A drug candidate for Alzheimer’s and Huntington’s disease, PBT2, can be repurposed to render Neisseria gonorrhoeae susceptible to natural cationic antimicrobial peptides

Background Neisseria gonorrhoeae is a Gram-negative bacterial pathogen that causes gonorrhoea. No vaccine is available to prevent gonorrhoea and the emergence of MDR N. gonorrhoeae strains represents an immediate public health threat. Objectives To evaluate whether PBT2/zinc may sensitize MDR N. gonorrhoeae to natural cationic antimicrobial peptides. Methods MDR strains that contain differing resistance mechanisms against numerous antibiotics were tested in MIC assays. MIC assays were performed using the broth microdilution method according to CLSI guidelines in a microtitre plate. Serially diluted LL-37 or PG-1 was tested in combination with a sub-inhibitory concentration of PBT2/zinc. Serially diluted tetracycline was also tested with sub-inhibitory concentrations of PBT2/zinc and LL-37. SWATH-MS proteomic analysis of N. gonorrhoeae treated with PBT2/zinc, LL-37 and/or tetracycline was performed to determine the mechanism(s) of N. gonorrhoeae susceptibility to antibiotics and peptides. Results Sub-inhibitory concentrations of LL-37 and PBT2/zinc synergized to render strain WHO-Z susceptible to tetracycline, whereas the killing effect of PG-1 and PBT2/zinc was additive. SWATH-MS proteomic analysis suggested that PBT2/zinc most likely leads to a loss of membrane integrity and increased protein misfolding and, in turn, results in bacterial death. Conclusions Here we show that PBT2, a candidate Alzheimer’s and Huntington’s disease drug, can be repurposed to render MDR N. gonorrhoeae more susceptible to the endogenous antimicrobial peptides LL-37 and PG-1. In the presence of LL-37, PBT2/zinc can synergize with tetracycline to restore tetracycline susceptibility to gonococci resistant to this antibiotic.

Regulatory networks in bile salt and ferric iron induced resistance of enterohemorrhagic Escherichia coli to cationic antimicrobial peptides

Enterohemorrhagic Escherichia coli (EHEC) possess the ability to respond to its surroundings though two-component systems. We hypothesized that gastrointestinal cues such as bile, iron and pH promote EHEC resistance to cationic antimicrobial peptides (CAMPs) during infection. Killing assays reveal that exposure to low pH, high iron or bile salt mixture results in increased CAMP resistance that is dependent on pmrB but independent of pmrD. Low Mg+2 -induced CAMP resistance is dependent on pmrD. pmrD promoter analysis indicate that EHEC responds to PhoPQ-inducing conditions by increasing pmrD expression. However, pmrD expression is repressed upon exposure to low acid, bile salt mix and iron. This study suggests that a complex interplay of PhoPQ, PmrAB and PmrD is involved in EHEC’s response to various microenvironmental signals and in the promotion of EHEC’s resistance to CAMPS. The results also provide intriguing evidence of both cooperation and redundancy in the mediation of CAMP resistance by these molecular players.

Regulatory networks in bile salt and ferric iron induced resistance of enterohemorrhagic Escherichia coli to cationic antimicrobial peptides

Enterohemorrhagic Escherichia coli (EHEC) possess the ability to respond to its surroundings though two-component systems. We hypothesized that gastrointestinal cues such as bile, iron and pH promote EHEC resistance to cationic antimicrobial peptides (CAMPs) during infection. Killing assays reveal that exposure to low pH, high iron or bile salt mixture results in increased CAMP resistance that is dependent on pmrB but independent of pmrD. Low Mg+2 -induced CAMP resistance is dependent on pmrD. pmrD promoter analysis indicate that EHEC responds to PhoPQ-inducing conditions by increasing pmrD expression. However, pmrD expression is repressed upon exposure to low acid, bile salt mix and iron. This study suggests that a complex interplay of PhoPQ, PmrAB and PmrD is involved in EHEC’s response to various microenvironmental signals and in the promotion of EHEC’s resistance to CAMPS. The results also provide intriguing evidence of both cooperation and redundancy in the mediation of CAMP resistance by these molecular players.

Conjugation of Silver Nanoparticles with De Novo Engineered Cationic Antimicrobial Peptides: Proposal for Study (Preprint)

BACKGROUND Cationic antimicrobial peptides have broad antimicrobial activity and provide a novel way of targeting multi drug resistant bacteria in an era of increasing antimicrobial resistance. Current developments show positive prospects for both antimicrobial peptides and silver nanoparticles individually. OBJECTIVE The primary objective is to propose another method of enhancing antimicrobial activity by conjugating silver nanoparticles with cationic antimicrobial peptides for a subsequent preliminary assessment on studying the minimum inhibitory concentration of multi drug resistant bacteria. The secondary objective would be to evaluate the safety of the conjugated compound to assess viability for in vivo use. METHODS The proposition is planned for approximately 3 overarching stages. Firstly, I propose synthesis of wlbu2c, a modified version of antimicrobial peptide wlbu2 with an added cysteine group, using standard Fmoc procedure. This will subsequently be attempted to stably conjugate with silver nanoparticles ideally through photochemical means. Secondly, the conjugate wlbu2c-AgNP will be tested for antimicrobial activity following Clinical & Laboratory Standards Institute Manual on standard minimum inhibitory concentration testing. If all of the above is completed the experiment can progress to the assessment of cytotoxicity using cell lysis assays. RESULTS I-TASSER simulation revealed that our modified peptide wlbu2c has similar secondary structure to original wlbu2 peptide. No other results have been obtained at this time other than aforementioned theoretical propositions. CONCLUSIONS The addition of silver nanoparticles to already developing de novo engineered antimicrobial peptides provide a second degree of freedom toward the development of potent antimicrobials. Future prospects include emergency last line therapy, treatment for current difficult to eradicate bacterial colonization such as in cystic fibrosis, implantable medical devices, cancer and immunotherapy. This proposal is intended to be provided to the public as I do not anticipate funding at this time.