Team:TU-Delft/Project

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Peptidor: Detection and killing of resistant S. aureus using antimicrobial peptides

The bacterium Methicillin-Resistant Staphylococcus aureus causes major problems, especially in hospitals, causing over half a million infections annually in the United States alone. Of the alternative treatments currently under investigation one of the more promising is antimicrobial peptides (AMPs). These small peptides, typically several dozen amino acids in length, bind to and aggregate on the outer-membrane of the target organism and are able to make holes in the membrane at sufficiently high concentrations. The variation in the membrane composition of different organisms makes these peptides highly specific, meaning that infectious bacteria can be killed with little effect on neighboring human cells. Thousands of AMPs are have been discovered in nature and unlike antibiotic chemicals, AMPs can easily evolve as pathogens develop resistance. Using multiple AMPs in combination, can reduce the probability of developing resistance. The Peptidor project consists of an E. coli that can detect S. aureus in order to locally produce and deliver AMPs at the site of infection. Detection is achieved using the native quorum sensing system of S. aureus. Upon detection, AMPs, inactivated by fusion to a SUMO-tag, are overexpressed. After a delay period, introduced through a negative transcriptional cascade, a SUMO protease is expressed which cleaves off the inactivating tag of the peptides. Additionally the timer activates a safety mechanism containing an E. coli kill switch. Using this mechanism, high concentrations of peptide can be delivered very locally in order to efficiently kill S. aureus.

Figure 1: Schematic diagram of the complete system

Finally, the complete system is splitted up into four different modules which were used as building blocks.The building blocks are:

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-<h2 align="center">Overview</h2>
+<h2 align="center">Peptidor: Detection and killing of resistant <i>S. aureus</i> using antimicrobial peptides</h2>
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-<p align="justify">Methicillin-Resistant Staphylococcus aureus is causing major problems in hospitals and nursing homes. It causes infections, over 0.5 million in 2002 in the United States and puts patients with open wounds and weakened immune systems at great risks. It is so problematic because it is very difficult to treat; these bacteria have developed resistance against most antibiotics available. Several newly discovered strains of MRSA show antibiotic resistance even to vancomycin and teicoplanin, which are now the one of the last antibiotics useful against MRSA. Although a lot of research is done on alternative treatments, not much success has been booked yet. One of the more promising methods though, seems to be by the use of antimicrobial peptides.  </p>
+The bacterium Methicillin-Resistant <i>Staphylococcus aureus</i> causes major problems, especially in hospitals, causing over half a million infections annually in the United States alone. Of the alternative treatments currently under investigation one of the more promising is antimicrobial peptides (AMPs). These small peptides, typically several dozen amino acids in length, bind to and aggregate on the outer-membrane of the target organism and are able to make holes in the membrane at sufficiently high concentrations. The variation in the membrane composition of different organisms makes these peptides highly specific, meaning that infectious bacteria can be killed with little effect on neighboring human cells. Thousands of AMPs are have been discovered in nature and unlike antibiotic chemicals, AMPs can easily evolve as pathogens develop resistance. Using multiple AMPs in combination, can reduce the probability of developing resistance. The Peptidor project consists of an <i>E. coli</i> that can detect <i>S. aureus</i> in order to locally produce and deliver AMPs at the site of infection. Detection is achieved using the native quorum sensing system of <i>S. aureus</i>. Upon detection, AMPs, inactivated by fusion to a SUMO-tag, are overexpressed. After a delay period, introduced through a negative transcriptional cascade, a SUMO protease is expressed which cleaves off the inactivating tag of the peptides. Additionally the timer activates a safety mechanism containing an <i>E. coli</i> kill switch. Using this mechanism, high concentrations of peptide can be delivered very locally in order to efficiently kill <i>S. aureus</i>.
-<p align="justify" margin: 20px >Therefore, our project focusses on designing an <i>E. coli </i>that can detect MRSA in order to locally produce and deliver antimicrobial peptides against gram-positive bacteria. Our <i>  E. coli</i> will detect <i>S. aureus</i> by its own quorum sensing system, and at that moment inactivated antimicrobial peptide are started to be produced. With the use of a timer the peptide will be activated by cleavage of an inactivating tag. With the use of a timer, high concentrations of peptide can be delivered very locally in order to efficiently kill MRSA. In order to ensure the safety of the system, the peptide-activating protease is combined with a kill switch that will kill our <i>E. coli</i>(Figure 1).
-</p>
-<p align="justify">This project is novel because we aim to fight a life threatening, hard to cure disease by incorporating and combining components of multiple kingdoms into one organism. We let a gram-negative bacterium sense the communication molecules of gram-positive bacteria and respond to this with an animal innate immune response. The protease activation of the antimicrobial peptide is from fungal origin.  </p>
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