Localization and assembly of the Vibrio cholerae type IV pilus secretin channel

Date created: 
2017-04-12
Identifier: 
etd10094
Keywords: 
Secretin
Lipoprotein
Co-chaperone
Type IV pili
Localization
Channel assembly
Vibrio cholerae
Abstract: 

Type 4 pili (T4P) are filamentous structures found on the surfaces of many Gram-negative bacteria, including Vibrio cholerae. The V. cholerae T4P are the toxin-coregulated pili (TCP), which mediate bacterial aggregation and exoprotein secretion, critical functions in colonization of the human intestine to cause the diarrheal disease cholera. TCP assemble at the inner membrane (IM), grow through a multiprotein conduit in the periplasm and through a secretin channel in the outer membrane (OM). The multimeric secretin channel is formed by secretin subunits, which are translocated across the IM by the Sec apparatus, and in most T4P systems, are transported to the OM with the help of a lipoprotein co-chaperone. In the V. cholerae TCP the secretin subunit itself, TcpC, is a lipoprotein, and its putative co-chaperone, TcpQ, is non-lipidated. Here we use mutagenesis, cellular fractionation and functional assays to investigate secretin channel assembly in V. cholerae. TcpC must be co-expressed with TcpQ to complement a ΔtcpC mutant in an assay of pilus functions, but the reciprocal is not true. TcpQ is necessary for pilus assembly but not for localization of TcpC to the outer membrane, demonstrating that TcpQ is not a co-chaperone for TcpC. The periplasmic domain of TcpC can be expressed on its own, localizes to the outer membrane, and localizes TcpQ at the outer membrane as well, provided TcpC is lipidated. When the periplasmic domain of TcpC is unlipidated it gets degraded and TcpQ accumulates in the periplasm, suggesting that the periplasmic domain of TcpC interacts with TcpQ and localizes it to the outer membrane via its lipid moiety. Our results lead to a model whereby TcpC Cys1 is lipidated at the IM by the Lgt machinery and transported to the OM in complex with TcpQ. TcpC inserts into the OM at two points: via its C-terminal portion, which forms a β-barrel channel with TcpC C-terminal domains of other secretin subunits, and via lipid moiety in its N-terminal domain, which interacts with TcpQ to link the OM channel to periplasmic pilus conduit.

Document type: 
Thesis
Rights: 
This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
File(s): 
Supervisor(s): 
Lisa Craig
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.
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