Abstract

                Enterococcus faecalis is a Gram-positive bacterium that is a normal part of the human intestinal flora. It is intrinsically resistant to cephalosporin antibiotics, helping to make it a leading cause of hospital acquired infections. The mechanism of cephalosporin resistance in E. faecalis is not fully defined, but one clue that is falling into place is a eukaryotic-like membrane-bound serine/threonine kinase (IreK). IreK has been shown to regulate an antibiotic resistance pathway in E. faecalis. A fragment of IreK that consists of the extracellular PASTA domains and the transmembrane domain of IreK was found to directly interact with RodZ_EF, a transmembrane protein proposed to be a part of the putative elongasome in E. faecalis. The elongasome is a protein machine found in rod shaped bacteria that performs localized peripheral peptidoglycan synthesis. Due to the evidence of direct interaction between the extracellular PASTA and transmembrane domains of IreK and RodZ_EF, we chose to ask the question of whether IreK and RodZ_EF, directly interact with other possible elongasome proteins. To test this question, the bacterial two hybrid assay was used to look for direct protein-protein interactions between the RodZ_EF and six E. faecalis elongasome homologues and direct protein-protein interactions between the extracellular PASTA and transmembrane domains of IreK and six E. faecalis elongasome homologues. We obtained evidence that homologues of the elongasome proteins MreC and MreD interact directly with RodZ_EF and the PASTA and transmembrane domains of IreK. These connections suggest that the elongasome is the means through which IreK senses cephalosporins in the environment, allowing IreK to react by activating the pathway for antibiotic resistance.