectiveness of this QSAR model. Therefore, we foresee that our developed model would be useful as a tool to cut short tedious trial and error processes in designing new amine-containing heterolipid vectors for delivery of nucleic acid therapeutics, especially siRNA.

Cell-Wall-Targeting Antibiotics Cause Lag-Phase Bacteria to Form Surface-Mediated Filaments Promoting the Formation of Biofilms and Aggregates.

Antibiotics are known to promote bacterial formation of enhanced biofilms, the mechanism of which is not well understood. Here, using biolayer interferometry, we have shown that bacterial cultures containing antibiotics that target cell walls cause biomass deposition on surfaces over time with a linear profile rather than the Langmuir-like profiles exhibited by bacterial adherence in the absence of antibiotics. We observed about three times the initial rate and 12 times the final biomass deposition on surfaces for cultures containing carbenicillin than without. Unexpectedly, in the presence of antibiotics, the rate of biomass deposition inversely correlated with bacterial densities from different stages of a culture. Detailed studies revealed that carbenicillin caused faster growth of filaments that were seeded on surfaces from young bacteria (from lag phase) than those from high-density fast-growing bacteria, with rates of filament elongation of about 0.58 and 0.13 µm min-1 , respectively. With surfaces that do not support bacterial adherence, few filaments were observed even in solution. These filaments aggregated in solution and formed increased amounts of biofilms on surfaces. These results reveal the lifestyle of antibiotic-induced filamentous bacteria, as well as one way in which the antibiotics promote biofilm formation.