Leyton Dominguez, Arely Fernanda; (2024) Study of putative aminotransferases from Staphylococcus aureus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The antimicrobial resistance (AMR) problem was responsible for 4.95 million deaths in 2019, with 10 million people predicted to die annually by 2050. Staphylococcus aureus is one of the six leading pathogens associated with AMR deaths, it is considered as a serious threat by the CDC and a high priority pathogen by the WHO. One of the ways to tackle AMR is by finding new targets. In the work described here, we focused on the study of three putative aminotransferases coded in the genome of S. aureus predicted to be involved in the aspartate and haem biosynthetic pathways as potential drug targets. Mutant strains from the Nebraska Transposon Mutant Library (NTML) with disruptions in the genes coding for a putative aspartate aminotransferase, SAUSA300_1916 (AspB), and for two putative glutamate-1-semialdehyde aminotransferases (GSA-ATs), SAUSA300_1614 (HemL1) and SAUSA300_1845 (HemL2), were used to evaluate the impact of the deficiency of the enzymes in S. aureus colonisation using planarian model, virulence using Galleria mellonella model, growth in different media, and biofilm formation using the crystal violet assay. Our results show that, although the putative AspB and HemL enzymes were found not to be essential for the growth or biofilm formation of S. aureus under the conditions tested, their deficiency led to a significant virulence attenuation in the G. mellonella model. This model had the advantage over the planarian model of being able to precisely inject the bacteria without the need for a sophisticated microinjection instrument and was used for further analysis. The genes coding the putative enzymes were cloned, expressed in Escherichia coli and the purified proteins were assayed for aminotransferase activity. SAUSA300_1916 (AspB) was confirmed to be a pyridoxal phosphate (PLP)-dependent aminotransferase showing a Km of 1.36 ± 0.13 mM for aspartate and was inhibited by the PLP-dependent aminotransferase inhibitors adapalene and PF-04859989, amongst others. SAUSA300_1614 and SAUSA300_1845 bound pyridoxamine phosphate (PMP) and PLP, respectively. Furthermore SAUSA300_1614 (HemL1) showed the bioinformatically predicted GSA-AT activity, while SAUSA300_1845 did not. SAUSA300_1614 had a Km of 8.6 ± 2.9 μM for its substrate, glutamate-1-semialdehyde, and was inhibited by γ-acetylenic GABA and γ-vinyl GABA. We showed that the enzymes AspB and HemL1 had the annotated activity, while the activity of the putative HemL2 protein remains to be elucidated. Both enzymes, AspB and HemL1, were inhibited by compounds approved to be used in humans that are safe and whose efficacy in S. aureus treatment might potentially be tested. Since these pyridoxal dependent enzymes have no homologs in humans, they represent specific targets for the development of new drugs against S. aureus.

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