Results
Results
With our project, we aimed to establish a cell-free endolysin expression platform that enables the reliable and rapid identification of lead endolysin variants, particularly against Gram-negative bacteria. In addition, we sought to develop a toolkit based on this system to facilitate fast and easy endolysin modification and the generation of optimized variants.
The standard plasmid pALiCE01 was used as the backbone for assembling our expression vector, with the endolysin PlyEc2 (bba-25h8ekrx) serving as the protein of interest (POI) (Xu et al., 2021; LenioBio®, 2024). The construct was assembled via restriction enzyme digestion using KpnI and NotI. The correct assembly was confirmed by Sanger sequencing (Figure 1).
Figure 1: Positive sanger sequencing result of the pALiCE01_PlyEc2 plasmid (Created with SnapGene).
For efficient endolysin production using the ALiCE® cell-free expression system, highly purified plasmid DNA was required. To achieve this, six individual plasmid purification samples were pooled and further purified using phenol-chloroform purification. The resulting purified plasmid was subsequently used for cell-free protein synthesis of our POI using the ALiCE® system, and the reaction was terminated after 48 hours. The centrifuged supernatant (SN) was collected and employed in growth inhibition assays using P. aeruginosa PAO1.
For the initial growth inhibition experiments, we estimated that the ALiCE® system produced approximately 2 mg/mL of endolysin per reaction, as described in (LenioBio®, 2024). Based on this assumption, final concentrations of 20 µg/mL and 60 µg/mL were tested, considering that the minimal inhibitory concentration (MIC) of PlyEc2 had been reported to be around 12.5 µg/mL. However, no growth inhibition was observed under any condition, neither for the PlyEc2 supernatants nor for the non-template control, without plasmid DNA added to the reaction mix. The treated P. aeruginosa PAO1 cultures exhibited growth comparable to that of the positive control (Figure 2).
Figure 2: Initial growth inhibition assay of P. aeruginosa PAO1 together with PlyEc2. The assay was conducted in triplicates at 37 °C, 250 rpm in LB medium with approximately 106 cells per well of exponentially grown P. aeruginosa PAO1. NTC: non-template control; SN: supernatant
Contrary to our expectations, the initial growth inhibition assays revealed no detectable inhibitory activity. Due to the lack of a reliable and feasible protein detection method, the actual concentration of the expressed endolysin could not be determined. Consequently, an insufficient protein yield, absence of expression, or loss of functionality could not be excluded at this stage. To overcome this limitation, we introduced a StrepII tag into our constructs, enabling both verification of protein expression and quantification of our POI.
For our second cloning approach we utilized a modified version of the pALiCE01 plasmid as the backbone, containing a StrepII-tag and a (GGGGS)2 linker. In addition to our initial endolysin PlyEc2, we selected PlyYouna2 (BBa-25uoafah), an endolysin primarily active against Gram-positive bacteria, but reported to exhibit activity against certain Gram-negative strains as well (Son et al., 2023). In order to allow for standardized cloning, we utilized a Golden Gate Assembly approach for our vector construction. The plasmid maps of our constructs can be viewed in Figure 3.
Figure 3: Plasmid maps of pALiCE01_StrepII_PlyEc2 and pALiCE01_StrepII_PlyYouna2 (Created with SnapGene).
The resulting plasmids were successfully assembled, verified by sanger sequencing (Figure 4), and purified as described previously. These constructs were subsequently used for cell-free expression of the respective endolysin via the ALiCE® system. Additionally, two controls were included: a non-template control, without plasmid DNA, and a positive control expressing StrepII-tagged eYFP from the standard pALiCE01 plasmid.
Figure 4: Positive sanger sequencing results of A) pALiCE01_StrepII_PlyEc2 and B) pALiCE01_StrepII_PlyYouna2 (Created with SnapGene).
To verify protein production, the supernatants (SN) of all ALiCE® reactions and the pellets of the endolysin samples (resuspended in 1× PBS) were analyzed by SDS-PAGE, accompanied by plate spot assays (Figure 5). The plate spot assay for PlyEc2 was performed with P. aeruginosa PAO1, whereas Bacillus megaterium ATCC 14581 was used for PlyYouna2, as both species are known to be susceptible to the corresponding endolysins (Xu et al., 2021; Son et al., 2023).
For the assays, bacterial lawns from exponentially grown liquid cultures were plated on LB agar plates. After a short drying period, 10 µL of the SN samples, resuspended pellets, NTC extracts, and appropriate antibiotics were applied. For PlyYouna2, additional samples of natively purified protein (isolated via MagStrep Strep-Tactin™ XT Beads and eluted in the appropriate elution buffer) were also tested.
A comparative analysis of the two approaches yielded analogous results. The endolysin-containing supernatants (SN) exhibited growth-inhibitory effects, similar to the negative control with ampicillin on B. megaterium ATCC 14581. As expected, ampicillin did not inhibit P. aeruginosa PAO1 growth due to naturalist intrinsic β-Lactam resistance (Alam et al., 2019). Repeating the assay with gentamicin confirmed the expected growth inhibition (data not shown). Unexpectedly, the NTC SN samples also caused growth inhibition, whereas the natively purified PlyYouna2 and the 1x BXT elution buffer showed no inhibitory effect.
Figure 5: Plate spot assay of PlyYouna2 on Bacillus megaterium ATCC 14581 (A) and of PlyEc2 on Pseudomonas aeruginosa PAO1(B). For A) the supernatant (SN), pellet and natively purified PlyYouna2 as well as 50 µg/µL Ampicillin, the non-template control (NTC) supernatant and the elution buffer of the purification (1x BXT) were tested for lysis. For B) the PlyEc2 supernatant, 50 µg/µL Ampicillin and the non-template control (NTC) supernatant were tested regarding lysis.
Protein expression of both endolysins was further analyzed via SDS-PAGE. For PlyEc2 (SN) and PlyYouna2 (SN and pellet), bands were visible at the expected molecular weights (PlyEc2: ~25 kDa; PlyYouna2: ~31.8 kDa). However, the NTC also displayed a discernible band at a comparable position, an unexpected observation under standard conditions (Figure 6).
Figure 6: SDS-PAGE analysis of different ALiCE® reactions, including supernatants (SN) and resuspended pellets of PlyEc2 (left), PlyYouna2 and control (right) samples. NTC = non-template control & eYFP = positive control with standard pALiCE01 vector. Protein sizes were quantified using PageRuler Prestained protein ladder (10 - 180 kDa).
These findings initially suggested a potential contamination of the NTC with one of the endolysin plasmids. However, since the purified PlyYouna2 no longer exhibited growth inhibition. This raised alternative hypotheses: defective purification, non-functional protein production, or inefficient expression.
To clarify the cause of the observed lack of activity, another SDS-PAGE was conducted for PlyYouna2, including both native and denaturing MagStrep Strep-Tactin™ XT Bead purification (Figure 7). The anticipated protein band at 31.8 kDa was not detected in any fraction (whole lysate (WL), SN, pellet, or native purified samples). A malfunction of the ALiCE® system was disproven, as the positive control (eYFP) showed strong fluorescence and a clear ~29 kDa band compared to the NTC.
Figure 7: SDS-PAGE analysis of different ALiCE® reactions, including supernatants (SN) and resuspended pellets of PlyEc2 (left), PlyYouna2 and control (right) samples. NTC = non-template control & eYFP = positive control with standard pALiCE01 vector. Protein sizes were quantified using PageRuler Prestained protein ladder (10 - 180 kDa).
For PlyEc2, an additional SDS-PAGE and Western blot (WB) were performed to verify the functionality of the ALiCE® system and to address the reliability of phenol-chloroform plasmid purification and MagStrep Strep-Tactin™ XT Beads purification. Alongside the standard eYFP plasmid, an ALiCE® reaction using phenol-chloroform-purified eYFP plasmid was included. As shown in Figure 8, clear protein bands at ~29 kDa were observed in the WL, SN, and natively purified protein for both eYFP reactions. These findings were confirmed by WB analysis. In contrast, no band at the expected size of 25 kDa was detected for PlyEc2 in either Ponceau staining or WB, indicating that no endolysin was produced. Nevertheless, these results confirm the overall functionality of the ALiCE® system and rule out phenol-chloroform plasmid purification or MagStrep Strep-Tactin™ XT Beads purification as sources of error.
Figure 8: Ponceau staining on the left and Western blot on the right. A) shows the results of the StrepII-tagged eYFP derived from the standard pALiCE01 plasmid already delivered purified. Further it exhibits the PlyEc2 samples of the whole lysate (WL), supernatant (SN), resuspended pellet (P), and the natively purified protein (A). B) shows the results of the StrepII-tagged eYFP derived from the standard pALiCE01 that was previously purified with the phenol-chloroform purification method. It contains the WL, SN, P and A as well as the input (IP) and the wash of the native protein purification. Additionally, IP and wash of PlyEc2 and the non-template control (NTC) are visible. The NTC is an ALiCE® reaction sample where no plasmid was used as template. It exhibits the negative control of the ALiCE® reaction. Further, the SDS-PAGE gel of B) contained a leaky pocket. As a result, a sample flowed across the gel, resulting in the slight continuous staining visible in the WB. Protein sizes were quantified using Precision Plus ProteinTM Standard protein ladder (10 - 250 kDa).
In parallel to the expression of individual endolysins, we began developing a modular toolbox approach based on the ALiCE® cell-free expression system. This toolbox is designed to enable the rapid generation of endolysin fusion constructs by allowing the exchange of individual genetic modules, thereby facilitating the efficient identification of new lead variants. The fusion of antimicrobial peptides (AMPs) with endolysins is of particular interest, as this combination promises to enhance lysis efficiency against Gram-negative bacteria (Carratalá et al., 2023). In this regard, our molecular dynamics (MD) simulations also contributed to this hypothesis (see model).
To implement this approach, we introduced position markers within the construct, allowing for the straightforward exchange of sequence elements without the need for new primer design. This modular design enables flexible combinations of AMP linkers and endolysins, allowing rapid testing of numerous variants in a standardized format (Figure 9). For more information see Contribution.
However, since no endolysin expression was achieved in the ALiCE® system, production of new fusion constructs could not be initiated. An alternative expression kit had been planned since May, but its implementation was ultimately not possible due to a lengthy approval process and time constraints.
Figure 9: Plasmid map of pALICE01_K14_PlyEc2_StrepII with required primers for the GGA. Further information about the primers is available under Contribution (Created with SnapGene).
During our literature review and consideration of potential applications for our endolysin variants, we identified biofilms as a major challenge in the fight against bacterial infections. Not only do they increase the resistance and persistence of bacteria in infectious diseases, but they are also a serious problem on implants, in food production, and in water and piping systems.
Biofilms form when numerous bacterial cells aggregate to produce a polymeric slime layer (extracellular matrix, ECM) around themselves. This matrix enables adhesion to surfaces and protects the cells from external influences such as antibiotics, disinfectants, and the immune system (Biofilms Research Group “Biofilms” Jena University Hospital (2025); Ban-Cucerzan et al., 2025). Therefore, a truly sustainable alternative to antibiotics must also overcome the protective barrier formed by biofilms, which is why we sought to expand our research to include this critical aspect.
Conveniently, bacteriophages have the ability to infiltrate biofilms. Their advantage lies in their autonomous replication within the biofilm, the efficient lysis of bacterial cells, and the secretion of hydrolases and endolysins, which weaken the biofilm matrix. This enables biofilms to be completely destroyed, or at least made significantly more sensitive to antimicrobial agents (Mayorga-Ramos et al., 2024). However, the composition of the biofilm matrix often limits the infiltration ability of phages, among other things due to its physical barrier effect and the heterogeneous metabolic states within the bacterial population (Meneses et al., 2023).
Therefore, we wanted to investigate ways of increasing the infiltration capacity of phages by using phage display, by generating various fusion proteins via cloning that were then to be expressed on the phage surface. In order to cover a broad spectrum of activity, we combined different approaches aimed at weakening the integrity of the biofilm.
As a transport vehicle for our endolysins, we chose the filamentous M13 phage, which, due to its high density of over 2,500 pVIII major coat proteins, offers an ideal platform for phage display and has already been extensively studied in this context (Chang et al., 2023). In addition, M13 binds to the F pilus of Escherichia coli strains, thereby inhibiting horizontal gene transfer, which in turn can reduce the rapid adaptation of bacteria to stress factors and the development of resistance. Furthermore, the M13 phage is not lytic but lysogenic, making the effect of endolysins easier to detect. At the same time, infection by M13 can slow down the bacterial cell cycle (Kok et al., 2024).
In parallel, we investigated various peptides. First, we used two antimicrobial peptides (AMPs): K14, a short, cationic peptide with amphiphilic properties (Sato & Feix, 2008), which, due to its positive charge, effectively binds to the negatively charged bacterial cell membrane, destabilizing and permeabilizing it.
In addition, we used Lf11-324, a derivative of lactoferricin, whose biofilm-inhibiting effect is based on interaction with the cell membrane, destabilization of the extracellular matrix (ECM), and an antibacterial effect mediated by iron deprivation (Zweytick et al., 2011; Gifford et al., 2005). Since lactoferricin is a component of the human immune system, this peptide seemed particularly suitable to us, as phage coating with this molecule could also attenuate possible undesirable immune reactions in future medical applications.
To specifically weaken the biofilm matrix, we also tested the lipid-bound peptide VFR12, a derivative of human thrombin, to investigate whether binding to lipophilic components of the biofilm could impair its stability (Singh et al., 2013).
Finally, we also wanted to specifically disrupt signal transduction processes that influence bacterial growth. For this purpose, we chose the immunomodulator peptide IDR-108 (de la Fuente-Núñez et al., 2014), which modulates the bacterial stress response, disrupts intercellular cohesion in the biofilm, and has been shown to activate the immune response in the host (Steinstraesser et al., 2012; Mansour et al., 2015).
In the end, we decided not to use genetically modified phages, but only wild type (WT) phages in combination with the raw peptides, since our HP feedback considered them more appropriate for application in humans from a regulatory point of view in Germany.
After overcoming a few minor, unexpected difficulties at the beginning (see Engineering), and after all legal requirements and the necessary administrative work for working in the S2 laboratory had been completed and personal safety was ensured through safety training and a medical clearance by the responsible physician.
The selected peptides were to be tested on biofilms of the enteroaggregative and pathogenic E. coli 55989 strain (S2) as well as, for the control group, on biofilms of E. coli K-12.
The measured absorption at 580 nm correlates with the amount of biofilm formed and therefore served as an indirect indicator of the effectiveness of our phage and peptide treatments. All measurements were performed in at least biological triplicates (n = 3). As expected, the enteroaggregative E. coli strain 55989 formed more biofilm on average than the non-pathogenic control strain K-12.
Interestingly, the samples treated with M13 phage alone showed a slight reduction in biofilm mass. However, when the phage was used in combination with one of the peptides IDR-108, Lf11-324, or VFR12, the biofilm-reducing effect of the peptides appeared to be partially reversed—both in the enteroaggregative and K-12 strains. However, these values showed a high standard deviation, indicating biological variability or minor technical fluctuations. The peptides alone, applied at a concentration ten times the minimum inhibitory concentration (MIC), thus led to a significantly greater reduction in biofilm quantities than in combination with M13 (Figure 10).
Contrary to our expectations, Lf11-324 caused only a slight reduction in biofilm compared to the pure M13 samples, while VFR12 achieved a biofilm reduction of over 20% and IDR-108 reduced the amount of biofilm by more than a third compared to the buffer control in the enteroaggregative strain(Figure 10).
Figure 10: Biofilm decimation via peptides and phages for the enteroaggregative E. coli strain 55989. Biofilms were grown for 48 h and treated with the respective test substance for 24 h. Then washed, dyed with crystal violet, washed again, and the absorption was measured at 580 nm. Values compared to buffer control treated with TBS.
The K-12 strain showed a differentiated picture: VFR12 had little effect on the amount of biofilm, Lf11-324 had a similar effect to that on the pathogenic strain, while IDR-108 led to a reduction of almost 50% (Figure 11).
Figure 11: Biofilm decimation via peptides and phages for the control E. coli strain K-12. Biofilms were grown for 48 h and treated for 24 h with respective substances, then washed, dyed, and absorption measured at 580 nm. Values compared to TBS buffer control.
The combinations of peptides yielded consistent results: in the enteroaggregative strain, the combination of Lf11-324 and VFR12 led to only a slight reduction, while Lf11-324 + IDR-108 reduced the biofilm by almost 20% and IDR-108 + VFR12 by almost 25% – in each case compared to the buffer control. In the K-12 strain, the combinations Lf11-324 + VFR12 and VFR12 + IDR-108 showed only minor effects, while the combination of Lf11-324 + IDR-108 led to a reduction in biofilm quantity of over 30% (Figure 12).
Figure 12: Biofilm decimation via peptide combinations for the enteroaggregative E. coli strain 55989 and control K-12. Biofilms grown for 48 h, treated for 24 h, then washed, dyed, and absorption measured at 580 nm. Values compared to buffer control treated with TBS.
We observed the most unexpected result with peptide K14. When applying twice the MIC concentration – based on the MIC previously described for E. coli BL21 – only a slight reduction in biofilm quantity was observed in both strains (55989 and K-12). However, at ten times the concentration, this effect was reversed: there was a significant increase in biofilm formation – by over 150% in the 55989 strain and even by more than 270% in K-12 compared to the buffer control. When M13 was added, the value for 55989 remained almost unchanged, while the biofilm value for K-12 continued to increase, reaching almost 350% of the control value (Figure 13).
Figure 13: Effects of the AMP K14 on biofilm quantity of enteroaggregative E. coli strain 55989 and control K-12. Biofilms grown for 48 h, treated for 24 h, then washed, dyed, and absorption measured at 580 nm. Values compared to buffer control treated with TBS.
The slight reduction in biofilm quantity caused by M13 WT phages, which actually replicate lysogenically and do not lyse bacteria, can be explained by their binding to the F pilus, which, in addition to interrupting horizontal gene transfer, can slow down cell division and bacterial growth. Although infection and insertion of the genetic phage material do not kill the bacteria, it does lead to altered gene expression, which negatively affects the bacterium's metabolism and thus the synthesis of extracellular polymeric substance (EPS) (Amankwah et al., 2021).
However, the fact that the peptides IDR-108, VFR12, and Lf11-324 have greater biofilm destruction properties on their own than in combination with phages may be due to the phages either occupying their binding sites on the bacterial surface or sterically hindering them, or simultaneously interacting with the peptides themselves, which could reduce their availability and effectiveness. The fact that Lf11-324 actually has little effect on biofilm, even though its effectiveness against bacteria was rated as high in previous studies, may be due to the fact that it is a cationic molecule, which means it can be bound by eDNA and other anionic molecules in the biofilm and at the same time may diffuse less easily to the bacterial membrane (Vieira-da-Silva, 2023).
VFR12, on the other hand, does not bind as easily to anionic molecules due to its chemical properties, allowing it to diffuse better to the bacteria and accumulate in the membrane, destabilizing it and making it permeable, which explains the lower biofilm values (Lei et al., 2019; Zhang et al., 2017). IDR-108 not only has an antimicrobial effect by penetrating and destabilizing the bacterial cell wall, which also has a negative impact on EPS synthesis, but also blocks the (p)ppGpp signal, thereby disrupting its protective mechanism and reducing the expression of biofilm-synthesizing genes (de la Fuente-Núñez et al., 2014). These two simultaneous attack pathways also explain why IDR-108 is so much more effective.
The different effects on K-12 biofilms could be explained as follows: Lf11-324 acts mainly directly on the bacterial membranes, which is why this effect does not have to differ significantly between different biofilms. VFR12, however, acts predominantly via lipid interactions, and K-12 biofilms are known to be less complex in structure than the biofilms of enteroaggregative bacteria (Beloin et al., 2008). If fewer lipids are available for interaction, this may explain the low effect. The fact that IDR-108 is more effective in K-12 may be because the stress response and intracellular regulatory mechanisms are highly conserved in K-12 strains and are also essential for biofilm formation (Hobley et al., 2015). Since K-12 generally forms less biofilm than enteroaggregative strains, the effect may be even stronger here (Beloin et al., 2008). This background also explains the different effects of the peptide combinations, and especially the good results of IDR-108 and VFR12 for 55989, since they disrupt the biofilm with multiple different attacking methods due to their different modes of action.
The slight biofilm-inhibiting function of K-14 at low concentrations was expected, as K-14 can insert itself in bacterial membranes due to its chemical properties and likely cause pore formation, as a result of which the cell can no longer maintain its chemical equilibrium (Lesniewski, 2022). However, the high absorption at high concentrations has not been described in the literature before and could have various reasons: Either complex formation, which distorts the result when stained with crystal violet, or aggregation, which binds the individual bacteria more strongly together (Domnin et al., 2025). The more likely explanation, especially given the differences in the strains, would be an effect on cellular stress signals. As a laboratory strain, K-12 is significantly more sensitive to stress and altered gene regulation than pathogenic strains (Bhatia et al., 2022). This also explains the sharp increase in biofilm formation in K-12, which is also a protective mechanism when high concentrations of K-14 are combined with M13 phages, as these also influence the cellular processes of bacteria and can trigger stress and altered gene regulation. However, another approach to explain the relatively little negative effect on biofilms in low concentrations compared to the other tested peptides is the high charge density of K14, as explained in our model part. There, the insertion in the membrane was shown in our model for the CM15 that has only one different amino acid to be much slower than for other AMPs. Many K14 molecules together could also hinder each other during the insertion process.
However, much more research is needed before clear statements can be made on this subject. In general, our method is very sensitive in its application, and the trends derived from it must be interpreted with caution. In order to obtain more meaningful results, the experiments must be repeated several times. In addition, further different concentrations should be tested, and kinetics should be applied. It would also be interesting to investigate the synergistic effects of different phages, AMPs, ABPs, and antibiotics, as well as changes in environmental factors that can significantly influence biofilm formation. However, this would require more time and money to continue the experiments than we had available.
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