Abstract
The rise in antibiotic resistance has led to an increased research focus on discovery of new antibacterial candidates. While broad-spectrum antibiotics are widely pursued, there is evidence that resistance arises in part from the wide spread use of these antibiotics. Our group has developed a system to produce protein affinity agents, called synbodies, which have high affinity and specificity for their target. In this report, we describe the adaptation of this system to produce new antibacterial candidates towards a target bacterium. The system functions by screening target bacteria against an array of 10,000 random sequence peptides and, using a combination of membrane labeling and intracellular dyes, we identified peptides with target specific binding or killing functions. Binding and lytic peptides were identified in this manner and in vitro tests confirmed the activity of the lead peptides. A peptide with antibacterial activity was linked to a peptide specifically binding Staphylococcus aureus to create a synbody with increased antibacterial activity. Subsequent tests showed that this peptide could block S. aureus induced killing of HEK293 cells in a co-culture experiment. These results demonstrate the feasibility of using the synbody system to discover new antibacterial candidate agents.
Highlights
While there is no perfect understanding of the forces directing evolution of antibiotic resistance a prominent view holds that these issues are in part the consequence of the widespread use of broad spectrum antibiotics [1,2]
In the first step, labeled bacteria are applied to the peptide microarray, which consists of 10,000 peptides of 20aa length
We found that the (3-aminopropyl)triethoxysilane (APTES) microarray surface chemistry used in earlier published reports of screening proteins, antibodies and carbohydrates [18,23,24,25] showed high levels of non-specific binding to the interstitial regions of the array and low level binding to peptide spots when whole bacterial cells were screened
Summary
While there is no perfect understanding of the forces directing evolution of antibiotic resistance a prominent view holds that these issues are in part the consequence of the widespread use of broad spectrum antibiotics [1,2]. It has been proposed that generation antimicrobial treatments must focus on: developing pathogen-specific antibiotics, greatly improving diagnostics, and expanding the role of immunotherapy [3]. Along these lines, there has been a resurgence of monoclonal antibody (mAbs) based therapeutic development [4,5]. Antibody therapies were the first effective anti-infective agents (e.g. for pneumonia, meningitis, erysipelas). Their wide usage is restricted by the high cost of development and production of pathogen specific mAbs and the large number of current antimicrobial drugs on the market. The second is that they generally have high toxicity and broad activity, which is consistent with their evolutionary origin [17]
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