The broad, long-term objective of this application is to further our global understanding of how nontypeable Haemophilus influenzae (NTHI) mediates resistance to components of the host immune response. For NTHI, understanding this process may explain how this organism persists in the host airway as a commensal microorganism and yet maintains the ability to cause disease of both the upper and lower respiratory tracts. Clearly, a better understanding of NTHI pathogenesis will augment health related research in diseases such as otitis media (OM). We have recently shown that the Sap (sensitivity to antimicrobial peptides) transporter system is a major virulence determinant of NTHI, is required for resistance to killing by antimicrobial peptides (APs) and plays a role in potassium uptake in this microorganism. We hypothesize that the Sap proteins protect the bacterial cytoplasmic membrane from permeabilization by host APs.
The aims of this application are to: 1) Determine whether APs are transported to the bacterial cytoplasm in a Sap-dependent manner, and determine whether transported APs are then degraded by bacterial peptidases and 2) Determine whether the NTHI SapD ATPase directly associates with the TrkA/H complex to mediate potassium uptake. We propose to measure transport of three distinct classes of APs into whole cells and thereby define the function of the Sap transporter in AP uptake. In order to confirm whole-cell transport we will localize APs in cells by immunogold electron microscopy. We will then determine the fate of transported APs by monitoring peptide contents and AP concentrations in the cytoplasmic fraction of whole cell lysates by reverse-phase HPLC analysis.
In Aim 2, we will determine whether TrkH is required for high rate potassium transport in NTHI and monitor the interaction of SapD with the TrkA/H protein complex by chemical cross-linking and affinity purification. Finally, we will determine whether rapid potassium uptake can alter AP transport via the Sap transporter. The data generated from these studies will specifically define, for the first time, the molecular mechanism of the Sap transport system in NTHI and, perhaps more importantly, provide insight into how NTHI mediates survival in response to host innate immune components in vivo. ? ? The Sap transporter provides a critical and necessary function in disease-causing bacteria of the middle ear, and mediates survival in response to attack by the host immune response. The information gained from fulfillment of these aims will advance our understanding of how this bacterium causes disease and better equip us to design therapeutic approaches to combat middle ear infections. ? ? ? ?
| Heimlich, Derek R; Harrison, Alistair; Mason, Kevin M (2014) Host Antimicrobial Peptides in Bacterial Homeostasis and Pathogenesis of Disease. Antibiotics (Basel) 3:645-76 |
| Raffel, Forrest K; Szelestey, Blake R; Beatty, Wandy L et al. (2013) The Haemophilus influenzae Sap transporter mediates bacterium-epithelial cell homeostasis. Infect Immun 81:43-54 |
| Vogel, Andrew R; Szelestey, Blake R; Raffel, Forrest K et al. (2012) SapF-mediated heme-iron utilization enhances persistence and coordinates biofilm architecture of Haemophilus. Front Cell Infect Microbiol 2:42 |
| Shelton, Catherine L; Raffel, Forrest K; Beatty, Wandy L et al. (2011) Sap transporter mediated import and subsequent degradation of antimicrobial peptides in Haemophilus. PLoS Pathog 7:e1002360 |
| Mason, Kevin M; Raffel, Forrest K; Ray, William C et al. (2011) Heme utilization by nontypeable Haemophilus influenzae is essential and dependent on Sap transporter function. J Bacteriol 193:2527-35 |
