Functional divergence of FimX in PilZ binding and type IV pilus regulation

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Pathogenesis

Background:

• Type IV pili (T4P) are essential surface structures involved in bacterial motility, biofilm formation, and pathogenicity.
• The protein FimX and its orthologs are known regulators of T4P biogenesis in bacteria, including the pathogen Pseudomonas aeruginosa.
• Previous studies reported a direct interaction between FimX(XAC2398) and PilZ(XAC1133) in Xanthomonas axonopodis pv. citri via FimX's EAL domain.

Purpose of the Study:

• To investigate the conservation of the FimX-PilZ interaction across different bacterial species, specifically comparing Xanthomonas and Pseudomonas.
• To elucidate the molecular basis for potential differences in FimX-PilZ binding affinity.
• To understand the implications of this interaction divergence on T4P formation and bacterial physiology.

Main Methods:

• In vitro and in vivo binding assays were employed to assess FimX-PilZ interactions in P. aeruginosa.
• Surface plasmon resonance (SPR) was utilized to quantify the binding affinity between homologous and heterologous protein pairs.
• Amide hydrogen-deuterium exchange and site-directed mutagenesis were used to map the interaction interface on FimX.

Main Results:

• The strong interaction observed between FimX and PilZ in X. axonopodis pv. citri is not conserved in P. aeruginosa, with binding below measurable limits.
• SPR analysis revealed that the P. aeruginosa FimX-PilZ interaction is at least 1000-fold weaker than the Xanthomonas pair.
• The N-terminal lobe of FimX(XAC2398) was identified as the binding site for PilZ(XAC1133), and key residues in this region are absent in P. aeruginosa FimX.

Conclusions:

• The FimX-PilZ interaction is not evolutionarily conserved between Xanthomonas and Pseudomonas species due to divergence in FimX orthologs.
• Variations in FimX structure, particularly the N-terminal lobe of the EAL domain, account for the differential binding affinities.
• The functional roles of FimX and PilZ in bacterial motility and T4P biogenesis likely vary significantly across different bacterial species.