Lytic transglycosylases LtgA and LtgD perform distinct roles in remodeling, recycling and releasing peptidoglycan in Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae releases peptidoglycan (PG) fragments during infection that provoke a large inflammatory response and, in pelvic inflammatory disease, this response leads to the death and sloughing of ciliated cells of the Fallopian tube. We characterized the biochemical functions and localization of two enzymes responsible for the release of proinflammatory PG fragments. The putative lytic transglycosylases LtgA and LtgD were shown to create the 1,6-anhydromuramyl moieties, and both enzymes were able to digest a small, synthetic tetrasaccharide dipeptide PG fragment into the cognate 1,6-anhydromuramyl-containing reaction products. Degradation of tetrasaccharide PG fragments by LtgA is the first demonstration of a family 1 lytic transglycosylase exhibiting this activity. Pulse-chase experiments in gonococci demonstrated that LtgA produces a larger amount of PG fragments than LtgD, and a vast majority of these fragments are recycled. In contrast, LtgD was necessary for wild-type levels of PG precursor incorporation and produced fragments predominantly released from the cell. Additionally, super-resolution microscopy established that LtgA localizes to the septum, whereas LtgD is localized around the cell. This investigation suggests a model where LtgD produces PG monomers in such a way that these fragments are released, whereas LtgA creates fragments that are mostly taken into the cytoplasm for recycling.

Figure 1

Soluble cell wall components liberated by LtgA and LtgD separated by HPLC. Reactions were performed by incubating 500 μg of purified gonococcal sacculi with purified LtgA, LtgD, or corresponding active site mutants. Soluble products were separated by reversed-phase HPLC using an ACN gradient. Sacculi from strains lacking O-acetylation and wild-type strains were digested. Peaks were identified by LC-MS of similarly digested sacculi. G, N-acetylglucosamine; aM, 1,6-anhydro-N-acetylmuramic acid; numbers represent the number of peptide stem amino acids (L-Ala, D-Glu, meso-DAP, D-Ala, D-Ala).

Figure 2

Liberated PG monomers from sequential digestion of sacculi with LtgA and LtgD. Soluble PG fragments from whole sacculi digests with LtgA or LtgD followed by boiling and a subsequent digest with LtgA or LtgD were separated by HPLC. The abundance of PG monomers (tripeptide (GaM-3) and tetrapeptide (GaM-4)) was determined by calculating the peak area (AUP) from three independent experiments. Horizontal bars indicate significance of P < 0.05, and (*) indicates P < 0.01 by Student’s t-test. Error bars represent standard deviation.

Figure 3

Digestion of a synthetic tetrasaccharide dipeptide. (A) A tetrasaccharide dipeptide was incubated with the indicated LT, and the resulting products were separated by reversed-phase HPLC using an ACN gradient. Incubation with LtgA and LtgD yielded two peaks corresponding to (i) reducing pentapeptide monomer and (ii) anhydro pentapeptide monomer. Digestion with LtgC and the no enzyme control yielded a single peak (iii) corresponding to undigested tetrasaccharide dipeptide. (B) Chemical structure of synthetic compound iii and observed digestion products with exact masses confirmed by mass spectrometry. The arrow indicates the site of LtgA and LtgD activity that results in the formation of a 1,6-anhydro bond indicative of LT activity.

Figure 4

Localization and lipidation of LtgA and LtgD. (A) Strains containing C-terminal 3XFLAG at the native site of ltgA or ltgD were fractionated into outer membrane (OM), total membrane (TM), and soluble (SOL) fractions using ultracentrifugation. Anti-FLAG antibodies were used to determine the subcellular localization of LtgA and LtgD, which localized to the same fractions as the OM control, PilQ. The strains used had a chromosomal cat gene to identify the cytoplasmic fraction and the presence of SecY was used as an inner membrane control. (B) The incorporation of the lipid [³H]-palmitate into recombinant LTs producing wild-type (WT), cysteine to alanine lipidation mutations (LM), or proteins lacking signal sequences (SM). The (*) indicates an active site mutant was used.

Figure 5

PG fragments released from growing ltgA and ltgD mutants. Cultures of gonococci were pulsed with [³H]-glucosamine to label PG, and released fragments were collected during a chase period. Radiolabeled fragments were separated by size-exclusion chromatography and measured by liquid scintillation counting. (A) Active site point mutants release PG fragments nearly identical to gene deletions with increased (1) tetrasaccharide dipeptide and (2) tetrasaccharide peptide, and decreased (3) anhydrodisaccharide peptide monomers and (4) free anhydrodisaccharide. (B) Disrupting the lipidation of LtgA had no effect on the amount of peptidoglycan fragments released, but disrupting lipidation of LtgD decreased the amount of released PG monomers. (C) Chemical structure of detected PG fragments released by N. gonorrhoeae. 1, tetrasaccharide dipeptide; 2, tetrasaccharide peptide; 3, anhydrodisaccharide peptide monomer; 4, free anhydrodisaccharide

Figure 6

Cellular PG fragments extracted from LT and ampG mutants. Hot water extracts from cultures labeled with [³H]-glucosamine were separated by size-exclusion chromatography to determine the abundance of PG precursors and fragments in the cell. (A) Mutation of ltgD resulted in increased cellular UDP-MurNAc-pentapeptide compared to wild-type. (B) Mutation of ampG decreased the amount of detectable PG precursors, but an ltgD ampG mutant increased UDP-MurNAc-pentapeptide and UDP-MurNAc-tripeptide compared to wild-type. (C) A double ltgA ampG mutant resulted in a decrease in the amount of radiolabeled PG fragments in the cell. (D) The ltgD(C19A) lipidation mutant contained wild-type levels of PG precursors, but the ltgD(C19A) ampG mutant contained high levels of UDP-MurNAc-tripeptide and -pentapeptide like the ltgD ampG mutant. UM5, UDP-MurNAc-pentapeptide; UM3, UDP-MurNAc-tripeptide.

Figure 7

PG fragments released from strains deficient in AmpG-mediated PG recycling. Strains were labeled with [³H]-glucosamine. (A) Deletion of ampG causes the release of significantly more PG monomers and free anhydrodisaccharide compared to wild-type. (B) The ltgA ampG and ltgD ampG mutants released 84.3% and 36.2% less PG monomer, respectively, compared to an ΔampG mutant. (C) Mutations affecting the lipidation of LtgA or LtgD had no significant effect on the amount of PG monomers released in an ampG deletion background. 1, tetrasaccharide dipeptides; 2, tetrasaccharide peptides; 3, anhydrodisaccharide peptide monomers; 4, free anhydrodisaccharide.

Figure 8

Cellular localization of LtgA and LtgD. LT deletion strains were complemented with chromosome insertions of inducible LtgA3XFLAG or LtgD3XFLAG. Localization of LTs was determined using an anti-FLAG antibody, and cells were counterstained with DAPI. (A) LtgA localized to the cell septum perpendicular to the plane of the diplococci. Monococci lacked localized signal. (B) LtgD is expressed at discrete focal points around the diplococci and is not predominantly localized at the cell septum. Scale bar = 2μm.