On the role of Yersinia LcrV alleles in type III secretion and protective immunity.

摘要

Type III secretion is a mechanism used by many bacterial species to evade and subvert the host immune system, allowing the bacteria to survive and replicate. This system is utilized by three species of Yersinia that are pathogenic to humans, Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis. While the first two species generally cause gastrointestinal disease and are rarely lethal, Y. pestis is the causative agent of plague and as such has a high incidence of mortality associated with it. Additionally, due to the possibility of airborne transmission of the pneumonic form of the disease, there has been an increasing concern over illegitimate use of the bacteria as an agent of biological warfare. Efforts at understanding the pathogenesis of each species, as well as attempts at engineering successful vaccines against Y. pestis, have focused on the type III secretion system utilized by the bacteria to elude the host immune system, as loss of functionality of the type III secretion system renders the bacteria avirulent.;In each species, the genes necessary for production and activation of the type III secretion system are harbored on an approximately 70 kb virulence plasmid. After perception of several host signals, the bacteria fully assemble the type III secretion apparatus, which bears some resemblance to the flagellar system. This system essentially acts as a needle through which effector proteins are translocated from the cytoplasm of the bacteria to the cytosol of host cells, where they perform a variety of functions that ultimately result in escape from host immune responses. In chapter II, the transport and visualization of this process is examined by adapting a fluorescent technology to type III secretion in Y. enterocolitica. The ability of the bacteria to inject type III substrates containing a 12 amino acid motif that binds to a fluorophore was interrogated, as well as the ability of the bacteria to inject primary immune cells. Chapter III explores the effect of fusion of impassable reporter proteins to type III substrates, including how such hybrids affect both secretion and injection of other type III substrates. This study revealed that while such hybrids appear to block transport of other type III substrates, in actuality they are initiated into the type III pathway and are subsequently rejected. However, this initiation prevents the secretion of negative regulatory proteins, which accounts for the apparent blockade. Mutations in the negative regulators indeed relieve the perceived "blockade.";The contribution of heterologous genes to protective immunity is investigated in chapter IV. Current vaccine efforts focus largely on LcrV, a protein that resides at the tip of the type III needle and is absolutely essential for type III injection and virulence. Earlier work reported that antibodies against Y. pestis LcrV cannot block type III injection by other Yersinia species and suggested that polymorphisms in lcrV may provide for escape from LcrV-mediated plague immunity. This chapter demonstrates that while antibodies directed against Y. pestis LcrV are unable to prevent infection by Y. enterocolitica, expression of Y. enterocolitica lcrV in Y. pestis does not in fact provide for escape from LcrV-mediated protective immunity. A potential explanation for the protective effect afforded by LcrV-specific antibodies is brought to light in chapter V. These studies reveal that purified LcrV is able to interact with YopD secreted by Y. enterocolitica, and that particular regions of LcrV are required for such an interaction to occur. Additionally, it was shown that antibodies directed against LcrV are able to perturb the interaction with YopD, an interaction that is thought to be essential for type III injection to occur, providing a possible mechanism by which LcrV-specific antibodies generate protective immunity.