Elektroforetyczna i immunologiczna analiza natywnych białek wydzielniczych wytwarzanych in vitro w warunkach indukcji Ysa (Yersinia secretion apparatus) przez izolaty Yersinia enterocolitica 1B/O8 wyosobnione od ludzi w Polsce
[Electrophoretic and immunological analysis of native proteins secreted in vitro under conditions inducing Ysa (Yersinia secretion apparatus) by clinical isolates of Yersinia enterocolitica 1B/O8 in Poland]

Streszczenie

Przeprowadzono elektroforetyczną analizę profili białkowych wybranych izolatów epidemicznego szczepu Yersinia enterocolitica bioserotypu 1B/O8 pochodzących od osób z jersiniozą zamieszkałych na terenie Polski. Badano zdolność wybranych białek do reakcji z surowicą królika immunizowanego referencyjnym szczepem Yersinia enterocolitica 1B/O8 WA-314 oraz próbkami surowicy uzyskanymi od osób z bakteriologicznie potwierdzonym zakażeniem pałeczkami Yersinia enterocolitica bioserotypu 1B/O8. Analiza uzyskanych elektroforegramów nie wykazała obecności białek Ysa i Ysp mimo zastosowania warunków indukcji umożliwiających wytwarzanie tych białek przez szczep referencyjny Yersinia enterocolitica 1B/O8 WA-314 oraz szczepy Yersinia enterocolitica 1B/O8 pochodzące z Instytutu Pasteura. Wyniki badań serologicznych nie potwierdziły obecności swoistych przeciwciał dla białek Ysa i Ysp zarówno w próbce surowicy immunizowanego królika, jak i próbkach surowicy uzyskanych od osób z jersiniozą.

Abstract

Introduction: The high pathogenicity Yersinia enterocolitica 1B/O8 produce variety of virulence
factors including chromosomal T3SS known as Ysa-Ysp system that is considered to act at the early stage of infection. The aim of the study was to examine the ability to produce Ysa-Ysp proteins in vitro by human clinical isolates of the epidemic Y. enterocolitica 1B/O8 strains in native conditions and immunological characterization of expressed proteins. Methods: Seven Y. enterocolitica 1B/O8 isolates with known epidemiological link and the reference high pathogenicity strain WA-314 and six strains from the Institute Pasteur (France) were examined for production of Ysa-Ysp proteins according with procedure described by Matsumoto and Young (Mol. Microbiol., 2006, 59:689-76). All the isolates and strains were characterized by SDS-PAGE to determined Ysa-Ysp proteins profile. The immunological characterization was performed by using western-immunobloting method using sera from two immunized rabbits and from two patients with bacteriology confirmed Y. enterocolitica 1B/O8 infection.Results: The reference strain WA-314 yielded typical Ysa-Ysp proteins profile. In contrast all the tested Y. enterocolitica 1B/O8 human isolates yielded the same SDS--PAGE profile that was apparently distinct from profile of Ysa-Ysp proteins of reference strain WA-314. Conclusions: The Y. enterocolitica 1B/O8 isolates of the epidemic strain circulating in Poland were found to be unable to produce Ysa-Ysp proteins in vitro under conditions sufficient to stimulate expression of the Ysa-Ysp proteins in the reference strain WA-314 and strains from the Institute Pasteur (France). Our results may suggest that the ability to produce Ysa-Ysp proteins in concentrations sufficient to induce production of specific antibodies is not indispensible for Y. enterocolitica 1B/O8 infection in humans. The western-immunoblotting analysis of human serum samples showed that the antibodies were not induced by Ysa and Ysp proteins during infection caused by the epidemic strain of Y. enterocolitica 1B/O8 circulating in Poland. Similar, negative result was found with serum of a rabbit immunized intravenously by the reference strain WA-314.