Researcher: Dr. Chris Colbert

Pathogenicity and the ability to form biofilms of Gram-negative bacteria are dependent on the acquisition of iron. Iron is imported from the environment via specific, high affinity, iron chelating molecules, called siderophores that bind to a set of tightly-regulated, high affinity, Ton-B dependent outer membrane transporters (TBDTs). The transcription of TBDTs is tightly regulated by multiple mechanisms, one of which up-regulates TBDT transcription in response to signals induced by extracellular iron-laden siderophore. This self-transcriptional regulation is conserved amongst all Gram-negative bacteria. Our central hypothesis is that siderophore binding on the cell surface activates TBDT transcriptional regulation by a series of protein-protein interaction events that cross both the outer and inner membranes to release an intracellular transcription factor that turns on operon transcription. The goal of this proposal is to investigate the structural basis of these protein-protein interaction events at the membrane interfaces as well as in the periplasmic space. We have selected the Gram-negative bacteria Pseudomonas putida to study these protein interaction events. To accomplish our goal we will use structural (X-ray crystallography and NMR spectroscopy) and biophysical methods to determine three-dimensional structures of the proteins involved and to investigate the mechanism of signal transduction that results in up-regulation of transcription of TBDTs.

Researchers: Nathan Fisher and Kendra Greenlee

Stenotrophomonas maltophilia is an emerging, multi-drug-resistant (MDR), opportunistic pathogen that frequently colonizes ventilator tubes and indwelling medical devices where it forms biofilms. Initial colonization can lead to severe, life-threatening infection. Approximately five percent of all cases of hospital-associated pneumonia in the United States are caused by this organism. Even more importantly, recent studies show that both incidence and prevalence are increasing, especially in immunocompromised, cystic fibrosis, chronic obstructive pulmonary disease (COPD), and cancer patients—demographic groups key to the research mission of the National Institutes of Health (NIH; Brook, 2012; Emerson et al., 2010; Metan et al., 2006). Clinical treatment is challenging because of S. maltophilia’s natural resistance to most classes of broad spectrum antibiotics. Moreover, resistance to antibiotics that first appear effective in vitro develops quickly in vivo due to upregulation of a plethora of efflux systems with broad specificity (Garrison, 1996). Based on successful approaches for other bacterial pathogens, our long-term goal is to develop treatment options that circumvent the S. malthophilia efflux systems by targeting secreted virulence factors or by pharmacologically bolstering the otherwise inadequate or inappropriate immune response. Currently, there is a critical knowledge gap with respect to how S. maltophilia avoids clearance by the immune system. Thus, our objective here is to identify the central components in both the bacterial virulence repertoire and the innate immune response to S. Maltophilia.

Researcher: Yagna Jarajapu

Diabetic vascular disease is the leading cause of mortality and morbidity worldwide. Impaired endothelial function and regeneration are the major causative factors for vascular disease. Bone marrow-derived stem/progenitor cells (BMPCs) play a potential role in vascular repair and offer a promising approach for the treatment of diabetic vascular disease. However, the success of the autologous cell therapies is mainly dependent on the efficiency of mobilization and collection of sufficient number of BMPCs, which is considerably lower in individuals with long-term diabetes. Furthermore, diabetic BMPCs are dysfunctional with reduced ability to induce regeneration therefore the approach of autologous cell therapies is currently not feasible in diabetic patients. Development of novel approaches is needed to boost mobilization and enhance the reparative potential of BMPCs in diabetes.

This investigator hypothesizes that the metabolite of angiotensin-converting enzyme (ACE)-2, Angiotensin (Ang)-(1-7), offers a novel target for boosting mobilization and function of BMPCs in diabetes basing on the following preliminary findings: 1) Ang-(1-7) increased the reparative potential of dysfunctional BMPCs from diabetic individuals. 2) Ang-(1-7) increased the circulating BMPCs in diabetic mice and stimulates their mobilization in response to vascular injury. 3) Ang-(1-7) decreased the oxidative stress and increased nitric oxide bioavailability in diabetic BMPCs. 4) Ang-(1-7) receptor, Mas is highly expressed in BM cells. The following specific aims are proposed to test the hypothesis. Specific Aim 1: Ang-(1-7) treatment enhances the mobilization of BMPCs by clinically used BM-mobilizers in diabetes. Specific Aim 2: Ang-(1-7) differentially modulates stromal and stem/progenitor cells to enhance the mobilization of BMPCs. The proposed aims will be pursued by a multidisciplinary approach using state-of-art-techniques in murine models of type 1 and type 2 diabetes.

Researchers: Guodong Liu and Kent Rodgers

The objective of this study is to design a rapid and near real-time detection method that exploits the bacterial requirement for iron as a means of capturing and identifying Gram-negative pathogens.

Researcher: Dr. Sangita Sinha

Autophagy, a conserved, catabolic eukaryotic pathway is essential for cellular homeostasis. Beclin 1 is a key conserved autophagy effector, but the mechanism by which it mediates autophagy is unknown. Our central hypothesis is that Beclin 1 is an interaction hub for autophagy, with various competing interactions either facilitating or inhibiting autophagy. The goal of this proposal is to investigate the structure and function of an uncharacterized Beclin 1 N-terminal region; establish the structure and conformation of multi-domain Beclin 1 constructs in the basal autophagy-inactive state; probe conformational changes upon binding of the autophagy inhibitor, Bcl-2; investigate how these different interactions compete with or complement each other; and investigate the role of highly conserved Beclin 1 residues in autophagy. This work will provide invaluable information about Beclin 1 structure, conformation and oligomerization, and elucidate how selected interactions modulate Beclin 1 structure and function. Pending funding of an R01 proposal, funds from this CoBRE Pilot proposal will support ongoing work and publication of our substantial results.