The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells

Abstract

The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode~1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalianpathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 6C and 37 6C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multinucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.

Role of Yersinia pestis Toxin Complex Family Proteins in Resistance to Phagocytosis by Polymorphonuclear Leukocytes

“…Our results indicate that although the Y. pestis Tc proteins are not insecticidal, they are active against PMNs. This is in agreement with previous studies which have shown that Y. pestis and Yersinia pseudotuberculosis Tc proteins are not toxic to Sf9 insect cells, M. sexta larvae, or X. cheopis (12,13) but are active against mammalian NIH 3T3 and Caco-2 cells (12). Additionally, Yersinia enterocolitica Tc protein homologues are only mildly insecticidal and nematocidal when fed orally (33)(34)(35) but are important in colonization of the gastrointestinal tract of mice (36).…”

“…Tc proteins of Y. pestis (9), Y. pseudotuberculosis (12,39), P. luminescens (1,40), Y. entomophaga (37), and X. nematophilus (41) have been shown to form large-molecular-weight protein complexes. In general, Tc protein complexes are composed of class A (TcA; Y. pestis YitA and YitB), class B (TcB; Y. pestis YitC), and class C (TcC; Y. pestis YipA and YipB) Tc proteins (9,37,(41)(42)(43)(44)(45)(46).…”

“…Unlike the insecticidal Tc proteins of P. luminescens, Y. pestis Tc proteins are not toxic to Manduca sexta larvae or active against Spodoptera frugiperda (Sf9) insect cells (12). Furthermore, although the Y. pestis Tc proteins are highly upregulated during infection of the rat flea, Xenopsylla cheopis (7,8), they are not toxic to fleas (13), nor are they required for infection of or survival within the flea digestive tract or for blockage of the proventriculus (8).…”

“…Furthermore, although the Y. pestis Tc proteins are highly upregulated during infection of the rat flea, Xenopsylla cheopis (7,8), they are not toxic to fleas (13), nor are they required for infection of or survival within the flea digestive tract or for blockage of the proventriculus (8). However, Y. pestis Tc proteins show some cytotoxicity to NIH 3T3 mouse fibroblast cells (12), and the Tc genes (yitA, -B, and -C) are upregulated when Y. pestis is within J774A.1 macrophages (14). Y. pestis isolated from infected fleas has increased resistance to uptake and killing by human and murine polymorphonuclear leukocytes (PMNs) (15,16) and murine macrophages (7).…”

Insecticidal Toxin Complex Proteins from Xenorhabdus nematophilus

“…Class B proteins are ϳ170 kDa, and class C proteins are ϳ110 kDa. There are many different varieties of class A, B, and C proteins in both Gram-negative and Gram-positive bacteria (12)(13)(14)(15).…”