All ETDs from UAB

Advisory Committee Chair

Carlos J Orihuela

Advisory Committee Members

William H Benjamin Jr

David Briles

Jessy Deshane

Hui Wu

Carlos J Orihuela

Document Type

Dissertation

Date of Award

2017

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

During invasive disease, blood circulating Streptococcus pneumoniae are capable of invading the heart and forming bacteria filled cardiac microlesions. These microlesions are devoid of immune cells and disruptive of cardiac functionality. While the mechanisms underlying pneumococcal invasion of the myocardium are well-described, how the heart-invaded pneumococci evade immune detection and clearance is unknown. Since its discovery in 1881, invasive pneumococcal disease has been associated with the presence of extracellular diplococci or short chains in affected tissues. Herein, we show that heart-invaded pneumococci replicate within cellular vesicles and transition into biofilms. Although the host cell permissible for intracellular replication is yet unknown, the finding that a prototypical extracellular pathogen like S. pneumoniae uses an intracellular replication phase to cause tissue damage in itself is very novel and significant. Furthermore, we show that pneumococci within mature cardiac microlesions exhibit salient biofilm properties including intrinsic resistance to antibiotic killing and the presence of an extracellular matrix; thus, providing the first report for pneumococcal biofilm formation during invasive disease. We also performed dual RNA-seq analysis on infected heart and blood samples to capture the in vivo gene expression profile for heart- and blood-isolated pneumococci. Our exhaustive RNA-seq data confirmed the biofilm phenotype in the heart and revealed stark anatomical site-specific differences in virulence gene expression in vivo; the latter having major implications on vaccine antigen selection. Our novel RNA-seq approach also identified genes within three genomic islands as exclusively expressed in vivo, wherein, deletion of one such island, Region of Diversity 12, resulted in a biofilm-deficient and highly inflammogenic phenotype. These observations supporting published reports that link the ability to form biofilms to a dampened host-response; although the mechanism for the same was unknown. We subsequently determined that biofilm-pneumococci pre-empted cytokine/chemokine production and neutrophil infiltration into the myocardium by rapidly killing heart resident macrophages in a pneumolysin-dependent manner. Taken together, our findings described herein provide a novel site, i.e. intracellular; previously unreported growth phenotype during invasive disease, i.e. biofilm formation; and a counter-intuitive molecular mechanism, i.e. rapid heart resident macrophage killing; for how heart invaded pneumococci establish themselves within myocardium without inflammation.

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