In symptomatic Neisseria gonorrhoeae infections, the inflammatory response to bacterial products results in tissue damage and pain in the human host. Peptidoglycan fragments released by the bacteria during growth represent a major pro-inflammatory factor in these infections. In particular, the peptidoglycan monomers are known to cause the death and sloughing of ciliated cells in human Fallopian tubes, and one of the peptidoglycan monomers is an agonist for the intracellular pattern-recognition receptor NOD1. Peptidoglycan dimers, also released by the bacteria, are processed by host lysozyme to create peptidoglycan fragments that are agonists for another peptidoglycan sensing receptor, NOD2. Peptidoglycan monomers and dimers are created by enzymes called lytic transglycosylases that function to degrade strands of peptidoglycan in the cell wall so that additional strands can be inserted to facilitate growth. The lytic transglycosylase LtgA produces nearly half of the peptidoglycan monomers released by gonococci and a vast majority of the peptidoglycan fragments that are recycled. LtgD produces the rest of the peptidoglycan monomers released by N. gonorrhoeae. Published transcriptomic studies indicate that ltgA is subject to transcriptional regulation, and this regulation may therefore affect gonococcal cell wall metabolism and release of pro-inflammatory peptidoglycan fragments. To understand how N. gonorrhoeae might control peptidoglycan fragment metabolism or release, we previously performed a proteomics analysis of five different mutants that have distinct defects in peptidoglycan fragment recycling. This analysis identified a putative transcriptional regulator, NGO1982, as significantly increased in three of the mutants. Deletion of ngo1982 resulted in 8-9 fold increased transcript levels for ltgA, suggesting that NGO1982 is a repressor of ltgA. Since published studies had identified MtrR as an activator of ltgA and the antisense RNA NgncR_246 as an additional regulator of ltgA expression, ltgA appears to be regulated by three distinct factors. To understand the consequences of this regulation for infection, we will: 1) Determine how growth in human tissues affect ltgA and ltgD regulation, and how this regulation affects cell wall-related phenotypes in infection, and 2) Use biochemical and genetic methods to determine the molecular mechanisms used by these regulators. Preliminary results demonstrate that ltgA is strongly regulated, and that regulatory factors can greatly increase or decrease peptidoglycan fragment release. In cervical infection, LtgA and LtgD levels decreased even as colony forming units increased. Significantly reducing or greatly increasing peptidoglycan fragment release may allow gonococci to cause asymptomatic infection in some tissues and highly inflammatory infections other tissues.
Bacteria produce small fragments of the cell wall as they grow, and a number of bacterial pathogens release those molecules and induce an inflammatory response in the host. These studies will examine how producers of those cell wall fragments are controlled in the pathogen Neisseria gonorrhoeae. These studies will determine in what human tissue infections those regulatory mechanisms are used and if lack of regulation makes the bacteria sensitive to host defenses.
