Functional Genomics of Heavy Metal Resistance in the Foodborne Pathogen Listeria monocytogenes.

PARSONS, CAMERON TYLER. Functional Genomics of Heavy Metal Resistance in the Foodborne Pathogen Listeria monocytogenes. (Under the direction of Dr. Sophia Kathariou). Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is found ubiquitously in nature. To be able to survive in diverse environments, L. monocytogenes has acquired adaptations such as the ability to grow in the cold, to form biofilms, and to tolerate a variety of toxic compounds. L. monocytogenes also possesses adaptations that enable it to survive and proliferate inside an animal host, such as the ability to evade the host immune system, spread cell-to-cell and invade normally sterile sites such as the placenta and central nervous system. One of the adaptations that plays a role in both environmental and in vivo survival of L. monocytogenes, is its tolerance to heavy metals. In the environment L. monocytogenes can encounter extremely low levels of the essential metals it needs to function, or extremely high levels of both essential and toxic metals; either can prove to be fatal for the cell. Inside an animal host, L. monocytogenes can also encounter extremely high or low levels of essential metals, making the ability to tightly regulate intracellular metal levels of key importance to both saprophyte and pathogen. Here, transposon (mariner-based) mutagenesis was employed to investigate the genetic basis for the tolerance of L. monocytogenes to both essential, as well as toxic metals. Mutant libraries for two produce-related strains F8027 and 2011l-2858 were screened for mutants that had an increased susceptibility to arsenic, cadmium, and copper. Four mutants were isolated with decreased tolerance to cadmium, and one mutant with a decreased tolerance to copper. One of the cadmium-sensitive mutants was found to have a transposon insertion in cadA4, a novel member of the CadA family of cadmium-effluxing P-type ATPases, harbored on a larger mobile genetic element (MGE). Genetic complementation of this mutant with chromosomally integrated cadA4 restored cadmium tolerance. Furthermore, heterologous expression in two unrelated cadmium-susceptible L. monocytogenes strains conferred cadmium tolerance comparable to that of F8027, confirming the role of cadA4 in cadmium resistance. Assessments in the Galleria mellonella model suggested that cadA4 was negatively correlated with virulence, serving to suppress virulence. Another transposon mutant of strain F8027 with decreased tolerance to cadmium was found to have a transposon insertion in an intergenic region between a previously uncharacterized cell wallassociated protein and a gene of unknown function, on the same MGE as cadA4. This mutant was found to have reduced tolerance to zinc as well. Construction of deletion mutants suggested that the cell wall-associated protein was responsible for the decreased tolerance to zinc, and genetic complementation suggested that the inactivation of one of the downstream genes was responsible for the decreased tolerance of this mutant to cadmium. Assessments in the Galleria mellonella model suggested that, in addition to its role in zinc tolerance, the cell wall-associated protein was required for full virulence potential. The cell wall-associated protein was also found to mediate wild type levels of biofilm formation. The one mutant found to have a decreased tolerance to copper, was a derivative of strain 2011L-2858, and harbored a transposon insertion in the intergenic region just before the start codon of pbp4, encoding a penicillin binding protein. While previous work had associated this gene with susceptibility to β-lactam antibiotics and bacteriocins, this was the first time that a link between this gene and copper tolerance was noted. This gene was also found to be required for full virulence potential in the Galleria mellonella model, and to impact biofilm formation at 25 and 37 ̊C. In summary, three novel determinants associated with metal homeostasis have been identified, and a previously unknown association has been established between a well-characterized gene for a penicillin-binding protein and copper homeostasis. Functional Genomics of Heavy Metal Resistance in the Foodborne Pathogen Listeria monocytogenes. by Cameron Tyler Parsons A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy