# A redundant isoprenoid biosynthetic pathway supports Staphylococcus aureus metabolic versatility

## TL;DR

This study reveals that Staphylococcus aureus can use multiple enzymes to make isoprenoids, which are essential for its survival and infection, suggesting new drug targets.

## Contribution

The discovery of a redundant isoprenoid biosynthetic pathway in S. aureus, with overlapping roles of IspA and HepT, and implications for drug development.

## Key findings

- HepT compensates for IspA in isoprenoid synthesis when IspA is absent in S. aureus.
- Mutants lacking both IspA and HepT can still survive, indicating a third PDS may exist.
- Deficiencies in IspA or HepT lead to colonization defects in a mouse infection model.

## Abstract

Isoprenoids are ubiquitous molecules that serve as fundamental building blocks for life. In bacteria, isoprenoids are precursors for carotenoid pigments, respiratory cofactors, and essential sugar carrier lipids, such as lipid II. Isoprenoid synthesis initiates via condensation of the five-carbon (C5) precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This initial reaction condenses one DMAPP and two IPPs, resulting in C15 farnesyl diphosphate (FPP), an intermediate that is sequentially elongated with IPP. FPP is thought to be synthesized exclusively by the prenyl diphosphate synthase (PDS), IspA. In Staphylococcus aureus, ispA mutants lack the golden carotenoid pigment, staphyloxanthin. The fact that ispA can be inactivated in S. aureus and other bacteria is surprising given the reliance of lipid II on FPP and supports the hypothesis that an additional enzyme produces the critical isoprenoid precursor. We isolated pigmented ispA suppressor mutants harboring single-nucleotide polymorphisms within a second PDS-encoding gene, hepT, suggesting that HepT and IspA have overlapping roles in S. aureus isoprenoid synthesis. Subsequent work determined that IspA and HepT support metabolic versatility, as a hepT ispA double mutant fails to aerobically respire partially due to a lack of prenylated heme cofactors. The finding that a hepT ispA double mutant is viable supports a model whereby a third PDS compensates in the absence of ispA and hepT to produce lipid II precursors. Lastly, we show that ispA and hepT mutants exhibit colonization defects in a murine model of systemic infection, demonstrating that isoprenoid biosynthesis is a potential drug target for combating S. aureus.

Isoprenoid synthesis is an essential process that is presumed to be initiated by the prenyl diphosphate synthase (PDS), IspA. However, our understanding of this pathway is incomplete considering that ispA mutants have been described in several bacterial species, leaving the mechanism for isoprenoid synthesis initiation uncertain in these genetic backgrounds. Using the opportunistic pathogen Staphylococcus aureus, we demonstrate that a second PDS, HepT, supports the production of isoprenoid-dependent molecules in the absence of IspA. Importantly, we show that mutants deficient for either IspA or HepT display colonization defects in a murine model of systemic infection. Furthermore, the simultaneous mutation of hepT and ispA is tolerated in S. aureus and suggests the presence of a third PDS capable of initiating isoprenoid synthesis. This study establishes PDSs as viable targets for the treatment of S. aureus infections and provides novel insights into the redundant nature of isoprenoid synthesis in this pathogen.