Molecular Determinants of Salmonella Cell-Envelope Copper Homeostasis

National Institute of Health. R01- AI150784. PI José Argüello. Co-PI Fernando Soncini and Susana Checca (IBR-Rosario, Argentina)

The goal of this project is to define the mechanisms of Cu homeostasis in the cell envelope of the pathogen Salmonella enterica. This organism is an important and frequent cause of gastroenteritis, as well as, systemic infections. Cu is required as a redox co-factor in the catalytic centers of enzymes. However, free Cu is highly reactive and deleterious to cells. Cu, along with the oxidative burst, is central in host-pathogen interactions as part of the innate immune response. As such, redox/Cu homeostasis is essential for bacterial virulence. While there has been significant progress in identifying cytoplasmic Cu homeostatic mechanisms, there is a lack of understanding of how the cell envelope handles and distributes Cu, whilst maintaining the associated redox balance. Our goal is to define and model the Cu distribution in the Salmonella cell envelope and identify its molecular links with the redox stress response. The aims of this proposal are: 1) Quantify Cu fluxes and equilibria among periplasmic, cytoplasmic and external compartments while defining the size and identity of the periplasmic Cu sink pool. 2) Define the role of CueP as the periplasmic Cu chaperone exchanging the metal with various targets. 3) Determine the role of the ScsABCD system at the interface of Cu^– and redox-homeostasis. The relation between ScsABCD activity and Cu binding to substrates or among ScsABCD enzymes will be established. To achieve these aims, laboratories will use a combination of approaches (modeling of metal fluxes, proteomics, metallomics, in vitro host/pathogen interaction).

Copper Homeostasis in the Opportunistic Pathogen Pseudomonas Aeruginosa

National Institute of Health. R01-GM114949. PI José Argüello.

Our goal is to define the mechanisms of Cu⁺ homeostasis in the pathogen Pseudomonas aeruginosa. This organism is an important and frequent cause of hospital acquired infections, especially in immune compromised patients. Free Cu⁺ is highly reactive and deleterious to cells and consequently copper tolerance systems contribute significantly to bacterial virulence. On the other hand,it is a micronutrient required as a redox co-factor in the catalytic center of enzymes. It is not known how cellular components interact and participate in ion distribution to achieve Cu⁺ targeting to essential cuproenzymes and tolerance to high Cu⁺ concentrations. We hypothesize that cells have two Cu⁺ sensing/distribution networks. One is responsible for targeting Cu⁺ to cuproproteins and responds to changes in cuproenzymes functionality when challenged by environmental stressors. The other network maintains a cellular Cu⁺ quota and responds to cytoplasmic Cu⁺ levels. We aim to define and model these Cu⁺ distribution networks by using transcriptomics and metalloproteomics approaches. In addition, we are characterizing the specificity and routes of Cu⁺ entrance, distribution in the cytoplasm, transport to the periplasm, and final targeting to efflux systems or cuproenzymes. Departing from reductionist approaches, the project shifts the analysis of heavy metal homeostasis by considering the full range of involved elements, the biochemical equilibria in which they participate, and the integrated system response to environmental challenges.

The Role of ZIP12 in Zinc Homeostasis and Associated Neurodegenerative Pathologies

National Institute of Health. 1R21NS125242. PI: Robert Demspki. Co-PI José Argüello.  

Zinc is an essential micro-nutrient that participates in catalytic and structural functions touching nearly every metabolic process in the cell. While alterations in neuronal Zn²⁺ distribution have been associated with multiple disease states including Alzheimer’s Disease (AD), ALS, schizophrenia, and depression, the molecular mechanism of Zn²⁺ homeostasis in neurons is largely unexplored. The Zinc and Iron-regulated transport Proteins (ZIP) mediate the entrance of first row transition metals into the cytoplasm. hZIP12, one of the fourteen human ZIPs, is a Zn²⁺ uptake transporter expressed in the brain. This proposal is based on the hypothesis that studies of hZIP12 and variants associated with neurodegenerative diseases will serve as an entrance point to build testable mechanistic models of neuronal Zn²⁺ homeostasis. Aim one tests the hypothesis that hZIP12 mutations associated with neuronal diseases impact transport activity and/or sur- face expression. Aim two tests the hypothesis that hZIP12 dysfunction modifies cellular Zn²⁺ pools and metal distribution. Cytosolic Zn²⁺ levels, as well as transmembrane transporter expression, will be assessed under early steady state and non- toxic Zn²⁺ levels. Results from these studies will initiate the definition of molecular and subcellular mechanisms of Zn²⁺ homeostasis in the brain, provide a detailed description of hZIP12 function in neuronal Zn²⁺ subcellular distribution, and elucidate the role of hZIP12 in relevant neurodegenerative diseases.