The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus

Dixon Ng,Caitlyn A Hauke,Ingrid Spielman,Rajan Lala,Subramania Kolappan,Tony Harn,Ronald K Taylor,Nicolas Biais,Tuba Altindal,Lisa Craig,Yang Gao,Zia Verjee,Gabriela Kovacikova,Jarrad M Marles,Andreas J Baumler

doi:10.1371/journal.ppat.1006109

Abstract

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system.

Highlights

Vibrio cholerae is a Gram-negative bacterial pathogen that causes the human diarrheal disease cholera, which afflicts millions of people each year [1]
Bacterial pathogens utilize a number of highly complex and sophisticated molecular systems to colonize their hosts and alter them, creating customized niches in which to reproduce. One such system is the Type IV pilus system, made up of dozens of proteins that form a macromolecular machine to polymerize small pilin proteins into long thin filaments that are displayed on the bacterial surface
These pili have a remarkable array of functions that rely on their ability to (i) adhere to many substrates, including host cell surfaces, pili from nearby bacteria, DNA and bacterial viruses, and (ii) to depolymerize or retract, which pulls the bacteria along mucosal surfaces, pulls them close together in protective aggregates, and can even draw in substrates like DNA and bacteriophage for nutrition and genetic variation

Summary

Introduction

Vibrio cholerae is a Gram-negative bacterial pathogen that causes the human diarrheal disease cholera, which afflicts millions of people each year [1]. The severe diarrhea is caused by cholera toxin, an ADP ribosylating enzyme that is transported from the cytoplasm across the inner membrane via the Sec machinery and from the periplasm across the outer membrane via the Type II secretion (T2S) system [1, 3,4,5]. Colonization of the small intestine by V. cholerae requires a second virulence factor, the toxin coregulated pilus (TCP), which self-associates to induce bacterial aggregation and microcolony formation [6,7,8]. TCP is the primary receptor for the lysogenic bacteriophage CTXφ, which carries the cholera toxin genes ctxAB and is responsible for converting V. cholerae from a harmless marine microbe to a deadly human pathogen causing pandemic disease [11]. Understanding TCP biology is essential for understanding V. cholerae pathogenesis and designing prevention and treatment strategies for cholera disease

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