Bioconjugation of Bacteriophage Pf1 and Extension to Pf1-Based Bionanomaterials

Background: Filamentous bacteriophages such as M13 are an important class of macromolecular assembly, rich in chemical moieties that can be used to impart modifiable positions at the nanoscale. Objective: To explore the structurally more complex Pf1 bacteriophage with respect to a diverse set of bioconjugation reactions and to prepare novel fluorescently-labelled Pf1-based composite biomembranes for future applications in areas such as nanoporous filtration biofilms and photoconducting nanocomposite materials. Methods: Pf1 was characterized with respect to amine (N-terminal, Gly1 and Lys20), carboxylate (aspartate, glutamate), and aromatic (tyrosine) modification and its extension to the creation of functional biomaterials. Modification with an amine reactive fluorophore was carried out with Pf1. Results: The reaction profiles between M13 and Pf1 differ, with M13 capable of modification at two primary amines on its major coat protein, while Pf1 is capable of a single reaction per coat protein. Subsequent to the production of dyefunctionalized Pf1, a biocomposite of wild type and functionalized Pf1 could be fabricated into a bulk material by glutaraldehyde (amine-reactive) crosslinking. These biomaterials were characterized by scanning electron and confocal microscopy, showing a distribution of patches of functionalized Pf1 within the main Pf1 construct. Conclusion: The current study provides a framework for future fabrication of advanced bionanomaterials based on the Pf1 bacteriophage.

Bactericidal Activities of Cathelicidin LL-37 and Select Cationic Lipids against the Hypervirulent Pseudomonas aeruginosa Strain LESB58

ABSTRACTPseudomonas aeruginosaLiverpool epidemic strain (LES) infections in cystic fibrosis (CF) patients are associated with transmissibility and increased patient morbidity. This study was designed to assess thein vitroactivities of cathelicidin LL-37 peptide (LL-37) and select cationic lipids againstPseudomonas aeruginosaLESB58 in CF sputum and in a setting mimicking the CF airway. We found that LL-37 naturally present in airway surface fluid and some nonpeptide cationic lipid molecules such as CSA-13, CSA-90, CSA-131, and D2S have significant, but broadly differing, bactericidal activities againstP. aeruginosaLESB58. We observed strong inhibition of LL-37 bactericidal activity in the presence of purified bacteriophage Pf1, which is highly expressed byP. aeruginosaLES, but the activities of the cationic lipids CSA-13 and CSA-131 were not affected by this polyanionic virus. Additionally, CSA-13 and CSA-131 effectively prevent LESB58 biofilm formation, which is stimulated by Pf1 bacteriophage, DNA, or F-actin. CSA-13 and CSA-131 display strong antibacterial activities against different clinical strains ofP. aeruginosa, and their activities againstP. aeruginosaLESB58 and Xen5 strains were maintained in CF sputum. These data indicate that synthetic cationic lipids (mimics of natural antimicrobial peptides) are suitable for developing an effective treatment against CF lungP. aeruginosainfections, including those caused by LES strains.

Proteomic, Microarray, and Signature-Tagged Mutagenesis Analyses of Anaerobic Pseudomonas aeruginosa at pH 6.5, Likely Representing Chronic, Late-Stage Cystic Fibrosis Airway Conditions

ABSTRACT Patients suffering from cystic fibrosis (CF) commonly harbor the important pathogen Pseudomonas aeruginosa in their airways. During chronic late-stage CF, P. aeruginosa is known to grow under reduced oxygen tension and is even capable of respiring anaerobically within the thickened airway mucus, at a pH of ∼6.5. Therefore, proteins involved in anaerobic metabolism represent potentially important targets for therapeutic intervention. In this study, the clinically relevant “anaerobiome” or “proteogenome” of P. aeruginosa was assessed. First, two different proteomic approaches were used to identify proteins differentially expressed under anaerobic versus aerobic conditions. Microarray studies were also performed, and in general, the anaerobic transcriptome was in agreement with the proteomic results. However, we found that a major portion of the most upregulated genes in the presence of NO3 − and NO2 − are those encoding Pf1 bacteriophage. With anaerobic NO2 −, the most downregulated genes are those involved postglycolytically and include many tricarboxylic acid cycle genes and those involved in the electron transport chain, especially those encoding the NADH dehydrogenase I complex. Finally, a signature-tagged mutagenesis library of P. aeruginosa was constructed to further screen genes required for both NO3 − and NO2 − respiration. In addition to genes anticipated to play important roles in the anaerobiome (anr, dnr, nar, nir, and nuo), the cysG and dksA genes were found to be required for both anaerobic NO3 − and NO2 − respiration. This study represents a major step in unraveling the molecular machinery involved in anaerobic NO3 − and NO2 − respiration and offers clues as to how we might disrupt such pathways in P. aeruginosa to limit the growth of this important CF pathogen when it is either limited or completely restricted in its oxygen supply.

Identification of lysine residues at the binding site of bacteriophage-Pf1 DNA-binding protein

The accessibility of NH2 groups in the DNA-binding protein of Pf1 bacteriophage has been investigated by differential chemical modification with the reagent ethyl acetimidate. The DNA-binding surface was mapped by identification of NH2 groups protected from modification when the protein is bound to bacteriophage-Pf1 DNA in the native nucleoprotein complex and when bound to the synthetic oligonucleotide d(GCGTTGCG). The ability of the modified protein to bind to DNA was monitored by fluorescence spectroscopy. Modification of the NH2 groups in the native nucleoprotein complex showed that seven out of the eight lysine residues present, and the N-terminus, were accessible to the reagent, and were not protected by DNA or by adjacent protein subunits. Modification of these residues did not inhibit the ability of the protein to bind DNA. Lysine-25 was identified by peptide mapping as being the major protected residue. Modification of this residue does abolish DNA-binding activity. Chemical modification of the accessible NH2 groups in the complex formed with the octanucleotide effectively abolishes binding to DNA. Peptide mapping established that, in this case, lysine-17 was the major protected residue. The differences observed in protection from acetimidation, and in the ability of the modified protein to bind DNA, indicate that the oligonucleotide mode of binding is not identical with that found in the native nucleoprotein complex with bacteriophage-Pf1 DNA.