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Antimicrobial inactivation in Cystic Fibrosis

Persistent airway infections take on an extreme form in Cystic fibrosis (CF), the most common fatal, inherited disease in the United States. CF is a genetic disorder resulting from the inheritance of a defective autosomal recessive gene. The average life expectancy of the 30,000 CF patients currently in the U.S. is about 35 years. The gene responsible for CF codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP regulated Cl- ion channel found in the apical membranes of secretory epithelial cells [1]. Mutations in CTFR disrupt epithelial ion transport and can lead to respiratory failure, pancreatic insufficiency, infertility, as well as a range of other defects. There is, at present, no cure for the disease.

The airway surface liquid (ASL) is a ~5 micron thick liquid layer on the surface of the airway epithelium. The ionic environment of the ASL is complex. Although some debate remains regarding the ASL ionic composition [2], there is no question that the ASL in CF is enriched in anionic polyelectrolytes. In addition to the anionic glycoproteins comprising normal mucus, CF mucus contains highly anionic polyelectrolytes such as extracellular filaments and DNA produced by colonizing bacteria [3-5], as well as F-actin [6] and DNA [7,8] released from lysed inflammatory cells. The concentration of DNA in CF sputum can be as high as 20 mg/ml, and comprises 4-10% of the dry weight of the sputum.

Most of the antimicrobials in the airway are cationic, such as lysozyme, lactoferrin, β-defensin, and LL37. Recent work has investigated the extent to which cationic antimicrobial peptides and inflammatory mediators, which are also present in sputum, are inactivated by the anionic polyelectrolytes. These results demonstrate that F-actin and DNA sequester antimicrobial peptides [10,11] as well as cytokines [12] in CF sputum. The antimicrobial activity of the cationic peptide cathelicidin LL37 is a useful case study. It has been shown that LL37 activity is clearly inhibited by DNA and F-actin in vitro, and in CF sputum [10,11].

Fig. 1. Schematic representation of cation induced F-actin bundles: Cations organize into density waves which twist the actin helix to optimize electrostatic contact [24].

The electrostatic behavior of polyelectrolytes such as DNA and F-actin is considerably more complex than uncharged polymer fluids, but much progress has been made in our understanding within the last 10 years. (See Glossary: Electrostatics in aqueous media) In the presence of oppositely charged multivalent cations, both DNA and F-actin can overcome their mutual electrostatic repulsion, attract one another and condense into new phases [13-23]. Examples from nature include the hierarchical ordering of DNA chains via histones within chromosomes, and the high-density liquid crystalline DNA packaging by protamines in bacteria and viral capsids. In our recent x-ray diffraction work, we experimentally establish a microscopic mechanism for cation-mediated attraction between F-actin filaments, and find that the cations organize into density waves that induce nanomechanical twist distortions of the actin helix (Fig.1) in order to enhance the zipper-like charge alignment [24].

Fig. 2. A bundle of actin filaments (blue) held together electrostatically by lysozyme (orange), as obtained from molecular dynamics calculations in conjunction with synchroton x-ray diffraction experiments (background). These complexes can contribute to persistent airway infections in cystic fibrosis by sequestering antimicrobials. 'Non-stick' versions of lysozyme can be made: reduction of lysozyme charge changes its geometric arrangement with actin and destabilizes the bundle.

DNA and F-actin are released into the ASL when neutrophils and other cells lyse as the result of inflammatory response. It has been shown that DNA is also directly released by colonizing bacteria before the formation of biofilms [5]. Together with mucins and salts native to mucus, these anionic polyelectrolytes impinge on antibacterial activity in the airway in a number of different ways. Condensed bundles composed of anionic polyelectrolytes are, in fact, a common feature of CF sputum [25]. It has been suggested that cationic antibacterial proteins constitute at least a portion of the ligands holding these anionic polyelectrolytes together [35], hence the availability of such proteins is significantly diminished. This is consistent with our preliminary investigations on the relation between antimicrobial-polyelectreolyte aggregation and antimicrobial activity. For example, we have studied F-actin-lysozyme complexes, and found that lysozyme close-packs into a 1-D columns in between a hexagonal arrangement of F-actin filaments, so that the active site of lysozyme is effectively obstructed by this dense packing [26, 27]. Moreover, F-actin-lysozyme self-assembly persists across the wide range of reported CF physiological conditions (~50-150 mM monovalent salt), and we have observed these self-assembled structures with purified molecules as well as in sputum. In a collaboration that involves soft matter physicists, theoretical and computational physicists, protein engineers, and medical doctors, we aim to develop different strategies to unbind sequestered antimicrobials, as well as design ‘non-stick’ analogs of existing antimicrobials that do not get sequestered [28].

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