Effect of the T-domain on intracellular transport of diphtheria toxin

Abstract

subunit b of diphtheria toxin (Dt), which consists of two domains: r (receptor-binding) and t (transmembrane), plays an important role in toxin-receptor binding on the cell-targets and in transportation of catalytic subunit a to the cell cytosol. recombinant analogues of the subunit b are promising representatives in the unique class of transporting proteins, able to deliver different types of biologically active molecules to cell cytosol. in the development of these protein constructs understanding of the role of each of the Dt fragments in determination of transporting pathways of endocytosed complex toxin-receptor is urgently required.We have studied in this work the t-domain effect on intracellular transport of recombinant fragments of Dt. We have compared intracellular transport of the r-domain and the subunit b, the last one consisted of both r-domain and t-domain. recombinant fragments of Dt used in this work were labeled with fluorescent proteins, which allowed applying colocalization technique for our study. application of confocal microscopy technique revealed differences in transportation of recombinant derivates of Dt in vero cells: r-domain moved faster than subunit b to tubular compartments. analysis of r-domain and subunit b transportation confirmed almost linear increase of their colocalization with the time regarding to Pearsons correlation coefficient (Pcc). however, amount of colocalized with r-domain subunit b were not linearly increased with time according to manders coefficient (m 1 ), this could indicate the ability of subunit b to transport to such compartments that r-domain do not reach. Possible role of the t-domain in intracellular transportation and compartmentalization of the toxin may be associated with the ability of the t-domain to form a proton channels and its ability to interact with coPi complex. K e y w o r d s: diphtheria toxin, t-domain of diphtheria toxin, endocytosis, fluorescent proteins, confocal microscopy, intracellular transport.

Enhancement of internalization of diphtheria toxin recombinant fragments in sensitive cells mediated by toxin’s T-domain

“…Creation of the relevant genetic constructs based on pET24(+) vector with kanamycin resistance and protocols for protein expression in e. coli Rosetta BL21 (DE3) cells on LB media by IPTG induction can be found in previous publications [5,9]. Purification of proteins by Ni-NTA agarose column chromatography with imidazole elution gradient and determination of protein concentration by tricine SDS-PAGE analysis with TotalLab TL120 software were also performed according to [5,9].…”

“…The protocol of sample preparation for flow cytometry, including cell detachment from Petri dish surface, assessment of cell density in a counting chamber, endocytosis suppression by NaN 3 and low temperature (4 °C), adjusting conditions of incubation medium to reduce a cell surface non-specific protein adsorbtion by 1% BSA, is described in detail in [4,5].…”

“…to cells from both of these organisms, as demonstrated in [4][5][6], suppose that the native toxin also enters both susceptible and resistant cells. It is believed that inside the resistant cells Cd is not transported to the cytosol from endosomal lumen [7], therefore these cells stay alive even in the presence of high doses of DT.…”

“…Therefore, our recombinant derivatives of toxin retain the fully functional Td. Initial evidence of Td involvement in the regulation of intracellular transport of the DT was obtained in [5]. We suggested in [5] that coat protein complex I (COPI) can be involved in this process.…”

“…Initial evidence of Td involvement in the regulation of intracellular transport of the DT was obtained in [5]. We suggested in [5] that coat protein complex I (COPI) can be involved in this process. The present work provides some new data on the intracellular DT transport.…”