A Single Dual-Function Enzyme Controls the Production of Inflammatory NOD Agonist Peptidoglycan Fragments by Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae gonococcus (GC) is a Gram-negative betaproteobacterium and causative agent of the sexually transmitted infection gonorrhea. During growth, GC releases lipooligosaccharide (LOS) and peptidoglycan (PG) fragments, which contribute significantly to the inflammatory damage observed during human infection. In ascending infection of human Fallopian tubes, inflammation leads to increased risk of ectopic pregnancy, pelvic inflammatory disease, and sterility. Of the PG fragments released by GC, most are disaccharide peptide monomers, and of those, 80% have tripeptide stems despite the observation that tetrapeptide stems make up 80% of the assembled cell wall. We identified a serine-protease l,d-carboxypeptidase, NGO1274 (LdcA), as the enzyme responsible for converting cell wall tetrapeptide-stem PG to released tripeptide-stem PG. Unlike characterized cytoplasmic LdcA homologs in gammaproteobacteria, LdcA in GC is exported to the periplasm, and its localization is critical for its activity in modifying PG fragments for release. Distinct among other characterized l,d-carboxypeptidases, LdcA from GC is also capable of catalyzing the cleavage of specific peptide cross-bridges (endopeptidase activity). To define the role of ldcA in pathogenesis, we demonstrate that ldcA disruption results in both loss of NOD1-dependent NF-κB activation and decreased NOD2-dependent NF-κB activation while not affecting Toll-like receptor (TLR) agonist release. Since the human intracellular peptidoglycan receptor NOD1 (hNOD1) specifically recognizes PG fragments with a terminal meso-DAP rather than d-alanine, we conclude that LdcA is required for GC to provoke NOD1-dependent responses in cells of the human host.IMPORTANCE The macromolecular meshwork of peptidoglycan serves essential functions in determining bacterial cell shape, protecting against osmotic lysis, and defending cells from external assaults. The conserved peptidoglycan structure, however, is also recognized by eukaryotic pattern recognition receptors, which can trigger immune responses against bacteria. Many bacteria can induce an inflammatory response through the intracellular peptidoglycan receptor NOD1, but Neisseria gonorrhoeae serves as an extreme example, releasing fragments of peptidoglycan into the environment during growth that specifically antagonize human NOD1. Understanding the peptidoglycan breakdown mechanisms that allow Neisseria to promote NOD1 activation, rather than avoiding or suppressing immune detection, is critical to understanding the pathogenesis of this increasingly drug-resistant organism. We identify a peptidoglycan l,d-carboxypeptidase responsible for converting liberated peptidoglycan fragments into the human NOD1 agonist and find that the same enzyme has endopeptidase activity on certain peptidoglycan cross-links, the first described combination of those two activities in a single enzyme.

Keywords: Neisseria gonorrhoeae; high-pressure liquid chromatography; innate immunity; peptidoglycan; peptidoglycan hydrolases.

FIG 1

LdcA is exported from the cytoplasm in N. gonorrhoeae gonococci (GC). (A) l,d-Carboxypepdidases catalyze the removal of the terminal d-alanine from PG peptide stems, leaving mDAP as the terminal amino acid. (B) ClustalW alignment of serine protease LdcA sequences from alphaproteobacteria, betaproteobacteria, and gammaproteobacteria. The sequences were obtained from NCBI. The solid underline indicates a consensus TAT motif, the dotted line indicates a lipobox motif, and the arrow indicates the location of the predicted signal peptidase cleavage site. K. kingae, Kingella kingae; B. cepacia, Burkholderia cepacia; B. cenocepacia, Burkholderia cenocepacia; B. pseudomallei, Burkholderia pseudomallei. (C) Subcellular fractionation and Western blot analysis of GC strain MS11 without native ldcA, expressing C-terminally HA-tagged versions of either full-length ldcA or ldcA with a deletion of the N-terminal leader sequence (ldcA_Δ4−75). Total membrane (TM), outer membrane (OM), and soluble (sol) fractions are indicated. The soluble fraction includes both periplasm and cytoplasm. All samples were probed with control antibodies for proteins located in the outer membrane (PilQ), inner membrane (SecY), and cytoplasm (chloramphenicol acetyltransferase [CAT]; expressed from an integrated plasmid).

FIG 2

Disruption of LdcA activity alters peptidoglycan fragment release. (A to C) GC strains with a signal peptide truncation (ldcA_Δ4−75), an in-frame deletion (ΔldcA), or an active site mutation (ldcA_S165A) were metabolically pulse-chase labeled with [³H]glucosamine alongside wild-type strain MS11 (WT) and the appropriate complementation strain (+ldcA⁺) in separate experiments. PG fragment release profiles were obtained by fractionating cell-free supernatant by size exclusion chromatography and measuring radiolabeling levels by liquid scintillation counting. Profiles for each set of three strains are representative of results from at least two independent growth, labeling, and size exclusion chromatography analyses. (D to I) PG monomers were obtained from size exclusion chromatography fractions, and data corresponding to 10,000 cpm were separated by reversed-phase HPLC on a C₁₈ column. One-minute fractions were collected and measured by liquid scintillation counting. GaM-3, GlcNAc-anhydro-MurNAc-tripeptide monomer; GaM-4, GlcNAc–anhydro-MurNAc–tetrapeptide monomer.

FIG 3

LdcA has l,d-carboxypeptidase activity on PG monomers. (A to C) Radiolabeled tetrapeptide monomer fragments were provided as the substrate in a 2-h reaction with 25 nM (100 ng) N-terminally His-tagged wild-type LdcA (B) or LdcA_S165A (C) or in a control reaction with no enzyme (A). Reaction products were separated by reversed-phase HPLC. One-minute fractions were collected and measured by liquid scintillation counting. Counts from each run are displayed as a percentage of the total cpm collected in all HPLC fractions from that run. Enzyme reaction results are representative of at least three independent tests of LdcA enzyme activity on PG monomer. GaM-3, GlcNAc-anhydro-MurNAc-tripeptide monomer; GaM-4, GlcNAc-anhydro-MurNAc-tetrapeptide monomer.

FIG 4

LdcA processes peptide-linked PG dimers to PG monomers. (A) The radiolabeled PG dimer fraction was obtained by size exclusion chromatography analysis of a ΔldcA strain (Fig. 2B), and 20,000 cpm of dimers were separated by reversed-phase HPLC. (B to D) Radiolabeled peptide-linked PG dimer from an ldcA mutant was provided as the substrate in 2-h reactions with no enzyme (B) or 25 nM (100 ng) N-terminally His-tagged wild-type LdcA (C) or LdcA_S165A (D). Reaction products were separated by reversed-phase HPLC. Fractions were collected and measured; count data are displayed as described for Fig. 3. Enzyme reaction results are representative of at least two independent tests of LdcA enzyme activity on PG dimers. (E) Peptide-linked PG dimers were isolated from an LtgA digestion of ldcA_S165A pacA_H329Q sacculi and provided as the substrate in 4-h reactions performed with 25 nM (100 ng) N-terminally His-tagged wild-type LdcA or LdcA_S165A or in a control reaction performed with no enzyme. mAU, milliabsorbance units. (F) Time of flight mass spectrometry analysis of the product peak appearing at ~25 min in the His-LdcA reaction whose results are presented in panel E. The m/z values for major observed ions are indicated on the graph. (G) Separate digestions of dimer with wild-type LdcA and a no-enzyme (No enz) control (as described for panel E) were performed and analyzed by LC/MS. The observed masses corresponding to peptide-linked dimers present after LdcA reactions are indicated with their intensity values (in arbitrary units). GaM, N-acetyl-glucosaminyl-1,6-anhydro-N-acetyl-muramyl. Numbers indicate the length of peptide chains attached to each GaM unit. LC/MS analysis was performed two times with similar results; one representative set of intensity values is displayed.

FIG 5

Free tripeptides are lost from GC sacculi without functional LdcA. (A) Mutanolysin-digested sacculi from wild-type strain MS11 with a pacA_H329Q mutation (nonacetylated PG) separated by reversed-phase HPLC. The indicated peaks represent the following PG monomers: peak 1, GlcNAc-MurNAc-tripeptide; peak 2, GlcNAc-MurNAc-tetrapeptide; peak 3, GlcNAc-anhydro-MurNAc-tripeptide; peak 4, GlcNAc-anhydro-MurNAc-tetrapeptide. (B) Mutanolysin digestions and reversed-phase HPLC of nonacetylated sacculi from ldcA_S165A, ldcA_S165A + ldcA⁺, ldcA_Δ4−75, and ldcA_Δ4−75 + ldcA⁺ strains. (C) Mutanolysin digestions and reversed-phase HPLC of sacculi from strain ldcA_S165A pacA_H329Q following an overnight reaction performed with 250 nM (1 μg) N-terminally His-tagged wild-type LdcA or LdcA_S165A or a control reaction performed with no enzyme.

FIG 6

LdcA is responsible for the release of human NOD1 agonist by GC. (A to D) HEK-293 cells overexpressing hNOD1 (A), hNOD2 (B), TLR4 (C), or TLR9 (D) and transfected with an secreted alkaline phosphatase (SEAP) reporter for NF-κB activity (HEK-Blue cells) were exposed to control ligands, cGCBL medium, or cell-free supernatant from growth of wild-type strain MS11, a ΔldcA strain, or a complemented mutant (ΔldcA + ldcA⁺). SEAP levels were measured colorimetrically at OD₆₅₀, and relative NF-κB activity levels are expressed as the OD₆₅₀ values from HEK-Blue reporter cells, with background OD₆₅₀ values subtracted from those determined for a matched cell line with a SEAP reporter but no construct for NOD/TLR receptor expression (HEK-Blue Null1/2 cells). Reporter cells and Null cells were grown, treated, and measured in parallel for each experiment. Values represent means ± standard errors of averages of results from three independent experiments. (E) HCT116 cells transfected with a secreted alkaline phosphatase (SEAP) reporter for NF-κB activity (HCT116-Dual cells) were exposed to control ligands or cGCBL medium or were infected with ~1 × 10⁶ CFU/ml of pilus⁺ Opa⁻ versions of the following strains: the wild-type strain (MS11), mutant ΔldcA, mutant ΔldcA + ldcA⁺, mutant ldcA_S165A, and mutant ldcA_S165A + ldcA⁺. Measurement of SEAP activity at OD₆₅₀ provides a measurement of relative NF-κB activity levels. Values displayed are the means ± standard deviations of results from triplicate wells determined in one experiment representative of three independent experiments. (A to E) Significance was determined for the bracketed comparisons by unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant. TriDAP is a NOD1 agonist (l-Ala-γ-d-Glu-mDAP), MDP is a NOD2 agonist (muramyl dipeptide), LPS-EK is a TLR4 agonist (lipopolysaccharide from E. coli K-12), and ODN2006 is a TLR9 agonist (CpG oligonucleotide). IL-1β, interleukin-1β; TNF-α, tumor necrosis factor alpha.