Dataset Information

The differential proteomic response of Pseudomonas aeruginosa and Staphylococcus aureus to the silver (1) compound, SBC3

ABSTRACT: The race to combat antibiotic resistance and develop novel therapies has triggered studies on novel metal-based formulations. Silver remains a strong candidate since ancient times due to its multimodal and broad-spectrum activity against bacterial and fungal pathogens. 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 lipophilicity and drug stability. The lead silver(I) acetate complex, 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene (NHC*) (SBC3) synthesised by the Tacke group has previously demonstrated promising growth and biofilm-inhibiting properties. As an extension of this, we examined the responses of two structurally different bacteria to SBC3 using label-free quantitative proteomic analysis. Multidrug resistant Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive) are associated with chronic wound infections and Cystic Fibrosis lung colonisation where co-infection often exacerbates disease. SBC3 increased the abundance of alginate biosynthesis, secretion system and drug detoxification proteins in P. aeruginosa whilst a multitude of pathways including anaerobic respiration, twitching motility, and ABC transport were decreased. This contrasted with affected pathways in S. aureus such as increased DNA replication/repair and cell redox homeostasis and decreased protein synthesis, lipoylation, glucose metabolism. Increased abundance of cell wall/membrane proteins were indicative of the structural damage induced by SBC3 to both cell types. These findings show the potential broad applications of SBC3 in treating Gram-positive and Gram-negative bacteria.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Pseudomonas Aeruginosa Pao1lm10 Staphylococcus Aureus

SUBMITTER: Magdalena Piatek

LAB HEAD: Professor Kevin Kavanagh

PROVIDER: PXD038616 | Pride | 2023-03-11

REPOSITORIES: Pride

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Pseudomonas aeruginosa and Staphylococcus aureus Display Differential Proteomic Responses to the Silver(I) Compound, SBC3.

Piatek Magdalena M O'Beirne Cillian C Beato Zoe Z Tacke Matthias M Kavanagh Kevin K

Antibiotics (Basel, Switzerland) 20230208 2

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 promi ...[more]

PMID: 36830259

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Project description:Unprecedented bacterial targets are urgently needed for the development of novel antibiotics to overcome the current resistance crisis. Challenges include the limited uptake of compounds as well as prioritizing proteins for their druggability and functional relevance. Especially, the wealth of uncharacterized proteins represents an untapped source for novel targets. However, tools to decipher their function are largely lacking. We here utilize the systematic mining of pyridoxal phosphate dependent enzymes (PLP DEs) in bacteria to focus on a target class, which is known to bind ligands, accesses PLP via active transport from the media and is involved in crucial metabolic processes. For this, we systematically exploited the chemical space of the pyridoxal (PL) scaffold and obtained eight PL probes bearing modifications at various ring positions. These probes were subsequently tested for phosphorylation by cognate kinases and labelling of PLP DEs in clinically relevant Gram-positive (Staphylococcus aureus) as well as Gram-negative (Escherichia coli and Pseudomonas aeruginosa) strains. Overall, the coverage of this diverse set of probes along with refined labelling conditions not only exceeded the performance of a previous probe generation, it also provided a detailed map of binding preferences of certain structural motifs. Although originally conducted in mutant cells devoid of PLP de novo biosynthesis, we here demonstrate efficient PLP DE labelling also in a wild type strain. Overall, the profiling revealed several putative PLP DEs with unknown function, of which we exemplarily characterized five via in-depth enzymatic assays. Finally, we screened a panel of putative PLP binders for antibiotic activity and unravelled the targets of hit molecules via competitive profiling with our probes. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors.

Project description:Aspergillus fumigatus is an opportunistic fungal pathogen of the lungs resulting in both chronic and acute infection. The fungus interacts with numerous bacteria that compose the microflora of the lung including Pseudomonas aeruginosa and Klebsiella pneumoniae, both common isolates from Cystic fibrosis sputum. It is well documented that bacterial and fungal interactions both physically and through production of secreted products which can alter the growth and virulence of other species. The interaction with P. aeruginosa has been characterised previously indicating decreased growth and increased gliotoxin production in the presence of cells and the opposite when only exposed to culture filtrate. In our analysis exposure of cell free culture filtrate inhibited fungal growth and increased gliotoxin production. This was supported with proteomic analysis indicating reduction in 1,3-beta-glucanosyltransferase (-3.97 fold), methyl sterol monooxygenase erg25B (-2.9 fold) and Calcium/calmodulin dependent protein kinase (-4.2 fold) involved in fungal development. Coupled with increased expression of Glutathione S-transferase gliG (6.17 fold), non-ribosomal peptide synthase gliP (3.67 fold), O-methyltransferase gliM (3.5 fold), Gamma-glutamyl acyltransferase gliK (2.89 fold) and thioredoxin reductase gliT (2.33 fold) involved in Gliotoxin production indicate a possible shift towards secondary metabolism. Qualitative proteomic analysis of the bacterial supernatant also elucidates possible mechanisms for these alterations including physical interaction by Outer membrane protein A, High-affinity zinc uptake system protein ZnuA and Iron uptake system component EfeO reducing essential nutrient availability, enzymatic activity including Periplasmic serine endoprotease DegP-like and redox active proteins including Alkyl hydroperoxide reductase C. These proteomic alterations to the fungus could have implications for the severity of virulence in the context of coinfection and may impact disease progression.

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Dataset Information

The differential proteomic response of Pseudomonas aeruginosa and Staphylococcus aureus to the silver (1) compound, SBC3

Dataset's files

Publications

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

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