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.

Diphtheria toxin is the main pathogenicity factor of causative agent of diphtheria Corynebacterium diphtheriae. Due to the small molecule size, it is of considerable interestfor the development of synthetic protein molecules with transporting function, e.g. immunotoxins. Expression and characterization of nontoxic recombinant fluorescent derivates of diphtheria toxin and its nontoxic mutant CRM 197 were described in this article. Obtained proteins may be applied in studies of receptor-binding and transporting functions of the toxin in cells, for determination of toxin receptor proHB-EGF expression level, immunization and antibody generation against the toxin and in development of diagnostic test-systems, detection of diphtheria toxin and antitoxic antibodies.

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