Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

William Goldman

Abstract

Disease progression of primary pneumonic plague is biphasic, consisting of a pre-inflammatory and a pro-inflammatory phase. Most lung pathogens induce a measureable inflammatory response within a few hours of infection, but Yersinia pestis is strikingly different: the first 36 hours of infection are characterized by rapid bacterial replication in the lung without generating appreciable levels of proinflammatory cytokines/chemokines, histological changes in infected tissue or outward disease symptoms. The ultimate goal of this thesis was to understand the processes that contribute to the pre-inflammatory phase of disease. Upon inhalation of infectious droplets, Y. pestis can interact with a myriad of different cell types, but the initial bacterial / host cell interactions during pulmonary infection are largely uncharacterized. I found that Y. pestis cannot be lavaged from the lung during the initial stages of infection but is freely lavageable during the later stages of infection, indicating that Y. pestis adheres tightly to lung tissues during the pre-inflammatory phase of disease. Although recent studies have implicated Ail as the Y. pestis adhesin that mediates this interaction, my data suggest that Ail acts to stabilize the outer membrane, and the loss of Ail alters other outer membrane proteins. Interestingly, the reintroduction of two major Yersinia adhesins, yadA and inv, into Y. pestis reduces the virulence of Y. pestis in both bubonic and pneumonic animal models of infection, offering an explanation as to why these genes were lost during the course of Y. pestis evolution. I also developed a negative selection screen termed transposon site hybridization: TraSH) to identify bacterial factors required for the two different phases of Y. pestis pulmonary infection. I validated the technique by defining the essential genes for Y. pestis viability, which largely coincided with essential genes found in Escherichia coli. However, I was unable to identify any known virulence factors using TraSH following an intranasal infection, including genes involved in the Ysc type-three secretion system: T3SS), which are known to be essential for pulmonary infection, suggesting that there is global trans-complementation of attenuated mutants by wild-type Y. pestis. Finally, using a co-infection model of pneumonic plague, I discovered that Y. pestis quickly creates a localized, dominant anti-inflammatory state that allows for the survival and rapid growth of both itself and normally avirulent organisms. In contrast, Y. pseudotuberculosis, the relatively recent progenitor of Y. pestis, and K. pneumoniae, another highly virulent lung pathogen, show no similar trans-complementation effect, which is unprecedented among other respiratory pathogens. Both the unbiased negative selection screen using a vast pool of Y. pestis mutants: TraSH) and the co-infection experiments revealed no selection against any known virulence genes, demonstrating the transformation of the lung into a globally permissive environment during the pre-inflammatory phase of pneumonic plague that allows not only wild-type Y. pestis to proliferate rapidly in the lung, but other microbes as well.

Comments

Permanent URL: http://dx.doi.org/10.7936/K71C1TW9

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