Surface displayed proteins on bacteria play key roles in pathogenesis as they promote bacterial adhesion to host tissues, acquisition of essential nutrients, evasion and suppression of the immune response and host cell entry. We will study how Gram-positive bacterial pathogens display and utilize virulence factors during infections, and seek to develop new antibiotics that work by inhibiting bacterial protein display. Research will concentrate on Staphylococcus aureus, a leading cause of lethal hospital- and community-acquired infections in the United States that kill more people than any other infectious agent annually. S. aureus and other Gram- positive bacteria covalently attach virulence factors to their cell wall using sortase transpeptidase enzymes. In S. aureus, surface proteins are displayed by the sortase A (Sa-SrtA) and sortase B (Sa-SrtB) enzymes. They work together to construct the Iron-regulated surface determinant (Isd) system that actively harvests the essential nutrient iron from human hemoglobin during infections. Sortases and Isd proteins are prime targets for the development of new anti-infective agents as both contribute to S. aureus pathogenesis.
In aim #1, we obtain broad mechanistic insight into how pathogenic bacteria display virulence factors by determining structures of representative enzymes bound to specially synthesized sorting signal analogs, and by using a newly developed in vivo transpeptidase assay to explore how sortases recognize the cross-bridge peptide.
In aim #2, several promising sortase inhibitors we have discovered will be further developed using NMR, computational and synthetic chemistry methods. These molecules are a potentially innovative approach to treat lethal infections, as they would prevent bacteria from displaying virulence factors on their surface, rendering them defenseless against the host's immune response. Once their potency and selectivity have been optimized, their therapeutic efficacy will be evaluated using a mouse model of S. aureus systemic infection. Research in aim #3 will study how S. aureus uses sortase attached Isd proteins to scavenge the essential nutrient iron from human hemoglobin, and will attempt to disrupt this process through targeted amino acid mutagenesis. Using NMR and biochemical methods we will elucidate the mechanism through which the Sa- SrtA target protein IsdH extracts heme from hemoglobin, and how the Sa-SrtB target protein IsdC relays heme from upstream hemoreceptors positioned near the cell surface to the IsdDEF heme transporter complex located in the membrane. Collectively, this research will increase our understanding of the molecular basis of S. aureus pathogenesis and it could lead to new therapeutics to treat bacterial infections.

Public Health Relevance

Staphylococcus aureus and other bacterial pathogens use surface displayed proteins to mount infections. We will study the enzymatic machinery that displays these proteins, and how they function to scavenge the essential nutrient iron from human hemoglobin. We will also continue to develop several promising small molecule protein display inhibitors that could function as potent anti-infective agents to treat infections caused methicillin-resistant strains of S. aureus, and other multi-drug resistant bacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI052217-11
Application #
8437143
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Huntley, Clayton C
Project Start
2002-06-01
Project End
2017-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
11
Fiscal Year
2013
Total Cost
$353,596
Indirect Cost
$118,596
Name
University of California Los Angeles
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Sjodt, Megan; Macdonald, Ramsay; Spirig, Thomas et al. (2016) The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin. J Mol Biol 428:1107-29
Amer, Brendan R; Macdonald, Ramsay; Jacobitz, Alex W et al. (2016) Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent. J Biomol NMR 64:197-205
Jacobitz, Alex W; Naziga, Emmanuel B; Yi, Sung Wook et al. (2016) The "Lid" in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site. J Phys Chem B 120:8302-12
Tekeste, Shewit S; Wilkinson, Thomas A; Weiner, Ethan M et al. (2015) Interaction between Reverse Transcriptase and Integrase Is Required for Reverse Transcription during HIV-1 Replication. J Virol 89:12058-69
Macdonald, Ramsay; Sarkar, Dibyendu; Amer, Brendan R et al. (2015) Solution structure of the PhoP DNA-binding domain from Mycobacterium tuberculosis. J Biomol NMR 63:111-7
Zhang, Jiang; Reza Malmirchegini, G; Clubb, Robert T Clubb T et al. (2015) Native top-down mass spectrometry for the structural characterization of human hemoglobin. Eur J Mass Spectrom (Chichester) 21:221-31
Zilbermintz, Leeor; Leonardi, William; Jeong, Sun-Young et al. (2015) Identification of agents effective against multiple toxins and viruses by host-oriented cell targeting. Sci Rep 5:13476
Dickson, Claire F; Jacques, David A; Clubb, Robert T et al. (2015) The structure of haemoglobin bound to the haemoglobin receptor IsdH from Staphylococcus aureus shows disruption of the native α-globin haem pocket. Acta Crystallogr D Biol Crystallogr 71:1295-306
Chan, Albert H; Yi, Sung Wook; Terwilliger, Austen L et al. (2015) Structure of the Bacillus anthracis Sortase A Enzyme Bound to Its Sorting Signal: A FLEXIBLE AMINO-TERMINAL APPENDAGE MODULATES SUBSTRATE ACCESS. J Biol Chem 290:25461-74
Malmirchegini, G Reza; Sjodt, Megan; Shnitkind, Sergey et al. (2014) Novel mechanism of hemin capture by Hbp2, the hemoglobin-binding hemophore from Listeria monocytogenes. J Biol Chem 289:34886-99

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