Gonococcal rfaF mutants express Rd2 chemotype LPS and do not enter epithelial host cells

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Pathogenesis

Background:

• The Isi-1 gene in Neisseria gonorrhoeae was previously linked to lipopolysaccharide (LPS) inner-core biosynthesis.
• Understanding LPS biosynthesis is critical for deciphering bacterial virulence mechanisms.

Purpose of the Study:

• To investigate the precise function of the Isi-1 gene in Neisseria gonorrhoeae.
• To determine the role of Isi-1 in LPS structure and its impact on gonococcal invasiveness.

Main Methods:

• Gene disruption of Isi-1 in Neisseria gonorrhoeae strain MS11.
• Complementation analysis of Salmonella typhimurium rfa mutants.
• Amino acid sequence comparison between gonococcal and Salmonella RfaF.
• Immunochemical analysis of LPS using monoclonal antibodies.
• In vitro invasion assays using human Chang epithelial cells.

Main Results:

• Isi-1 disruption altered LPS structure, causing faster migration consistent with inner-core defects.
• Isi-1 functionally complements Salmonella rfaF mutants, identifying it as the gonococcal RfaF homolog.
• High amino acid similarity (70%) between gonococcal and Salmonella RfaF proteins was observed.
• LPS from the Isi-1 mutant showed impaired invasion of Chang epithelial cells, despite opacity protein expression.

Conclusions:

• The Isi-1 gene encodes a functional heptosyltransferase II (RfaF) in Neisseria gonorrhoeae.
• LPS structure, influenced by Isi-1/RfaF, is a critical determinant of gonococcal invasion.
• This study establishes a significant genetic and functional homology between gonococcal and enteric bacterial LPS biosynthesis pathways.