The evolvability of environmental beta-lactamases
Abstract
Antibiotic resistance in human pathogens poses an enormous threat to public health. The history of antibiotic use since the 1940s shows that resistance typically arises within a few years of the introduction of a new antibiotic into the clinic. One of the most striking Òsuccess storiesÓ in antibiotic resistance - from the point of view of microbes Ð has been the rapid evolution of the TEM and SHV families of beta-lactamases that have spread rapidly throughout the world since the introduction of penicillin in the 1940s. These families are remarkably evolvable; only a few mutations have led to the ability to destroy second- and third-generation ?-lactam antibiotics. Within the past few years, it has become evident that genes encoding beta-lactamases, as well as many other antibiotic resistance proteins, are found not only in clinical isolates, but in soil and water microbes as well. Genetic transfer between pathogens and environmental bacteria clearly occurs. Although its extent is unknown, the possibility that a vast reservoir of antibiotic resistance genes may be accessible to pathogens is a serious concern. A further unknown is whether the large number of environmental beta-lactamases are as evolvable as the TEM and SHV families that are so prevalent in clinical isolates. The level of threat posed by environmental beta-lactamases hinges on the answer to this question. The goal of this seed proposal is to conduct an initial investigation into the evolvability of beta-lactamases from bacteria living in environments that have not been exposed to clinically used beta-lactam antibiotics. Libraries of variants of two environmental beta-lactamases that confer resistance to ampicillin will be generated by site-saturation mutagenesis and error-prone PCR. Enzymes with enhanced activity toward cefotaxime will be identified by selecting for bacteria that grow on concentrations of cefotaxime that kill bacteria expressing the progenitor enzyme. The degree of improvement in catalytic activity toward cefotaxime achieved by a few mutations will be compared to that achieved in the TEM family. Additional beta-lactamases from environmental samples will be expressed and characterized in preparation for a future study that will examine the evolvability of a larger number of enzymes and will identify the structural and dynamic features that contribute to high or to low evolvability. The outcome of the broader project that will follow this small-scale project will be an assessment of the degree to which environmental beta-lactamases pose a threat to the use of beta-lactam antibiotics. In a broader sense, this work will provide a greater understanding of how evolvability can vary in enzymes from different organisms that catalyze the same chemical reaction. This issue is of great importance for synthetic biology and metabolic engineering endeavors for which it is necessary to evolve enzymes with novel catalytic capabilities.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Apr 22, 2019
- Source ID
- W911NF1510100
Entities
People
- Shelley Copley
Organizations
- Army Contracting Command
- United States Army
- University of Colorado Boulder