Combating Intracellular Respiratory Infections

Abstract

Several of the major commensal pathogens including Streptococci and Staphylococci, which cause severe respiratory diseases, are known to invade host tissues, generating an intracellular reservoir of bacteria. Intracellular bacteria are protected from host immunity and treatment by major classes of antibiotics. The intracellular niche promotes bacterial survival, antibiotic resistance, and disease recurrence. Major antibiotics such as penicillin, cephalosporins and aminoglycosides poorly penetrate mammalian cells, and thus are ineffective against intracellular bacteria. The aim of this study was to develop technologies to enable the cell penetrating peptide (CPP) Xentry, a novel drug delivery reagent, to deliver the anti-bacterial protein lysozyme and antibiotics to respiratory epithelial cells. If successful these approaches could be used to target and destroy intracellular reservoirs of the respiratory pathogens Streptococcus pneumoniae, Klebsiella pneumoniae and Streptococcus pyogenes that cause severe respiratory diseases. The intial goal was to conjugate Xentry to bovine serum albumin (BSA) and lysozyme, with the notion that serum albumins and lysozyme could be used to carry antibiotics to cells, given their natural propensity to bind drugs. Delivery of antibiotics bound to lysozyme provides a two-pronged attack on bacteria due to the anti-bacterial activity of lysozyme itself and the antibiotics it would carry. Here it was demonstrated that the TAMRA-labelled Xentry peptide LCLRK could be crosslinked to FITC-labelled BSA using the amine-to-amine crosslinkers dimethylsuberimidate (DSS) and bis-N-succinimidyl-nanopolyethylene glycol [BS(PEG)9]. The conjugate that was produced was readily identifiable on SDS polyacrylamide gels due to its increase in size and orange colour resulting from the mix of pink-coloured TAMRA-Xentry and yellow-coloured FITC-BSA. Its shift in size could also be detected from visualizing the fluorescent protein bands, and by staining them with Coomassie blue. In contrast, TAMRA-Xentry could not be conjugated to FITC-lysozyme, presumably because all of the free amine groups in lysozyme were already bound by the FITC-label. In contrast, TAMRA-Xentry could be conjugated to unlabeled recombinant human lysozyme using DSS. Further, a XentryQQQ peptide which had been modified to contain an optimal transglutamination site could be efficiently crosslinked to unlabeled recombinant human lysozyme using transglutaminase. The fluorescent conjugate was predominantly of similar size to lysozyme on SDS-gels, but higher molecular weight forms were apparent suggesting formation of lysozyme dimers and trimers. A turbidimetric assay to measure lysozyme activity was established based on the ability of lysozyme to degrade the bacterium Micrococcus luteus and reduce the turbidity of bacterial suspensions. The turbidimetric assay was employed to determine whether conjugation of Xentry to lysozyme inhibits the anti-bacterial activity of lysozyme. The enzymic activity of lysozyme was not significantly inhibited by DSS- and transglutaminase-mediated conjugation to Xentry. In an alternative approach, a chimeric peptide was synthesized in which Xentry was fused to the Chariot™ peptide, which is able to noncovalently deliver proteins to cells. It was hoped that Xentry would improve the cell uptake of the peptide. The Chariot-Xentry fusion peptide was shown to retain the ability of the parental peptides to penetrate the human lung adenocarcinoma epithelial cell line A549. The Chariot-Xentry fusion peptide appeared to be able to noncovalently deliver FITC-labeled BSA into A549 cells, with the protein appearing to accumulate in endosomes. However, follow-up controls revealed that FITC-labeled BSA can enter A549 cells on its own, presumably via megalin, a multi-ligand receptor which is proposed to clear excess protein from the alveolar spaces. A novel approach was devised to employ a highly negatively-charged form of Xentry to deliver positively-charged gentamicin to cells. Previous work in the laboratory had revealed that fusion of a negatively-charged heparin-mimetic peptide to the N-terminus of Xentry prevented its ability to penetrate cells. The idea here was to determine whether gentamicin which has a +3 charge would bind the negatively-charged fusion peptide (here designated GSY-Xentry) via electrostatic interaction, thereby neutralizing Xentry and restoring its ability to penetrate cells. It was shown that while the GSY-Xentry peptide could not penetrate A549 cells, it could readily do so after pretreatment with 50 mM gentamicin. In summary, a variety of novel approaches were tested to develop vehicles for the delivery of anti-bacterial reagents to combat intracellular infection. Time did not allow for the approaches to actually be tested for their ability to kill intracellular bacteria. Attempts were made to establish a bacterial invasion assay, but the assay will require further optimization. Potentially the most promising approach is using the GSY-Xentry peptide to electrostatically deliver negatively-charged aminoglycosides to cells, but this approach requires further experimentation to determine whether the aminoglycosides are rendered bioavailable once inside the cell in order to kill the intracellular bacteria.