Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Pathogenesis

Background:

• Type II secretion systems (T2SS) are essential for protein secretion in Gram-negative bacteria, impacting nutrient acquisition, adaptation, and virulence.
• T2SS function is linked to the assembly of type 4 pilus (T4P)-like fibers known as pseudopili.
• Pseudopilin and T4 pilin subunits share conserved structural features, including a critical glutamate residue (E5) in their transmembrane segments.

Purpose of the Study:

• To investigate the role of the conserved E5 glutamate residue in pseudopilin assembly and T2SS function.
• To determine the specific molecular interactions affected by E5 mutations in pseudopilins.

Main Methods:

• Site-directed mutagenesis to create E5 variants of pseudopilins PulG and PulH.
• Mass spectrometry to analyze pili composition and rule out effects on pilin processing or N-methylation.
• Bacterial two-hybrid analysis to assess protein-protein interactions within the T2SS machinery.
• Co-purification experiments to confirm specific interactions.

Main Results:

• Mutations at the E5 position in major pseudopilin PulG abolished pseudopilus assembly and function.
• Minor pseudopilin PulH also required E5 for secretion of its substrate, pullulanase.
• E5 mutations in PulG and PulH impaired their interaction with the assembly factor PulM, but not PulF.
• Co-purification confirmed an E5-dependent interaction between PulM and PulG.

Conclusions:

• The E5 residue plays a critical role in the recruitment of pseudopilins to the assembly machinery, specifically through interaction with PulM.
• This E5-dependent recruitment is a key step for the assembly of functional T2SS and is likely relevant for T4P assembly systems.
• The findings elucidate a specific molecular mechanism underlying T2SS assembly.