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 #
5R01AI052217-12
Application #
8627105
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
2014-03-01
Budget End
2015-02-28
Support Year
12
Fiscal Year
2014
Total Cost
$376,166
Indirect Cost
$126,166
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
Amer, Brendan R; Clubb, Robert T (2014) A sweet new role for LCP enzymes in protein glycosylation. Mol Microbiol 94:1197-200
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
Jacobitz, Alex W; Wereszczynski, Jeff; Yi, Sung Wook et al. (2014) Structural and computational studies of the Staphylococcus aureus sortase B-substrate complex reveal a substrate-stabilized oxyanion hole. J Biol Chem 289:8891-902
Ran, Yanchao; Malmirchegini, G Reza; Clubb, Robert T et al. (2013) Axial ligand replacement mechanism in heme transfer from streptococcal heme-binding protein Shp to HtsA of the HtsABC transporter. Biochemistry 52:6537-47
Spirig, Thomas; Malmirchegini, G Reza; Zhang, Jiang et al. (2013) Staphylococcus aureus uses a novel multidomain receptor to break apart human hemoglobin and steal its heme. J Biol Chem 288:1065-78
Chan, Albert H; Wereszczynski, Jeff; Amer, Brendan R et al. (2013) Discovery of Staphylococcus aureus sortase A inhibitors using virtual screening and the relaxed complex scheme. Chem Biol Drug Des 82:418-28
Sam, My D; Clubb, Robert T (2012) Preparation and optimization of protein-DNA complexes suitable for detailed NMR studies. Methods Mol Biol 831:219-32
Spirig, Thomas; Weiner, Ethan M; Clubb, Robert T (2011) Sortase enzymes in Gram-positive bacteria. Mol Microbiol 82:1044-59
Januszyk, Kurt; Fleissner, Mark R; Atchabahian, Lara et al. (2011) Site-directed spin labeling electron paramagnetic resonance study of the ORF1 protein from a mouse L1 retrotransposon. Protein Sci 20:1231-43
Anderson, Timothy D; Robson, Scott A; Jiang, Xiao Wen et al. (2011) Assembly of minicellulosomes on the surface of Bacillus subtilis. Appl Environ Microbiol 77:4849-58

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