Abstract

Peptidosomes are the new fundamental approach for generating and applying encapsulated bacteria. By the creation of spherical compartments containing a liquid environment inside, bacteria are still able to grow and fulfil a given task. The mesh-like structure of the sphere allows the selective exchange of compounds via diffusion, but holds the bacteria trapped inside. Therefore, we are able to benefit from the entrapped cells’ abilities, while still ensuring that they are not released into their surroundings. Peptidosomes can be further enhanced by incorporating magnetic or biological beads – which themselves can be functionalized with proteins – into their peptide-based fibrillary shell.

Background

Bacteria are omnipresent in biotechnology and applied projects. They can be used as hosts to produce nearly any biological compound of interest such as: drugs, vaccines, enzymes, antibiotics or even fuels and solvents. Their fast life cycle and comparable low requirements of living conditions highlight their industrial relevance. Over the last decades, the main focus to increase yields laid on extensive metabolic engineering and optimizing growth conditions.

Yet, there are more aspect which need to be considered when producing a compound of interest. First, where is the product of interest found: inside of the producing strain or will it be secreted to the surrounding media? Second, what is necessary to separate the valuable end-product from the bacteria? And maybe most important, how to assure a safe use of genetically engineered production strains? If you would like to know more on encapsulated bacteria in Peptdisomes and boosting the production of a compound of interest check out our: Signal Peptide Toolbox and Peptide Secretion sections.

To address these major biological and technical questions, the TU Dresden iGEM team presents EncaBcillus. Using Bacillus subtilis as model organism we introduce a new fundamental approach for cultivation of bacteria: the Peptidsomes. These Peptidosmes are buildup of self-assembled Fmoc dipeptide phenylalanines (Fmoc-FF) and are able to form spherical cages. The cages hold back the bacteria from the surrounding but are freely diffusible for smaller molecules. We show, that B. subtilis is able to grow, while encapsulated inside of the Pepdiosomes and demonstrate the applicability of two (fluorescence and luminescence) reports for the use with Pepdiosomes.

While evaluating this new method of bacterial immobilization, we established two applications using Peptidsomes:

• An encapsulated β-lactam Biosensor
• Communication between encapsulated bacteria and their surrounding

Design

Fmoc-FF

Nanostructures are gaining great attention, due to their endless possible applications. In general, nanostructures clusters are ordered within nanoscopic dimensions. In some cases, the building blocks of the nanostructures (which can be of organic or inorganic sources) can self-assemble. This process can be driven by non-covalent forces (e.g. hydrogen bonds, van der waals forces or aromatic interactions) that dictate the organization into supramolecular structures [1]. For our project EncaBcillus, we investigated the encapsulation of bacteria by the self-assembling building block Fmoc-FF (9-fluorenylmethoxycarbonyl diphenylalanine) (Figure 1).

While studying the mechanisms that direct the self-assembly of amyloid fibrils by short aromatic peptides, it was observed that the dipeptide diphenylalanine (FF), the core recognition motif of the Alzheimer’s β-amyloid polypeptide, self-assembled into nanotubular structures in aqueous solution [2][3]. Later, while studying the interactions in the process, the chemical group 9-fluorenylmethoxycarbonyl (Fmoc) was added at the N terminal of the dipeptide, facilitating the self-assembly into typical amyloid-like fibrils [4].

Follow-up studies discovered, that it is possible to trigger the self-assembly of the dipeptide in solution. At a pH higher than 8, the dipeptide stayed in solution, but when slowly adding hydrochloric acid to reach pH levels below 8, a clear gel was formed [5].

At alkaline pH, the molecules are negatively charged and therefore repealing each other. If the pH drops, protonation of the molecules occurs, neutralizing the negative charge and permitting the self-assembly of the dipeptide into fibers [6]. The formation of this mesh was shown to be induced by exposing a solution of Fmoc-FF with gaseous CO₂ [7].

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Peptidosome creation

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Bacteria encapsulated in Peptidosomes

In EncaBcillus, we wanted to demonstrate and characterise functional Peptidosome encapsulating Bacillus subtilis. This fundamental new approach of applying Fmoc-FF with bacteria, will create endless new possibilities for bacterial immobilisation.

To encapsulate the bacteria we first needed to establish a lab protocol. We created Fmoc-FF solutions in water, adjusted the pH to 10.5 and responded B. subtilis cells either directly taken from overnight cultures or day cultures.

To monitor bacterial growth in Peptidosomes, day cultures were grown till an OD₆₀₀ of 0.02. Cells were pelleted by centrifugation for 5 min at 16000g. The supernatant was discarded and the pellet resuspended in Fmoc-FF solution. Droplets of 15 μl of this solution were placed on an ultrahydrophobic membrane and exposed to CO₂.

We performed multiple assays to prove the encapsulating and growth inside the Peptidsosomes. Detailed protocols can be found below. Adjustments regarding bacterial used concentrations are stated in the specific experiments.

• Plate Reader Well Scan
• Stereo Fluorescence Microscopy

Results

Characterization of Peptidosomes

Before we could encapsulate bacteria, we had to establish a robust and reproducible protocol of Peptidosome generation under lab conditions. To tackle this task, we used pH indicator cresol red solutions to visualise generated Peptidosomes (Figure 4). In addition to ease the handling of the Peptidosomes, the pH indicator colour also correlated with the status of the Peptidosomes formation. Prior the CO₂ exposure the indicator was red (Figure 4, A), after sufficient CO₂ exposure (will be discussed later) the solution changed to yellow (Figure 4, B), indicating a self-assembled Fmoc-FF layer and thus ready-to-use Peptidosomes.

These generated Peptidosomes stayed stable after CO₂ treatment and we were able to transfer them into liquid filled petri-dishes or well plates.

After we having established the protocol for the Peptidosome creation, we evaluated if we could vary the size of the Peptidosomes. For testing this, we simply generated Peptidosomes of different volumes and could demonstrate successful created and stable Peptidosomes ranging from 1 to 20 µl (Figure 5).

For discovering the optimal exposure time to CO₂ we varied it between 30 sec and 20 min. We found out, that the ones with an exposure time of 10 min were the most stable ones, showing stability after several days, even under 37°C incubation with shaking (Figure 6).

The final aim of the project is the encapsulation and cultivation of bacteria inside the Peptidosomes. For this, it is crucial that while the Fmoc-FF network should not let the organisms escape, it should allow the interchange and communication with the surrounding environment, for example, to make available fresh nutrients for the organism, and/or to allow the release of secreted molecules of interest out of the Peptidosome while keeping the bacteria inside.

With a really straight forward experiment, including the help of the pH-indicator we were able to prove that a diffusion between the inside of the peptidosome and the surrounding environment is possible. We transferred Peptidosomes to water or LB-Media and measured the time it took for the colour to disappear, or in other words, the time it takes for the coloured solution inside the cage to diffuse out of it.

The yellow color of both Paptidosomes faded over time. From the Peptidosom in water, the yellow color completely disappeared after 25-30 minutes, in LB after 45-50 minutes. As shown in Figure 7, the Peptidosome in water is no longer recognizable after 30 minutes, while the Peptidosome in LB medium is still visible as a light yellow spot. This observation also coincides with expectations, as the Fmoc-FF membrane is a network of pores. The pores allow media exchange with the surrounding liquid.

Proving that diffusion between the inside of the Peptidosome and the environment is possible, we wanted to know next if diffusion between two Peptidosomes which are in direct contact is possible. For this experiment only one of the two connected Peptidosomes contained coloured solution. Over the lapse of around 1.5 hours, it was possible to observe how the colour was disseminating to the non-coloured peptidosome until it reached an equilibrium (Figure 8), proving that there is diffusion, or intercommunication, between the two Peptidosomes.

Additionally, to the previous described method, we used a microinjection-technique to inject a coloured solution to the inside of the Peptidosome (Figure 9). To close the hole that was probably opened on the membrane by the introducing the glass capillary, the Peptidosome was exposed again for 5 min to CO₂. Afterwards it was transferred to water. As shown in Figure 9 E) the peptidosome was stable.

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Encapsulation of Bacillus subtilis in Peptidosomes

Before the encapsulation of B. subtilis inside the Peptidosomes, it was necessary to perform experiments to study possible interactions between the organism and the dipeptide, for example, whether the bacteria can use Fmoc-FF as a nitrogen source, or if the organism can survive the process of encapsulation, since the bacterial pellet is resuspended in the alkaline Fmoc-FF solution.

Can Fmoc-FF use B. subtilis as a nitrogen source?

Special culture plates were created using Jensen’s medium. This type of medium has all the basic nutrients that bacteria needs for its development, with exception of nitrogen.

Four treatments were tested:

1. Nitrogen free Jensen’s medium
2. Jensen’s medium supplemented with nitrogen (adding Fe [III] ammonium citrate and Potassium glutamate)
3. Jensen’s medium mixed with Fmoc-FF
4. Jensen’s medium with Fmoc-FF added over the dried plates

Treatment 1 was a negative control, since B. subtilis (wildtype) cannot grow in absence of nitrogen, so no growth at all was expected. Treatment 2 was the positive control: with the nitrogen supplement, B. subtilis should be able to grow. These expectations corresponded with reality: colonies grew in treatment 2 but none in treatment 1. Treatments 3 and 4 did not result in colony growth either, meaning that the Fmoc-FF cannot be used as a source of nitrogen for the bacteria.

Tolerance of B. subtilis against alkaline pH

The experiment to test the tolerance was performed as follows: B. subtilis culture was divided in four treatments, varying the pH of the culture in each one; 7 (normal growth condition as a control), 8, 9, and 10. NaOH was used to induce the different changes in the pH for each treatment, and was added after one hour of cultivation. The experiment was carried out in a plate reader to follow the change of the optical density (OD₆₀₀) which correlates with bacterial growth.

The graph shows the growth of the organisms under each condition. It can be observed that from pH 7 to 9, no significant change in the growth is observed. However, under pH 10, the organism stops its growth. To check if the bacteria was still viable after the high pH treatment, it was transferred to growth plates with neutral pH. There, it was observed that B. subtilis was able to recover and growth normally, meaning that the organism can survive the process of encapsulation.

Bacillus subtilis in Peptidosomes

Once it was proven that B. subtilis can survive the designed encapsulation process, and that the organism cannot use the building block Fmoc-FF as a nitrogen source, experiments to entrap the bacteria started.

For this we resuspended an appropriate amount of bacteria in the Fmoc-FF solution. Afterwards droplets of this were deposited on the ultrahydrophobic membrane and exposed to CO₂ for 10 minutes and afterwards transferred to water or LB media.

You can find the detailed protocols here.

Well Scan experiment

For the first experiments of encapsulation, we used the B. subtilis strains TMB4131 W168 lacA::erm Pveg_sfGFP and TMB3090 W168 sacA::cat Pveg_luxABCDE. The first strain has the characteristic of expressing sfGFP in a constitutive way, which was useful to prove the presence of bacteria in the Peptidosomes by detecting the fluorescence expressed by the cells. TMB3090 expresses luciferase in a constitutive way, which makes a detection of a luminescence signal possible. We performed a plate reader assay using the well-scan-mode. In this mode, the whole well is scanned to detect the exact position of a signal, either fluorescence or luminescence. Its absence is displayed in a map with a green colour, while the position of the fluorescence/luminescence source appears as red. Please check ot the concerning protocol.

In figure 11 examples of the well scans of different samples are displayed.

The well scan maps appear completely green where fluorescence/luminescence is absent, i.e. water and empty Peptidosome. Wells containing a sample of the day culture and lyophilized eGFP solved in water show a red colour in the whole map. However, a localized red spot over a green background is observed where the source of fluorescence/luminescence is contained: the cells trapped inside the Peptidosomes. This shows that when the Peptidosome with cells inside is transferred to water, bacteria cannot diffuse away, but its kept contained within the structure, resulting in a localized red signal.

Fluorescence microscopy

The encapsulation of B. subtilis expressing sfGFP in a constitutive way was also demonstrated by the fluorescence microscopy. In this experiment, the bacteria were encapsulated as described. As shown in Figure 12, the Peptidosome emitted green light what proved the existence of sfGFP-expressing bacteria inside of the Peptidosome.

The Growth of B.subtilis in Peptidosomes

The method we used to check the growth and reproduction of bacteria inside the Peptidosome was performed by generating Peptidosomes loaded with a known amount of bacteria. Some Peptidosomes were plated on LB agar right after being generated, while other Peptidosomes were incubated in LB broth at 37°C for 3.5 and 7 hours and afterwards plated and incubated overnight. An increase in the number of colonies formed by the incubated Peptidosomes means that bacteria can grow inside the structure. The result is shown in the next table.

Time [h]	0	3.5	7
Colonies	370.67 ± 57.42	593.33 ± 74.44	Lawn

As observed on the results, the number of colonies in the plates are increasing after 3.5 h of incubation, until finally after 7 hours it is not possible to count the amount of colonies anymore, meaning that the cells can reproduce inside the cages, therefore, Peptidosomes are indeed suitable for the establishment of bacterial cultures.

Cryo-SEM

When performing that experiment, we discovered bacterial growth in the supernatant where the Peptidosomes were incubated. To figure out the reason for this, cryo-Scanning Electron Microscopy (cryo-SEM) was used to image bacteria-loaded Peptidosomes. The results are showed below.

As observed in the images, some cells are completely or only partially integrated into the membrane, composed by a mesh of fibers. This cells found on the outer surface of the membrane, if released, can be responsible for the bacterial growth on the supernatant. Therefore we designed a second growth experiment, in which also the whole supernatant of each Peptidosome was plated with the purpose of observing the amount of bacteria released there.

Two extra treatments were tested, the first one consisted in adding “washing” steps, meaning that the Peptidosomes were transferred twice to fresh media before their incubation. We hypothesized that the bacteria on the outer membrane would be released and left behind after the transfers. The second treatment that we tested consisted in pre-incubating the Peptidosome in LB broth adjusted to have an acid pH, this with the finality of closing the membrane to entrap even more the cells, triggering the self-assembly of the unreacted Fmoc-FF solution still present in the Peptidosome.

Treatment	Normal	Washing	pH treatment
Colonies	203.7 ± 309.1	0.7 ± 1.2	1.7 ± 1.9

By introducing the treatments, the number of colonies was reduced from several hundreds to an average of .7 for the washing steps and 1.7 for the low pH treatment. After analysing the results, we concluded that the washing steps are important to reduce the number of cells released in the supernatant, whereas the treatment with acid broth does not add a significant effect on this. In that way, by introducing the washing steps and also reducing the concentration of cells in the Peptidosomes, the amount of bacteria present on the outer surface of the membrane was importantly diminished, fact that was corroborated by performing a second cryo-SEM and showed below.

In the second measurement the number of detectable bacteria that lie or are integrated on the membrane is significantly lower. Only a few individual fibers are visible and the surface is wrinkled. (figure XX A), B))

Only on the Peptidosome with the low pH treatment, a cell that was not integrated in the membrane was observed (D).

The results of the growth experiment with the additional treatments were confirmed microscopically. There was no significant improvement in the Peptidosomes treated with acid LB medium compared to the sole integration of wash steps. Only a single cell, which could relieve from the Peptidosome, was detected. This confirms the reduced number of colonies in the supernatant of the growth experiment when carrying out the additional treatment methods.

The one loosely attached cell might be a problem for some applications where the surrounding medium is expected to be free of bacteria, therefore, the manufacturing technique of Peptidosomes should be adapted accordingly. An adequate method for this could be introducing the cells by microinjection, since there is not a chance that bacteria can be attached to the outer part of the membrane.

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3. Surface decoration

The properties of self-assembled Fmoc-FF membrane allow tiny objects, such as Dynabeads, to be enveloped in the wall of the Peptidosome. Dynabeads envelopment enables the control of the Peptidosome movements in the magnetic field, that in combination with other features like producing strain encapsulation and small molecules or proteins diffusion, provides a powerful delivery tool, which can be used in various applications.

Nevertheless, the surface of DynaBeads can be decorated with His-Tag protein. For the “proof of principle” experiments His-Tag GFP were selected due to its availability and easy imaging procedure. First, the Invitrogen Protocol was optimized for our goals and tested for DynaBeads decoration with histidine-tagged GFP (Figure 15).

To prove the surface of the Dynabeads enveloped to the Peptidosome membrane availability for the decoration the labeling procedure was applied after the Peptidosome formation in both Binding/Wash Buffer and LB media (figure 16).

In conclusion, here we proved the possibility of Peptidosome surface decoration with histidine-tagged molecules that can be used for various applications where enzymatic activity of the surface of Peptidosome or Peptidosome immobilization is required. Surface decoration protocol is compatible with almost any goals due to its flexibility, as the decoration procedure can be performed before and after Peptidosome formation in the specific Binding/Washing buffer or just in the LB media.

References

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