Difference between revisions of "Part:BBa K2976004"

Latest revision as of 22:14, 21 October 2019

Granulysin

Granulysin is a member of the saposin-like protein (SAPLIP) family and primarily secreted by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, which possesses antimicrobial activity against M. tuberculosis (Mtb) and other organisms [1].

Usage and Biology

It is generally recognized that cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells play vital roles in host defense against pathogenic bacteria, which could exhibit cytolytic activity against microbes via producing various antimicrobial substances, such as perforin, granzyme and granulysin [2]. Granulysin is a cationic protein possessing broad antimicrobial activity, especially fatal to Mtb. It directly kills extracellular Mtb by damaging membrane integrity and disrupting lipid metabolism of the bacillus [3]. Moreover, granulysin significantly decreased the number of bacteria in Mtb H37Rv-infected mice model and there was no obvious cytotoxicity of granulysin [4]. Therefore, in 2019 CPU_CHINA project, we choose granulysin as a weapon to directly attack extracellular Mtb in blood circulation due to its excellent anti-TB activity.

Characterization

2019 CPU_CHINA attempted to develop a novel strategy to treat tuberculosis based on immune-like cells. Since granulysin is a major part of our project, we conducted research on it and obtained valuable results.

Figure 1.Induced secretion of granulysin.

Plasmid (TLR2-P2A-TLR1-T2A-CD14) and plasmid (NF-κB induced promoter-granulysin) were co-transfected into HEK293T cells. Then, we utilized Pam3Cys-Ser-(Lys)4, TLR1/TLR2 agonist to stimulate Toll like receptor (TLR) and its downstream signaling pathway. In our project, the activation of NF-κB can eventually leads to the expression of granulysin. Since the granulysin contains signal peptides, it is eventually destined to be secreted out of cells. Therefore, we determine the concentration of granulysin by ELISA assay. As shown in Figure 1, the concentration of granulysin in the cell culture medium is significantly higher than other groups at 24h (A), 36h (B) and 48h (C).

Figure 2.Time-Killing Curve of granulysin against M. smegmatis.

Since M.tuberculosis is quite a dangerous species, we utilize a safer strain, M.smegmatis as a substitute to test in vitro antimicrobial activity of granulysin. The same amount of M.smegmatis was seeded into the cell culture medium with or without granlysin. Then, bacteria and cell medium were incubated at 37℃ with 200 rpm and OD600 of the medium was determined every 10 hours. The results demonstrate that granulysin exhibits good antimicrobial activity against M.smegmatis.

Figure 3.MTT assay of HEK293T cells after granulysin treatment.

To further determine the safety of granulysin to normal human cells, MTT assay was conducted to examine the survival rate of HEK293T cells after incubated with the supernatant containing different concentration of granulysin for 12 hours. The value of survival rate was steady, about 100%, proved the good safety of granulysin.

For more details, please check out our demonstrate page.

References

[1] Dotiwala, F. , Lieberman, J. (2019). Granulysin: killer lymphocyte safeguard against microbes. Curr Opin Immunol, 60:19-29.

[2] Dotiwala, F. , Sen Santara, S. , Binker-Cosen, A. A. , Li, B. , Chandrasekaran, S. , & Lieberman, J. . (2017). Granzyme b disrupts central metabolism and protein synthesis in bacteria to promote an immune cell death program. Cell, 171(5):1125-1137.

[3] Stenger, & S. (1998). An antimicrobial activity of cytolytic t cells mediated by granulysin. Science, 282(5386), 121-125.

[4] Ma, J. , Lu, J. , Huang, H. , Teng, X. , Tian, M. , & Yu, Q. , et al. (2015). Inhalation of recombinant adenovirus expressing granulysin protects mice infected with mycobacterium tuberculosis. Gene Therapy.

Sequence and Features