Date on Master's Thesis/Doctoral Dissertation

8-2016

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Warawa, Jonathan

Committee Co-Chair (if applicable)

Lawrenz, Matthew

Committee Member

Lawrenz, Matthew

Committee Member

Graham, James

Committee Member

Kosiewicz, Michele

Committee Member

Scott, David

Author's Keywords

Klebsiella pneumoniae; sequencing; bioluminescence; optical diagnostic imaging; therapeutics; virulence determinants

Abstract

Klebsiella pneumoniae causes an acute respiratory infection in human with severe outcomes and high mortality rates even with antibiotic treatment. Even with its critical clinical importance, few virulence systems have been identified for K. pneumoniae limiting the development of new therapeutic strategies. Accordingly, we performed Next Generation sequencing for the strain ATCC 43816, a virulent strain in mouse respiratory disease models, and compared its genomic data with two previously sequenced strains NTUH-K2044 and MGH 78578 for the purpose of identifying genes required for colonizing host lungs. Furthermore, the virulence potential of the three K. pneumoniae strains were tested in a mouse model of pulmonary disease uniquely generated by our group to insure the specific delivery of an inoculum into host lungs allowing for studying diseases associated specifically with the lower respiratory tract. To monitor disease progression noninvasively, a bioluminescent K. pneumoniae strain was engineered which allowed for monitoring meropenem therapeutic efficacy against the bacteria in real time. A transposon mutant library was generated in the bioluminescent strain and introduced into mice lungs in order identify critical fitness factors required by K. pneumoniae to survive the selective pressure of host lung. The attenuation of known and potential virulence factors, including capsular polysaccharide (CPS) and type 6 secretion systems (T6SSs), were tested in our lung-specific murine model of respiratory disease. Similar to previous findings, manC capsule mutant was attenuated in our lung-specific disease model whereas for the vgrG T6SSs mutants, only cluster one illustrated some potential attenuation in the host, and future studies will be conducted to confirm these outcomes. K. pneumoniae is thought to be an extracellular pathogen but we have provided the first evidence suggesting that this dogma might not be entirely true by demonstrating the capability of the bacteria to proliferate within cultured macrophages in addition to the ability of a subpopulation of K. pneumoniae to become intracellular within mice lungs. Further studies will need to be conducted to identify the role(s) of the intracellular lifestyle for K. pneumoniae during the pulmonary disease.

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