RESUMO
Enterotoxigenic Escherichia coli (ETEC) colonize the human gut, causing severe cholera-like diarrhoea. ETEC utilize a diverse array of pili and fimbriae for host colonization, including the Type IVb pilus CFA/III. The CFA/III pilus machinery is encoded on the cof operon, which is similar in gene sequence and synteny to the tcp operon that encodes another Type IVb pilus, the Vibrio cholerae toxin co-regulated pilus (TCP). Both pilus operons possess a syntenic gene encoding a protein of unknown function. In V. cholerae, this protein, TcpF, is a critical colonization factor secreted by the TCP apparatus. Here we show that the corresponding ETEC protein, CofJ, is a soluble protein secreted via the CFA/III apparatus. We present a 2.6 Å resolution crystal structure of CofJ, revealing a large ß-sandwich protein that bears no sequence or structural homology to TcpF. CofJ has a cluster of exposed hydrophobic side-chains at one end and structural homology to the pore-forming proteins perfringolysin O and α-haemolysin. CofJ binds to lipid vesicles and epithelial cells, suggesting a role in membrane attachment during ETEC colonization.
Assuntos
Escherichia coli Enterotoxigênica/química , Escherichia coli Enterotoxigênica/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Células CACO-2 , Sequência Consenso , Cristalografia por Raios X , Escherichia coli Enterotoxigênica/genética , Proteínas de Escherichia coli/genética , Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Óperon , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The type IV pili are helical filaments found on many Gram-negative pathogenic bacteria, with multiple diverse roles in pathogenesis, including microcolony formation, adhesion, and twitching motility. Many pathogenic enterotoxigenic Escherichia coli (ETEC) isolates express one of two type IV pili belonging to the type IVb subclass: CFA/III or Longus. Here we show a direct correlation between CFA/III expression and ETEC aggregation, suggesting that these pili, like the Vibrio cholerae toxin-coregulated pili (TCP), mediate microcolony formation. We report a 1.26-Å resolution crystal structure of CofA, the major pilin subunit from CFA/III. CofA is very similar in structure to V. cholerae TcpA but possesses a 10-amino-acid insertion that replaces part of the α2-helix with an irregular loop containing a 3(10)-helix. Homology modeling suggests a very similar structure for the Longus LngA pilin. A model for the CFA/III pilus filament was generated using the TCP electron microscopy reconstruction as a template. The unique 3(10)-helix insert fits perfectly within the gap between CofA globular domains. This insert, together with differences in surface-exposed residues, produces a filament that is smoother and more negatively charged than TCP. To explore the specificity of the type IV pilus assembly apparatus, CofA was expressed heterologously in V. cholerae by replacing the tcpA gene with that of cofA within the tcp operon. Although CofA was synthesized and processed by V. cholerae, no CFA/III filaments were detected, suggesting that the components of the type IVb pilus assembly system are highly specific to their pilin substrates.
Assuntos
Escherichia coli Enterotoxigênica/metabolismo , Proteínas de Fímbrias/classificação , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Sequência de Aminoácidos , Escherichia coli Enterotoxigênica/genética , Proteínas de Fímbrias/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas , Vibrio choleraeRESUMO
Vibrio cholerae relies on two main virulence factors--toxin-coregulated pilus (TCP) and cholera toxin--to cause the gastrointestinal disease cholera. TCP is a type IV pilus that mediates bacterial autoagglutination and colonization of the intestine. TCP is encoded by the tcp operon, which also encodes TcpF, a protein of unknown function that is secreted by V. cholerae in a TCP-dependent manner. Although TcpF is not required for TCP biogenesis, a tcpF mutant has a colonization defect in the infant mouse cholera model that is as severe as a pilus mutant. Furthermore, TcpF antisera protect against V. cholerae infection. TcpF has no apparent sequence homology to any known protein. Here, we report the de novo X-ray crystal structure of TcpF and the identification of an epitope that is critical for its function as a colonization factor. A monoclonal antibody recognizing this epitope is protective against V. cholerae challenge and adds to the protection provided by an anti-TcpA antibody. These data suggest that TcpF has a novel function in V. cholerae colonization and define a region crucial for this function.
