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Mutations in apt (adenine phosphoribosyltransferase) affect vancomycin susceptibility in vancomycin-intermediate Staphylococcus aureus
* 1 , 2, 3 , 2 , 2 , 1, 2
1  Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater OK 74078, USA
2  Department of Biology, New Mexico State University, Las Cruces, NM 88003, USA
3  Department of Biology, Saint Mary’s College, Notre Dame, IN 46556, USA

Published: 19 April 2021 by MDPI in The 1st International Electronic Conference on Antibiotics session Poster (registering DOI)

The gene apt encodes the purine salvage enzyme adenine phosphoribosyltransferase that catalyzes the conversion of adenine and phosphoribosyl pyrophosphate into AMP and also plays a role in the uptake of adenine. We previously reported on the laboratory selection of stable vancomycin-intermediate Staphylococcus aureus (VISA) mutants (MM66-3 and MM66-4) from a hetero-VISA strain (MM66). We now report that one mutation observed in the VISA mutants characterized was in apt. Compared to MM66, growth of MM66-3 and MM66-4 in the presence of adenine and 2-fluoroadenine was less impaired than the MM66 VISA mutants, which indicated a greater reduction in the accumulation of these toxic compounds in the VISA mutants compared to MM66. In addition to increased adenine and 2-flouroadenine resistance, we also noted that several genes required for purine (purLFMNHD) and pyrimidine (pyrABFE) biosynthesis were upregulated in both MM66 VISA mutants compared to MM66. It has been reported that selection with 2-flouroadenine resulted in 2-flouroadenine reduced susceptibility (FARS) mutants harboring mutations in apt. To ascertain a role for apt mutations on vancomycin susceptibility, FARS mutants of MM66 were selected and their apt regions were sequenced. Suspected MM66 FARS mutants arose on media containing 5 mM 2-flouroadenine at a mutation frequency of 4.7 X 10-7 and all randomly selected MM66 colonies demonstrated higher 2-flouroadenine MICs than MM66. In addition, all FARS MM66 mutants harbored one of a variety of mutations (e.g. deletion, insertion and nonsense mutations) within the apt gene. FARS mutants MM66-FARS-1 and MM66-FARS-6 demonstrated identical growth curves and slightly decreased tolerance to 5 mM adenine growth inhibition compared to the parent strain MM66. Furthermore, compared to MM66, all MM66-FARS mutants demonstrated increased resistance to Congo red, similar to apt mutants previously reported on. In contradiction to these findings, MM66-3 and MM66-4 actually demonstrated reduced resistance to Congo red compared to parent strain MM66. The FARS MM66 mutants also demonstrated increased distances grown on the vancomycin gradients investigated. These findings suggest that apt mutations can alter vancomycin susceptibility and suggests a role for altered purine metabolism in a VISA mechanism.

Keywords: Staphylococcus aureus, vancomycin, VISA, adenine phosphoribosyltransferase, 2-fluoroadenine