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The first barrier the Troygenic has to cross to enter eukaryotic cells is the cellular membrane. To enable our system to enter the cell, we equiped it with the ability to exploit a mechanism used by viruses for thousands of years: Endocytosis.
In the case of the chosen model organisms S. cerevisiae and Aspergillus niger, we studied four different cell specific ligands that might be taken up by endocytosis. To get inside Saccharomyces cerevisiae, we tested the mating factor alpha, the extracellular cysteine-rich domain of Opy2, and the N-terminal domain of Flo11. For infiltrating A. niger we used a proline-peptide that blocks the proline transporters and gets internalized when A. niger replaces the blocked transporters on its surface.
After cloning and purifying specific ligands fused to the fluorescence marker protein mCherry, we were able to show that the mating factor and the cysteine-rich domain of Opy2 are able to enter S. cerevisiae. Flo11 however, seemed to form aggregates outside of the cell and is not taken up by the target cell. The fusion of a short proline-peptide to the N-terminus of mCherry enables the protein to infiltrate A. niger. In conclusion, we successfully characterized different endocytosis ligands which are sufficient to be used for endocytosis of our Troygenics.
The functionality of our Troygenics depends on a specific and efficient uptake into the target organism. To overcome the barrier of the cell membrane, we used the same approach as many non-enveloped viruses do. A common way into their target is to exploit endocytotic pathways of the host cells (Thorley et al. 2010). Apart from the relatively unspecific ways of macropinocytosis many viruses bind their target cells through host-specific proteins presented on their surface and induce endocytosis into the host cell. Those proteins are often ligands for cell specific surface receptors or transporters (Cossart and Helenius 2014).

Cloning detectable fusion proteins

To gain access to our model organism S. cerevisiae, we examined three different ligands. The first one is the mating factor alpha (Mat), which specifically binds the mating pheromone receptor Ste2 that is taken up into the cell upon binding to the pheromone (Bardwell 2004). The second ligand is the extracellular cysteine-rich domain of the S. cerevisiae membrane receptor Opy2 (Opy). Opy2 binds extracellularly to the receptor Hkr1 in the osmoregulatory pathway (Tatebayashi et al. 2015). And finally, the N-terminal domain of the surface protein Flo11 (Flo) was investigated. This domain is able to bind to other Flo11-proteins on the yeasts surface (Douglas et al. 2007; Goossens and Willaert 2012; Karunanithi et al. 2010).
While specific endocytosis of the Mat-Ste2-complex is described in literature, the uptake of Opy- or Flo-bound Troygenics would rely on constitutive endocytosis which is an important mechanism to maintain membrane-homeostasis in every living organism (Besterman and Low 1983; Samaj et al. 2004).
Since there are no known pheromones for A. niger, we use a different, virus-inspired approach. Many viruses use target cell specific transporters to be actively internalized by their host (Olah et al. 1994; Fujisawa and Masuda 2007; Tailor et al. 1999). Knowing this, we fused three prolines interspaced by a glycine-linker to mCherry (Pro_mCherry) to take advantage of the Aspergillus-specific proline transporter PrnB suggested by Prof. Diallinas from the Department of Biology of the National and Kapodistrian University of Athens. The fusion-protein will block the proline-transporter which triggers endocytosis of the blocked PrnB.

For closer investigation of our selected ligands, we fused them to mCherry, a red fluorescent protein. This enables us to detect the fusion-proteins Mat_mCherry(BBa_K2926049), Flo_mCherry (BBa_K2926050), Opy_mCherry (BBa_K2926051) and Pro_mCherry (BBa_K2926068) inside and outside the cell via fluorescence measurement (Fig. 1).
The fusion of the fluorescent protein mCherry to the cell specific ligand allows detection of the protein of interest inside and outside the cell.

Protein purification

First, the marker protein mCherry (BBa_J06504) was cloned into the expression- and purification-vector pTXB1. To express the desired fusion-proteins the coding sequence of the specific ligands, containing a short C-terminal glycine-serine-linker was successfully cloned into the pTXB1-mCherry plasmid upstream of mCherry. This resulted in four different pTXB1-constructs coding for the fusion-proteins Mat_mCherry(BBa_K2926049), Flo_mCherry (BBa_K2926050), Opy_mCherry (BBa_K2926051) and Pro_mCherry (BBa_K2926068), each time fused to the intein-chitin binding domain, thus ready for protein purification. Those fusion-proteins were expressed in E. coli ER2566. The expression was easily detectable by the red color of the culture (Fig. 2 and 3).
Expression culture of the fusion-proteins.
Opy_mCherry, Mat_mCherry, Flo_mCherry and Pro_mCherry (from left to right) in pTXB1 expressed in E. coli ER2566. Expression cultures were cultivated at 37 °C in LB containing 100 mg ampicillin per L, to an OD of 0.6. Expression was induced by addition of IPTG to a final concentration of 0.4 mM. After additional 30 minutes at 37 °C, cultures were transferred to 17 °C and protein was expressed over night.

The expression cultures showed different intensities of red which indicated varying levels of expression or a different fluorescence intensity of the expressed proteins.
Harvested expression culture of the fusion-proteins.
Expression cultures were harvested via centrifugation for 20 min at 4 °C and 4 000 rpm.

After cultivation we compared two different protocols for cell lysis. Lysis via Ribolyzer resulted in a much lower yield than lysis via French Press (Fig. 4).
Ribolyzer (left) and French Press (right).
Harvested cells were lysed using Zirconia metal beads (1 mm) in a Ribolyzer at 8 000 rpm for 15 s. Lysis via French Press was performed two times at 16 000 psi with a flow rate of around 1 mL per minute. The lysate was cleared by centrifugation at 4 °C for 1 h and 4 500 rpm.

Purification of the fusion-proteins from the cell-lysate was performed using the IMPACT-Kit from NEB. The protein of interest was C-terminally fused to an intein tag and a chitin-binding domain. The resulting protein was loaded onto a chitin column (Fig. 5) and washed with a buffer with a high salt concentration.
Purification columns loaded with the fusion-proteins.
Cleared lysate was loaded onto a chitin column and washed with a buffer with high salt concentration. After washing the protein split off the chitin column, it was washed in PBS and concentrated.

To cleave the protein of interest from the column, it was incubated with DTT for 20-24 hours. After purification the different fusion proteins were analyzed on a SDS-PAGE to determine the purity as well as the correct molecular weight of the fusion-proteins (Fig. 6).
SDS-PAGEs of the purification process.
The purification process and the purified proteins were analyzed via SDS-PAGE. E. coli lysate of the expression culture, flow-through- and wash-fraction of the column purification as well as the purified protein were denatured by heating the samples to 98 °C for 10 min in SDS-PAGE loading buffer containing DTT and loaded on an polyacrylamide-gel (12 %). The proteins were separated through electrophoresis (25 mA). Protein bands in the purified protein samples likely originating from the target protein are marked in red.

The SDS-PAGE and a subsequent Bradford assay showed that we were able to purify Mat_mCherry with a molecular weight of 28.7 kDa and a yield of 2.35 mg, Opy_mCherry with a molecular weight of 31  kDa and a yield of 1.48 mg, Flo_mCherry with a molecular weight of 48.3 kDa and a yield of 40.9 µg and Pro_mCherry with a molecular weight of 27.7 kDa and a yield of 67.9 µg. To verify that the correct proteins were purified the marked bands were excised from the SDS-PAGE, washed, digested with trypsine and analyzed in a MALDI-ToF MS (Fig. 7).
Mass spectrum of the fusion proteins Mat_mCherry (1), Opy_mCherry (2), Flo_mCherry (3) and Pro_mCherry (4) after tryptic digestion compared to the theoretical mass spectrum.
Excised bands from the SDS-PAGEs of Mat_mCherry, Opy_mCherry, Flo_mCherry and Pro_mCherry were washed, digested over night with trypsine and co-crystallized with a α-Cyano-4-hydoxycinnamic acid-matrix on a MALDI target. The mass spectrum was recorded in a MALDI-ToF MS from Bruker Daltronics and data was evaluated using the software BioTools (Bruker). The upper panel for each sample shows the comparison between the obtained and the theoretical mass spectrum. The lower panels show individual matches (red) between the detected peptides and the amino acid sequence of the fusion-proteins.

The generated mass spectra and mass lists were evaluated using the software BioTools (Bruker). To compare the experimentally determined data to the theoretical protein sequence we performed an in silico trypsine-digestion of the expected protein sequence and compared the generated mass spectrum and mass list to the measured ones. We were able to match the obtained spectra for all four investigated fusion-proteins to the theoretically determined spectra.

Protein characterization

A very important property of the fusion-proteins is the ability to fluoresce unaffected by the fusion at the N-terminus. To verify this, we measured the fluorescence- and absorbance spectra of all four fusion-proteins (Fig. 8).
Fluorescence- and absorbance-spectra of the fusion proteins.
Emission- (dashed lines) and absorption-spectra (solid lines) of Mat_mCherry (dark red), Opy_mCherry (dark purple), Flo_mCherry (purple) and Pro_mCherry (blue) were measured (λEx=570 nm, λEm=600 nm to 800 nm) using the TECAN infinite M200 and normalized to their maximum.

All four fluorescence spectra look very similar. The absorbance spectra of all four fusion proteins are matching each other as well. Overall, the fluorescence- and absorbance-spectra of the fusion-proteins are very similar to the ones measured for mCherry (Fig. 9).
Fluorescence- and absorbance-spectra of mCherry and Mat_mCherry as an example for a fusion-protein.
Emission- (dashed lines) and absorbance-spectra (solid lines) of Mat_mCherry (dark red) and mCherry (grey) were measured (λEx=570 nm, λEm=600 nm to 800 nm) using the TECAN infinite M200 and normalized to their maximum.

To further characterize the fluorescence properties of the purified proteins, we diluted the proteins from 0.01 µM to 0.5 µM and compared the fluorescence intensity to the one of mCherry standardized to the fluorescence of 0.5 µM Texas Red (Fig. 10)
Fluorescence intensity of a dilution series of the fusion-proteins.
Fluorescence intensity of the dilution series of the fusion-proteins Mat_mCherry (dark red), Opy_mCherry (dark purple), Flo_mCherry (purple), Pro_mCherry (blue) and mCherry (grey) were measured (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red) using the TECAN infinite M200 and normalized to the fluorescence intensity of 0.5 µM Texas Red at the same wavelength.

As a result, we observed that Pro_mCherry showed the highest fluorescence intensity followed by Flo_mCherry, Mat_mCherry and Opy_mCherry. Compared to mCherry, the fluorescence intensity of the fusion-proteins has been decreased (Fig. 10). The fluorescence intensity of 1 µmol Flo mCherry equals the fluorescence of 0.49 µmol Texas Red, the fluorescence intensity of 1 µmol Mat_mCherry equals the intensity of 0.47 µmol Texas Red, the fluorescence intensity of 1 µmol Opy_mCherry equals the intensity of 0.41 µmol Texas Red and the fluorescence intensity of Pro_mCherry equals the fluorescence intensity of 0.54 µmol Texas Red. Normalizing the fluorescence intensity to a reference dye like Texas red enables the comparability of data, measured in different experimental setups and labs. After normalizing the data to a fixed value, the determination using a comparable relative fluorescence unit (RFU) is possible.

Endocytosis assays

The function of the ligands in the final Troygenics is to facilitate their binding and uptake by endocytosis. To demonstrate this functionality, we used the ligand-mCherry fusion-proteins in different endocytosis assays.

Fluorescence in the supernatant

With the purified fusion-proteins Mat_mCherry, Opy_mCherry, Flo_mCherry and Pro_mCherry, as well as mCherry, we performed an endocytosis-assay (Fig. 11). S. cerevisiae was incubated for one hour with 1 µM fusion-protein. Every 15 minutes a sample was taken, cells were pelleted by centrifugation and the fluorescence intensity in the supernatant was determined using a plate reader (Fig. 12).
Schematic overwiev of the endocytosis assay
Target cells are incubated with the fusion-protein. Over the time the cells specifically take up the proteins from the media. This results in a measurable decrease of fluorescence in the medium.

Mat_mCherry, Opy_mCherry and mCherry are taken up by S. cerevisiae
S. cerevisiae was incubated in SD medium (30 °C, 180 rpm, OD aro 0.4, dark) over 1 h with 1 µM mCherry (grey), Mat_mCherry (dark red), Opy_mCherry (dark purple) and Flo_mCherry (purple). Every 15 minutes, a sample was taken, the cells were pelleted by centrifugation, and the remaining fluorescence in the supernatant was measured using the TECAN infinite M200 (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red). The results were normalized to the fluorescence intensity at t=0 and the fluorescence intensity of the negative control without cells.

The results show that the fluorescence intensity in the supernatant of the samples with Mat_mCherry, Opy_mCherry and mCherry decreases over the time. This indicates that Opy_mCherry, Mat_mCherry and even mCherry alone seem to interact with and might be taken up by S. cerevisiae. The specific ligands Mat and Opy seem to enhance endocytosis as shown by the faster decrease of fluorescence in the medium. In contrast, the fluorescence intensity of Flo_mCherry in the supernatant did not decrease over the time which led us to the conclusion that the fusion-protein is not taken up by the cell.

The same assay described above for S. cerevisiae was carried out for A. niger as a model organism for filamentous fungi to verify the uptake of Pro_mCherry into the cells. Additionally, to investigate the specificity of the tested ligands, A. niger was also incubated with the S. cerevisiae-specific Mat_mCherry (Fig. 13).
The proline-fused mCherry is selectively taken up by the target organism A. niger.
A. niger was incubated in minimal medium (30 °C, 180 rpm, dark) over 1 h with 0.5 µM mCherry (grey), Mat_mCherry (dark red) and Pro_mCherry (blue). After 60 minutes, a sample was taken and the remaining fluorescence in the supernatant was measured using the TECAN infinite M200 (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red). The results were normalized to the fluorescence intensity at t=0 and the fluorescence intensity of the negative control without cells.

Due to the lower growth rate of A. niger compared to S. cerevisiae, only one sample after 60 minutes was taken. The results show no change in fluorescene in the supernatant after 60 min for mCherry or the S. cerevisiae-specific Mat_mCherry. This indicates that neither mCherry nor Mat_mCherry were taken up by A. niger. In contrast the assumed ligand of the Aspergillus-specific proline transporter Pro_mCherry was able to enter A. niger successfully as seen by the 20 % decreased fluorescence readout in the supernatant.
In conclusion our results show that it is possible to find organism-specific ligands that selectively enhance endocytosis into the targeted cell while not binding or entering cells from other organisms.

Fluorescence microscopy

In addition to the described endocytosis assays, we showed that our ligands specifically enhance endocytosis in their target cells, using fluorescence microscopy (Fig. 14). In detail we show the uptake of the fusion-proteins by S. cerevisiae (Fig. 15). Using fluorescence microscopy we can verify that the fusion-proteins are truly entering the target cell and are not just attaching to the cell wall or degraded by secreted proteases.
Fluorescence microscope.
To verify the specific uptake of our fusion-proteins into the target cell, we investigated fusion-protein-treated target cells under the fluorescence microscope LSM700 (Zeiss).

Fluorescence microscopy of S. cerevisiae after incubation with different fusion-proteins shows their specific uptake into the cells.
S. cerevisiae (0.35 OD) was resuspended in YPD (60 µL) and incubated (30 min, 30 °C, 450 rpm, dark) with mCherry (upper left), Mat_mCherry (upper right), Opy_mCherry (lower left) or Flo_mCherry (lower right). After washing with PBS, the cells were visualized using a fluorescence microscope (Fig. 14) (LSM 700 (Zeiss), magnification: 100 x, filters: Texas Red [λEx=555 nm, λEm=570 nm to 800 nm], transmitted light).

In the fluorescence microscopy Mat_mCherry (upper right) and Opy_mCherry (lower left) were detectable within the cells. Mat_mCherry was taken up with a slightly higher efficiency than Opy_mCherry (data not shown). In contrast Flo_mCherry (lower right) seemed to form precipitates outside the cells while the negative control mCherry without any fusion-partner was not taken up by S. cerevisiae.

In conclusion, we showed by an endocytosis assay as well as fluorescence microscopy that our S. cerevisiae- ligands mating factor alpha and the cysteine-rich domain of Opy2 as well as the A. niger ligand, a short proline-peptide, were able to enhance endocytosis in the targeted cells. We also showed that Mat_mCherry is target-specific for S. cerevisiae and is not taken up into A. niger cells. As such, we were able to proof our initial concept of using organism-specific ligands to introduce proteins and ultimately our Troygenics specifically into the targeted organism as the specific uptake into the targeted organisms is our first mechanism to ensure specificity for our system in potential application.

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