Team:Costa Rica/Results

The results shown here are product of our work in different laboratories of three universities: Costa Rica Institute of Technology (ITCR), National University of Costa Rica (UNA) and the University of Costa Rica (UCR). Each test was made in compliance to the security standards needed to work with each microorganism and reagent.

Endolysin CD27L_1-179

CD27L_1-179 is the catalytic domain of a bacteriophage endolysin that infects Clostridium difficile. It is homologous to the domain of the N-acetyl-muramoyl-L-alanine amidase. This truncation mutation was selected instead of the complete protein because, according to previous studies, it shows a faster lysis to different strains of the pathogen and a high level of selectivity against Clostridia compared to the complete protein (Mayer et al., 2011).

We designed a construct that expresses CD27L_1-179 in the plasmid pTwist-Amp-High Copy. We used the T7 promoter for this construct due to its high transcription level and its general inactivation in the absence of IPTG. This promoter is commonly used in the strain selected, E. coli BL21(DE3). Also, the protein was tagged with 6 histidines in the C-terminal to facilitate its purification by affinity chromatography. Is worth to mention that, for our proposed solution, this lysin should be expressed in L.casei. However, as we were not able to transform Lactobacillus yet, we designed the aforementioned construct to express it in E.coli and test the protein activity.

The 6xHis-lysin is a soluble intracellular protein, therefore this fraction was the one used in the Ni-NTA resin purification. The protein of interest was eluted in 500 mM Imidazole. As shown in the next SDS-PAGE, considering the size of the band, we confirmed the presence of our purified protein in elution number 1 (F1).

Figure 1.Polyacrylamide gel electrophoresis of endolysin CD27L_1-179 purification.

Before lysis assays, the protein was dialyzed to remove Imidazole.

Lysis assay

The lysis activity of CD27L_1-179 was tested on Clostridium difficile NCTC 13307, Escherichia coli, Staphylococcus sp. and Salmonella abatetuba. The Kirby-Bauer method was used, bacterias were inoculated in a Mueller-Hinton agar, using different concentrations of lysin (0.12 mg/mL, 0.06 mg/mL and 0.03 mg/mL). Amoxicillin (AMX, 10 µg) and chloramphenicol (CHL, 30 µg) were used as positive controls and PBS as negative control. As shown in Figure 2, at these lysin concentrations, inhibitory halos were not observed. Thus, we concluded that this protein CD27L1-179 was not able to inhibit their growth in the conditions tested. Nevertheless, we keep working in the laboratory improving test conditions, such as lysin and zinc concentrations.

Figure 2. Lysis assay of endolysin CD27L_1-179 on: A) Clostridium difficile, B) Escherichia coli, C) Staphylococcus sp. and D) Salmonella abatetuba. Amoxicillin (AMX, 10 µg) and chloramphenicol (CHL, 30 µg) were tested as positive control agents and PBS as negative control (-); both antibiotics were used by recommendation of Andino-Molina and colleagues (2019). Lysin was assayed with three different concentrations (1: 120 µg/mL, 2: 60 µg/mL and 3: 30 µg/mL) to evaluate its growth inhibition capacity.

Auto Inducer Peptide

AIP is the Quorum Sensing signaling protein of C. difficile. Nevertheless, the pre signal protein (AgrD) suffers an unknown modification by AgrB and so, AIP’s sequence is still undiscovered. Therefore, we produced both proteins.

This proteins, AgrD and AgrB, have a pLac promoter to produce AIP only when IPTG is added. Also, a GFP reporter is coded polycistronically after the AgrD peptide, as a way to indirectly quantify AIP expression. Is important to note that nickel affinity chromatography isn't used as a purification and quantification method. This consideration was based on the previously reported insoluble properties of the peptide and the possible interference of a his tag during the processing of AgrD by AgrB.

After induction, proteins were extracted and analyzed on a SDS-PAGE. As you can see in the next figure, GFP (intracellular protein, 26 kDa) (Slade et al., 2009), AgrB (transmembrane protein, 21 kDa) (Zhang et al., 2002) and AIP protein (~5 kDa) are located in the intracellular fraction.

Figure 3. Polyacrylamide gel electrophoresis of intracellular proteins of a recombinant AIP bacterial culture. Protein with 26 kDa correspond to GFP, 21 kDa to AgrB and approx. 5 kDa to AIP (modified AgrD).

It is worth to mention that, considering the size of the annotated bands in figure 1, we hypothesize that our desire proteins are present in this fraction. Nevertheless, sequencing is necessary to confirm this argument.

In other hand, the extracellular fraction didn’t show any band related to our desired proteins (Figure 4).

Figure 4.Polyacrylamide gel electrophoresis of extracellular and intracellular proteins of a recombinant AIP bacterial culture. Protein with 26 kDa correspond to GFP, 21 kDa to AgrB and approx. 5 kDa to AIP (modified AgrD).

Although the GFP and the AgrB should be in the intracellular fraction, the AIP is supposed to be transferred to the extracellular medium. Therefore, we recommend modifications to our system and further characterization in order to transfer AIP to the extracellular space.

References

Andino-Molina, M., Barquero-Calvo, E., Seyboldt, C., Schmoock, G., Neubauer, H., Tzoc, E., Rodríguez, C. & Quesada-Gómez, C. (2019). Multidrug-resistant Clostridium difficile ribotypes 078 and 014/5-FLI01 in piglets from Costa Rica. Anaerobe, 55, 78-82.

Mayer, M. J., Garefalaki, V., Spoerl, R., Narbad, A., & Meijers, R. (2011). Structure-based modification of a C. difficile-targeting endolysin affects activity and host range. Journal of bacteriology, 193(19), 5477–5486. doi:10.1128/JB.00439-11

Slade, K. M., Baker, R., Chua, M., Thompson, N. L., & Pielak, G. J. (2009). Effects of recombinant protein expression on green fluorescent protein diffusion in Escherichia coli. Biochemistry, 48(23), 5083–5089. doi:10.1021/bi9004107.