In Vivo Activity of Metal Complexes Containing 1,10-Phenanthroline and 3,6,9-Trioxaundecanedioate Ligands against Pseudomonas aeruginosa Infection in Galleria mellonella Larvae

Drug-resistant Pseudomonas aeruginosa is rapidly developing resulting in a serious global threat. Immunocompromised patients are specifically at risk, especially those with cystic fibrosis (CF). Novel metal complexes incorporating 1,10-phenanthroline (phen) ligands have previously demonstrated antibacterial and anti-biofilm effects against resistant P. aeruginosa from CF patients in vitro. Herein, we present the in vivo efficacy of {[Cu(3,6,9-tdda)(phen)2]·3H2O·EtOH}n (Cu-tdda-phen), {[Mn(3,6,9-tdda)(phen)2]·3H2O·EtOH}n (Mn-tdda-phen) and [Ag2(3,6,9-tdda)(phen)4]·EtOH (Ag-tdda-phen) (tddaH2 = 3,6,9-trioxaundecanedioic acid). Individual treatments of these metal-tdda-phen complexes and in combination with the established antibiotic gentamicin were evaluated in vivo in larvae of Galleria mellonella infected with clinical isolates and laboratory strains of P. aeruginosa. G. mellonella were able to tolerate all test complexes up to 10 µg/larva. In addition, the immune response was affected by stimulation of immune cells (hemocytes) and genes that encode for immune-related peptides, specifically transferrin and inducible metallo-proteinase inhibitor. The amalgamation of metal-tdda-phen complexes and gentamicin further intensified this response at lower concentrations, clearing a P. aeruginosa infection that were previously resistant to gentamicin alone. Therefore this work highlights the anti-pseudomonal capabilities of metal-tdda-phen complexes alone and combined with gentamicin in an in vivo model

Exposure of Candida parapsilosis to the silver(I) compound SBC3 induces alterations in the proteome and reduced virulence

The antimicrobial properties of silver have been exploited for many centuries and continue to gain interest in the fight against antimicrobial drug resistance. The broad-spectrum activity and low toxicity of silver have led to its incorporation into a wide range of novel antimicrobial agents, including N-heterocyclic carbene (NHC) complexes. The antimicrobial activity and in vivo efficacy of the NHC silver(I) acetate complex SBC3, derived from 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene (NHC*), have previously been demonstrated, although the mode(s) of action of SBC3 remains to be fully elucidated. Label-free quantitative proteomics was applied to analyse changes in protein abundance in the pathogenic yeast Candida parapsilosis in response to SBC3 treatment. An increased abundance of proteins associated with detoxification and drug efflux were indicative of a cell stress response, whilst significant decreases in proteins required for protein and amino acid biosynthesis offer potential insight into the growth-inhibitory mechanisms of SBC3. Guided by the proteomic findings and the prolific biofilm and adherence capabilities of C. parapsilosis, our studies have shown the potential of SBC3 in reducing adherence to epithelial cells and biofilm formation and hence decrease fungal virulence

Continuous flow synthesis and antimicrobial evaluation of NHC* silver carboxylate derivatives of SBC3 in vitro and in vivo

N-heterocyclic silver carbene compounds have been extensively studied and shown to be active agents against a host of pathogenic bacteria and fungi. By incorporating hypothesized virulence targeting substituents into NHC–silver systems via salt metathesis, an atom-efficient complexation process can be used to develop new complexes to target the passive and active systems of a microbial cell. The incorporation of fatty acids and an FtsZ inhibitor have been achieved, and creation of both the intermediate salt and subsequent silver complex has been streamlined into a continuous flow process. Biological evaluation was conducted with in vitro toxicology assays showing these novel complexes had excellent inhibition against Gram-negative strains E. coli, P. aeruginosa, and K. pneumoniae; further studies also confirmed the ability to inhibit biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) and C. Parapsilosis. In vivo testing using a murine thigh infection model showed promising inhibition of MRSA for the lead compound SBC3, which is derived from 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene (NHC*).European Commission - European Regional Development FundScience Foundation IrelandSchool of Chemistry & the College of Science of University College Dubli

Evaluation of the mode of action of novel metal-based antimicrobial compounds

The global threat of antimicrobial resistance (AMR) is steadily on the rise jeopardising the efficacy of conventional antimicrobial agents. This crisis is driven by the inappropriate use of antimicrobials and decline in novel drug discovery. The most threatening pathogens have developed resistance to multiple drug classes and demand particular attention. Part of the ESKAPE pathogen list, Staphylococcus aureus and Pseudomonas aeruginosa are major causes of severe nosocomial infection and frequent colonisers of cystic fibrosis patient lungs. Moreover, Candida and Aspergillus spp. contribute to the vast majority of fungal infections and exacerbate morbidity and mortality in the immunocompromised. Metal ions have a long history of antimicrobial use however, the advent of AMR and the desire for more innovative strategies have revived interest. Metal ions possess unique (and multiple) modes of action. In addition, the diverse physiochemical properties of metal-based complexes (e.g. coordination number, geometry, reactivity and type and number of ligands) enables the production of vast libraries of agents with differing properties. Indeed, the design, synthesis, characterisation and microbiological assessment relies upon an interdisciplinary approach in the fields of inorganic chemistry and biology. This project conducted microbiological assessment of novel gallium(III)- and silver(I)-based agents whereby drug candidates were screened against a range of bacterial and fungal pathogens. The most susceptible of which were subjected to label-free quantitative proteomic analysis to uncover the likely mechanistic roles. The iron-mimicking capabilities of gallium and resulting disruption in iron ion homeostasis has led to the generation of therapeutic formulations including gallium maltolate (GaM). Anti-Pseudomonal assessment in vitro translated in vivo using Galleria mellonella as an insect model whilst subsequent proteomic studies revealed a number of affected pathways which would contribute to the attenuated growth and virulence of the pathogen. The poor bioavailability of gallium compounds prompted the development of water-soluble heteroleptic Ga(III) polypyridyl compounds ([Ga(bipy)2(2,3-DHBA-2H)](NO3) (1), [Ga(bipy)2(3,4-DHBA-2H)](NO3) (2) and [Ga(bipy)2(2,3,4-THBA-2H)](NO3) (3)) bearing a catecholate moiety. These siderophore conjugates, which aimed to enhance gallium uptake, were evaluated against Aspergillus fumigatus. Proteomic and biochemical analyses revealed the superior activity of these novel agents to existing gallium nitrate and effect on mitochondria. Subsequent work analysed the antimicrobial activity of the silver(I) N-heterocyclic carbene (NHC) complex, 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene (NHC*), termed SBC3. Alterations in the Candida parapsilosis proteome revealed a significant decrease in protein synthesis and directed further studies on the anti-virulence capacities of SBC3 in terms of cell adhesion, biofilm formation and cell morphology. Possessing broad-spectrum activity, SBC3 was also examined against two structurally different bacteria: P. aeruginosa (Gram-negative) and S. aureus (Gram-positive). Both were susceptible to SBC3 treatment however, proteomic findings identified distinct variations in affected proteins and associated pathways. The results presented in this thesis offer novel insights on the activity of these selected metal complexes for the treatment of resistant microbial pathogens. Proteomic analysis is a useful tool in drug discovery and development, which is key in addressing escalating rates of AMR

Galleria mellonella: The Versatile Host for Drug Discovery, In Vivo Toxicity Testing and Characterising Host-Pathogen Interactions

Larvae of the greater wax moth, Galleria mellonella, are a convenient in vivo model for assessing the activity and toxicity of antimicrobial agents and for studying the immune response to pathogens and provide results similar to those from mammals. G. mellonella larvae are now widely used in academia and industry and their use can assist in the identification and evaluation of novel antimicrobial agents. Galleria larvae are inexpensive to purchase and house, easy to inoculate, generate results within 24–48 h and their use is not restricted by legal or ethical considerations. This review will highlight how Galleria larvae can be used to assess the efficacy of novel antimicrobial therapies (photodynamic therapy, phage therapy, metal-based drugs, triazole-amino acid hybrids) and for determining the in vivo toxicity of compounds (e.g., food preservatives, ionic liquids) and/or solvents (polysorbate 80). In addition, the disease development processes are associated with a variety of pathogens (e.g., Staphylococcus aureus, Listeria monocytogenes, Aspergillus fumigatus, Madurella mycotomatis) in mammals are also present in Galleria larvae thus providing a simple in vivo model for characterising disease progression. The use of Galleria larvae offers many advantages and can lead to an acceleration in the development of novel antimicrobials and may be a prerequisite to mammalian testing

Quantitative proteomic reveals gallium maltolate induces an iron-limited stress response and reduced quorum-sensing in Pseudomonas aeruginosa

Gallium-based drugs have been repurposed as antibacterial therapeutic candidates and have shown significant potential as an alternative treatment option against drug resistant pathogens. The activity of gallium (Ga3+) is a result of its chemical similarity to ferric iron (Fe3+) and substitution into iron-dependent pathways. Ga3+ is redox inactive in typical physiological environments and therefore perturbs iron metabolism vital for bacterial growth. Gallium maltolate (GaM) is a well-known water-soluble formulation of gallium, consisting of a central gallium cation coordinated to three maltolate ligands, [Ga(Maltol-1H)3]. This study implemented a label-free quantitative proteomic approach to observe the effect of GaM on the bacterial pathogen, Pseudomonas aeruginosa. The replacement of iron for gallium mimics an iron-limitation response, as shown by increased abundance of proteins associated with iron acquisition and storage. A decreased abundance of proteins associated with quorum-sensing and swarming motility was also identified. These processes are a fundamental component of bacterial virulence and dissemination and hence suggest a potential role for GaM in the treatment of P. aeruginosa infection

Quantitative proteomic reveals gallium maltolate induces an iron-limited stress response and reduced quorum-sensing in Pseudomonas aeruginosa

Gallium-based drugs have been repurposed as antibacterial therapeutic candidates and have shown significant potential as an alternative treatment option against drug resistant pathogens. The activity of gallium (Ga3+) is a result of its chemical similarity to ferric iron (Fe3+) and substitution into iron-dependent pathways. Ga3+ is redox inactive in typical physiological environments and therefore perturbs iron metabolism vital for bacterial growth. Gallium maltolate (GaM) is a well-known water-soluble formulation of gallium, consisting of a central gallium cation coordinated to three maltolate ligands, [Ga(Maltol-1H)3]. This study implemented a label-free quantitative proteomic approach to observe the effect of GaM on the bacterial pathogen, Pseudomonas aeruginosa. The replacement of iron for gallium mimics an iron-limitation response, as shown by increased abundance of proteins associated with iron acquisition and storage. A decreased abundance of proteins associated with quorum-sensing and swarming motility was also identified. These processes are a fundamental component of bacterial virulence and dissemination and hence suggest a potential role for GaM in the treatment of P. aeruginosa infection. </p

<i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i> Display Differential Proteomic Responses to the Silver(I) Compound, SBC3

The urgent need to combat antibiotic resistance and develop novel antimicrobial therapies has triggered studies on novel metal-based formulations. N-heterocyclic carbene (NHC) complexes coordinate transition metals to generate a broad range of anticancer and/or antimicrobial agents, with ongoing efforts being made to enhance the lipophilicity and drug stability. The lead silver(I) acetate complex, 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene (NHC*) (SBC3), has previously demonstrated promising growth and biofilm-inhibiting properties. In this work, the responses of two structurally different bacteria to SBC3 using label-free quantitative proteomics were characterised. Multidrug-resistant Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive) are associated with cystic fibrosis lung colonisation and chronic wound infections, respectively. SBC3 increased the abundance of alginate biosynthesis, the secretion system and drug detoxification proteins in P. aeruginosa, whilst a variety of pathways, including anaerobic respiration, twitching motility and ABC transport, were decreased in abundance. This contrasted the affected pathways in S. aureus, where increased DNA replication/repair and cell redox homeostasis and decreased protein synthesis, lipoylation and glucose metabolism were observed. Increased abundance of cell wall/membrane proteins was indicative of the structural damage induced by SBC3 in both bacteria. These findings show the potential broad applications of SBC3 in treating Gram-positive and Gram-negative bacteria

In Vivo Activity of Metal Complexes Containing 1,10-Phenanthroline and 3,6,9-Trioxaundecanedioate Ligands against Pseudomonas aeruginosa Infection in Galleria mellonella Larvae

Drug-resistant Pseudomonas aeruginosa is rapidly developing resulting in a serious global threat. Immunocompromised patients are specifically at risk, especially those with cystic fibrosis (CF). Novel metal complexes incorporating 1,10-phenanthroline (phen) ligands have previously demonstrated antibacterial and anti-biofilm effects against resistant P. aeruginosa from CF patients in vitro. Herein, we present the in vivo efficacy of {[Cu(3,6,9-tdda)(phen)2]&middot;3H2O&middot;EtOH}n (Cu-tdda-phen), {[Mn(3,6,9-tdda)(phen)2]&middot;3H2O&middot;EtOH}n (Mn-tdda-phen) and [Ag2(3,6,9-tdda)(phen)4]&middot;EtOH (Ag-tdda-phen) (tddaH2 = 3,6,9-trioxaundecanedioic acid). Individual treatments of these metal-tdda-phen complexes and in combination with the established antibiotic gentamicin were evaluated in vivo in larvae of Galleria mellonella infected with clinical isolates and laboratory strains of P. aeruginosa. G. mellonella were able to tolerate all test complexes up to 10 &micro;g/larva. In addition, the immune response was affected by stimulation of immune cells (hemocytes) and genes that encode for immune-related peptides, specifically transferrin and inducible metallo-proteinase inhibitor. The amalgamation of metal-tdda-phen complexes and gentamicin further intensified this response at lower concentrations, clearing a P. aeruginosa infection that were previously resistant to gentamicin alone. Therefore this work highlights the anti-pseudomonal capabilities of metal-tdda-phen complexes alone and combined with gentamicin in an in vivo model