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Type IV Pilus Alignment Subcomplex Proteins PilN and PilO Form Homo- and Heterodimers in Vivo
Tiffany L Leighton1, Daniel H Yong1, P Lynne Howell2
1From the Department of Biochemistry and Biomedical Sciences and the Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario L8S 4K1 and.
Abstract:
Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Type IV pili (T4P) are among the key virulence factors used by P. aeruginosa for host cell attachment, biofilm formation, and twitching motility, making this system a promising target for novel therapeutics. Point mutations in the conserved PilMNOP alignment subcomplex were previously shown to have distinct effects on assembly and disassembly of T4P, suggesting that it may function in a dynamic manner. We introduced mutations encoding Cys substitutions into pilN and/or pilO on the chromosome to maintain normal stoichiometry and expression levels and captured covalent PilNO heterodimers, as well as PilN and PilO homodimers, in vivo Most covalent PilN or PilO homodimers had minimal functional impact in P. aeruginosa, suggesting that homodimers are a physiologically relevant state. However, certain covalent homo- or heterodimers eliminated twitching motility, suggesting that specific PilNO configurations are essential for T4P function. These data were verified using soluble N-terminal truncated fragments of PilN and PilO Cys mutants, which purified as a mixture of homo- and heterodimers at volumes consistent with a tetramer. Deletion of genes encoding alignment subcomplex components, PilM or PilP, but not other T4P components, including the motor ATPases PilB or PilT, blocked in vivo formation of disulfide-bonded PilNO heterodimers, suggesting that both PilM and PilP influence the heterodimer interface. Combined, our data suggest that T4P function depends on rearrangements at PilN and PilO interfaces.
Insights
Pseudomonas aeruginosa uses Type IV pili (T4P) to infect hosts. This study reveals that specific arrangements of PilN and PilO proteins are crucial for T4P function and bacterial motility.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- Pseudomonas aeruginosa is a significant cause of hospital-acquired infections, often exhibiting antibiotic resistance.
- Type IV pili (T4P) are critical virulence factors in P. aeruginosa, mediating host cell attachment, biofilm formation, and twitching motility.
- The PilMNOP alignment subcomplex of T4P is implicated in dynamic assembly and disassembly processes.
Purpose of the Study:
- To investigate the dynamic role of the PilMNOP alignment subcomplex in T4P function.
- To explore the in vivo formation and functional impact of PilN and PilO homo- and heterodimers.
- To elucidate the influence of PilM and PilP on PilNO heterodimerization.
Main Methods:
- Introduction of cysteine substitutions into pilN and pilO genes to capture covalent dimers in vivo.
- Analysis of twitching motility in P. aeruginosa strains with engineered PilN/PilO dimers.
- Purification and characterization of soluble N-terminal truncated PilN and PilO fragments.
- Gene deletion studies of alignment subcomplex components (pilM, pilP) and their effect on PilNO heterodimer formation.
Main Results:
- Covalent PilN and PilO homodimers were generally found to have minimal impact on P. aeruginosa function.
- Specific PilNO homo- and heterodimers were observed to abolish twitching motility, indicating their essential role.
- PilM and PilP deletions, but not other T4P components, prevented in vivo formation of disulfide-bonded PilNO heterodimers.
- Purified PilN/PilO fragments formed homo- and heterodimers consistent with a tetrameric complex.
Conclusions:
- The PilMNOP alignment subcomplex likely functions dynamically, with specific PilNO configurations being essential for T4P-mediated motility.
- PilM and PilP play a regulatory role in mediating the PilNO heterodimer interface.
- T4P functionality is dependent on rearrangements occurring at the PilN and PilO interfaces.
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