# The staphylococcal type VII secretion system protein EsxC impacts daptomycin sensitivity through controlling bacterial cell envelope integrity

## TL;DR

This study shows that the EsxC protein in Staphylococcus aureus affects how well daptomycin works by controlling the bacteria's cell membrane structure.

## Contribution

The study reveals a novel role of EsxC in modulating membrane integrity and antibiotic sensitivity in S. aureus.

## Key findings

- EsxC-deficient S. aureus strains are more sensitive to daptomycin and other membrane-targeting antibiotics.
- The absence of EsxC alters membrane properties, including charge and fluidity, increasing daptomycin binding.
- EsxC influences calcium ion interactions with the membrane, affecting antibiotic activity and infection outcomes.

## Abstract

The human pathogen Staphylococcus aureus encodes a specialized type VII secretion system (T7SS), which plays an important role in bacterial virulence during infection. However, the functions of the T7SS during infection and in bacterial physiology remain unclear. Here, we demonstrate that S. aureus strains lacking a T7SS effector, EsxC (ΔesxC), were highly sensitive to the important last resort drug, daptomycin, as well as other membrane-targeting antibiotics, including gramicidin and bithionol. To understand how EsxC mediates increased antibiotic sensitivity, we investigated the ΔesxC cell envelope. Scanning electron microscopy analysis of the esxC mutant revealed a distinct cell surface morphology. ΔesxC also displayed a decrease in membrane fluidity, altered membrane protein profiles, and altered cell wall synthesis. The esxC mutant membranes demonstrated an increased negative charge compared to the WT, which was associated with an enhanced daptomycin binding. We then showed that calcium ions, which are required for daptomycin activity, impacted growth of ΔesxC and sensitivity to daptomycin, suggesting that EsxC is involved in modulating calcium binding to the membrane. Furthermore, the esxC mutant displayed a heightened susceptibility to daptomycin during intracellular infection and in a murine skin infection model. Thus, our data show that the T7SS protein EsxC, in association with other T7SS proteins, contributes to the sensitivity of S. aureus to membrane-acting drugs, such as daptomycin, through modulation of cell membrane integrity.

Type VII secretion system (T7SS) has a range of functions in bacteria, including specific roles in bacterial physiology, including DNA uptake, membrane integrity, and bacterial development. In S. aureus, T7SS has been shown to be critical for bacterial virulence, intra-species competition, and in host cell interactions, although their functions in bacterial physiology are not clear. Here, we report a role of the staphylococcal T7SS component EsxC in the modulation of the cell membrane and surface integrity, likely in association with co-dependent effectors, which impacts the activity of membrane targeting drugs like daptomycin. Our data indicate that targeting the T7SS could provide a new approach to enhancing activity of existing therapeutic agents.