Date of Award

2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

Matthew J. Wargo

Abstract

Nosocomial lung infections are a growing concern in the United States, with more than 300,000 cases reported annually. More than 30 % of which are caused by the Gram-negative bacteria, Pseudomonas aeruginosa and Klebsiella pneumoniae. Similarly, Gram-negative bacteria establish chronic infections in individuals with cystic fibrosis (CF) that are difficult or impossible to eradicate.P. aeruginosa has historically been one of the most prevalent pathogens of adults with CF. However, as antipseudomonal therapy has improved, more antibiotic resistant species have taken hold, including Stenotrophomonas maltophilia, which now colonizes more than 10 % of individuals with CF. Regardless of the cause or source, Gram-negative respiratory infections are becoming increasingly difficult to treat due to the rising incidence of multiple drug resistance among these organisms. To aid in the development of new therapeutics, a greater understanding of how these organisms transition from the environment to the host lung is needed. Here we utilized a combination of transcriptomics and molecular genetics to examine how P. aeruginosa, K. pneumoniae, and S. maltophilia, recognize and exploit the host lung milieu during the initiation of infection.

One of the first components of the host lung environment that aspirated bacteria are exposed to is pulmonary surfactant (PS). This phospholipid-rich substance coats the distal airways of the lung and is thought to contain molecular cues that facilitate lung colonization by pathogenic bacteria. Here, we characterized the transcriptional response of K. pneumoniae to purified PS to examine how this organism interreacts with the host lung during colonization. This work revealed numerous virulence and colonization-related genes that are expressed by K. pneumoniae under these conditions. We also tested the contribution of other surfactant-induced transcripts to K. pneumoniae pathogenesis using engineered gene deletion strains and a mouse model of pneumonia. This work revealed the polyamine efflux pump, MdtJI, and glycine betaine transporter, ProU are required for K. pneumoniae virulence.

Phosphatidylcholine is the primary constituent of PS. P. aeruginosa is capable of completely metabolizing the phosphocholine head group of this lipid, and readily does so when exposed to PS. We previously observed that the most highly expressed genes in P. aeruginosa in response to PS were those involved in the catabolism of a downstream choline metabolite, sarcosine. Although our group had previously characterized the choline catabolic pathway of P. aeruginosa, the transcriptional regulation of sarcosine catabolism was not known. We utilized a genetic screen to identify the regulator controlling the expression of the sarcosine catabolic genes in P. aeruginosa. This regulator, which we named SouR (Sarcosine oxidase utilization Regulator) is the first sarcosine-responsive regulator to be characterized.

The thick, viscous mucus (sputum) that accumulates within the CF lung serves as the primary nutrient source for microbes colonizing the CF lung. Here, we characterized the transcriptional responses of three S. maltophilia strains during growth in synthetic CF sputum media (SCFM2) to gain insight into how this organism interreacts with the host lung. We also compared the SCFM2 transcriptomes of two S. maltophilia CF isolates with the SCFM2 transcriptome of the acute infection model strain, K279A. This revealed CF isolate-specific signatures in gene expression that reflect adaptation to the CF lung.

Language

en

Number of Pages

300 p.

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