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DNAzymes for control of bacterial biofilms

P. aeruginosa biofilm formation and Fe:

In addition to signaling, micronutrients and their spatial and temporal distributions can strongly affect biofilm development. For example, the ability of P. aeruginosa to form biofilms is dependent upon the supply of available Fe3+. [1] When deprived of Fe3+, it has been observed that P. aeruginosa undergoes twitching motility and meander across a surface rather than form stable clusters and biofilms. Moreover, in an Fe3+ poor environment, it produces the siderophores pyoverdin and pyochelin and membrane receptors for the Fe3+ - siderophore complexes.

Fig. 1. The growth of P. aeruginosa in the presence and absence of lactoferrin, measured by confocal microscopy [1].

The need for Fe3+ can potentially be exploited as a strategy for biofilm control. For example, lactoferrin is a naturally occurring antibiotic found in CF sputum that prevents biofilm formation by chelating Fe3+ (Fig 2). However, there are several disadvantages to the use of lactoferrin as an antibiotic to prevent biofilm formation in the context of airway infections in CF. First, stressed P. aeruginosa secretes proteases that cleave lactoferrin in order to recover Fe3+. There are approved drugs that mimic lactoferrin and chelate Fe3+, such as deferoximine. However, as it is a siderophore, a significant number of bacterial species have receptors for deferoximine, and can thereby recover the chelated Fe3+. The second reason is related to the fact that lactoferrin, like most naturally occurring antibiotics (as well as cation chelators), is cationic.

There is broad agreement that the Airway Surface Liquid (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 produced by colonizing bacteria, [2, 3] as well as F-actin [4] and DNA [5, 6] released from lysed inflammatory cells. Condensed bundles composed of F-actin and of DNA are, in fact, a common feature of CF sputum. [7] There is strong evidence that cationic antibacterial proteins constitute at least a portion of the ligands holding these polyelectrolytes together. [8] Lactoferrin, like other cationic antimicrobials such as lysozyme, beta defensins, and LL-37, can be sequestered by the anionic polyelectrolytes and thereby inactivated. There are many examples of inhibition of enzymatic activity in the lung due to the existence of anionic polymers, such as the finding that human neutrophil elastase is inhibited by an anionic polymer, thought to be RNA released in pneumococcal extracts. [9]

We envision a new class of antibiotics that would eliminate the disadvantages inherent to lactoferrin. First, we would need an antibiotic that is anionic instead of cationic so that it would not become sequestered by anionic biopolymers in CF sputum. Moreover, it will be designed to be a short oligomer so that counterion-mediated binding to cationic proteins is minimized. Second, we would need an antibiotic that is not protein based so that it would not be cleaved by secreted bacterial proteases or recognized by bacterial receptors for siderophores. One possible solution is DNA based antibiotics via directed molecular evolution (DME). DME is the cyclical process of selecting from a large pool of randomized DNA those strands that best satisfy a given set of selection criteria that define the properties of the desired DNA. [10-12] The initial pool of randomized DNA contains ~1015 different sequences of DNA. The sequences of DNA that best satisfy the selection criteria are selected out of the initial pool of randomized DNA using physical and chemical methods. These strands of DNA are then be amplified using the polymerase chain reaction (PCR) and then used for the next round of selection. The DME algorithm is repeated 10x-15x in order to produce DNA that best meets the selection criteria. Our preliminary results suggest that we can employ DME to pre-program a Fe-specific binding site in ssDNA, and create a DNA-based lactoferrin mimic with four potential advantages: 1) it is negatively charged so will not be sequestered in CF mucus, 2) it will be invulnerable to the lactoferrin-specific proteases that Pseudomonas secrete, 3) it may bind Fe tighter than lactoferrin, and 4) it is nucleic acid based, and therefore can be scaled up easily for clinical use.

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