ABSTRACT
Type IV pili (T4P) are critical to virulence for Vibrio cholerae and other bacterial pathogens. Among their diverse functions, T4P mediate microcolony formation, which protects the bacteria from host defences and concentrates secreted toxins. The T4P of the two V. cholerae O1 disease biotypes, classical and El Tor, share 81% identity in their TcpA subunits, yet these filaments differ in their interaction patterns as assessed by electron microscopy. To understand the molecular basis for pilus-mediated microcolony formation, we solved a 1.5 A resolution crystal structure of N-terminally truncated El Tor TcpA and compared it with that of classical TcpA. Residues that differ between the two pilins are located on surface-exposed regions of the TcpA subunits. By iteratively changing these non-conserved amino acids in classical TcpA to their respective residues in El Tor TcpA, we identified residues that profoundly affect pilus:pilus interaction patterns and bacterial aggregation. These residues lie on either the protruding d-region of the TcpA subunit or in a cavity between pilin subunits in the pilus filament. Our results support a model whereby pili interact via intercalation of surface protrusions on one filament into depressions between subunits on adjacent filaments as a means to hold V. cholerae cells together in microcolonies.
Subject(s)
Fimbriae Proteins/chemistry , Fimbriae Proteins/metabolism , Fimbriae, Bacterial/metabolism , Vibrio cholerae/chemistry , Vibrio cholerae/growth & development , Amino Acid Motifs , Amino Acid Sequence , Fimbriae Proteins/genetics , Fimbriae, Bacterial/chemistry , Fimbriae, Bacterial/genetics , Molecular Conformation , Molecular Sequence Data , Sequence Alignment , Vibrio cholerae/genetics , Vibrio cholerae/metabolismABSTRACT
The bacterial pathogen Vibrio cholerae uses toxin-coregulated pili (TCP) to colonize the human intestine, causing the severe diarrheal disease cholera. TCP are long, thin, flexible homopolymers of the TcpA subunit that self-associate to hold cells together in microcolonies and serve as the receptor for the cholera toxin phage. To better understand TCP's roles in pathogenesis, we characterized its structure using hydrogen/deuterium exchange mass spectrometry and computational modeling. We show that the pilin subunits are held together by tight packing of the N-terminal alpha helices, but loose packing of the C-terminal globular domains leaves substantial gaps on the filament surface. These gaps expose a glycine-rich, amphipathic segment of the N-terminal alpha-helix, contradicting the consensus view that this region is buried in the filament core. Our results explain extreme filament flexibility, suggest a molecular basis for pilus-pilus interactions, and reveal a previously unrecognized therapeutic target for V. cholerae and other enteric pathogens.
Subject(s)
Bacterial Toxins/toxicity , Fimbriae, Bacterial/ultrastructure , Vibrio cholerae/ultrastructure , Amino Acid Sequence , Bacterial Toxins/chemistry , Cholera/microbiology , Conserved Sequence , Diarrhea/microbiology , Fimbriae, Bacterial/chemistry , Humans , Intestines/microbiology , Models, Molecular , Molecular Sequence Data , Protein Conformation , Vibrio cholerae/pathogenicityABSTRACT
OBJECTIVES: The objective of this study was to determine the effects of carbamide peroxide (CP) and hydrogen peroxide (HP) bleaching on the flexural strength (FS) and flexural modulus (FM) of dentin. METHODS: 2x2x20mm bovine dentin specimens were immersed in the bleaching agents to simulate overnight (10 or 15% CP, 6h daily, 2 weeks), exaggerated overnight (10% CP, 6h/day, 5 days/week, 2 months), daytime (6.5 or 7.5% HP, 1h daily, 3 weeks) and in-office (35% HP, 1h/day, 2 days/week, 3 weeks) treatment protocols. Distilled water (DW) and a placebo gel acted as control immersion materials. After immersion, the specimens were rinsed and stored in DW. Mechanical testing was performed 24h after the last treatment using an Instron Universal Testing Machine with a crosshead speed of 0.75 mm/min. The results were analyzed by ANOVA and Tukey's tests (p<0.05). RESULTS: There were significant reductions in the FS and FM of dentin after 2-week and 2-month exposures to CP. There were no significant differences in the FS or the FM of the dentin among the HP treatment and control groups. CONCLUSIONS: Direct in vitro application of CP bleaches caused significant decreases in dentin FS and FM. Similar decreases were not observed among the HP-treated dentin groups, which were exposed to shorter treatment times. Further research is needed to determine the effect of CP and HP on dentin in vivo.
