Peptidoglycan metabolism and fragment release

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As bacteria grow and divide, they constantly break down peptidoglycan strands to allow for cell wall expansion or reshaping the cell wall for septation and cell separation. Unlike most Gram-­negative bacteria, Neisseria gonorrhoeae releases significant amounts of the peptidoglycan fragments produced during these processes. The released peptidoglycan fragments induce inflammatory responses in multiple cell types and cause sloughing of ciliated cells in the Fallopian tube. The ciliated cell sloughing is characteristic of gonococcal pelvic inflammatory disease and leads to the loss of the ability to move an egg down the oviduct. We have worked to characterize the enzymes that act in peptidoglycan breakdown so that we can understand the process of peptidoglycan fragment production. We are characterizing these enzymes to determine their substrates and products as well as subcellular localization and location relative to the septum on the gonococcal cell. Using mutants lacking genes for certain peptidoglycan degradation enzymes, we determined that ciliated cell sloughing occurs in response to peptidoglycan monomers with a three amino acid peptide, implicating pattern-­recognition receptor NOD1 in the cellular response. We also discovered that gonococci release a second immunostimulatory muropeptide, a glycosidically-­linked peptidoglycan dimer that stimulates NOD2. In this study we will characterize lytic transglycosylases for the generation of the peptidoglycan dimers and characterize the enzymes that create the free peptide that serves as a NOD1 agonist. We will examine AmpG function in peptidoglycan fragment recycling and determine how gonococci sense peptidoglycan fragments in the cytoplasm and regulate peptidoglycan fragment release or uptake. Finally we will characterize cellular responses to released peptidoglycan fragments to understand how Fallopian tube tissue is damaged in pelvic inflammatory disease and how neutrophils respond to released eptidoglycan fragments.