β-lactamase Mediated Antibiotic Resistance
The most common mechanisms of resistance to β-lactam antibiotics in Gram negative bacteria is the hydrolysis of the antibiotics by a group of enzymes collectively called β-lactamases. Antibiotic resistance by bacteria harboring these enzymes results in 23,000 deaths in the United States every year, and the number of antibiotics that can be effectively used in the clinics is shrinking. There are two broad classes of β-lactamases: serine-β-lactamases, which use a nucleophilic serine proceed through a covalent intermediate, and metallo-β-lactamases, which catalyze a direct attack of the water on the β-lactam ring. The Palzkill Lab studies major enzymes from both the serine and metallo-β-lactamase groups.
- Evolution of TEM-1 β-lactamase
- Characterizing mutations that increase protein stability and alter substrate specificity in CTX-M-14 β-lactamase
- Structural basis of carbapenem resistance in OXA β-lactamases
- Structural and molecular basis of carbapenem resistance by KPC-2 β-lactamase
- Sequence requirement for the function of metallo-β-lactamases CphA and NDM-1
- Understanding the inhibition of β-lactamases by BLIP (β-lactamase inhibitor protein) and BLIP-II
The lab uses a combination of site-directed mutagenesis, protein purification, steady state and pre-steady state kinetics, and X-Ray crystallography to understand the molecular basis of β-lactamase catalysis and substrate specificity.
β-lactam Resistance in MRSA
In contrast to Gram negatives, the most prevalent mechanism of β-lactam resistance in the Gram positive bacteria Staphylococcus aureus is the production of PBP2a, which is able to avoid the inhibitory effects of the antibiotics. This is the means by which methicillin-resistance S. aureus (MRSA) is able to persist despite treatment with multiple β-lactam antibiotics. Previous research in the Palzkill lab found that the protein BLIP-II (β-lactamase inhibitory protein) was able to weakly bind and inhibit PBP2a, thus making the S. aureus susceptible to β-lactam antibiotics. Current research is focused on identifying mutations that increase the affinity of BLIP-II to PBP2a.
Norovirus Diagnostics and Therapeutics
Noroviruses are the leading cause of acute gastroenteritis and foodborne illness, resulting in about 21 million cases in the United States each year. Infections spread rapidly in areas of close human contact and most severely affect children, the elderly, and immunocompromised patients who can become chronically infected. Current diagnostics--RT-PCR and enzyme immunoassays--are severely limited for use at the point-of-care, and there are currently no treatments available besides rehydration. The lab is involved in the development of antibody and peptides as rapid diagnostic tools for Norovirus infections using phage display technology, as well as single chain antibodies that bind and inhibit Norovirus.
Molecular Recognition in Protein-protein Interactions
The Palzkill Lab is studying the basis of molecular recognition in these protein-protein interactions, which serve critical roles in cellular function. For this purpose, we are using BLIP (β-lactamase Inhibitory Protein) and BLIP-II and their interactions with serine β-lactamases as a model system. The methods used for these studies include stop-flow kinetics, enzyme inhibition assays, and X-Ray crystallography.