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. CD27L1-179 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).
Before lysis assays, the protein was dialyzed to remove Imidazole. The lysis activity of CD27L1-179 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) and chloramphenicol (CHL) were used as positive controls and PBS
as negative control.
As shown in Figure 2 inhibitory halos were not observed. Thus, we concluded that this protein
CD27L1-179 was not able to inhibit their growth in the concentrations used. Nevertheless, we keep
working in the laboratory improving test conditions, such as lysin and zinc concentrations. Figure 2. Lysis assay of endolysin
CD27L1-179 on: A) Clostridium difficile, B) Escherichia coli,
C) Staphylococcus sp. and D) Salmonella abatetuba. Amoxicillin (AMX) and
chloramphenicol (CHL) 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: 120µg/mL, 2: 60µg/mL and 3: 30µg/mL) to evaluate its growth
inhibition capacity. 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 Clostridium difficile-targeting endolysin affects activity and
host range. Journal of bacteriology, 193(19), 5477–5486. doi:10.1128/JB.00439-11
Endolysin CD27L1-179
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 final solution, this lysin should be expressed in L.casei. However, as we were not able to transform Lactobacillus, we designed the aforementioned construct to express it in E.coli an test the protein activity.
The lysin is a soluble intracellular protein, therefore this fraction was the one used
in the Ni-NTA resin purification. Wash 1 had 10 mM Imidazole and Wash 2, 20 mM Imidazole. Then, 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).
Lysis assay
References