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<h1>Ag43 Secretion System</h1>

 <h2 id="overview">Overview</h2>

  <p>Antimicrobial-peptides (AMPs) have wide range of toxicity to microbes, gram-negative / positive bacteria, archaea, fungi and viruses. However, even if we hope that <i>E. coli</i> would produce host-toxic AMPs, it would have harmful result for host cells owing to their broad spectrum of toxicity. It would be painful task for <i>E. coli</i> to produce AMPs. If we introduce gene expressing host-toxic proteins into <i>E. coli</i>, they would die of their toxicity. So, in order to yield host-toxic protein without killing host cells, we iGEM HokkaidoU Japan designed a safe secretion system of AMPs, and using this system, we made <i>E. coli</i> produce thanatin, one of AMPs derived from shield bug, <i>Podisus maculiventris</i>.</p>

  <h3>Tandem-multimerization of thanatin</h3>
  <p>A broad-spectrumed antimicrobial-peptide with  from shield bug, thanatin, is short polypeptide composed of 21 amino acids. Thanatin contains 2 cysteine residues and disulfide bond forms between these 2 residues (Cys11 and Cys18). The disulfide bond between 2 cysteine residues is the core of active thanatin (1). This disulfide bridge stabilizes the anti-parallel β-sheets (Fig. 1). Thanatin is very stable short polypeptide and has wide range of activity against bacteria, archaea and fungi. The detailed activation mechanism of thanatin is still unknown. However, it is known that thanatin attacks the membrane of target cells and causes pore-forming. </p>

<img src="https://static.igem.org/mediawiki/2015/6/63/Hokkaidou_thanatin_Fig.1.jpg" class="figure">
<p class="caption">Fig. 1 Schematic of thanatin structure. Thanatin is a 21-residue short polypeptide. It has disulfide bond between 2 cysteine residues and anti-parallel β-sheets.</p>

  <p>C-terminal residues of thanatin are crucial to its activity rather than N-terminus. Thanatin N-terminus is comparatively tolerant towards modifying; adding or deletion of residues. However, in case of C-terminus, newly added or removed amino acid residues might be catastrophic, perhaps causing loss or remarkable decreasing of activity. Taking it into account, ligating some thanatin tandem would make it possible to yield detoxicated form of thanatin without killing host cells (Fig. 2). </p>
<br>

<img src="https://static.igem.org/mediawiki/2015/c/c0/Hokkaidou_thanatin_multimer_Fig.2.jpg" class="figure">
<p class="caption">Fig. 2 Image of thanatin multimer. We designed our thanatin fragment having aspartic acid at it C-terminus. Amino side of aspartic acids are cleaved by endoproteinase AspN. Multimerized thanatin do not have toxicity, but, when it is cleaved to monomers, each monomer regains wide-spectrum toxicity. </p>

  <p>Then, we need to recover the toxicity of thanatin after expressing detoxicated tandem-repeated thanatin. In order to realize re gaining of toxicity, we utilized endoproteinase AspN, which specifically cleaves the peptide bonds at amino side of aspartic acid residues (purchased at New England Biolabs. website). We added aspartic acid at carboxy-terminal side of thanatin. Thanatin-Asp-thanatin-Asp-thanatin… multimer is cleavable and monomerizable with AspN. Monomerized thanatin would regain pristine toxicity. 
We appreciate Dr. Seiichi Taguchi, a professor of Biosystems Engineering, Hokkaido University, for offering us much information about thanatin function. </p><br>


  <h3>Secretion of Thanatin by auto-transporter; Antigen43</h3>
  <p>An easy and rapid Collection method of expressed protein is desired. It would be laborious task to homogenize cells in order to collect inner protein or polypeptide. To collect proteins and polypeptides easily and rapidly, we utilized Antigen43 (Ag43), which is an autotransporter protein of <i>Escherichia coli</i>. Usually, Ag43 is surface-displayed and used for autoaggregation of <i>E. coli</i> (2).
Autotranspoter proteins, including Ag43, are commonly composed of N-terminal signal peptide following passenger domain (α-domain), and C-terminal β-barrel translocator domain (β-domain) (Fig. 3). Translocator domain is inserted in outer membrane (OM) and forms pathway across the OM. This pathway is necessary for translocation of N-terminal passenger domain from periplasmic space to bacterial surface across the outer membrane. Therefore, passenger domain (α-domain) are displayed on the surface of bacterial cell. Signal peptide is indispensable for translocation of unfolded Ag43 into periplasmic space across the inner membrane. In addition, β-domain of Ag43 contains autochaperon domain, which mediates α-domain folding. After the secretion of passenger domain, passenger is autocatalytically cleft from β-domain, but then non-covalent bond betweenα-domain and β-domain arrows passenger remain on the surface of bacterial cells. Passenger domain is non-covalently bound to translocator domain with β-barrel, so easily released from β-domain with heat treating (60 degrees C) (3).</p><br>

<img src="https://static.igem.org/mediawiki/2015/d/d5/Hokkaidou_Ag43_Fig.3.jpg" class="figure">
<p class="caption">Fig. 3 Structure of Antigen43. Ag43 contains N-terminal signal peptide, α-domain (passenger), autochaperon domain and β-barrel translocator domain.</p>

  <p>Fig. 4 shows the mechanism of Ag43 insertion into OM and secretion of thanatin on the surface of bacterial cell. First, N-terminal signal peptide is inserted into inner membrane (IM) and then, following polypeptide are translocated towards periplasmic space across IM. Next, β-domain is inserted into OM, forming β-barrel structure and finally, thanatin multimer is displayed through β-domain on the bacterial surface. 
 Replacing α-domain with thanatin multimer, remaining signal peptide and β-barrel domain, would arrow the display of thanatin multimer on the bacterial surface. So, we inserted thanatin as passenger between signal peptide and β-barrel domain. After secretion of multimerized thanatin, AspN treating cause monomerization of thanatin. Thanatin monomer would regain antimicrobial activity (Fig. 5).</p>

<br>
<img src="https://static.igem.org/mediawiki/2015/2/2a/Hokkaidou_the_mechanism_of_thanatin-Ag43_secretion_Fig.4.jpg" class="figure">
<p class="caption">Fig. 4 Image of secretion mechanism of thantin-Ag43 fusion protein from expression to surface-display.</p>

<br>
<img src="https://static.igem.org/mediawiki/2015/e/e3/Hokkaidou_activation_of_thanatin_Fig.5.jpg" class="figure">
<p class="caption">Fig. 5 A method for thanatin-activation. On cleaving surface-displayed thanatin-multimer with AspN, thanatin-monomer will be active form.</p>

 <h2 id="design">Design</h2>

  <p>We have designed sequence of thanatin with C-terminal Aspartic acid (Asp). Thanatin fragment also contains BamHI restriction enzyme site (N-terminus) and BglII restriction enzyme site (C-terminus) (Fig. 6a). Autotransporter biodevice (Fig.6b) is composed of pBAD promoter + araC (BBa_00000), N-terminal signal peptide, Ag43 translocator β-domain, and double terminator (BBa_00000). BglII restriction enzyme site is located between signal peptide and β-domain. We built a construct for thanatin detoxication and secretion by ligate thanatin fragment (BamHI / BglII cut) with BglII cut autotransporter biodevice (Fig. 7).</p>

<br>
<img src="https://static.igem.org/mediawiki/2015/2/25/Hokkaidou_biodevice_Fig.6.jpg" class="figure">
<p class="caption">Fig. 6 Schematics of fragment and plasmid that we used in this project. (a)Thanatin fragment. It contains BamHI and BglII restriction enzyme site. Aspartic acid at C-terminal side of thanatin is important for thanatin activation. (b)Autotransporter biodevice. This plasmid contains the gene of Ag43, but lacks α-domain so we can insert any fragment between signal peptide and β-domain.</p>

<br>

<img src="https://static.igem.org/mediawiki/2015/e/eb/Hokkaidou_construct_Fig.7.jpg" class="figure">
<p class="caption">Fig. 7 Image of complete version of construct.</p>

  <p>Thanatin secretion biodevice is under control of one of inducible promoters; P<sub>BAD</sub> promoter. In the absence of L-arabinose, AraC protein act as repressor and bind to promoter region, therefore expression of downstream gene of P<sub>BAD</sub> is negatively controlled. However, on adding L-arabinose, this inducer interact with AraC and cause the conformation change of repressor. Finally, AraC is released from promoter region, leading recruiting of RNA polymerase and initiation of transcription. 
 Thanatin - Ag43 fusion protein is inducible by adding L-arabinose. And then, AspN treating of surface displayed thanatin multimer would realize collection of antimicrobial active thanatin.</p></ul>


<h2 id="experiments">Experiments</h2>

  <h3>Thanatin multimerization</h3>
  <p>How can we create thanatin multimer and β subunit fusion protein correctly and securely? One of some methods to create thanatin multimer is inserting thanatin fragment into thanatin - Ag43 fusion protein. Once we have got thanatin monomer inserted autotransporter biodevice, we can make thanatin multimer and Ag43 fusion protein from this plasmid. Thanatin-monomer-inserted autotransporter biodevice contains BamHI/BglII scar (between signal peptide and thanatin) and BglII restriction enzyme site (between thanatin and β subunit). So, after cutting thanatin-inserted autotransporter with BglII, ligation this plasmid with thanatin fragment (BamHI/BglII cut) would make it possible to create thanatin multimer (Fig. 8). However, in this method, there is a possibility of reverse insertion of thanatin because C-terminal BglII of thanatin might be ligated with C-terminal BglII of autotransporter.</p>

<br>
<img src="https://static.igem.org/mediawiki/2015/4/41/Hokkaidou_insertion_of_thanatin_Fig.8.jpg" class="figure">
<p class="caption">Fig. 8 Insertion of thanatin into autotransporter biodevice. (a)Forward insertion. It is desired pattern of insertion. (b)Reverse insertion. This insertion will happens by accident. We cannot insert thanatin only in forward direction selectively.</p>

  <p>To prevent thanatin from reverse-insertion, we utilized another method. Fig. 9 shows the outline of alternative thanatin multimerization method.</p>

<br>
<img src="https://static.igem.org/mediawiki/2015/7/75/Hokkaidou_2_fragments_for_thanatin_multimerization_Fig.9.jpg" class="figure">
<p class="caption">Fig. 9 Flowchart of thanatin multimerization. (a)First, we amplified 2 types of fragment with 2 different primer sets. (b)Second, we digested 1st fragment (BamHI / SpeI cut) and 2nd fragment (BglII / SpeI cut). And finally, we ligated these 2 fragments. (c)Complete form of thanatin multimer secretion system.</p>

<ol>
 	<li><p>Amplify the autotransporter plasmid containing 1 thanatin by PCR with 2 primer sets below.</p>
 		<ul style="list-style:none;">
			<li><p>1st primer set: for making 1st fragment</p>
				<ul style="list-style:none;">
					<li><p>Forward: a primer binding to pBAD region, which has an ability to regenerate BamHI restriction enzyme site.</p></li>
					<li><p>Reverse: a primer binding to 200bp-downstream region of suffix.</p></li>
				</ul>
			</li>
			<li><p>2nd primer set: for making 2nd fragment</p>
				<ul style="list-style:none;">
					<li><p>Forward: a primer binding to 100bp-upstream region of suffix.</p></li>
					<li><p>Reverse: a primer binding to 200bp-downstream region of β subunit.</p></li>
				</ul>
			</li>
		</ul>
	</li>
	<li><p>Digest the amplified 2 fragments. Cut the 1st fragment with BamHI and SpeI, and cut the 2nd fragment with SpeI and BglII.</p></li>
	<li><p>Ligate 1st and 2nd fragment and complete the creation of thanatin-multimer.</p></li>
	<li><p>Repeat these experimental operation to increase the number of inserted thanatin.</p></li>
</ol>
  
<h2 id="result">Result</h2>

<p>Even if we hoped that <i>E. coli</i> produce thanatin, it is painful task for them to do so owing to its broad-spectrum toxicity (1). Produced active thanatin would kill its host cells.
 In order to yield thanatin, we designed autotransporter biodevice containing thanatin multimer (1mer, 2mer, 3mer, and 4mer) and introduced them into <i>E. coli</i>. After enough cultivation (10hours) in liquid lysogeny broth (LB) medium, we resuspended cells in LB medium to optical density at 600nm (OD<sub>600</sub>) =0.1 (total volume of LB: 2ml). Before initiating cultibation, 200µl of L-arabinose (final concentration; 0.1%) was added for P<sub>BAD</sub> induction. Then, we measured temporal change of OD<sub>600</sub> every hour over 10 hours (until cultured cell reached stationary phase). We also determined OD<sub>600</sub> of <i>E. coli</i> containing autotransporter biodevice alone as negative control. This control is for examination of the influence of expression of Ag43. In addition, we prepared 5 types of <i>E. coli</i> above without L-arabinose adding in order to examine whether gene expression is correctly induced. (Fig.10).</p>

<img src="" class="figure">
<p class="caption">Fig. 10 Growth curves of <i>E. coli</i> expressing different number of thanatin tandem multimer. We measured temporal change of OD<sub>600</sub> and draw growth curve. As negative control, we also draw growth curve of <i>E. coli</i> containing only Ag43 β subunit.</p>

<img src="https://static.igem.org/mediawiki/2015/archive/1/14/20150918053632%21Hokkaidou_ara%2B.png" style="width:auto; height:500px; class="figure">
<img src="https://static.igem.org/mediawiki/2015/archive/1/14/20150918053651%21Hokkaidou_ara%2B.png"style="width:auto; height:500px; class="figure">
<img src="https://static.igem.org/mediawiki/2015/archive/1/14/20150918053706%21Hokkaidou_ara%2B.png"style="width:auto; height:500px; class="figure">
<img src="https://static.igem.org/mediawiki/2015/1/14/Hokkaidou_ara%2B.png"style="width:auto; height:500px; class="figure">
<img src="https://static.igem.org/mediawiki/2015/archive/1/14/20150918053606%21Hokkaidou_ara%2B.png"style="width:auto; height:500px; class="figure">

<p>Fig. 10 shows that as the number of thanatin repeat increases, growth of <i>E. coli</i> is more inhibited.</p>

<p>In order to make sure that transformed E. coli secrete thanatin outside the cell, we carried out an operation to collect thanatin. 
Collection method is as below;</p>

<ul><p>1. Resuspend the cells in LB medium to OD<sub>600</sub> =0.1 (total volume: 2ml) after 10-hour-cultivation. At the same time, induce the gene expression adding L-arabinose.</p>

<p>2. After OD<sub>600</sub> reaches 0.5, centrifuge at 5000rpm for 1minute and remove the cells. The host <i>E. coli</i> at this phase is considered to be the best stage to collect thanatin efficiently. </p>

<p>3. Remove the supernatant, add 1µl of AspN with 20µl of reaction buffer, resuspend it and then, incubate the mixture at 37 degrees C for 30minutes. </p>

<p>4. Centrifuge the suspension at 5000 rpm for 1 minute again. If <i>E. coli</i> secreted thanatin, we would be able to collect active thanatin monomer from the second supernatant. </p></ul>

<p>After collecting operation, we needed to examine that if collected supernatant include thanatin and have toxicity.  In order to determine thanatin’s toxicity, we carried out MIC (Minimum Inhibition Concentration) test. <br>
MIC test is one of the most general examination of an antimicrobial activity against microorganism. The lowest concentration of antimicrobial agent that inhibits visible growth of microorganisms is defined as MIC. In order to assay thanatin’s activity, we prepared dilution range of the supernatant including thanatin from 1/2 to 1/100000 (total volume is 20µl each) with 96-well microtiter plates. Wild type <i>E. coli</i> (DH5α) was cultivated in LB medium until OD<sub>600</sub> reached 0.4 and diluted 100,000 folds. After that, 80 µl of the suspension was added to dilution range of supernatant. We incubated the cells at 120 rpm, 30 degrees C for 18 hours. After incubation, the OD<sub>600</sub> was determined with a microtiter plate reader. The result is shown below.
</p>



<h2 id="conclusion">Conclusion</h2>

<h2 id="reference">Reference</h2>

<p>(1) Seiichi Taguchi, Kanako Kuwasako, Atsushi Suenaga, Miyuki Okada and Haruo Momose, Functional Mapping against Escherichia coli for the Broad-Spectrum Antimicrobial peptide, Thanatin, Based on an In Vivo Monitoring Assay System, J. Biochem. 2000, 128:745-754</p>

<p>(2) van der Woude, Marjan W Henderson, Ian R. Regulation and function of Ag43 (flu). Annu. Rev. Microbiol. 2008, 62:153–69</p>

<p>(3) Glen C. Ulett, Jaione Valle, Christophe Beloin, Orla Sherlock, Jean-Marc Ghigo and Mark A. Schembri. Functional Analysis of Antigen 43 in Uropathogenic Escherichia coli Reveals a Role in Long-Term Persistence in the Urinary Tract Infect. Immun. 2007, 75(7):3233. DOI: 10.1128/IAI.01952-06.</p>

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