Difference between revisions of "Part:BBa K811000"

Latest revision as of 14:29, 10 October 2022

Cytolysin A (ClyA) Cytolysin A (ClyA) pore forming protein causes cell lysis.

Sequence and Features

Sequence and Features

Usage and Biology

ClyA is a protein native to E. coli, Shigella flexneri, and Salmonella typhi that is capable of forming 13-mer pore complexes in a redox-independent manner. Expression of clyA in the absence of other hemolytic toxins is sufficient to induce hemolysis experimentally, and is therefore considered to be a potent cytolytic agent. Unlike a similar protein, HlyA, ClyA is not synthesized as a protoxin, which requires further posttranslational modifications to become active. ClyA functional immediately following translation of mRNA to protein.

ClyA is a 34kDa protein that is composed primarily of α-helical bundles that form a rod-shaped molecule. The membrane insertion domain is known as a β tongue and is critical for hemolytic activity. If the β tongue is mutated, the hemolytic activity of clyA is abrogated.

The regulation of clyA secretion is not well characterized. To date, the method by which newly synthesized clyA is localized to the periplasm is unknown. All that is known about this process is that the secretion of clyA is not dependent on any known signal sequence, nor does it require cytolytic activity of clyA.

Characterization

The clyA gene was synthesized commercially and cloned into pDawn, a newly developed expression system that induces protein expression in response to irradiation from blue light. The pDawn-clyA construct was then transformed into E. coli. BL21 for subsequent expression.

~50 cfu of BL21 containing pDawn-clyA were plated onto Colombia Agar+ 5% Sheep's Blood plates and incubated for 48 hours under blue light at 25C. As shown below in figure 1, clyA was secreted from BL21 and lysed the red blood cells in the blood agar.

Figure 1: The cytolytic capacity of clyA-his in BL21 cells under blue light induction on 5% Sheep's Blood Agar for 48 hours at 25C

In addition, cytotoxicity was quantified using a Promega Cytotox Fluor Kit. This kit qualifies cell lysis at a fluorescence output of 520nm. It was shown that ClyA successfully lysed cells more effectively at higher concentrations. Furthermore, in both HEK293T and SKBR3 cells, there was a significant cell-dependent effect on cytotoxicity (positive correlation). Comparing 10ug/ml ClyA with the negative control in the cytotoxicity assay against cancerous SKBR3 cells shows over a 14 fold increase in lysis with the presence of ClyA

Contribution by the ELTE team (2022)

Biological Usage

Team ELTE from 2022 provided new documentation on the properties of Cytolysin-A, which could broaden the usage of this protein.

Cytolysin-A is an alpha pore-forming protein expressed in Escherichia Coli and Salmonella typhii which upon oligomerization kills the cells as it forms pores in their membrane [1]. This pore-forming trait can be enhanced if cholesterol is added [2] [3]. The presence of this molecule stabilizes the intermediates of the pore-forming process. This is caused by amino acids found on the N-terminal helix of the protein, which are very familiar to the loosely defined consensus CRAC motif (Cholesterol Recognition and Consensus Motif). This phenomenon has been described as cholesterol-dependent-cytolysis (CDC) and other pore-forming toxins have been acting similarly [4]. They also contain this CRAC motif. This motif has not been clearly described but it has an important Thr-Leu pair. Possibly this motif interacts with the cholesterol found in the membrane of the eukaryotic cells and this interaction stabilizes the intermediates of the pore formation.

Another important aspect of ClyA is the way it is released into the bacterial environment. Although it is still not clear what is the signal sequence in the protein that prompts the protein to leave the cell, however, it is now well described that it leaves in outer membrane vesicles (OMVs) [5]. These are vesicles formed from the outer membrane, and cholesterol also plays a role in this [6]. OMVs often have receptors on their surface as a segment of the membrane is basically excised from the bacteria. Their content is mainly molecules from the periplasmic space.

Characteristics

Team ELTE from 2022 integrated this part into their project. They aimed to create a blue-light inducible drug delivery device, similarly to Team Upenn from 2012. For this, they used a modified arabinose-operon-based expression system, called the BLADE Expression System. BBa_K4375019

Figure 3: lue-light induced Cytolysin A production and characterisation of the BLADE Expression System; A) Blue-light induced Cytolysin A production characterisation via SDS-PAGE; B) Blue-light induced Cytolysin A production characterisation via SDS-PAGE.

Team ELTE aimed to characterise the Cytolysin A production BLADE Expression System using SDS-PAGE, and study its hemolytic activity on blood agar plates. (Fig 3.)

For the Cytolysin A production characterisation, they obtained the supernatant of the bacterial culture after 4 h of blue light illumination and precipitated its protein content. They observed a band indicating Cytolysin A production, where it was expected, which was absent in the case of the dark control sample. (Fig. 3/B) They implemented the protocol of Chiang et al. for this aim, but it should be noted, that this experiment needs to be repeated with a construct to which an affinity tag, such as 6xHis-Tag, is added. [7] In this way, the protein of our interest could be purified lessening the number of aspecific bands.

They also meant to assess the hemolytic activity of Cytolysin A, using the BLADE Expression System. They spread the overnight cultures on Blood Agar plates, grew them for 8 h in dark at 37°C, which was followed by an 4 h long illumination with blue light at 25 °C. (The dark control plate was kept in dark at 25 °C for that time.) For additional negative control, they also spread BLADE-mCherry sample on the plates.) As They expected, no hemolytic activity was observed in the case of mCherry. Regarding the BLADE-ClyA samples, the biological activity of Cytolysine A was undoubtful since the blood agar discoloured where the samples were plated. Unfortunately, they could not observe a significant difference between dark and illuminated plates. This could also be explained by the considerations they beforehand mentioned. (Temperature, frozen bacterial culture)

Reference

[1] Peraro, M. D.; van der Goot, F. G. Pore-Forming Toxins: Ancient, but Never Really out of Fashion. Nature Reviews Microbiology 2015, 14 (2), 77–92. https://doi.org/10.1038/nrmicro.2015.3

[2] Sathyanarayana, P.; Maurya, S.; Behera, A.; Ravichandran, M.; Visweswariah, S. S.; Ayappa, K. G.; Roy, R. Cholesterol Promotes Cytolysin A Activity by Stabilizing the Intermediates during Pore Formation. Proceedings of the National Academy of Sciences 2018, 115 (31). https://doi.org/10.1073/pnas.1721228115.

[3] Li, Y.; Li, Y.; Mengist, H. M.; Shi, C.; Zhang, C.; Wang, B.; Li, T.; Huang, Y.; Xu, Y.; Jin, T. Structural Basis of the Pore-Forming Toxin/Membrane Interaction. Toxins 2021, 13 (2), 128. https://doi.org/10.3390/toxins13020128.

[4] Morton, C. J.; Sani, M.-A.; Parker, M. W.; Separovic, F. Cholesterol-Dependent Cytolysins: Membrane and Protein Structural Requirements for Pore Formation. Chemical Reviews 2019, 119 (13), 7721–7736. https://doi.org/10.1021/acs.chemrev.9b00090.

[5] Murase, K. Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins 2022, 14 (2), 78. https://doi.org/10.3390/toxins14020078.

[6] Schwechheimer, C., Kuehn, M. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol 13, 605–619 (2015). https://doi.org/10.1038/nrmicro3525

[7] Chiang, C.-J.; Huang, P.-H. Metabolic Engineering of Probiotic Escherichia Coli for Cytolytic Therapy of Tumors. Scientific Reports 2021, 11 (1). https://doi.org/10.1038/s41598-021-85372-6.