Cytotoxic Necrotizing Factor Is an Effective Immunogen in a Yersinia pseudotuberculosis Aerosol Mouse Model

Abstract

There appears to be a link between bacteria producing certain Rho-activating toxins and respiratory disease. This class of toxins includes the cytotoxic necrotizing factor (CNF) of Escherichia coli, the dermonecrotic toxin (DNT) of certain Bordetella sp., and the CNF homologue of Yersinia pseudotuberculosis (CNF^Y). The host targets for these toxins are the GTP-binding proteins of the Rho family that function in a number of signaling pathways, including the modification of actin regulators. The toxins’ effect on Rho activity is a result of deamidation of the Rho proteins, leading to constitutive activation of Rho GTPases that are key regulators of the actin cytoskeleton. Because the cytoskeleton is involved in migration of phagocytes, phagocytosis, and the secretion of cytokines, this permanent activation of Rho proteins impairs host immune responses. Necrotoxic (CNF⁺) E. coli strains are associated with necrotizing pneumonia. Another member of this toxin family, DNT, is essential for virulence in a porcine pneumonia model (Bordetella bronchiseptica) and appears to be responsible for the severe lung damage in laboratory mice infected with this pathogen. The CNF^Y homologue is present in all strains of Y. pseudotuberculosis and Y. pestis that have been examined to date, although Y. pestis and many strains of Y. pseudotuberculosis do not produce the toxin due to a truncation in the cnf locus. We found that a Y. pseudotuberculosis strain expressing CNF^Y was virulent in the aerosol mouse model, while the CNF-negative isogenic strain did not cause mortality even at 100 LD₅₀. We expressed CNF^Y in E. coli and vaccinated mice with the purified toxoided protein. Vaccinated animals were protected from >600 aerosol LD₅₀ of a virulent CNF^Y+ strain but were not protected from a virulent strain carrying the truncated toxin gene. Thus, although CNF^Y is not required for aerosol virulence of all Y. pseudotuberculosis strains, it is an effective immunogen for CNF^Y+ strains. The Rho-activating toxin family may be an attractive target for development of medical countermeasures for necrotizing pneumonias.