Physical and functional S-layer reconstitution in Aeromonas salmonicida

Area of Science:

• Microbiology
• Biochemistry
• Cell Biology

Background:

• The S-layer of Aeromonas salmonicida is a proteinaceous outer cell envelope with various proposed functions.
• Previous studies identified S-layer functions by analyzing S-layer-defective mutants.

Purpose of the Study:

• To establish methods for reconstituting the Aeromonas salmonicida S-layer.
• To investigate the functional competence of the reconstituted S-layer in vitro and in vivo.

Main Methods:

• Purified S-layer protein (A-protein) was reconstituted on inert membranes, immobilized lipopolysaccharide (LPS), or viable cells.
• Reconstitution was performed with purified A-protein or preassembled S-layer sheets.
• The effects of divalent cations (Ca2+) and LPS on A-protein assembly and binding were analyzed.

Main Results:

• Purified A-protein assembled into tetragonal sheets, with Ca2+ promoting regular organization and high-affinity LPS binding.
• Reconstitution on viable A. salmonicida cells required LPS and Ca2+ for ordered assembly and enhanced attachment.
• Reconstituted S-layer sheets on mutant cells restored porphyrin binding, hydrophobicity, autoaggregation, adherence, invasion, and cytotoxicity resistance, but not antibiotic resistance or complement resistance.

Conclusions:

• Successful reconstitution of the Aeromonas salmonicida S-layer was achieved using various methods.
• The reconstituted S-layer partially restored key surface-associated functions, confirming its multifunctional nature.
• Incomplete functional recovery indicates that some S-layer properties may depend on specific cellular contexts or interactions.