Mutational analysis of LtgC, a lytic transglycosylase required for cell separation in Neisseria gonorrhoeae

Abstract

Lytic transglycosylases function to degrade peptidoglycan strands that comprise the bacterial cell wall. Degradation of peptidoglycan at the septum following cell division is necessary for cell separation, and a deletion of ltgC in Neisseria gonorrhoeae results in growth in clusters of around 6-20 cells rather than as normal diplococci or monococci. N. gonorrhoeae LtgC is a homolog of Escherichia coli MltA, and comparison of the two proteins shows that LtgC has an extra domain not found in MltA, referred to as domain 3. To better understand the function of LtgC, we characterized N. gonorrhoeae mutants with substitutions in amino acids predicted to be necessary for enzymatic activity or amino acids predicted to be on the surface of domain 3, and we characterized a mutant lacking domain 3. All the mutants showed defects in cell separation, and the bacteria failed to release peptidoglycan-derived disaccharides into the medium. Purified LtgC proteins with the amino acid substitutions had reduced peptidoglycan degradation activity. LtgC was found to bind AmiC in bacterial 2-hybrid assays, and domain 3 mutations reduced binding. In human blood, an ltgC mutant showed decreased survival, suggesting the cell wall defects in the mutant make the bacteria more sensitive to innate immune system components. ImportanceNeisseria gonorrhoeae uses a smaller set of proteins for peptidoglycan breakdown compared to Escherichia coli or other model systems. The peptidoglycan breakdown that occurs at the septum following cell division in N. gonorrhoeae requires three proteins, amidase AmiC, amidase activator NlpD, and lytic transglycosylase LtgC. LtgC has an unusual structure that includes a third domain not found in related proteins. Using mutants that lacked LtgC activity or had amino acid changes in the third domain, we found that the extra domain is involved in interaction of LtgC with AmiC and that it is required for LtgC function for cell separation. All of the ltgC mutants examined showed reduced survival in blood, indicating the importance of LtgC activity for infection.