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
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
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
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).
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).
The expression cultures showed different intensities of red which indicated varying levels of expression or a
different fluorescence intensity of the expressed proteins.
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.
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).
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).
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.
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).
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).
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)
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.
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).
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).
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.
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.
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
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
as the specific uptake into the targeted organisms is our first mechanism to ensure specificity for our system in potential