Date of Award

Summer 8-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Urinary tract infections (UTIs) are some of the most common bacterial infections worldwide and are increasingly complicated by high antibiotic resistance and recurrence rates. Explanations for the marked individual differences in UTI susceptibility remain incomplete. In this thesis we show that urinary colonization by uropathogenic E. coli (UPEC) is influenced by urine composition and the activity of an important innate immune protein, siderocalin (SCN; also called lipocalin 2 or neutrophil gelatinase-associated lipocalin/NGAL). During UTI, host factors limit the availability of iron, an essential nutrient for the invading pathogen. In response, UPEC modify the urinary environment with metal binding siderophores, some of which are bound by the soluble protein SCN. Interactions between these opposing factors during early UPEC colonization determine the pathogen’s ability to successfully acquire iron and grow to a density sufficient to cause infection.

SCN has been described at length as an antimicrobial protein, exerting its effect by sequestering certain ferric siderophores. This has led to the hypothesis that a pathogen’s additional, non-SCN-binding siderophores are adaptations to this host pressure; however, the role of individual siderophores has been shown in some models to depend greatly on the infection environment. Because human urine is chemically complex and distinct from other sites of infection, we first investigated SCN’s effect on uropathogenic E. coli (UPEC) growth in human urine from a healthy reference population. Using genetic deletions, chemical inhibition, and chemical complementation, we observed enterobactin siderophore expression to be a key factor permitting UPEC growth in SCN-supplemented human urine from a subset of individuals. Because SCN neutralizes enterobactin in non-urinary experimental systems, this result suggests a determinative role for urine-specific components in manipulating antimicrobial paradigms.

Our initial inquiry showed dramatic variability in SCN’s antimicrobial activity between individuals’ urine specimens. We next used these individual differences as an independent variable, defining groups of high and low activity, in order to investigate the urinary factors controlling SCN activity. Chemical and demographic comparisons yielded a significant positive correlation between SCN activity and elevated urine pH. To determine whether further individual differences arose from differences in urinary small molecule composition (the urinary metabolome), we compared individuals using a mass spectrometry-based metabolomic approach. This approach identified aryl alcohols as significant correlates with SCN activity. These results support a model in which the urinary environment is able to influence urinary tract colonization by pathogens.

To further understand how these urinary metabolites may contribute to SCN antimicrobial activity, we sought to identify key metabolite cofactors present in restrictive urine specimens that actively participate in SCN’s antimicrobial mechanism as observed above. We developed a robust biophysical screen that allowed us to look for urine fractions containing iron-dependent SCN ligands. A biophysical validation process identified several elevated aryl alcohols that bound SCN and were able to reconstitute SCN’s antimicrobial activity in simple, defined media with limited iron. Demonstrating that urinary metabolites confer elevated SCN activity in a defined media provides mechanistic validation for our proposed urinary model, and further supports a dietary component to preventative UTI therapies. The human metabolome may thus represent an underappreciated contributor to disease susceptibility and pathogen evolution, and a potential target for future therapeutic interventions.

Collectively, the work presented in this thesis describes an emerging host-pathogen axis, where urinary composition plays a pivotal role in the efficacy of an innate immune response, and suggests targeted avenues for improved clinical control of UTI.

Language

English (en)

Chair and Committee

Jeffrey P Henderson

Committee Members

Thomas J Brett, Michael G Caparon, Daniel E Goldberg, Clay F Semenkovich, Timothy A Wencewicz,

Comments

Permanent URL: https://doi.org/10.7936/K7W957BP

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