Soares, Sarah Aparecida; (2019) Natural products as inhibitors of bacterial type IV secretion systems. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Antimicrobial resistance is an increasing threat for global public health as it prevents the effective treatment of infectious diseases with available antimicrobials. Such diseases are the second leading cause of death worldwide, especially in developing countries. One of the main ways bacteria can become resistant to antimicrobials is through acquisition of exogenous DNA, particularly plasmids, containing antimicrobial resistance genes. Plasmids, are DNA molecules that are independent of the chromosome and can be transferred from one bacterium to another in a process called conjugation. Conjugation is commonly performed by type IV secretion systems (T4SSs), the genes for which are encoded on the plasmids themselves. The search for conjugation inhibitors is paramount in the fight against the spread of antimicrobial resistance genes. Plants were the main source of medication for thousands of years for all types of disease, including infections caused by bacteria that use the T4SSs as virulence factor delivery systems, such as H. pylori. In addition, plants produce compounds that inhibit phytopathogens T4SSs which are homologues to human pathogens T4SSs. Despite this, plants are still an underexplored resource for new medications. Considering these facts, the primary aim of the present work was to identify and characterize specific conjugator inhibitors of plant origin. In order to accomplish this an automated conjugation assay was implemented using lux reporter genes on the plasmid of interest that would give rise to bioluminescence once conjugation occurred. In that way the presence of a conjugation inhibitor would be detected by the inhibition of light emission. The automated conjugation assay not only made the processing of a large quantity of plant extracts possible also allowed the characterization of compounds as specific or unspecific conjugation inhibitors, which is not possible with a traditional mating assay. With the automated assay in place, plant extracts from Sarawak, Borneo in Malaysia were screened for their conjugation inhibition activities against clinically relevant plasmids. Five plant extracts were identified as specific inhibitors of R388 (IncW) transfer. These were bark extracts of Myristica villosa (hexane, chloroform and methanol extracts), hexane bark extract of Myristica papyracea and hexane bark extract of Knema percoriacea. All of which belong to the Myristicaceae species. iii Three ω-phenyl fatty acids, S70-1 (Ph-11:0), S70-2 (Ph-13:1, 4c) and S70-3 (Ph- 13:0) were isolated and characterized from the Myristica villosa hexane bark extract. S70- 2 is a novel compound and it was the most active one, with an IC50 of 5 μg/mL (17μM). This is the first report of ω-phenyl fatty acids occurrence in Myristicaceae plants. The discovery of specific conjugation inhibitors of plant origin opens a new horizon for the development of a new combination therapy for better and innovative medical treatments. Conjugation inhibitors could be used as co-adjuvants of antibacterial compounds to inhibit the acquisition of resistance genes by the pathogens from commensal non-pathogenic bacteria. Furthermore the reported results provides evidence that plant-based treatments may target T4SS which are the delivery systems of virulence factors of certain pathogens, such as H. pylori. This could also be used for the development of medications for these diseases caused by these organisms.

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