Zvi Hayouka's Lab
Fighting pathogenic bacteria using novel peptide-based antimicrobial agents
Research topics
1. Random peptide mixtures (RPMs):
Antimicrobial Peptides (AMPs) are produced by eukaryotes as part of their innate immune response to bacterial infections. Many of them contain cationic and hydrophobic residues that possess the ability to disrupt bacterial cell membranes. The broad molecular diversity among AMPs suggests that their activity is not dependent on specific amino acid sequences or peptide conformation. However, they might have undesired characteristics including development of resistance by target bacteria and high costs of production, which limiting their application. This inspired us to generate random cationic peptide mixtures using stepwise solid-phase peptide synthesis, an approach that has not been previously applied for the synthesis of random peptide mixtures. Instead of using a single amino acid for each coupling step, we use a defined mixture of two amino acids to generate 2^n sequences of random peptides with a defined composition and chain length. This modification significantly reduces the production cost since no purification is needed after each coupling step, as required in the conventional method. Our analyses demonstrate that random peptides composed of hydrophobic and cationic a-amino acids, such as phenylalanine and lysine, display potent antimicrobial activity. Moreover, altering the amino acid stereochemistry adds a high degree of selectivity for bacterial membranes to the peptide mixture.
RPMs as food preservative
Another direction that we are exploring is the use of RPMs as food preservative to replace or reduce the amount of harmful food preservative as sodium nitrite. Meat products contain valuable nutrients that are important for human health and development but are also highly susceptible to colonization by microorganisms. This can lead to spoilage and serious foodborne illnesses. We have explored the effect of RPMs on food spoilage bacteria in minced turkey meat. Amendment of RPMs to meat led to significant reductions in bacterial abundance in experimental tests, and RPMs worked synergistically with nitrite to reduce bacterial loads and extend the shelf life. We showed strong antimicrobial activity for RPMs against spoilage bacteria in meat, including Listeria monocytogenes and Pseudomonas putida. These results demonstrate the potential of RPMs as a safer preservative for reducing spoilage in meat and other food products (Palman et al. Food Microbiology, 2020).
RPMs as promising antibiotic alternatives
Antibiotic resistance is a daunting challenge in modern medicine, and novel approaches that minimize the emergence of resistant pathogens are desperately needed. Recently, we have demonstrated that RPMs have strong safety and pharmacokinetic profiles in mouse models. RPMs rapidly killed both Pseudomonas aeruginosa and Staphylococcus aureus efficiently and disrupted preformed biofilms by both pathogens. Importantly, RPMs were efficacious against both pathogens in mouse models of bacteremia and acute pneumonia. Our results demonstrate that RPMs are potent broad-spectrum therapeutics against antibiotic-resistant pathogens (Bennett et al. ACS infectious disease, 2021).
Standard Fmoc-based solid-phase synthesis methods were used, but a mixture of protected amino acids rather than a single protected amino acid was added for each coupling step. The results of three coupling steps are illustrated. In this process, each bead of the solid support bears many growing chains with many different sequences.
2. Bacterial toxin-antitoxin systems:
Our research involves the development of new approaches for the design and synthesis of chemical entities that are targeted toward unexplored bacterial biological interactions. We are study the toxin-antitoxin (TA) system in bacteria. These systems are present in many bacterial species, including major human pathogens. Each TA system comprises an antitoxin protein that binds to and inactivates its corresponding toxin protein. Upon degradation of the antitoxin in response to external stress, the toxin is released to induce bacterial death. Inhibition of TA interactions offers an opportunity for the development of selective antibacterial compounds because no mammalian homologs of TA pairs are known.
Toxin (T, blue) antitoxin (A, gray) interaction and inhibition. (Upper panel) When toxin binds the antitoxin, its toxic activity is inhibited so the bacteria will grow. (Lower panel) Upon addition of our designed inhibitors (IN, red) TA interaction is inhibited and the toxin is free to induce bacterial death.
3. Bacterial quorum sensing systems:
Bacteria use cell-cell communication systems termed quorum sensing to coordinate adaptive properties and developmental stages such as virulence, sporulation and biofilm formation to cell density. Bacillus cereus (Bc) is a Gram-positive food borne pathogen that causes foodborne illnesses, by forming biofilms and producing heat resistant spores. Understanding quorum sensing systems is important for the design of novel antimicrobial agents. PlcR is the major transcriptional regulator of Bc quorum sensing and responsible for encoding major virulence factors genes. PlcR activity is controlled by the signaling peptide PapR, derived from a 48 amino acid peptide which is secreted and matured as heptapeptide ADLPFEF. Together with Avishag Yehuda (PhD student), we have characterized this model system at the molecular level by designing and characterizing direct quorum sensing synthetic peptide derivatives. Our findings reveal several amino acids that are important for the activation or inhibition of this system. These results are the first step for future development of a new generation of antimicrobial, antibiofilm and anti-sporulating agents with many possible applications in the fields of food safety and human health (Yehuda et al. Chem comm 2018).