Inactivation of the elongation factor Tu by mosquitocidal toxin-catalyzed mono-ADP-ribosylation

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Bacillus sphaericus produces a mosquitocidal toxin (MTX), a 97-kDa protein with distinct enzyme and binding domains.
• The 27-kDa enzyme domain exhibits ADP-ribosyltransferase activity but is cytotoxic to bacterial expression systems.
• A truncated, non-toxic MTX fragment can be expressed and cleaved to yield the active enzyme.

Purpose of the Study:

• To investigate the molecular mechanism of the 27-kDa MTX enzyme fragment's cytotoxicity.
• To identify the specific bacterial protein targeted by the MTX enzyme domain.

Main Methods:

• Expression and cleavage of a truncated MTX fragment in E. coli.
• In vitro ADP-ribosylation assays using E. coli lysates.
• Identification of ADP-ribosylated proteins using mass spectrometry (MALDI-TOF) and peptide mapping.
• Analysis of EF-Tu function after ADP-ribosylation.

Main Results:

• The 27-kDa MTX enzyme fragment ADP-ribosylated numerous E. coli proteins, predominantly a 45-kDa protein.
• Mass spectrometry identified the targeted protein as E. coli elongation factor Tu (EF-Tu).
• ADP-ribosylation of EF-Tu inhibited ternary complex formation but not GTP binding.
• Inhibition of EF-Tu function led to the cessation of bacterial protein synthesis.

Conclusions:

• The MTX enzyme fragment targets and inactivates bacterial EF-Tu via ADP-ribosylation.
• Inhibition of protein synthesis by inactivating EF-Tu is a key mechanism of MTX cytotoxicity.
• This study elucidates a novel bacterial protein synthesis inhibition pathway mediated by a mosquitocidal toxin.