AFM reveals the interaction and nanoscale effects imposed by squalamine on Staphylococcus epidermidis

The Gram-positive bacterium Staphylococcus epidermidis is responsible for important nosocomial infections. With the continuous emergence of antibiotic-resistant strains, the search for new treatments has been amplified in the last decades. A potential candidate against multidrug-resistant bacteria is squalamine, a natural aminosterol discovered in dogfish sharks. Despite its broad-spectrum efficiency, little is known about squalamine mode of action. Here, we used atomic force microscopy (AFM) imaging to decipher the effect of squalamine on S. epidermidis morphology, revealing the peptidoglycan structure at the bacterial surface after the drug action. Single-molecule force spectroscopy with squalamine-decorated tips shows that squalamine binds to the cell surface via the spermidine motif, most likely through electrostatic interactions between the amine groups of the molecule and the negatively-charged bacterial cell wall. We demonstrated that - although spermidine is sufficient for the initial attachment of squalamine to S. epidermidis - the integrity of the molecule needs to be conserved for its antimicrobial action. A deeper analysis of the AFM force-distance signatures suggests the implication of the accumulation-associated protein (Aap), one of the main adhesins of S. epidermidis, in the initial binding of squalamine to the bacterial cell wall. This work highlights that AFM -combined with microbiological assays at the bacterial suspension scale- is a valuable approach to better understand the molecular mechanisms behind the efficiency of squalamine antibacterial activity.

Automated summary

This study used atomic force microscopy (AFM) to analyze the morphological changes of Staphylococcus epidermidis after squalamine treatment, revealing the peptidoglycan structure on the bacterial surface. Single-molecule force spectroscopy demonstrated that squalamine binds to the cell surface via the spermidine motif, likely through electrostatic interactions. The study also found the involvement of accumulation-associated protein (Aap) in the initial binding of squalamine to the bacterial cell wall. This work highlights the value of combining AFM with microbiological assays to understand the molecular mechanisms of squalamine's antibacterial activity.