Author: Ayub Sidra
Date: February 2008
Sidra Ayub is an undergraduate student at UC Davis. Thanks for contributing, Sidra!
Due to the widespread use of antibiotics and antibacterial products, many bacterial strains have developed a resistance against conventional therapeutics such as the penicillins methicillin and amoxicillin. Recent outbreaks of methicillin resistant Staphylococcus aureus (MRSA) in hospitals and schools have resulted in numerous fatalities which demonstrate the urgency for developing alternative antibiotics (Appelbaum, 2006). Short, naturally produced peptides which range from 5 to 100 amino acids, called antimicrobial peptides (AMPs), present a possible solution for the MRSA epidemic. Recent research has discovered the presence of AMPs in many organisms suggesting their evolutionary effectiveness in providing hosts with a defense against bacteria, viruses, fungi, and parasites.
Experimentally determined benefits of these peptides include their toxic selectivity towards pathogens, bactericidal properties, and low susceptibility to resistance (Taylor, 1993). The most accepted hypothesis explaining the selectivity of AMPs towards bacteria is the Shai,Matsuzaki,Huang (SMH) model (Shai, 1999). According to the SMH model, cationic peptides are attracted to negatively charged bacteria membranes. This microbial Achilles heel' distinguishes broad species of microbes from multi-cellular plants and animals with their uncharged cell surfaces (Zasloff, 2002). Once recognized, the surface of the bacterial membrane becomes surrounded with AMPs and the SMH model proposes a flip of the phospholipid bilayer, resulting in the entrance of the peptide into the cell. The peptide forms numerous pores on the membrane causing the bacterial cell to rupture (Shai, 1999). With accordance to the SMH model, peptidomimetics with an overall positive net charge are more selective towards pathogens (Wilcox, 2007). Due to the net zero charge of animal cells, at low concentrations host cells remain unaffected by AMPs.
Another benefit of AMPs differ is their abundance in all organisms, enabling us to isolate and use them as therapeutic agents. However, natural AMPs are difficult to purify and therefore impractical for mass production as antibiotics. In addition, linear structures and peptides consisting of only natural -amino acid sequences increase the susceptibility to proteolysis. Modified AMPs, called peptidomimetics, have altered sequences to decrease recognition by proteases and enhance biological activity (Lee and Hodges, 2003). Scientists have recognized the huge potential of antimicrobial peptidomimetics and are undertaking various efforts for their optimization
Decreased Effectiveness of β-Lactam Antibiotics
Bacteria readily evolve to form resistance against β-lactam antibiotics, compounds which contain a beta-lactam ring (Zasloff, 2002). Molecular evolution of Staphylococcus aureus is caused by mec-genes located on the Staphylococcal Cassette Chromosome (Lowey, 2003). β-lactam antibiotics such as penicillin, act on pathogens by recognizing penicillin-binding proteins (PBPs) in the cell wall of the bacterium. Next, the antibiotic disrupts the synthesis of peptidoglycan layers, inhibiting the cell from further growth and reproduction. Consistent use of antibiotics causes mec-A genes to encode PBP2a. Unlike normal PBPs, PBP2as are proteins that β-lactam antibiotics cannot bind to and therefore bacteria expressing them are not inhibited from synthesizing the peptidoglycan layers of their cell walls (Duerenberg et al., 2007). Due to their unique structural motifs, AMPs largely prevail over microbial mechanisms of resistance (Marshall, 2003).
Stability and Non-natural Amino Acids
In 1998, J.E. Oh and K.H. Lee designed and synthesized novel non-natural amino acids and incorporated them into an AMP via solid phase peptide synthesis (Je & Lee, 1999). They determined that the unusual amino acids, consisting of various functional groups, increased the resistance of the peptide against serum proteases more than three times with out a decrease in the activity. Their results showed the potential of non-natural amino acids as novel building blocks for antimicrobial peptides, and emphasized future studies. Currently, the lab of K.P. Nambiar is synthesizing a non-natural amino acid with a long hydrocarbon chain and β-turn conformation (Nambiar, 2001). Incorporation of this non-natural amino acid into a peptidomimetic followed by bacteria testing will reveal the antimicrobial properties of lab-synthesized peptidomimetics incorporating a stable secondary structure and a non-natural amino acid in their structural motif.
Peptidomimetics consisting exclusively of natural amino acids are often recognized by proteolytic enzymes in an organism and destroyed before they are able to target pathogens. Non-natural amino acids have unique R-chains which enzymes can not recognize and therefore proteolysis is avoided. Incorporation of aromatic rings, long hydrophobic carbon chains, and various functional groups in non-natural amino acids aids in structural motif stabilization of antimicrobial peptidomimetics. Their results determined the design and synthesis of non-natural amino acids to be a promising area in the structure,activity study of AMPs.
Stability and Structure of AMPs
AMPs avoid proteolysis by alternation of non-polar and cationic amino acids, disulfide bridge stabilization, and with alpha-helix, β-ribbon, or β-turn secondary structures (Benincasa, 2003). Despite their relative selectivity AMPs and peptidomimetics can be potentially toxic towards host cells. This toxicity is also determined by the stability of peptidomimetics. Recently, the role of disulfide bridges on conformation, bioactivity, and protein stability was investigated using the AMP Gomesin and variants (Fazio et al., 2006). Biological activity of Gomesin analogs was examined with the removal of one disulfide bond then both disulfide bonds. Through circular dichroism analysis, it was determined that the lack of disulfide bridges resulted in a decrease of antimicrobial and hemolytic activity of the peptide. Consequently, the additional stability induced by disulfide bridges was directly proportional to the effectiveness of the peptide.
A study done by Igor Zelezetsky and colleagues tested the conformational stability of alpha-helical cell-lytic peptides (Zelezetsky, 2005). It was discovered various modes of interaction were initiated for peptides with stable preformed helical conformations compared with others that form secondary structures only on membrane binding. This experiment revealed the fundamental relationship between secondary structure and mechanism of interaction between AMPs and microbial membranes, reinforcing the importance of structure stability.
The Test Against Resistance
Despite their relative insensitivity to resistance, pathogenic bacteria have developed several mechanisms to evade attacks by AMPs all the same. In a study conducted at the Laboratory of Human Bacterial Pathogenesis, it was determined that the antimicrobial peptide-sensing system aps is efficient in promoting resistance to a variety of AMPs in a strain of Staphylococcus. aureus (Li et al., 2007). These mechanisms include alterations of the net surface charge, proteolytic inactivation by secreted exoproteases, and AMP transporters that remove the AMP's from the bacterial cell and membrane (Harder and Schroder, 2005). These control mechanisms maintain the colonization of bacterial cells on populated regions such as human skin and mucosal surfaces.
However, low susceptibility to resistance between pathogen and AMP is vital in determining therapeutic properties against MRSA. A selection experiment was conducted by Michael Zasloff and Graham Bell to determine if resistance would be formed after prolonged exposure to a therapeutic dose of a potential AMP drug under extremely favourable conditions (Zasloff, 2005). According to the Zasloff,Bell challenge, the AMP Pexigangan, used to treat diabetic foot ulcer infections, was tested on bacterial environments over a course of 600 generations. Isogenic lineages of Pseudomonas fluorescens and Escherichia coliwere cultured and tested against Pexiganan. Finally, the experiment revealed resistance-formation of the bacteria towards the AMP. However, conditions of this experiment were modified to increase all possible resistance factors. Isogenic lineages were chosen because of their high tendency to form resistance and moderately high levels of AMPs were administered to bacteria, resulting in a higher probability of resistance mechanisms to develop. In addition, the experiment was repeated continuously until resistance inevitably occurred, and in a physiological environment would have been toxic for the organism. Before its widespread use organisms had not developed a resistance to penicillium, found in dirt, because it was present in only small quantities and didn't cause an evolutionary pressure. Therefore, concentration is an important factor for antibiotic,pathogen interaction, which is unaccounted for in the Zasloff,Bell experiment. Despite eventual resistance formation under these extreme conditions, AMPs remain to be potential biotech tools and pharmaceutical properties of Pexiganan are still being investigated.
Glimpse Into The Future
Three years after the pharmaceutical availability of Penicillin, hospitals began to notice a prevalence of antibiotic-resistant bacterial infections (Appelbaum, 2006). Fortunately, research over the past few decades has revealed excellent potential for AMPs as pharmaceutical therapeutics and has provided insight on beneficial characteristics of peptide stability, resulting in an increase of effectiveness. A deeper understanding of these properties will allow researchers to modify future peptidomimetics to even attack resistant strains of bacteria. One concern of pharmaceutical companies regards the high minimum inhibitory concentrations AMPs require, which increases toxicity towards host cells. Addressing this issue entails further research on the effective and inhibiting properties of antimicrobial peptidomimetics, resulting in a novel class of potent therapeutic agents. In addition, pathogen resistance to AMPs should be further tested to understand how they have been evolutionarily effective. As MRSA continues to effect our hospitals and communities, the need for new antibiotics intensifies. Consequently due to the vast potential of antimicrobial peptides, the next several years may prove AMPs to serve as our greatest weapon against resistant microbial infections.
Appelbaum, PC. 2006. MRSA,Tip of the iceberg. Clin Microbiol Infect. April 12 Suppl 2:3-10.
Benincasa, B. 2003. In vitro and in vivo antimicrobial activity of two alpha-helical cathelicidin peptides and of their synthetic analogs. Peptides. 24. 1723,1731.
Deurenberg, R.H., et al. 2006. The molecular evolution of Staphylococcus aureas. European Society of Clinical Microbiology and Infectious Diseases. 13: 222-235.
Fazio, MA. 2006. Structure,activity relationship studies of gomesin: importance of the disulfide bridges for conformation, bioactivity, and serum stability. Biopolymers. 84(2): 205-18.
Harder, J and Schroder, JM. 1997. A peptide antibiotic from human skin. Nature 387:861.
Lee, DL and Hodges, RS. 2003. Structure,activity of de novo designed cyclic antimicrobial peptides based on gramicidin S. Biopolymers. 71(2): 28-48.
Li, Min. Cha, Davis. Et al. 2007. The antimicrobial peptide-sensing system aps of Staphylococcus aureus. Molecular Microbiology.
Lowey, FD. 2003. Antimicrobial resistance: the example of Staphylococcus aureus. J Clin Invest; 111: 1265-1273.
Marshall, S. 2003. Antimicrobial peptides: A natural alternative to chemical antibiotics and a potential for applied biotechnology. Electronic Journal of Biotechnology. Vol. 6. No. 2.
Nambiar, KP, et al. 2001. Design, synthesis and incorporation in peptides of a beta-turn mimetic. American Peptide Society, 2001.
Oh, JE and Lee, KH. 1999. Synthesis of novel unnatural amino acid as a building block and its incorporation into an AMP. Biorg med chem. (12): 2985-90.
Shai, Y. 1999. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochem. Biophysics. Acta 1462, 55-70.
Taylor, R. 1993. Drugs "R" Us: Finding new antibiotics at the endogenous pharmacy.
Journal of NIH research. Vol 5, June.
Wilcox, S. 2007. Cationic peptides: A new hope. The science quarterly. August-04.
Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature. vol. 415. No. 6870.
Zasloff, M. Bell, Graham. Et al. 2005. Experimental evolution of resistance to an antimicrobial peptide. Proc Biol Sci. 273(1583): 251-256.
Zelezetsky, I., et al. 2005. Controlled alteration of the shape and conformational stability of alpha-helical cell,lytic peptides: Effect on mode of action and cell specificity. Biochem J: 390(P1): 177-85.
Written by Sidra Ayub
Reviewed by Antje Heidemann, Pooja Ghatalia
Published by Pooja Ghatalia.