Dataset Information

Mutations in a Membrane Permease or hpt Lead to 6-Thioguanine Resistance in Staphylococcus aureus.

ABSTRACT: We recently discovered that 6-thioguanine (6-TG) is an antivirulence compound that is produced by a number of coagulase-negative staphylococci. In Staphylococcus aureus, it inhibits de novo purine biosynthesis and ribosomal protein expression, thus inhibiting growth and abrogating toxin production. Mechanisms by which S. aureus may develop resistance to this compound are currently unknown. Here, we show that 6-TG-resistant S. aureus mutants emerge spontaneously when the bacteria are subjected to high concentrations of 6-TG in vitro. Whole-genome sequencing of these mutants revealed frameshift and missense mutations in a xanthine-uracil permease family protein (stgP [six thioguanine permease]) and single nucleotide polymorphisms in hypoxanthine phosphoribosyltransferase (hpt). These mutations engender S. aureus the ability to resist both the growth inhibitory and toxin downregulation effects of 6-TG. While prophylactic administration of 6-TG ameliorates necrotic lesions in subcutaneous infection of mice with methicillin-resistant S. aureus (MRSA) strain USA300 LAC, the drug did not reduce lesion size formed by the 6-TG-resistant strains. These findings identify mechanisms of 6-TG resistance, and this information can be leveraged to inform strategies to slow the evolution of resistance.

SUBMITTER: Chin D

PROVIDER: S-EPMC8370250 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Publications

Mutations in a Membrane Permease or <i>hpt</i> Lead to 6-Thioguanine Resistance in Staphylococcus aureus.

Chin Denny D Goncheva Mariya I MI Flannagan Ronald S RS Heinrichs David E DE

Antimicrobial agents and chemotherapy 20210817 9

We recently discovered that 6-thioguanine (6-TG) is an antivirulence compound that is produced by a number of coagulase-negative staphylococci. In Staphylococcus aureus, it inhibits <i>de novo</i> purine biosynthesis and ribosomal protein expression, thus inhibiting growth and abrogating toxin production. Mechanisms by which S. aureus may develop resistance to this compound are currently unknown. Here, we show that 6-TG-resistant S. aureus mutants emerge spontaneously when the bacteria are subje ...[more]

PMID: 34125595

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Project description:Host defense proteins (HDPs), aka defensins, are a key part of the innate immune system that functions by inserting into the bacterial membranes to form pores to kill invading and colonizing microorganisms. To ensure survival, microorganism such as S. aureus has developed survival strategies to sense and respond to HDPs. One key strategy in S. aureus is a two-component system (TCS) called GraRS coupled to an efflux pump that consists of a membrane permease VraG and an ATPase VraF, analogous to the BceRS-BceAB system of Bacillus subtilis but with distinct differences. While the 9 negatively charged amino acid extracellular loop of the membrane sensor GraS has been shown to be involved in sensing, the major question is how such a small loop can sense diverse HDPs. Mutation analysis in this study divulged that the vraG mutant phenocopied the graS mutant with respect to reduced activation of downstream effector mprF, reduction in surface positive charge and enhanced 2 hr. killing with LL-37 as compared with the parental MRSA strain JE2. In silico analysis revealed VraG contains a single 200-residue extracellular loop (EL) situated between the 7th and 8th transmembrane segments (out of 10). Remarkably, deletion of EL in VraG enhanced mprF expression, augmented surface positive charge and improved survival in LL-37 vs. parent JE2. As the EL of VraG is rich in lysine residues (16%), in contrast to a preponderance of negatively charged aspartic acid residues (3 out of 9) in the EL of GraS, we divulged the role of charge interaction by showing that K380 in the EL of VraG is an important residue that likely interacts with GraS to interfere with GraS-mediated signaling. Bacterial two-hybrid analysis also supported the interaction of EL of VraG with the EL of GraS. Collectively, we demonstrated an interesting facet of efflux pumps whereby the membrane permease disrupts HDP signaling by inhibiting GraS sensing that involves charged residues in the EL of VraG.

Dataset Information

Mutations in a Membrane Permease or hpt Lead to 6-Thioguanine Resistance in Staphylococcus aureus.

Publications

Mutations in a Membrane Permease or <i>hpt</i> Lead to 6-Thioguanine Resistance in Staphylococcus aureus.

Similar Datasets

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