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Antimicrobial peptide discovered from the Lactobacillus spp. in the human vaginal flora Antimicrobial peptide ORF

Usage and Biology

Antimicrobial peptides (AMP) are a large class of active oligopeptides with antimicrobial properties against harmful life forms (or pathogens) such as bacteria, fungi, parasites and viruses. In addition to their antimicrobial ability, some of these peptides can directly or indirectly participate in the immunomodulation of the host and protect the host from infection. Because they generally have a sufficient amount of positive charge and are often accompanied by hydrophobicity, they can bind to negatively charged biological membranes under electrostatic force, penetrate and destroy the membrane structure to cause cell death. Different from the traditional antibiotic single-target sterilization principle, antimicrobial peptide can carry out multi-target destruction in pathogens, which can greatly reduce the production of drug-resistant bacteria, and has a broad-spectrum antimicrobial properties, which is one of the best choices for replacing antibiotics in the future. In the current global situation, antimicrobial peptides have been used in many clinical treatment cases in bacterial infection, wound healing and cancer treatment. With the development of synthetic biology and biomedical engineering and other interdisciplinary disciplines, the molecular design and biosynthesis of antimicrobial peptides will have more ideas and technical support. Imbalance of intestinal flora and loss of tolerance to local microbiota are possible causes of inducing chronic enteritis. Therefore, regulating the balance of intestinal flora can help alleviate intestinal inflammation and improve the therapeutic effect of chronic enteritis.

We constructed suitable engineered bacterial strains to characterize a novel antimicrobial peptide newly isolated from Lactobacillus from the human vaginal flora in our Host lab (unpublished data). The antimicrobial peptide has been shown to have some bacteriostatic properties after validation with our constructed engineered bacteria. It has a wide range of bacteriostatic activity against Gram-positive and Gram-negative bacteria.

Plasmid construction of a novel antimicrobial peptide AMP

Constitutive plasmid construction

We obtained a novel antimicrobial peptide (AMP) from our host lab, and sent it to the company for gene synthesis to obtain a usable AMP gene sequence. We selected Escherichia coli BL21 (DE3) and strong constitutive promoter J23119 as chassis cell and promoter to enable long-term and efficient antimicrobial peptide expression. On this basis, we will isolate and purify the AMP expressed by our constructed engineered strains and use them as biological agents.

Based on these design principles, we designed plasmid pET29a-pJ23119-RBS-AMP-T7, as shown in Figure 2-1-1. Using homologous recombination integration, expression plasmid pET29a-pJ23119-RBS-AMP-T7 was constructed and transformed into DH5α. We picked several single colonies of E. coli on the transfected plates and then extracted the recombinant plasmid and performed PCR to verify that the primers were specific and the target fragment was 587bp, and the results are shown in Figures 2-1-2. We sent the plasmids with the correct positions of the bands to GENEWIZ Co. for sequencing. As shown in the figure 2-1-3, the results of the sequencing were correct. The recombinant plasmid pET29a-pJ23119-RBS-AMP-T7 was successfully constructed.

Figure 2-1-1 AMP expression plasmid pET29(a)-pJ23119-RBS-AMP-T

Figure 2-1-2 M：DL5000 DNA Marker(Vazyme)

plasmid pET29a-pJ23119-RBS-AMP-T7(587bp)

Figure 2-1-3 plasmid pET29a-pJ23119-RBS-AMP-T7 sequencing result

Nitric-Oxide-inducible plasmid construction

Since we changed the therapy from extracting antimicrobial peptides as biological agents for injection to directly taking engineered bacteria for treatment in our project and to validate the part's performance in vitro as much as possible in real treatment scenarios, we optimised and upgraded the engineered bacteria.

We considered the suggestions made by relevant experts and upgraded the engineered bacteria design in two parts, chassis cell and promoter:

1. Replacement of chassis cells from BL21 (DE3) to the safe and harmless E. coli Nissle 1917 (EcN)
2. Adding the responsive promoter SoxR/SoxS(BBa_K554003 BBa_K554000) after the strong constitutive promoter J23119

In order to be taken orally, we replaced the chassis cells with EcN, an FDA-approved human probiotic. EcN has been widely used in therapeutic applications and is endotoxin-free, which avoids the problem of endotoxin-containing BL21 (DE3) strains. Adding a responsive promoter ensures that our gene line is turned on and releases antimicrobial peptides in specific enteritis environments, avoiding accidental expression by engineered bacteria. (https://2024.igem.wiki/zqt-nanjing/engineering)

On these design principles, we designed the plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7 as shown in Figure 2-2-1, and using homologous recombination integration, we constructed the expression plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-AMP-T7. We transformed it into DH5α and extracted the recombinant plasmid after picking several single colonies of E. coli on the transformed plates for inoculation. We performed PCR to verify that the primers were specific primers with a target fragment of 2019bp, and the results are shown in Figures 2-2-2. We sent the plasmids with correct band positions to GENEWIZ Co. for sequencing. As shown in the figure 2-2-3, the sequencing results were all correct, which verified that the recombinant plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7 was successfully constructed.

Figure 2-2-1 NO-inducible plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7

Figure 2-2-2 M：DL2000 DNA Marker(Vazyme)

plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7(2019bp)

Figure 2-2-3 plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7 sequencing result

Protein expression and purification validation of the novel antimicrobial peptide AMP

Figure 3-1-1 Process of AMP protein purification by nickel columns

Figure 3-1-2 Process of performing SDS-PAGE to verify the AMP protein

SDS-PAGE validation

We extracted the correctly sequenced NO-inducible plasmid and transformed it into EcN for expression. Since NO is hazardous, we utilized SNP (sodium nitroprusside) as a nitric oxide (NO) donor for inducible expression. SNP can decompose and release NO in the presence of light, thus initiating downstream gene expression.

We picked a single colony, added 5ml LB culture solution in a test tube, added one thousandth (5µl) of the resistance gene Kana, picked the single colony into the test tube, and put it into a 37℃ incubator at 220rpm to incubate for 16 hours. Two tubes of bacteria were raised as experimental group and control group; OD₆₀₀ of the bacterial solution was measured, and the OD₆₀₀ of the bacterial solution was diluted to 0.1 (0.1×(50+colonization amount) = OD₆₀₀×colonization amount), which was added into 250ml conical flasks with 50ml of LB+50µl of Kana, and the culture was amplified for four to five hours, until both flasks of the bacterial solution had an OD₆₀₀ value close to 1.0. We initially set the induction temperature at 16°C and the induction time at 24 hours, and the final concentration of SNP (sodium nitroprusside) 100µM was replenished every 12 hours. At the end of induction, the protein fragmentation step was carried out, 200 µl of total protein samples from experimental and control groups were dispensed before ultrasonic fragmentation; after fragmentation, 200 µl of supernatant was aspirated as post-fragmentation supernatant, and 200 µl of precipitate was resuspended in ultrapure water as post-fragmentation precipitate. Concentrate by ultrafiltration, take up 200µl of concentrated supernatant as a sample; protein gel sample, 5-10µl for each well site, electrophoresis running gel; Thomas Brilliant Blue medium-high fire staining for two minutes, decolorization.

Because the Marker indication range is 8-200KD, and the AMP size is only 8.5KD after adding His tag, so the bands are not obvious. Therefore, we performed Western Blot protein blotting (WB) to verify whether the protein was expressed.

Figure 3-1-3 SDS-PAGE characterization of antimicrobial peptide containing NO-inducible promoter

M: Protein Marker; 1: Whole bacteria after induction; 2: Supernatant after induction and sonication; 3: Precipitate after induction and sonication

Western Blot Initial Validation

According to the previous results, we ran the remaining protein samples on the gel, transferred them, and conducted the WB experiment. We applied the primary antibody (1:7000) overnight, washed with TBST, applied the secondary antibody (1:5000) for 1 hour, continued washing, prepared the protein-developing solution in a 1:1 ratio, and developed the results. The results are shown in Figure 3-2-1. We found that the baseline expression level of AMP was relatively high, and inclusion bodies were formed with a high content in the precipitate. We decided to characterize the NO-inducible promotor SoxR/SoxS to determine the optimal inducer concentration and induction time, optimize the conditions, and obtain more AMP than the baseline expression.

Figure 3-2-1 Western Blot result of AMP expression of plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7

1-3 represent uninduced total protein, concentrated supernatant, and precipitation, respectively; well M is the Marker (at 8KD); 4-6 represent induced total protein, concentrated supernatant, and precipitation, respectively.

Western Blot Re-validation

Through the characterization of the promoter SoxR/SoxS (more details of the characterization can be found on the main page of BBa_K554000), we got the optimal induction time of p-SoxR-pSoxS-eGFP-RBS-AMP plasmid in EcN is 10 hours, and the optimal induction concentration of SNP is 100µM.

So we carried out the induction of AMP expression according to the above experimental conditions, picked single colonies, added 5 ml of LB culture medium to the test tube, added one thousandth (5µL) of the resistance gene Kana, picked the single colonies into the test tube, and put them into the 37℃ incubator at 220rpm for 16 hours. Two tubes of bacteria were raised as experimental group and control group. Measure the OD₆₀₀ of the bacterial solution, dilute the OD₆₀₀ of the bacterial solution to 0.1 (0.1×(50+colonization amount)=OD₆₀₀×colonization amount), add it into 250ml conical flasks of 50ml LB+50µl Kana, and amplify the incubation for four to five hours until the OD₆₀₀ values of both flasks were close to 1.0. Set the induction time to 10 hours, and SNP (sodium nitroprusside) 100µM final concentration was added. Protein fragmentation step was carried out at the end of induction, 200 µl of total protein samples from experimental and control groups were dispensed before ultrasonic fragmentation; after fragmentation, 200µl of supernatant was aspirated as post-fragmentation supernatant sample solution, and 200µl of precipitate was resuspended in ultrapure water as post-fragmentation precipitate. Take 20µl of sample plus 5µl of SDS-PAGE protein sample buffer (5×) cook protein at 100℃ for 10min, protein gel sample, run the gel; run the gel of the rest of the protein sample, transfer the membrane, and do the WB experiment; apply the primary antibody (1:7000) overnight, wash with TBST, apply the secondary antibody (1:5000) for 1hour, continue to wash, and then 1:1 configure the protein developer solution to develop the image.

As can be seen in Figure 3-3-1 WB development graph, the antimicrobial peptide containing NO-inducible promoter formed inclusion bodies, and the precipitate contained a large amount of AMP, but the induced AMP developed deeper and clearer bands than the background expression level. This indicates that the expression of the antimicrobial peptide mediated by the NO-inducible promoter was successful, and the precipitate contained a large amount of AMP.

Figure 3-3-1 WB characterisation of antimicrobial peptides containing NO-inducible promoter

1-3 are uninduced precipitate, supernatant and total protein, respectively; 4-6 are induced precipitate, supernatant and total protein, respectively; M wells are at Marker 6.5-14.4 KD

Performance testing of the antibacterial effect of the novel antimicrobial peptide AMP

Inhibition zone test

To verify and evaluate the antibacterial effectiveness of our new AMP, we will conduct zone of inhibition experiments and bacterial growth inhibition tests.

We will use E. coli BL21 (DE3) and Lactococcus lactis as indicator bacteria for the zone of inhibition experiment. The EcN strain containing the NO-induced plasmid will be induced for expression, and the supernatant and precipitate of the novel AMP protein will be extracted for the experiment.

The induced supernatant and precipitate will be gradient diluted to 1000 times, and compared with the supernatant and precipitate of the uninduced EcN strain containing the NO-induced plasmid.

As shown in Figure 4-1-1, even when diluted 1000 times, the antimicrobial peptide still exhibits a clear zone of inhibition against E. coli BL21 (DE3). The induced supernatant shows a more significant antibacterial effect compared to the control group's supernatant. This indicates that our novel AMP not only undergoes induced expression but also has strong antibacterial performance. However, regardless of the dilution factor, the formed inhibition zones are relatively small, suggesting low protein expression in the EcN vector. We will attempt to address this issue in subsequent experiments.

Since our novel antimicrobial peptide is screened from Lactobacillus spp., its antibacterial performance is less effective against Lactococcus lactis, as expected.

Figure 4-1-1 Results of the inhibition zone test with AMP

The left column shows E. coli BL21 (DE3), and the right column shows Lactococcus lactis; the first row to the fourth row respectively represent dilutions of 1, 10, 100, and 1000 times of the bacterial liquid; the left side of each plate is the experimental group, and the right side is the control group, with the supernatant at the top and the precipitate at the bottom.

Bacterial growth inhibition assay

We added 10ml of LB medium and 100ul of overnight cultured E. coli BL21 (DE3) into five test tubes. In the following four test tubes, AMP total protein was added at dilutions ranging from 1 to 1000 times. The OD value was measured every two hours to obtain the growth curve, as shown in Figure 4-2-1. Since proteins at different dilutions still have a certain absorbance effect, the final growth rate of the growth curve without AMP (the final OD value compared to the initial OD value) was set as 1. The final growth rates of other groups were calculated and compared with the former to determine the growth inhibition rate, as shown in Figure 4-2-2. The results indicate that even after a 1000-fold dilution, our new antimicrobial peptide still exhibits close to a 40% inhibition rate, demonstrating its strong antibacterial effectiveness.

As shown in Figures 4-2-3 and 4-2-4, for Lactococcus lactis, since our novel antimicrobial peptide is screened from Lactobacillus spp., it is less effective for Lactococcus lactis, and there is a part of bacteria that are not broken completely in our extracted AMP, so it appears to have some promoting effect.

Figure 4-2-1 BL21（DE3）growth Curves

Figure 4-2-2 BL21（DE3）Inhibition Rate

Figure 4-2-3 Lactococcus lactis Growth Curves

Figure 4-2-4 Lactococcus lactis Inhibition Rate

Figure 4-2-5 Sterile Control Growth Curves

Sequence and Features