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Role of beta-lactamase Mutations in Antibiotic Resistance

This protein structure and function project is concentrated on how mutations can alter the substrate specificity and thus the evolution of b-lactamase enzymes. b-lactam antibiotics, such as the penicillins and cephalosporins, are among the most often used antimicrobial agents. Because of their widespread use, bacterial resistance to these antibiotics has become an increasing problem. The most common mechanism of resistance is the production of b-lactamases. The b-lactamase enzyme provides resistance by catalyzing the hydrolysis of the penicillins and cephalosporins to inactive products. Because of the strong selective pressure of antibiotic therapy, b-lactamases evolve rapidly by acquiring amino acid substitutions that allow the enzymes to degrade newly developed penicillins and cephalosporins.

We are using in vitro mutagenesis to randomize the coding sequences of b-lactamases to create libraries of mutants that contain all possible amino acid substitutions for the region randomized. The random libraries are screened for mutations that alter the substrate specificity of the enzymes and the relevant mutants are characterized biochemically to understand how the substitutions change substrate specificity. These studies provide information for the design of new antimicrobials that are less susceptible to the rapid evolution of resistance.

Functional Genomics Study of Treponema pallidum

A functional genomics approach is being used to identify proteins important for the Treponema pallidum host-pathogen interaction. T. pallidum is the causative agent of syphilis. The complete genome sequence of this organism has been completed. During the previous funding period, we have used a topoisomerase-based method to clone PCR products encoding 1008 of the 1031 open reading frames identified in the genome sequence of T. pallidum. In addition, the plasmid vector system used for cloning the open reading frames, the univector system, permits the rapid conversion of the original plasmid clone set to other functional vectors containing various promoters or tag sequences.

The conversion to functional vectors is based on a single step Cre-loxP site-specific recombination reaction. Using Cre-loxP recombination, the T. pallidum clone set has been converted to specialized vectors for large scale protein expression, phage display and two-hybrid analysis. These plasmid collections will be used in a functional genomics approach to i) identify proteins involved in adhesion to host cells, ii) systematically identify T. pallidum antigenic proteins, and iii) establish a large-scale protein-protein interaction network among periplasmic and surface localized proteins.

Protein Interaction Complex of beta-lactamase with BLIP

Lab Website

Recent Publications (PubMed)

Hyser, J.M., Zeng, C.Q., Beharry, Z., Palzkill, T. and Estes, M.K. (2008). Epitope mapping and use of epitope-specific antisera to characterize the VP5 binding site in rotavirus SA11 NSP4. Virology 373:211-228.

Gao, H., Wang, X., Zang, Z.K., Palzkill, T. and Zhou, J. (2008). Probing the regulon of ArcA in Shewanella oneidensis by integrated genomic analysis. BMC Genomics 9:42.

Brinkman, M.B., McGill, M.A., Petterson, J., Rogers, A., Matejkova, P., Smajs, D., Weinstock, G.M., Norris, S.J., and Palzkill, T. (2008). A novel Treponema pallidum antigen, TP0136, is an outer membrane protein that binds human fibronectin. Infect. Immun. 76: 1848-1857.

Maejkova, P., Strouhal, M., Smajs, D., Norris, S.J., Palzkill, T., Petrosino, J.F., Sodergren, E., Norton, J.E., Singh, J., Richmond, T.A., Molla, M.N., Albert, T.J., and Weinstock, G.M. (2008). Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays. BMC Microbiology 8:76.

Titz, B., Rajagopala, S.V., Goll, J., Hauser, J., McKevitt, M.T., Palzkill, T., and Uetz, P. (2008). The binary interactome of Treponema pallidum- the syphilis spirochete. PLoS One 3:e2292.

Wang, X., Gao, H., Shen, Y., Weinstock, G.M., Zhou, J., and Palzkill, T. (2008). A high-throughput percentage-of-binding strategy to measure binding energies in DNA-protein interactions: Application to genome scale site-discovery. Nucl. Acids Res., 36:4863-4871.

Gao, H., Pattison, D., Yan, T., Klingemann, D.J., Wang, X., Petrosino, J., Hemphill, L., Wan, H., Leaphart, A.B., Weinstock, G.M., Palzkill, T. and Zhou, J. (2008). Generation and validation of a Shewanella oneidensis MR-1 clone set for protein expression and phage display. PLoS One, 3:e2983.

Marciano, D.C., Pennington, J.M., Wang, X., Wang, J., Chen, Y., Thomas, V.L., Shoichet, B.K. and Palzkill, T. (2008). Genetic and structural characterization of an L201P global suppressor substitution in TEM-1 beta-lactamase. J. Mol. Biol. 384: 151-164.

Wang, J., Palzkill, T., and Chow, D.-C. (2009). Structural insight into the kinetics and DCp of interactions between TEM-1 b-lactamase and BLIP. J. Biol. Chem. 284: 595-609.

Yuan, J., Huang, W., Chow, D.-C. and Palzkill, T.  (2009) Fine mapping of the sequence requirements for binding of b-lactamase inhibitory protein (BLIP) to TEM-1 b-lactamase using a genetic screen for BLIP function. J. Mol. Biol. 389: 401-412.