Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

Abstract

The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-L-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc (pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release. Peptidoglycan (PG)2 is a critical structural macromolecule that makes up the cell wall surrounding the cytoplasmic membrane of nearly all bacteria. This cage-like structure is made up of long polysaccharide strands that are composed of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) with short peptide stems (3-5 amino acids) covalently attached to the MurNAc moiety (1). Depending on the organism, some proportion of peptide stems are crosslinked to peptides on adjacent strands, whereas others remain non-cross-linked. Cross-linking provides structural integrity for the macromolecule and contributes to the determination of bacterial shape.Neisseria gonorrhoeae (the gonococcus or GC) is a Gramnegative bacterium, obligate human pathogen, and etiologic agent of the sexually transmitted infection gonorrhea. Gonorrhea is the second most common reportable bacterial infection in the United States with an estimated 820,000 cases annually (2). A robust inflammatory response is the cause of much of the damage observed during GC infection and is triggered by the release of lipo-oligosaccharide, porin proteins, and fragments of PG (3-5). In particular, N. gonorrhoeae release a greater abundance of PG fragments than Escherichia co...

The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence

“…Here we show that the GNA2091 mutant has a mild cell separation phenotype resembling that of Neisseria spp. with defects in AmiC amidase protein (43). Interestingly, although E. coli have 3 amidase proteins, AmiC is the unique cell separation amidase in Neisseria.…”

“…However, in the absence of YraP in the Δ2091 mutant strains, the outer membrane phospholipid composition of N. meningitidis is altered, and outer membrane protein assembly or function is altered for a number of proteins, including ZnuD, which in turn affects efficient zinc uptake. Interestingly, the cell separation AmiC protein uses a zinc cofactor (43). The zinc transport deficiency that we observed in the YraP mutant may therefore result in reduced function of AmiC and thereby give 1 plausible explanation for the cell separation phenotype of the mutant.…”

Neisseria gonorrhoeae Lytic Transglycosylases LtgA and LtgD Reduce Host Innate Immune Signaling through TLR2 and NOD2

“…Peptidoglycan structures were confirmed by mass spectrometry. LC-MS was performed as described by Lenz et al with an Agilent 1200 series LC/MSD TOF using electrospray ionization in the positive mode 48 . Peptidoglycan was quantified using a Fluoraldehyde OPA (o-phthalaldehyde) Reagent solution (Thermo Scientific Pierce), which reacts with the single primary amine present on the diaminopimelic acid of a monomeric PGN unit.…”

The cell cycle in Staphylococcus aureus is regulated by an amidase that controls peptidoglycan synthesis

“…2). We then searched for muropeptides consistent with amidase processing and identified a species having an exact mass that suggested structure X 14 (Supplementary Fig. 3a).…”