Generic mechanisms of adaptation to cationic antimicrobials & their effects on human host defence peptides

Abstract

Increasing rates of bacterial antimicrobial resistance (AMR), associated with almost 5 million deaths per annum as of 2019, mean that it has never been more crucial that all facets of this phenomenon are explored. An underappreciated and hence understudied aspect of the AMR crisis is the potential risk of cross-resistance. This is when bacteria develop resistance to another antimicrobial which may be related to one to which they have been exposed, or which they may never have encountered. Previous work by colleagues in the United Kingdom Health Security Agency Technology Development Group has established that cross-resistance to clinical antibiotics can occur in Gram-negative bacteria upon long-term bacterial exposure to biocides. Notably, upon exposure to the antiseptics octenidine and chlorhexidine, mutations are found not only in in genes controlling the expression of efflux pumps but also membrane remodelling genes and this causes wholesale changes in the bacterial cell envelope.

However, it remains unclear whether exposure to biocides may lead to cross-resistance to human innate immune effectors, which may also act on the bacterial cell surface, and if so whether the same mechanisms might be involved. In humans, host defence peptides (HDPs) act to guide the early stages of infection by exerting both immunomodulatory and direct antimicrobial effects which they are thought to achieve, like antiseptics, by disrupting the bacterial cell envelope.

LL-37 is a very well-studied human HDP and the sole human cathelicidin. In contrast, there are far fewer studies of its physiologically occurring N-terminally truncated fragments: RK-31, KS-30, and KR-20. These potentially play key roles in widening the antimicrobial activity of cathelicidins in humans, and due to their truncation their antibacterial properties and mechanism of action may be significantly distinct from that of the parent peptide.

Here therefore we first present studies that explain the effect of LL-37 truncation on peptide biophysical properties & antimicrobial behaviour and then ask whether and how Gram-negative bacteria are susceptible to cathelicidin cross-resistance in the event of biocide exposure–as well as whether previously identified genes of interest may play a role in this phenomenon.

Chapter 3 comprises a thorough characterisation of the conformational behaviour of the human cathelicidins, using circular dichroism spectroscopy to understand how the different HDPs respond to their environment. This includes temperature, pH, solvent, as well as bacterial metabolites which have a coordinating effect. Since these are differently produced by commensal vs pathogenic bacteria, they may modulate antibacterial activity. LL-37 and its fragments are variably sensitive to different bacterial metabolites, with the greatest induction of α-helix conformation occurring at low pH and in the presence of disodium succinate. To complement this approach, we employed molecular dynamics simulations to probe the conformational behaviour of the peptides in a model membrane environment.

Chapter 4 describes changes in antibacterial activity caused by the truncation of LL-37. In vitro results showing the inability of any human cathelicidin peptides to kill Staphylococcus aureus are validated in vivo using Galleria mellonella infection models. In contrast, LL-37 and its fragments are active against Acinetobacter spp. and differences in in vitro pharmacodynamics properties such as cooperativity & maximum bacterial killing rate are revealed for the first time, demonstrating that the shortest fragment is the most cooperative across tested Acinetobacter species. N-terminal truncation of the parent peptide is shown to affect bactericidal rate, but not potency. We also demonstrate the consistently superior maximum bacterial killing rate and cooperativity of octenidine vs chlorhexidine against these organisms.

In Chapter 5 we describe adaptation experiments, in which Gram-negative bacteria were serially passaged through increasing concentrations of antimicrobials to identify the genomic modifications arising from long-term exposure. In Pseudomonas aeruginosa, mainly pmrB mutations were identified upon adaptation to (non-cathelicidin) HDPs. Cross-resistance to HDPs was identified in biocide-adapted strains but not vice versa. The experiment was repeated with cathelicidins against a panel of four Acinetobacter strains, confirming that similar typologies of genes (involved in cell envelope remodelling and efflux) are mutated upon adaptation to these compounds.

Finally, in the Appendix, we describe progress made in characterising the P. aeruginosa protein SmvR. The protein was cloned into a plasmid vector, expressed, purified, and studied by 2D nuclear magnetic resonance (NMR) spectroscopy. Preliminary spectra enabled detection of part of the expected resonances, opening a new avenue of research into these bacterial mediators.

Date of Award	1 Jul 2023
Original language	English
Awarding Institution	King's College London
Supervisor	Kenneth Bruce (Supervisor), James Mason (Supervisor), Lucy J. Bock (Supervisor) & John Sutton (Supervisor)