Team:Utah State/Project


Antimicrobial peptides (AMPs) are known for their ability to kill bacteria that are harmful to its host and are part of the innate immune system of all multicellular organisms (Andreu & Rivas, 1998). AMPs from various organisms have shown the ability to inhibit many types of gram-positive/gram-negative bacteria, protists, fungi, and other organisms. The outstanding diversity of AMPs found in all organisms on the planet contribute to a constantly growing list of peptides with varying properties. The size, amino acid sequence, charge, conformation, hydrophobicity, and amphipathicity all contribute to the activity of an AMP. Most AMPs contain fewer than 100 amino acids and are positively charged (Giuliani et al., 2007).

Organisms across our planet produce antimicrobial peptides that allow them to thrive in their distinct environments. For example, crocodiles by nature are extremely territorial and engage in vicious battles with other competing members of their community, sustaining open wounds in the process. The AMPs that are naturally produced in their blood protect them from infections due to the innumerable bacteria and organisms that could otherwise cause them great harm. King penguins produce unique AMPs in their stomach to allow for extended storage of food to later regurgitate and feed to their young. Marsupials have shown to be unique hosts of AMPs. The tammar wallaby relies on the production of AMPs in their pouches to protect their young who do not have a developed immune system for the first few months of their lives (Wang et al., 2011).

The diversity of AMPs is likely another example of adaptations occurring in organisms due to environmental pressures. It has been determined that most AMPs are derived from precursor sequences that are quite similar. Conserved regions on AMPs even between different species of organisms has led to this belief (Zasloff, 2002). The changes present in the current AMP sequence are probably due to the thriving of organisms containing mutations in the AMP sequence that allowed them to gain an advantage over other individuals lacking the mutation (Zasloff, 2002). These AMPs have been tested over many generations in their host organisms and makes them fascinating for studies in which their activity is tested against a variety of organisms.

With so many AMPs, it is no wonder that a variety of mechanisms are possible for bacterial cell killing (Epand et al., 1999). Most AMPs that have been studied follow the Shai-Matsuzaki-Huang (SMH) model (Zasloff, 2002) (Lai & Gallo, 2009). This model overviews the interaction of a peptide with the outer layer of a cell membrane. The general flow of this model includes the “carpeting” of the membrane with the AMP followed by the displacement of lipids in the membrane, and finally the lysing of the membrane. The SMH model can be seen below.

Shai-Matsuzaki-Huang model (Zasloff, 2002)

The SMH model demonstrates that one of two outcomes is achieved to reach cell lysis: (1) the peptide will physically break the membrane to cause cell lysis or (2) the peptide will enter the cell to perform various interior mechanisms of action against the cell (Zasloff, 2002). This model demonstrates a general explanation of the mechanism of how AMPs operate. How exactly they act in killing a cell varies greatly from peptide to peptide. Some examples may be: depolarization of charged cell membranes, pore formation in cell membranes, and degrading of important cell structures. However, it is generally agreed that most AMPs work to achieve cell membrane permeabilization (Andreu & Rivas, 1998). Many organisms use a series of different AMPs from various classes to work more effectively against bacteria (Zasloff, 2002). AMPs are usually not produced in high concentrations until injury or illness occurs to the organism. Other AMPs are stored as inactive compounds and are controlled by cellular receptors (Lai & Gallo, 2009).

AMPs are characterized according to their structure and function. Our team selected AMPs from most of the classes of AMPs. These include anionic peptites (Scygonadin), linear cationic alpha-helical peptides (LL37, Grammistin Pp1, EcAMP1), and anionic and cationic peptides that contain cysteine and form disulphide bonds (Cg-Defh1) (Brogden, 2005). Generally, AMPs are alpha helical or contain beta-sheets. However, some are extended in structure or form loops.

Drug resistance is especially important in health settings in modern society. The use of antibiotics to treat bacterial diseases has led to the emergence of “superbugs”, drug resistant bacteria that infect humans. These superbugs are often untreatable with current antibiotics and are the cause of an increasing number of deaths each year. However, it appears as if resistance is harder to come by for bacteria against AMPs. This is likely due to the mechanism of most AMPs acting on bacterial cell membranes instead of its cell walls (Peters et al., 2010). AMP resistance would require a complete re-design of the bacterial membrane to prevent action of the AMP. While still possible, the likelihood of resistance being developed is much smaller, making AMPs a potential source of much more effective bacterial killers (Zasloff, 2002) (Epand et al., 1999).

An extremely important trait of an antimicrobial agent is that it has selective mechanisms against harmful bacteria. For use in the human healthcare system, AMPs must not target mammalian cells or demonstrate hemolytic activity. Due to the mechanism of most AMPs acting on bacterial membranes, it has been proposed that AMPs could work in tandem with current antimicrobials. By letting AMPs degrade the microbe’s membrane, an antimicrobial could easily gain access to the inside of the cell, making it much more effective (Peters et al., 2010). In vitro, it has been observed that minimum inhibitory concentrations (MIC) of AMPs against bacteria are much higher than it appears to be in vivo. It is thought that AMPs work in conjunction with other AMPs to act as more effective inhibitors of bacteria. LL37, a model peptide used in our study, has been shown to work in tandem with other peptides as well as lysozyme to increase toxicity to bacteria (Lai & Gallo, 2009). It has even been observed that AMPs may possess the potential to increase overall immune response, neutralize endotoxins, and aid in wound healing (Peters et al., 2010). The Figure below outlines many of the processes that AMPs accomplish in vivo.


                  Functions of AMPs in vivo ( modified from Lai & Gallo, 2009)

AMPs selected by Utah State

Since there is such a wide diversity of AMPs in nature, we decided to select our peptides from eight different species. The map below shows all of the organisms that we decided to draw from.


Figure shows the diversity of AMPs used by Utah State iGEM team 2013

EcAMP-1 (BBa_K1162001)

EcAMP-1 is derived from the grass species Echinochloa crus-galli. It consists of 37 amino acids and is helical in structure, held together by two disulfide bonds. This feature enables a unique hairpin structure which is not found in most other AMPs. EcAMP-1 has shown to have a toxic affect against a variety of fungi and protists. The mode of action for EcAMP-1 against fungi appears to be the prevention of hyphae elongation (Nolde et al., 2011).

Spheniscin-2 (BBa_K1162002)

Spheniscin-2 is a potent antimicrobial peptide whose DNA sequence of the king penguin. Studies have shown that in nature the King Penguin secrete Spheniscin-2 into its stomach to preserve food for long periods of time by preventing bacterial decomposition of the food. In the wild the male usually incubates the egg for the last 3 months before the egg hatches. In general the female will then come and feed her young shortly after. However, if the mother is delayed the survival of the young can still be insured by the male (Thouzeau et al 2003). However, storing food for long periods of time in a warm stomach would lead one to assume that bacterial degradation would be a limiting factor. However, studies have shown that Spheniscin-2 has a wide range of activity against many gram positive, gram negative, and even fungal microbes, which allows the male King Penguin to store food for long periods of time (2.). Thus, there is a wide range of use associated with Spheniscin-2. The potential of interweaving Spheniscin-2 with spider silk holds many promising results.

Grammistin-Pp1 (BBa_K1162003)

Grammistin-Pp1 is an AMP found in skin secretions from the clown grouper fish and is made up of thirteen amino acid residues. This AMP is used primarily to fend off predators. Although initially thought to have little antimicrobial properties, studies proved that grammistin Pp1 target cell membranes by interacting with phospholipids. Grammistin Pp1 has been shown to be lethal against many gram-negative and gram-positive bacteria including B. subtilis and S. aureus. A primary reason for our selection of grammistin Pp1 was the high concentration needed to inhibit the growth of E. coli. (Yokota et al., 2001)

CG-Defh1 (BBa_K1162004)

CG-Defh1 originates from immune cells in an oyster species. As a defensin AMP, CG-Defh1 contains four disulfide bonds and is most effective against gram-positive bacteria. The specific mode of action for this AMP is to inhibit peptidoglycan synthesis in the bacterial cell wall. This makes CG-Defh1 unique from many AMPs in that it does not target cell membranes. In fact, it was the first defensin from an invertebrate to exhibit antibacterial properties. Most importantly, CG-Defh1 inhibits the growth of S. aureus, a dangerous cause of nosocomial infections in the world.

Scygonadin (BBa_K1162005)

Scygonadin is produced in a species of mangrove crab to prevent bacterial invasion. It is the primary defense against dangerous microbes that thrive in marine environments. Scygonadin has been shown to be effective in inhibiting both gram-positive and gram-negative bacteria including M. leteus, S. aureus, C. glutamicum, and A. hydrophila. This AMP has been previously studied for its production via recombinant DNA in E. coli using a pTrc-CKS vector and a C-terminus 6x His-tag. No studies were done with a 10x His tag or spider silk inserts. (Peng et al., 2010)

LL-37 (BBa_K1162006)

LL-37 is an antimicrobial peptide (AMP) from the cathelicidin family of antimicrobial peptides, isolated from the Human (Homo sapiens). It is a 37-residue helical peptide found throughout the human body, exhibiting a broad spectrum of antimicrobial activity as well as a variety of immunomodulating effects (Dürr, Ulrich H.N., et. al 2006). It has the distinction of being the first and only cathelicidin member discovered from humans, and is extremely well studied and characterized for this reason. The peptide is produced via epithelial cells as well as leukocytes in places such as the skin, gastrointestinal tract, and the respiratory tract. This AMP is is cleaved from a larger protein, hCAP-18, in a series of modifications to yield its active form (Sorensen, O.E, et. al 2001).

WAM-1 (BBa_K1162007)

An antimicrobial peptide from the cathelicidin family of peptides, isolated from the tammar wallaby (Macropus eugenii). WAM-1 is one of several identified peptides that are produced in the mammary gland throughout lactation in order to supply protection from infection to the young wallabies that have underdeveloped immune systems. This is evident through the down-regulation of these peptides in the tammar wallaby milk at around 100 days after birth, the same period that the wallaby young develop immunocompetence (Wang, et. al 2011). Similar to other members of the cathelicidin family, the WAM-1 antimicrobial peptide has been shown to be effective against a broad range of bacterial pathogens (Kastin, A. J. 2013). Specifically, a group of researchers from Australia (Wang, et. al 2011) have tested this peptide, isolated from its natural source, against several gram-negative (E. coli, P. aeruginosa, among others) and gram-positive (B. subtilis, S. aureus, among others) bacterium with minimum inhibitory concentration (MIC) values that indicate highly effective antibiotic activity. To our knowledge, this antimicrobial peptide has not been expressed and purified recombinantly in E. coli.

OH-CATH(3-34) (BBa_K1162008)

OH-CATH(3-34) is an antimicrobial peptide isolated and characterized from the king cobra (Ophiophagus hannah)(Zhao et al 2008). A member of the cathelicidin family, OH-CATH Cathelicidins in general have been well characterized and are known to have a diverse C-terminal antimicrobial domain connected to a conserved cathelin-like N-terminal domain (Nizet et al 2003). Specifically, OH-CATH has been studied for its toxicity against a wide range of gram negative and gram positive bacteria. OH-CATH has also been characterized for its potent activity against multi-drug resistant bacterial strains including MRSA (Li et al 2012). Further studies by (Zhang et al 2010) have shown that removing the first few amino acid residues of the peptide dramatically reduced the hemolytic activity while having only minute effects on antimicrobial properties. This attribute would be highly desirable in any usage that would interact with humans and helped to determine our choice our peptides. The combination of high bacterial toxicity with low hemolytic activity justify more testing into the precise mechanisms of OH-CATH and its ability to be used as a scaffold for other antibiotic purposes.

AMP production in E. coli

There are many problems with producing AMPs on a large scale. Natural purification of these AMPs is a slow process and is generally inefficient. A more viable option is chemical synthesis. However, this process is extremely costly (Li, 2011). Finding a way to overcome these problems is key to the development of a new antimicrobial product. Producing AMPs in bacteria such as E. coli using recombinant DNA techniques could provide an answer to increasing production rates and decreasing costs.

The first obstacle faced in producing AMPs in E. coli is how to avoid inhibiting E. coli growth itself before satisfactory AMP concentrations have been achieved. E. coli is easy to engineer, is quick to grow, and inexpensive to use. This makes it the most attractive bacteria to use for protein production. To prevent the culture from dying before producing the desired AMP, strong transcriptional promoters must be included to reduce gene expression to a very low basal level (Sorensen & Mortensen, 2004). This promoter allows the E. coli culture to reach stationary growth phase before being induced to begin high-level gene expression of the AMP. In this and most studies, isopropyl-β-D-thiogalactopyranoside (IPTG) is used as an inducer.

We have chosen our antimicrobial peptides because they belong to several families of proteins and have the ability to inhibit a broad range of pathogenic microorganisms.

Antimicrobial Spider Silk

Antimicrobial spider silk proteins (functionalized spider silk) could be used to make antimicrobial spider silk fibers or films. A previous study by Gomes et al. 2011 demonstrated that it is feasible to produce antimicrobial spider silk films. The Utah State iGEM team in 2012 was able to successfully produce spider silk proteins in E. coli with the use of BioBricks and successfully spin spider silk protein that was approximately 25.4 kDa in size. The parts that were submitted to the registry by Utah State in 2012 are RFC 23 compatible which allows for protein fusions, hence the addition of RFC 23 compatible AMPs to spider silk protein at the C or N terminal would be highly desirable.

The spider silk generator (BBa_K844016) created by Utah State in 2012 was able to produce proteins of 25.4 kDa. An increase of spider silk subunit repeats at the genetic level would enable a larger (and therefore ‘stronger’ fiber) to be produced. This year we proposed to double the number of spider silk repeat units to 8 and attach AMPs to either the C or N terminal ends of the spider silk. The purification of this antimicrobial functionalized silk would be performed with a 10x His-Tag fused to the spider silk, at the opposite terminus as the AMP. The possibility of "masking" the properties of the AMP were taken into account with our genetic design, which is why we did not create this fusion protein with the AMP located in between the spider silk and 10x His-Tag proteins. The figures below demonstrate the design of antimicrobial functionalized spider silk.

All constructs were assembled (with RFC 23 standards) in pSB1C3 an E. coli as the chassis. AMP amino acid sequences were first back translated to obtain DNA sequences. In all cases a methionine (atg) had to be added. These DNA sequences were then codon optimized for expression in E. coli using the Life Technologies GeneArt ® software program. Sequences were verified not to contain any illegal restriction sites (EcoRI, XbaI, SpeI,and PstI). AMPs were designed with RFC 23 under consideration to allow for protein fusions. In this project protein fusions were important for AMPs to be purified with 10x His Tag, GFP, and also to allow AMPs to be fused to spider silk.


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