Hemoglobin Induces Invasion of Streptococcus pneumoniae into Human Lung Cells Open Access
Rao, Shambavi (Spring 2018)
Abstract
Streptococcus pneumoniae (Spn) are Gram-positive bacteria known to cause many debilitating and invasive pneumococcal diseases such as pneumonia, bacteremia, and meningitis. Lethal infection is mediated by the invasion of cells primarily located in the lower respiratory tract, permitting bacteria to translocate into the bloodstream. We studied the mechanism of pneumococcal invasion by establishing an in vitro model to replicate invasion using cultures of human lung Calu-3 cells. Pneumococcal invasion was tested in the presence and absence of hemoglobin (Hb). This in vitro model was utilized to determine invasion of pneumococci through polarized human respiratory epithelial cells by counting bacteria translocated to the basolateral side of the cells. A potential invasion mechanism mediated by disruption of tight-junction (TJ) proteins was explored by measuring the trans-epithelial electrical resistance (TEER) pre-, and post- pneumococcal, infection. Through a series of experiments, I observed that hemoglobin induced pneumococcal invasion within 8 hours of incubation but a decrease in TEER within 24 hours post- infection. Whereas invasion and disruption of the TJ belt was triggered by incubation with Hb, the effectors appeared to be different given that invasive pneumococci translocated before TEER was affected. Furthermore, a mutant in a potent toxin, pneumolysin, and a mutant in the competence stimulating peptide, were unable to translocate but both mutants disrupted the TJ belt at the same extent as that observed with the wild type strain. In this thesis, we concluded that Hb triggers in pneumococci invasion of human lung cells and disruption of the TJ belt and that the effectors of this Hb-induced phenotypes may be different and required further investigation.
Table of Contents
Abstract .......................................................................................... 1
Significance ................................................................................... 2
Introduction .................................................................................... 3
Specific Aims ................................................................................. 8
Experimental Methods .................................................................... 9
Bacterial Strains and Inoculum Preparation .............................. 9
Cell Culture ................................................................................ 9
Trans Epithelial Electrical Resistance (TEER)...........................10
Hemoglobin, Heme, and Iron Preparation .................................11
Bacterial Infection with or without Hemoglobin........................12
Statistical Analysis .....................................................................13
Results ............................................................................................. 13
Discussion ....................................................................................... 19
Future Directions ............................................................................. 24
References ....................................................................................... 26
Appendix ......................................................................................... 30
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