Revision as of 20:09, 1 October 2024

CMV-EGFP-PknB

Usage and Biology

PknB is a eukaryote-like serine/threonine kinase in Staphylococcus aureus that plays an important role in the bacterial response to antibiotics, particularly beta-lactams, via its PASTA domain (Stehle et al.,2012). PknB is a membrane-localized protein consisting of an N-terminal cytosolic kinase domain, a central transmembrane segment and three C-terminal extracellular PASTA domains. The PASTA (penicillin-binding protein and serine/threonine kinase-associated) domain plays a critical role in the recognition and binding of beta-lactam antibiotics (Stehle et al.,2012). Upon binding these compounds, the PASTA domain initiates a signaling cascade by inducing autophosphorylation of the N-terminal kinase domain. This activation leads to the initiation of downstream signaling pathways (Cheung et al.,2010). In S. aureus, this mechanism is critical for early detection of antibiotics and helps the bacteria adapt to antibiotic stress (Sauer et al.,2018). We utilized the PknB protein as the beta-lactam detector that passes the signal by phosphorylating one of our three transcription factors ATF2 (K5317016), GraR (K5317015) or CcpA (K5317014).

The composite part includes the upstream positioned constitutive active promoter CMV and the reporter gene EGFP (K3338006) to charactarize the PknB regarding it's cellular localization pre and post antibiotics stimulation.

Cloning

Theoretical Part Design

The CMV promoter was chosen to ensure a constitutive expresssion of the PknB in HEK293T cells and placing the PknB kinase upstream of the reporter gene EGFP allows the visualization of localisation of PknB. The PknB gene sequence itself was codon-optimized for expression in mammalian systems.

Sequence and Features

Cloning

The PknB insert was synthesised and the PknB sequence was amplified using the primers outlined in Table 1. The primers ensured that the 5' and 3' ends of the amplicons exhibited approximately 20 base pair (bp) overhangs, rendering them compatible with the backbone. The EGFP-C2 plasmid was linearised with BamHI and SalHI. The composite part-containing plasmid was assembled using the NEBBuilder® HIFI assembly kit, ensuring the correct positioning of the insert in the backbone through the use of overhangs.

Table1: Primers used to create matching overhangs of PknB amplicon to digested pEGFP-C2 backbone

Figure 1: Assembled vector map with CMV-EGFP-PknB integrated into the pEGFP-C2 backbone.

Characterization

Transfection experiments in mammalian HEK293T cells assessed functionality of our PknB kinase localisation and sensitivity. The composite part carrying plasmid was introduced via transfection to establish cellular localization of PknB before performing co-transfection experiments with the CMV-ATF2-mRuby2 (K5317016) and 3xCre3xAP1-miniCMV-miRFP670 promoter K5317017) under varying ampicillin concentration for stimulation. The EGFP fluorescence signal was analyzed for localization by microscopy and intensity by FACS analysis.

Single-transfection experiments

Figure 2: Single-transfected HEK293T cells with the PknB-EGFP-C2 plasmid depicted no EGFP-signal under unstimulated conditions. Scale bar = 20 µm.

As shown in figure 2, EGFP-PknB is correctly expressed in HEK293T cells. As intended, the EGFP-PknB depicts a membrane localized signal indicating a successful codon-optimization and correct implementation of a prokaryotic membrane protein into the eukaryotic cell membrane. With this, we were able to continue to find a functional detection protein, able to transfer the signal intracellularly.

Co-transfection experiments with ATF2

To pass the detected signal intracellularly, a transcription factor is neccessary that interacts with the kinase domain of PknB, and is activated by phosphorylation and transfers the signal on the level of expression regulation. Therefore, HEK293T cells were double-transfected with CMV-EGFP-PknB-C2 and CMV-ATF2-mRuby2 to analyse possible interactions by their fluorescence signals. Scale bar = 20 µM

Figure 3: Representative microscopy image of HEK293T cells expressing EGFP-PknB and ATF2-mRuby2. Shown are brightfield (left), fluorescence channels for eGFP and mRuby2 (both images in the center) and an overlay of the three channels (right).

The co-transfection of the functional EGFP-PknB and ATF2-mRuby2 is shown in figure 3. The expression of both parts was detectable, also located in one cell, indicating successful double-transfection. The EGFP signal, indicating the localization of PknB, was again membrane-closly localized. ATF2-mRuby2 on the other side demostrated a rather nuclear-cytoplasmic localization, both as exprected.

Stimulation of Co-transfected PknB-EGFP and ATF2-mRuby2 HEK cells with ampicillin

To show correct localization and interaction of PknB-eGFP and ATF2-mRruby2, both parts were transfected in HEK cells and incubated with ampicillin present in the culture media. We evaluated the fluorescence signal of these two proteins and their alteration of signal intensity compared to unstimulated, basal levels.

Figure 4: The montage shows representative images of double-transfected CMV-PknB-EGFP and CMV-ATF2-mRuby2 HEK293T cells with and without ampicillin stimulation. Shown are brightfield (left), fluorescence channels for eGFP and mRuby2 and an overlay of the three channels with and without coloured signals (right). Scale bar = 100 µm.

The Co-transfection experiments showed that PknB and ATF2 do not inhibit each other's co-expression and that both are possibly interacting. Under ampicillin stimulating conditions, both signals increase slightly. This paring of kinase and transcription factor can be used for further experiments with the 3xCre3xAP1-miniCMV promoter to fully assemble the cell-based antibiotic sensor and test its responsiveness to beta-lactam exposure.

References

Pensinger, D. A., Schaenzer, A. J., & Sauer, J. D. (2018). Do Shoot the Messenger: PASTA Kinases as Virulence Determinants and Antibiotic Targets. Trends in microbiology, 26(1), 56–69. https://doi.org/10.1016/j.tim.2017.06.010

Rakette S, Donat S, Ohlsen K, Stehle T (2012) Structural Analysis of Staphylococcus aureus Serine/Threonine Kinase PknB. PLOS ONE 7(6): e39136. https://doi.org/10.1371/journal.pone.0039136

Tamber, S., Schwartzman, J., & Cheung, A. L. (2010). Role of PknB kinase in antibiotic resistance and virulence in community-acquired methicillin-resistant Staphylococcus aureus strain USA300. Infection and immunity, 78(8), 3637–3646. https://doi.org/10.1128/IAI.00296-10