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

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

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

<div><p>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 <i>Vibrio cholerae</i> 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 <i>V</i>. <i>cholerae tcpB</i> 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.</p></div

<i>V</i>. <i>cholerae</i> requires TcpB for efficient pilus assembly and functions.

<p><b>(A)</b> Immunoblots of <i>V</i>. <i>cholerae</i> whole cell culture (WCC), sheared culture supernatant (SS) and cleared culture supernatant (CS) fractions probed with antibodies against TcpA, TcpF and TcpB, as indicated by the labeled protein bands. SS for the TcpA and TcpB blots was prepared by homogenizing the cells to shear the pili then removing intact cells by centrifugation. The SS fractions were topped up with PBS to a volume matching that of the WCC, and equal volumes (25 ul) of each sample were loaded onto the gel. TcpA in the SS fraction of the Δ<i>tcpB</i> and Δ<i>tcpC</i> mutants may represent contamination from cell membranes due to the shearing method or membrane blebbing due to high levels of TcpA that accumulate in the inner membrane. Secreted TcpF is detected in the CS fraction, obtained by removing <i>V</i>. <i>cholerae</i> cells by centrifugation and filtration without shearing. TcpF often appears as a doublet in immunoblots. The lower band of the doublet appears to be a proteolysed form of TcpF lacking the N-terminal ~25 amino acids. This segment is not resolved in the TcpF crystal structure suggesting it is flexible, which may explain its protease sensitivity [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006109#ppat.1006109.ref010" target="_blank">10</a>]. The Δ<i>tcpB</i> strain is complemented with p<i>tcpB</i> without (-) and with (+) 0.001% rhamnose inducer. The loading control is an unknown ~60 kDa protein present in the WCC fraction and detected by the <i>Strep</i>-Tactin-HRP conjugate. <b>(B)</b> <i>V</i>. <i>cholerae</i> autoagglutination as a function of TcpB expression. Cell cultures are shown after overnight growth followed by 15 min stationary incubation at room temperature (top panel). The OD₆₀₀ of the culture supernatant after the cells have settled is plotted for each sample in the bottom panel. The more complete the autoagglutination the lower the OD₆₀₀ value. Values are averaged for 3 experiments; error bars represent standard deviations.</p

TCP are produced in very low numbers in the <i>V</i>. <i>cholerae</i> Δ<i>tcpB</i> strain.

<p><b>(A)</b> The left TEM image is representative of WT O395, in which TCP bundles are abundant, indicated by arrows. In contrast, very few TCP bundles are observed for the Δ<i>tcpB</i> strain. One small bundle is shown in the image on the right. Flagella are indicated with arrowheads. <b>(B)</b> TEM images of TCP labeled with anti-TcpA primary antibody and gold-labeled secondary antibody. The gold particles are 6 nm in diameter. A section of each image is magnified in the inset to show the gold particles attached to the pili.</p

Complementation of <i>V</i>. <i>cholerae</i> Δ<i>tcpB</i> with <i>tcpB</i>-E5V mutants results in impaired autoagglutination and TcpF secretion without disrupting pilus assembly.

<p><b>(A)</b> TEM images of <i>V</i>. <i>cholerae</i> Δ<i>tcpB</i> complemented with <i>tcpB</i> mutants encoding Glu5 substitutions show abundant pilus production. Arrows point to TCP bundles and arrowheads point to flagella. <b>(B)</b> Immunoblots of <i>V</i>. <i>cholerae</i> fractions for the Δ<i>tcpB</i> strain complemented with WT <i>tcpB</i> or <i>tcpB</i> mutants. TcpF secretion is disrupted for the TcpB Glu5 variants despite them producing TcpB levels comparable to that of the WT <i>tcpB</i>-complemented Δ<i>tcpB</i> strain. <b>(C)</b> Autoagglutination is impaired in the <i>V</i>. <i>cholerae</i> Δ<i>tcpB</i> strain rescued with TcpB Glu5 mutants. The lower the OD₆₀₀ values the more complete the autoagglutination. Values are averaged for three replicates; error bars represent standard deviations.</p

Comparison of TcpB and the ETEC minor pilin CofB and model for TcpB-mediated assembly and retraction.

<p><b>(A)</b> Crystal structure of N-terminally truncated CofB (4QS4 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006109#ppat.1006109.ref098" target="_blank">98</a>]) shown in cartoon representation (top), and as a schematic (bottom) with the full N-terminal α-helix. Cysteines are colored cyan. <b>(B)</b> Amino acid sequence alignment of TcpB and CofB (NCBI Accession BAB62898). Alignment was first performed using Clustal Omega [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006109#ppat.1006109.ref110" target="_blank">110</a>] then adjusted to align the cysteines. Identical residues are shown in boldface type. The conserved Glu5 (red with white text) and cysteines (cyan) are indicated. Discrete domains are shaded based on the coloring of CofB shown in (A). <b>(C)</b> Proposed schematic of the TcpB structure based on sequence alignment with CofB. <b>(D)</b> Model for TcpB-mediated initiation of pilus assembly and retraction. TcpB is represented as a pilin domain (red stick and small oval) with an additional C-terminal domain (large oval). Incorporation of TcpB into the growing pilus may block passage of the pilus through the secretin complex as shown (Steps 3, 4) or may prevent further incorporation of TcpA. If pilus assembly cannot proceed, the pilin subunits will melt back into the membrane, one subunit at a time, retracting the pilus.</p

Autoagglutination, TcpF secretion and retraction but not TCP assembly are impaired in a chromosomal <i>tcpB</i>-E5V <i>V</i>. <i>cholerae</i> mutant.

<p><b>(A)</b> TEM images of piliated <i>V</i>. <i>cholerae</i> chromosomal <i>tcpB</i>-E5V/<i>flaA</i> mutants. <b>(B)</b> TCP bundles in TEM images were counted to quantify pilus expression in the various <i>V</i>. <i>cholerae</i> strains. Sixteen or more low magnification (1850X) images were counted for three growth replicates for each strain; error bars represent standard deviations. <b>(C)</b> Immunoblots of TcpF and TcpB in whole cell culture and cleared culture supernatant for <i>tcpB</i>-E5V/<i>flaA</i> mutants. The Glu5Val substitution disrupts TcpF secretion without affecting TcpB production. <b>(D)</b> The Glu5Val substitution in TcpB disrupts autoagglutination, which is restored in the <i>tcpB</i>-E5V/<i>flaA</i> double mutant. The lower the OD₆₀₀ values the more complete the autoagglutination. Values are averaged for three replicates; error bars represent standard deviations. <b>(E-G)</b> Micropillars assay data plots for <i>V</i>. <i>cholerae tcpB</i>-E5V/Δ<i>flaA</i> showing <b>(E)</b> the displacement of a representative micropillar as a function of time, which is not detected above noise level, <b>(F)</b> the projection in X and Y of the movement of this same micropillar over time, and <b>(G)</b> the force exerted on this micropillar over the same time period.</p

List of bacterial strains, plasmids and primers

<p>List of bacterial strains, plasmids and primers</p

<i>V</i>. <i>cholerae tcp</i> operon and comparison of the minor pilin TcpB and the major pilin TcpA.

<p>(A) <i>tcp</i> operon with the major pilin gene <i>tcpA</i> colored grey and the minor pilin gene <i>tcpB</i> colored red. The putative rho-independent transcription terminator is indicated with an asterisk. <b>(B)</b> Amino acid sequence alignment between the signal peptide and the N-terminal 30 residues of TcpB and TcpA (NCBI accession, CAA45456 and CAA45455, respectively). <b>(C)</b> Schematic of TcpB and TcpA pre-proteins.</p