Vibrio cholerae are Gram-negative bacteria responsible for cholera, a severe and fatal gastrointestinal disease. The ability of V. cholerae and many other bacterial pathogens to cause disease is dependent on type IV pili. V. cholerae use toxin-coregulated pili (TCP) to colonize the human intestine. TCP are long, thin, flexible polymers of the TcpA subunit that self-associate to hold cells together in microcolonies, serve as the receptor for the cholera toxin bacteriophage CTXφ and secrete colonization factor proteins. To better understand TCP’s roles in pathogenesis, its structure was characterized using hydrogen/deuterium exchange mass spectrometry, computational modeling, electron microscopy (EM) and three-dimensional image reconstruction. The V. cholerae TcpA pilin crystal structure was docked into the pilus EM reconstruction to generate a pseudo-atomic resolution TCP structure. Tight packing of the hydrophobic N-terminal α-helices holds the pilin subunits together, but loose packing of the globular domains leaves gaps on the filament surface. These findings explain filament flexibility, suggest a molecular basis for pilus:pilus interactions and reveal a potential therapeutic target. TCP are members of the type IVb pilus subclass, which is distinguished from the type IVa subclass by differences in amino acid sequence, length and topology of the pilin globular domains. To understand the biological significance of the distinct pilin folds, circular dichroism spectroscopy was used to compare the stability of the V. cholerae type IVb TCP with that of the Neisseria gonorrhoeae type IVa gonococcal (GC) pilus together with their pilin counterparts. We show that TcpA pilin monomers are more stable than GC pilins but surprisingly GC pili are more stable than TCP filaments. Thus, while the type IVb pilin fold appears to be more stable than the type IVa fold, differences in quaternary structures, including tighter packing and stacking of aromatic side chains appear to contribute to the extreme stability of the GC pili. The robustness of GC pili may be necessary to withstand high stress forces in the urogenital tract. This may also be a common feature of type IVa pili as an adaptation to the niches occupied by the bacteria, biological demands and functions of these filaments.
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Thesis advisor: Craig, Lisa
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