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pBad/araC-RBS B0034-Ag43 thanatin 4mer as alpha domain-double terminator

Ag43 is a kind of outer membrane protein that is composed of some domains. First one is signal peptide that transport Ag43 to outer membrane. After transportation, the signal peptide is cleaved and left at inner membrane. Second one is alpha domain that has connection peptide to another Ag43 alpha domain. Last one is beta domain that has beta barrel structure to let the barrel there. Due to the function of a part of beta domain, called auto-chaperon domain, alpha domain is let outside of the membrane. This part has thanatin, a kind of antimicrobial peptide 4mer as alpha domain. This thanatin has Asp(Aspartic acid) as the last residue. So if thanatin domain is digested by AspN endoproteinase (NEB), thanatin regain own toxicity.

Experiments

Thanatin multimerization

How can we create thanatin multimer and β domain 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 auto-transporter biodevice, we can make thanatin multimer and Ag43 fusion protein from this plasmid. Thanatin-monomer-inserted auto-transporter biodevice contains BamHI/BglII scar (between signal peptide and thanatin) and BglII restriction enzyme site (between thanatin and β domain). So, after cutting thanatin-inserted auto-transporter 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 auto-transporter.

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

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.

1. Amplify the auto-transporter plasmid containing 1 thanatin by PCR with 2 primer sets below.

   • 1st primer set: for making 1st fragment

     • Forward: a primer binding to P_BAD region, which has an ability to regenerate BamHI restriction enzyme site.

     • Reverse: a primer binding to 200 bp-downstream region of suffix.

   • 2nd primer set: for making 2nd fragment

     • Forward: a primer binding to 100 bp-upstream region of suffix.

     • Reverse: a primer binding to 200 bp-downstream region of β domain.

2. Digest the amplified 2 fragments. Cut the 1st fragment with BamHI and SpeI, and cut the 2nd fragment with SpeI and BglII.

3. Ligate 1st and 2nd fragment and complete the creation of thanatin-multimer.

4. Repeat these experimental operation to increase the number of inserted thanatin.

Result

Even if we hoped that E. coli 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 auto-transporter biodevice containing thanatin multimer (monomer, dimer, trimer, tetramer and introduced them into E. coli. After enough cultivation (10 hours) in liquid LB medium, we resuspended cells in LB medium to optical density at 600 nm (OD₆₀₀) =0.1 (total volume of LB: 2ml). Before initiating cultivation, 200 µL of L-arabinose (final concentration; 0.1%) was added for P_BAD induction. Then, we measured temporal change of OD₆₀₀ every hour over 10 hours (until cultured cell reached stationary phase). We also determined OD₆₀₀ of E. coli containing auto-transporter biodevice alone as negative control. This control is for examination of the influence of expression of Ag43. In addition, we prepared 5 types of E. coli above without L-arabinose adding in order to examine whether gene expression is correctly induced. (Fig.10).

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Growth curve of transformant expressing thanatin-tetramer fused with Ag43

Fig. 10 Growth curves of E. coli expressing different number of thanatin tandem multimer. We measured temporal change of OD₆₀₀ and draw growth curve. (a) Thanatin-monomer, (b) Thanatin-dimer, (c) Thanatin-trimer, (d) Thanatin-tetramer, (e) Growth curves of 5 transformants. As negative control, we also draw growth curve of E. coli containing only Ag43 β domain.

Fig. 10 shows that as the number of thanatin repeat increases, growth of E. coli is more inhibited. Growth inhibition is monitored 2 hours after induction on every transformants. Theransformant expressing thanatin-monomer reaches stationary phase about 11 hours after initiation of cultivation. As length of thanatin polymer increased, time to reach stationary phase were shortened. Thanatin-dimer, trimer, tetramer take 10, 7, 3 hours to be grown to stationary phase respectively.

In order to make sure that transformed E. coli secrete thanatin towards outside of the cell, we carried out an operation to collect thanatin. We needed to produce thanatin as much as possible for determination of thanatin’s activity. So, we cultivated transformant with thanatin-tetramer and Ag43 β subunit fusion protein. Collection method is as below;

• Resuspend the cells in LB medium to OD₆₀₀ =0.1 (total volume: 2 mL) after 10-hour-cultivation. At the same time, induce the gene expression by adding L-arabinose.

• 3 or 6 hours after induction, centrifuge at 5000 rpm for 1 minute and remove the cells.

• Remove the supernatant, add 5 µL of AspN with 100 µL of reaction buffer and 100 µL of DW, resuspend it and then, incuvate the mixture at 37℃ for 30 minutes. As negative control, we prepared mixture of 1 µL of AspN and 20 µL of reaction buffer. If AspN alone had toxicity, the result of negative control would indicate the toxicity of endoproteinase AspN.

• Centrifuge the suspension at 5000 rpm for 1 minute again. If E. coli secreted thanatin, we would be able to collect active thanatin monomer from the second supernatant.

After collecting operation, we needed to examine that if collected supernatant really include thanatin and have toxicity. In order to determine its toxicity, we carried out Minimum Inhibition Concentration (MIC) test. 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 evaluate thanatin’s activity, we concentrated supernatant containing thanatin 5-fold with centrifugal concentrator and prepared dilution range of the supernatant including thanatin from 1/2 to 1/32 (total volume is 20 µL each) with 96-well microtiter plates. Wild-type E. coli (DH5α) was cultivated in LB medium until OD₆₀₀ 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℃ for 18 hours. After incubation, the OD₆₀₀ was determined with a microtiter plate reader. The result is shown in Fig. 12.

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Fig. 11 Antimicrobial activity assay of thanatin. Growth inhibition of E. coli DH5α was assayed by adding 20 µl of serial dilutes containing thanatin into 80 µL of PB containing bacterial cells (cultivated to OD₆₀₀ values of 0.4 and diluted 100,000 folds).

The minimum inhibitory concentration (MIC) of collected thanatin lies between 5-folds and 2.5-folds dilution range (Table 1). Take the MIC value of purified thanatin (between 1.6 and 3.1 µM) into account, it is indicated that the concentration of collected thanatin would be about 7.8 µM.

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Table1 Antimicrobial activity assay.

Conclusion

Although 2 growth curves of transformants (arabinose induced or not) slightly differs, it is concluded that the expression of Ag43 does not affect the growth of transformants (Fig. 10). In the absence of L-arabinose, the growth curves of different thanatin-copy-number variants resemble that of transformant with Ag43 alone. We succeeded in inhibiting the expression of thanatin-Ag43 β subunit fusion protein with P_BAD promotor. Thus, we concluded that we succeeded in inactivation of thanatin by multimerizing and fusing with Ag43. It is indicated that we will be able to cultivate trsnsformants under safe condition and make them produce thanatin at the most appropriate point for thanatin mass-production.

As shown in Fig. 10, as the number of thanatin-repeat increases, the growth of E. coli is more inhibited. At first, we expected that tandem-multimerization would detoxicate thanatin because one thanatin or Ag43 β subunit connected to C-terminus of neighboring thanatin inactivate its toxicity. However, this result indicates that tandem-multimerized thanatin is host-toxic. In addition, the more the number of thanatin is increased, the more its toxicity is strengthened.
There is a possible reason why toxicity is strengthened as copy-number of thanatin is increased. It is possible that modifying of C-terminus of thanatin does not completely detoxicate it, so each fragment of thanatin has toxicity to some extent. Because of increase of thanatin’s copy number, surface-displayed part of fusion protein is lengthened. Consequently, the probability that thanatin will interact with outer membrane is increased.
Although surface-displayed thanatin remains to have host-toxicity in some extent, it is possible that transformant is grown without intensive host-killing, compared to transformant with thanatin without modification.

The result of MIC test indicates that we succeeded in cleaving thanatin by AspN, collecting thanatin from bacterial suspension, and to determining the antimicrobial activity of collected thanatin.

Our conclusion is that expression and secretion of thanatin to bacterial surface, cleavage and activation of thanatin by endoproteinase AspN, and determination of thanatin’s antimicrobial activity succeeded. Transformants secreting thanatin grew up without extinction. Multimerization and fusion with Ag43 is one of the useful methods to produce host-toxic antimicrobial peptide. We revealed that thanatin that is once inactivated certainly regain toxicity against microbes after treated by AspN. We estimated the concentration of thanantin using the result of purified thanatin’s MIC test. A possibility of mass-production of thanatin is implied in our experiment. What we have to do next is to improve the yield and purify the thanatin. There would be plenty of room for improvement. We will try harder to establish methods of thanatin mass-production. Perhaps, thanatin will be used as an alternative of antibiotics in the future.

Sequence and Features