Targeting multi-drug resistant pathogens with novel antimicrobial peptides

Mohamed Fathy Kamel Mohamed, Purdue University

Abstract

The emergence of antibiotic resistance is a notorious problem worldwide. In the United States alone, antibiotic-resistant bacteria infect at least two million people and kill around 23,000 patients each year. Half of these infections are attributed to the bacterial pathogen, methicillin resistant Staphylococcus aureus (MRSA) according to a report published in 2013 by the Centers for Disease Control and Prevention (CDC). Similarly, S. pseudintermedius is a leading cause of opportunistic infections in pet animals and has zoonotic potential. Furthermore, the marked increase in the incidence of infections due to extensively drug-resistant and pandrug-resistant isolates of Pseudomonas aeruginosa and Acinetobacter baumannii presents an alarming challenge that necessitates the development of novel therapeutic alternatives to traditional antibiotics in order to address this scourge. Currently, there is significant interest in antimicrobial peptides (AMPs) as novel therapeutic alternatives to conventional antibiotics. AMPs are major constituents of the host innate defense system for most organisms. AMPs are composed of a short sequence of amino acids (less than 50 residues), with a cationic charge and amphipathic character. AMPs possess several unique features that support their utility as antibacterial agents including possessing rapid bactericidal activity against a wide spectrum of pathogenic bacteria. Additionally, AMPs are typically not susceptible to the same mechanisms that confer resistance to traditional antibiotics. However, naturally-derived AMPs do possess limitations that have hindered their translation as clinically useful agents including, moderate antimicrobial activity, toxicity to host tissues, intolerance to physiological conditions, sensitivity to enzymatic degradation, and high manufacturing costs due to their complex design and long length of amino acids. Thus, there is a critical need for finding small peptides with strong antibacterial activity, salt tolerance and low toxicity. To address these issues, we screened a library of short synthetic peptides that were developed for alternative therapeutic applications and identified four peptides, namely RR, RRIKA, WR12 and D-IK8, with potent antimicrobial activity against a panel of drug-resistant staphylococci including MRSA. In contrast to many natural AMPs; these peptides retained their activity in the presence of high salt concentrations. WR12 and D-IK8 were able to eradicate persister cells, MRSA in stationary growth phase, and showed significant clearance of intracellular MRSA in comparison to antibiotics of choice (vancomycin and linezolid). In addition to their antibacterial activity against MRSA, these peptides were found to possess more potent activity against clinical isolates of methicillin-resistant S. pseudintermedius (MRSP) isolated from infected dogs. Topical application of investigated peptides effectively reduced both the bacterial load and inflammatory cytokines in a murine model of MRSA skin lesions. Next, we designed modified derivatives of the RR peptide to enhance the peptide’s potency and spectrum of antimicrobial activity against Gram-negative pathogens (P. aeruginosa and A. baumannii). From the peptides designed in this study, RR4 and its D-enantiomer, D-RR4, were the most potent analogues with at least 32-fold improvement in antimicrobial activity observed. Interestingly, D-RR4 demonstrated potent activity against colistin-resistant, cystic fibrosis strains of P. aeruginosa indicating a potential therapeutic advantage of this peptide over several AMPs. Of note, D-RR4 was able to bind to LPS to reduce the endotoxin-induced proinflammatory cytokine response in macrophages. Furthermore, D-RR4 protected Caenorhabditis elegans worms from lethal infections of P. aeruginosa and A. baumannii and enhanced the activity of colistin in vivo against colistin-resistant P. aeruginosa. The ability of bacterial pathogens to form biofilms that are highly tolerant to antibiotics further aggravates the situation and can lead to recurring infections. To address this significant problem, we conjugated the antibiotic kanamycin with a novel antimicrobial peptide (P14LRR) to develop a kanamycin peptide conjugate (P14KanS). P14KanS was superior in disrupting adherent bacterial biofilms when compared to conventional antibiotics. Furthermore, P14KanS protected C. elegans from lethal infections of biofilm-forming Gram-positive and Gram-negative pathogens. In conclusion, this study supports the potential of investigated peptides and the kanamycin peptide conjugate for the treatment of multi-drug resistant bacterial infections.

Degree

Ph.D.

Advisors

Seleem, Purdue University.

Subject Area

Microbiology|Health sciences|Veterinary services

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