Molecular Pathogenesis

Neisseria gonorrhoeae PBP3 and PBP4 Facilitate NOD1 Agonist Peptidoglycan Fragment Release and Survival in Stationary Phase

Ryan E. Schaub, Krizia M. Perez-Medina, Kathleen T. Hackett, Daniel L. Garcia, Joseph P. Dillard
Andreas J. Bäumler, Editor
DOI: 10.1128/IAI.00833-18

ABSTRACT

Neisseria gonorrhoeae releases peptidoglycan fragments during growth, and these molecules induce an inflammatory response in the human host. The proinflammatory molecules include peptidoglycan monomers, peptidoglycan dimers, and free peptides. These molecules can be released by the actions of lytic transglycosylases or an amidase. However, >40% of the gonococcal cell wall is cross-linked, where the peptide stem on one peptidoglycan strand is linked to the peptide stem on a neighboring strand, suggesting that endopeptidases may be required for the release of many peptidoglycan fragments. Therefore, we characterized mutants with individual or combined mutations in genes for the low-molecular-mass penicillin-binding proteins PBP3 and PBP4. Mutations in either dacB, encoding PBP3, or pbpG, encoding PBP4, did not significantly reduce the release of peptidoglycan monomers or free peptides. A mutation in dacB caused the appearance of a larger-sized peptidoglycan monomer, the pentapeptide monomer, and an increased release of peptidoglycan dimers, suggesting the involvement of this enzyme in both the removal of C-terminal d-Ala residues from stem peptides and the cleavage of cross-linked peptidoglycan. Mutation of both dacB and pbpG eliminated the release of tripeptide-containing peptidoglycan fragments concomitantly with the appearance of pentapeptide and dipeptide peptidoglycan fragments and higher-molecular-weight peptidoglycan dimers. In accord with the loss of tripeptide peptidoglycan fragments, the level of human NOD1 activation by the dacB pbpG mutants was significantly lower than that by the wild type. We conclude that PBP3 and PBP4 overlap in function for cross-link cleavage and that these endopeptidases act in the normal release of peptidoglycan fragments during growth.

INTRODUCTION

The bacterial cell wall provides strength and shape to the cell, resisting osmotic pressure and serving as an anchor for proteins, molecular machines, and membrane components (1–4). The cell wall is made of peptidoglycan (PG), a repeating disaccharide of N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) with short peptides attached to each MurNAc. Peptide bonds formed between portions of the peptide side chains function to cross-link the glycan strands together, making the cell wall a single interconnected macromolecule. In order for the cell wall to expand during growth, strands of PG must be degraded to allow for the insertion of more PG strands. Similarly, for the bacterium to divide and separate, it must build a PG wall to form the septum and subsequently break PG bonds to split the septal wall and allow the separation of the daughter cells (5–7).

In the Gram-negative species Neisseria gonorrhoeae, three functional classes of enzymes act to degrade cell wall material in the periplasm. Lytic transglycosylases cleave the glycan strands, cutting the MurNAc-(β-1,4)-GlcNAc bond (8, 9). An N-acetylmuramyl-l-alanine amidase cleaves the peptide chains from the glycan strands (10, 11). PG peptidases, including endopeptidases and carboxypeptidases, cut the peptide cross-links and shorten the peptide stems (12, 13). The PG fragments produced by these enzymes are released in significant amounts by growing N. gonorrhoeae cells (8, 14). An inflammatory response to the PG fragments results in host cell damage. In particular, PG fragments induce damage to cells of human fallopian tubes, causing the death and sloughing of ciliated cells in the organ culture model of pelvic inflammatory disease (15).

During infection, PG fragments are bound by pattern recognition receptors NOD1 and NOD2 (16, 17). Human NOD1 recognizes PG fragments that contain the second and third amino acids of the peptide chain (isoglutamate [iGlu]–diaminopimelic acid [DAP]) and terminate with the DAP (18). Among the PG fragments released by N. gonorrhoeae, the NOD1 agonists are the disaccharide tripeptide monomer (GlcNAc-anhydroMurNAc-l-Ala-d-Glu-meso-DAP) and the free tripeptide (l-Ala-d-Glu-meso-DAP) (11, 19). NOD2 recognizes whole sacculi, PG containing MurNAc and terminating with the dipeptide (MurNAc-l-Ala-d-Glu), and monomeric PG fragments that have a free hydroxyl at the reducing end (20–22). For N. gonorrhoeae, the NOD2 agonists include large PG fragments generated by autolysis and disaccharide-dipeptide monomers (GlcNAc-anhMurNAc-l-Ala-d-Glu). A third NOD2 agonist is generated by host lysozyme acting on glycosidically linked PG dimers and thereby creating PG monomers with a reducing end (22). Previous studies have demonstrated the roles of lytic transglycosylases LtgA and LtgD in producing toxic, monomeric PG fragments, including the disaccharide tripeptide monomer (8). Also, amidase AmiC has been shown to produce the proinflammatory free tripeptide that activates NOD1 (11). ld-Carboxypeptidase A (LdcA) has been shown to cleave DAP-DAP cross-links and also to produce NOD1 agonist PG fragments, by trimming tetrapeptides to tripeptides (12).

The roles of other endopeptidases and carboxypeptidases in PG fragment release and the induction of inflammation have not been described. The gonococcal PG-degrading enzymes PBP3 and PBP4 were characterized for biochemical function by Stefanova et al. and were found to act on purified PG sacculi or PG-related peptides. Both PBP3 and PBP4 were able cleave d-Ala–d-Ala at the C-terminal end of a peptide and were able to cleave d-Ala–d-DAP cross-links in sacculi (see Fig. S1 in the supplemental material) (13, 23). Thus, we predict that these enzymes will cleave dd-cross-links in the cell wall to separate strands during growth. The carboxypeptidase activities of PBP3 and PBP4 would be predicted to cleave the fifth amino acid from peptide side chains that are not cross-linked. These side chains with four amino acids are the substrates for an ld-transpeptidase that serves to make DAP-DAP linkages (24, 25). Here we have characterized the roles of PBP3 and PBP4 in PG composition, autolysis, PG fragment release, and NOD1 signaling. Our studies show that bacteria lacking either enzyme have few overt differences from wild-type (WT) strains but that double mutants are deficient in PG fragment release and NOD1 signaling. Also, double mutants are deficient in autolysis but exhibit increased cell death.

RESULTS

Mutants defective in dacB and pbpG are deficient in peptidoglycan fragment release.N. gonorrhoeae releases a variety of small PG fragments as it grows, and the released PG fragments can be readily monitored by metabolic labeling with [3H]glucosamine and separation of the fragments by size exclusion chromatography (14). To determine if PBP3 or PBP4 acts in PG fragment release, we characterized the dacB and pbpG mutants produced by Stefanova et al. (13, 23), comparing fragments released by the mutants to those released by the wild-type (WT) parent strain FA19. The dacB mutant (lacking PBP3) showed a level of release of monomeric PG fragments similar to that of the WT but had increased release of PG dimers (Fig. 1A). In addition, an extra monomer-sized peak was observed for the dacB mutant, migrating at a larger size than the GlcNAc-anhydroMurNAc-tetrapeptide monomer seen in WT PG profiles. Thus, this additional monomer in the dacB mutant is likely the GlcNAc-anhydroMurNAc-pentapeptide. Examination of PG fragment release from the pbpG mutant (lacking PBP4) showed no significant differences from WT FA19 (Fig. 1B). The apparent decrease in free disaccharide release by the pbpG and dacB single mutants was not consistently detected in multiple analyses.

FIG 1
FIG 1

PG fragment release from gonococcal dacB and pbpG mutants compared to that from the wild type. N. gonorrhoeae strain FA19 (WT) was compared to the dacB mutant (A), the pbpG mutant (B), the dacB pbpG double mutant (C), and the double complemented strain RS623 (D). Gonococcal strains were pulse-labeled by growth in a medium containing [6-3H]glucosamine and lacking glucose. Supernatants containing radiolabeled PG released by the cells were collected after 2.5 h of growth, and PG fragments were separated by size exclusion chromatography. Labeled PG was detected by scintillation counting. Symbols are used after the manner of Jacobs et al. (47).

PG fragment release from the dacB pbpG double mutant showed substantial differences from that for the WT parent strain (Fig. 1C). The peak on the sizing column representing the two PG monomers, disaccharide-tetrapeptide and disaccharide-tripeptide, was significantly diminished, particularly in the half of the peak that contains the disaccharide-tripeptide. Dimer release was greatly increased in the mutant, and a number of additional PG fragment peaks were detected, including a peak representing fragments larger than PG dimers, a peak at the size for a disaccharide-pentapeptide PG monomer, and a peak slightly smaller than that for the disaccharide-tripeptide monomer. The appearance of the large dimer peak and the new multimer peak is to be expected if it is assumed that PBP3 and PBP4 are the major enzymes involved in cutting PG peptide cross-links. However, the changes in the disaccharide-tripeptide monomer amounts were not predicted. The FA19 ΔdacB ΔpbpG double mutant was complemented with inducible dacB and pbpG constructs (complement). The complemented strain was restored to WT levels of released PG dimers and larger multimers (Fig. 1D). PG Monomer release was also restored in the complemented strain, although the amount of PG monomers released by the complemented strain was somewhat greater than that released by the WT.

PG fragment release from the mutants and the WT was also assessed by metabolic labeling of the peptide chains using [3H]DAP. Labeling with [3H]DAP allows the monitoring of free-peptide release in addition to monitoring of most of the PG fragments detected with [3H]glucosamine labeling. As with the [3H]glucosamine-labeled samples, the [3H]DAP-labeled samples showed only slight changes for the dacB mutant (Fig. 2A) and no changes for the pbpG mutant (Fig. 2B). The dacB mutant showed an increase in the disaccharide-pentapeptide monomer and a slight increase in dimers. However, the dacB pbpG double mutant showed significant increases in multimers and dimers (Fig. 2C). Also, very little PG monomer was released by the dacB pbpG mutant, and free peptide 1 release was abolished.

FIG 2
FIG 2

Profile of DAP-containing PG fragments released by dacB and pbpG mutants. N. gonorrhoeae strain FA19 (WT) was compared to dacB (A) pbpG, (B), and double mutant (dacB pbpG) (C) strains. Gonococcal strains were pulse-labeled by growth in a medium containing [2,6-3H]diaminopimelic acid. Supernatants containing radiolabeled PG released by the cells were collected after 2.5 h of growth, and PG fragments were separated by size exclusion chromatography. Labeled PG was detected by scintillation counting.

PG composition.We evaluated the effects of the dacB and pbpG mutations on the composition of the cell wall PG using high-performance liquid chromatography (HPLC) and mass spectrometry. Sacculi were prepared from each strain and were digested with mutanolysin to degrade the glycan strands. PG composition analysis for WT N. gonorrhoeae has been reported before, including the commonly studied strains MS11 and FA1090 (8, 26). Those studies showed a sacculus containing mostly tetrapeptide side chains, a substantial amount of tripeptide side chains, and a minority of pentapeptides and dipeptides. Our analysis of the PG of WT strain FA19 showed that it was mostly similar to other strains. However, strain FA19 showed a much larger amount of dipeptide than was found in other strains (Fig. 3).

FIG 3
FIG 3

Composition of the PG components in the sacculus. Macromolecular PG was purified from the wild type (FA19) and the dacB, pbpG, and dacB pbpG mutants. Sacculi were digested with mutanolysin, and PG fragments were separated by reversed-phase HPLC. Labels on the indicated peaks describe the length of the peptide attached to the glycan. For example, the Tri label represents GlcNAc-MurNAc-Ala-Glu-DAP. Hyphenated labels describe peptide cross-linked PG fragments. Modifications to the PG fragments are indicated in parentheses as follows: OAc, O-acetylation at the MurNAc C-6 carbon; Gly, Gly instead of Ala at position 4 or 5 of the peptide; Anh, 1,6-anhydro bond on MurNAc; DAP, DAP-DAP cross-linked peptides.

As reported previously for a dacB mutant of strain FA1090, mutation of dacB in FA19 resulted in a substantial decrease in the amount of tetrapeptides, a finding consistent with the idea that PBP3 acts as a carboxypeptidase to remove the fifth amino acid of the peptide (Fig. 3) (26). In agreement with this conclusion, there was a corresponding increase in the amount of pentapeptide fragments. Fragments containing a tripeptide were less abundant, which may indicate that PBP3 produces most of the tetrapeptide side chains in the sacculus and that this function is a necessary step before LdcA acts to create tripeptides. dacB compositional analysis also showed a substantial increase in peptide cross-links, including most peaks eluting after 75 min, a finding consistent with the function of PBP3 as an endopeptidase. Surprisingly, the largest peak in the HPLC analysis was for dipeptide fragments. The enzyme that creates these fragments is unknown, and it is not clear why loss of PBP3 function would increase dipeptide side chains.

Mutation of pbpG only slightly altered PG composition, with decreases in tetrapeptides and increases in pentapeptides and dipeptides (Fig. 3). The dacB pbpG double mutant showed a PG composition very similar to that of the dacB mutant. However, the increase in dipeptides relative to the WT was even larger. Also, tripeptide-containing PG fragments were absent from the profile. Complementation restored tetrapeptide, tripeptide, and dipeptide amounts as well as the amounts of cross-linked species. The sacculus of the complemented double mutant was nearly identical to the WT sacculus (see Fig. S2 in the supplemental material).

The PG composition analysis (Fig. 3) is in agreement with the PG fragment release profiles (Fig. 1 and 2). Pentapetides and dipeptides were increased in the sacculi, and PG monomers with pentapeptide or dipeptide side chains were increased in the released PG fragment pool of the dacB pbpG mutant. Similarly, tetrapeptides were decreased and tripeptides were absent in the cell wall of the double mutant. Tetrapeptide monomer release was greatly reduced, and tripeptide monomer release was greatly reduced or absent. The large increase in dimer and multimer release in the dacB and dacB pbpG mutants is likewise in agreement with the increase in peptide cross-links in the sacculi of these strains.

Growth, death, and autolysis.We examined the growth and autolysis of the dacB, pbpG, and dacB pbpG mutants. During growth in liquid medium, neither the dacB mutant nor the pbpG mutant showed differences in growth from the WT FA19 strain (data not shown). However, the dacB pbpG double mutant showed differences in death in liquid culture. The growth of the WT, double mutant, and double complemented strains in gonococcal base liquid (GCBL) medium was nearly identical as measured by optical density (OD) (Fig. 4A). Upon reaching stationary phase, after 4 h postinoculation, the WT and complemented strains began to show decreases in CFU counts per milliliter (Fig. 4B). The CFU counts per milliliter for the dacB pbpG mutant were lower than those for the WT at all time points, and CFU counts decreased faster for the mutant than for the WT or complemented strain.

FIG 4
FIG 4

Growth curves for the dacB pbpG double mutant and its complemented strain in comparison to that of wild-type strain FA19. (A) Growth was measured by optical density at 540 nm. Error bars represent standard deviations. (B) CFU counts per milliliter were determined by dilution plating. Measurements were made on three separate occasions. OD measurements and CFU measurements were made from the same cultures in parallel. Error bars represent the standard errors of the means. Asterisks mark values where the double mutant was statistically different from the WT (P, <0.05 by Student’s t test).

N. gonorrhoeae undergoes spontaneous lysis and death under conditions that are not favorable for growth (27). To examine the effects of the dacB and pbpG mutations on autolysis, we transferred the bacteria to a nutrient-free buffer and measured lysis over time using optical density as described previously (28). WT strain FA19 lysed rapidly, decreasing to <50% of the initial optical density in the first 60 min of incubation (Fig. 5). Each of the mutants lysed more slowly, with the pbpG mutant showing moderately reduced lysis, the dacB mutant showing substantially less lysis, and the double mutant showing the least autolysis. The double mutant only decreased in optical density by about one-third over the 120-min incubation period.

FIG 5
FIG 5

Autolysis under nongrowth conditions. Gonococcal cells grown in a rich medium were first washed in Tris-HCl buffer (pH 6.0) and then transferred to Tris-HCl buffer at a pH of 8.0 to trigger autolysis. The OD540 of each sample was measured over time using a spectrophotometer. Values are averages from four separate replicates. Error bars represent standard deviations. Asterisks indicate values that were statistically different (P, <0.05 by Student’s t test).

The growth curves and the assay of autolysis in a nutrient-free buffer appeared to be giving contradictory information; i.e., the dacB pbpG mutant was dying more than the WT in stationary phase, but in the buffer, the mutant was lysing less than WT. Therefore, we evaluated the morphology of the bacteria by use of thin-section transmission electron microscopy for samples of the bacteria grown in broth culture (Fig. 6). At the 2-h time point, during log-phase growth, the WT, double mutant, and complemented strain appeared identical. However, at the 6-h time point, after the bacteria were beginning to die, micrographs for the dacB pbpG mutant showed dead ghost cells with little electron density and few live cells. In contrast, the micrographs for the WT and complement strains showed very few dead cells at 6 h. These data suggest that the dacB pbpG mutant is not significantly defective in growth but rather that it has a defect in stationary-phase survival.

FIG 6
FIG 6

Thin-section electron micrographs of WT strain FA19, the dacB pbpG double mutant, and the complemented strain grown in broth culture. This image has been digitally lightened to improve its clarity.

We also evaluated growth by measuring the number of CFU per colony grown on GCB agar plates. For each time point, five colonies were lifted from the plate using Whatman paper pieces, and the bacteria were suspended in medium, diluted, and plated for CFU counts. For both the WT and the mutant strains, each colony contained around 106 CFU at 18 h of growth on agar, and the number of CFU per colony increased to >107 by 42 h (Fig. 7). Significant differences were seen between the WT and the dacB pbpG double mutant at all time points.

FIG 7
FIG 7

CFU counts per colony for the WT and mutants grown on GCB agar plates. At the indicated time points, colonies were lifted from the plate, diluted in GCBL, and plated in order to determine the number of live CFU present. The values reported are geometric means from three separate experiments. Error bars represent standard errors. Asterisks indicate values that are statistically different by Student’s t test (P < 0.05).

We further evaluated autolysis using an assay for RNA release during growth in liquid culture. The RNA was metabolically labeled with [3H]adenine. By use of this method, 93% of the material labeled is RNA, and 7% consists of DNA and other molecules (29). The amount of RNA released from the WT strain was the same as that from the dacB pbpG mutant, or slightly higher, throughout growth (Fig. 8A). Since the electron micrographs had shown a significant number of dead cells during late points in growth, we also determined the amount of protein in the cells in the culture in order to normalize the released RNA values (Fig. 8B). The ratios of released RNA to cell protein in the cultures did not differ for the mutant and WT strains (Fig. 8C). These data indicate that although the dacB pbpG mutant bacteria die more than the WT in stationary phase, the increased death does not occur by autolysis.

FIG 8
FIG 8

Autolysis during growth. Autolysis was measured by the release of [3H]adenine-labeled RNA. (A) RNA release over time. (B) Protein content in the cell pellet. (C) RNA released per unit of protein in the cell pellet. Values are averages for four biological replicates. Error bars represent standard deviations. Asterisks indicate values that are statistically different by Student’s t test (P < 0.05).

NOD1 and NOD2 activation.The dacB pbpG mutant exhibits altered PG fragment release, which suggested that human cells exposed to these PG fragments might show altered signaling through the PG fragment receptors NOD1 and NOD2 relative to that for cells from WT gonococci exposed to PG. The dacB pbpG mutant did not release free tripeptide or disaccharide-tripeptide and thus would be expected to show less NOD1 activation than the WT. Supernatants from gonococcal cultures were added to HEK293 cells overexpressing human NOD1 or NOD2 and carrying an alkaline phosphatase reporter controlled by NF-κB. For these experiments, we used both FA19 and a second gonococcal strain, MS11, since we found that the FA19 cell wall contains an unusually large amount of dipeptide and might show signaling differences from most gonococcal strains. Gonococcal sacculi from strain MS11 or FA19 stimulated NOD1 (Fig. 9A). Similarly, supernatants from both strains showed substantial stimulation of NOD1. However, supernatants from the dacB pbpG mutant of strain FA19 induced a significantly lower level of NOD1 signaling than the parent strain.

FIG 9
FIG 9

NOD1 and NOD2 responses to PG fragments present in gonococcal culture supernatants. HEK293 cells overexpressing human NOD1 (A) or human NOD2 (B) were treated with gonococcal supernatants (sup), purified gonococcal sacculi (PG), or control agonists. The NF-κB-dependent response was measured by determining secreted alkaline phosphatase activity. Abbreviations: tridap, Ala-Glu-DAP; trilys, Ala-Glu-Lys; MDP, muramyl dipeptide. The MDP control was MurNAc-l-Ala-l-Glu. Values are averages from at least three separate experiments. Error bars represent standard deviations. Asterisks indicate values that are statistically significant by Student’s t test (P < 0.05).

In assays of NOD2 signaling, sacculi from both strain MS11 and strain FA19 showed stimulatory activity (Fig. 9B). However, FA19 sacculi were significantly more active. Similarly, FA19 supernatants were stimulatory for NOD2, while MS11 supernatants were not. In contrast to the results with NOD1, supernatants from dacB pbpG mutants were not significantly less stimulatory for NOD2 than supernatants from the WT strains. For strain MS11, mutation of dacB and pbpG led to increased NOD2 stimulation by supernatants. An increase in the amount of dipeptide monomers, as noted in the FA19 dacB pbpG composition, would explain this increased NOD2 stimulation.

DISCUSSION

Our analysis of the composition of the N. gonorrhoeae cell wall in the dacB and pbpG mutant strains compared to that in the WT and complemented strains demonstrates that PBP3 and PBP4 act on the cell wall to cut cross-links and remove the fifth amino acid from peptide stems (Fig. S1 in the supplemental material). In N. gonorrhoeae, approximately 40% of PG peptide chains are cross-linked, and thus, 60% would carry a pentapeptide until acted on by PBP3 or PBP4 (30). The increases in pentapeptide-containing fragments in the sacculus for dacB, pbpG, and dacB pbpG mutants are consistent with the notion that these enzymes are almost exclusively responsible for breaking the d-Ala–d-Ala bonds, along with the biosynthetic transpeptidases PBP1 and PBP2. The decreases in tetrapeptide fragments in the sacculi of dacB and dacB pbpG mutants correlate with the increases in pentapeptides and are much more prominent in these mutants than in the pbpG mutant. Similarly, increases in the amounts of peptide-cross-linked fragments are prominent in the sacculi of the dacB and dacB pbpG mutants, but the amounts are relatively unchanged for the pbpG mutant. These data suggest that PBP3 performs most of the work of cutting d-Ala–d-Ala bonds and peptide cross-links in the periplasm, while PBP4 performs the same biochemical function, but to a lesser extent. These data are consistent with the results of Stefanova et al. showing high levels of carboxypeptidase activity for PBP3 in vitro and lower levels for PBP4 (23).

Mutation of dacB alone or dacB plus pbpG had significant effects on the release of soluble PG fragments. The explanations for some of these changes are readily apparent. Mutation of dacB resulted in an increase in pentapeptide side chains in the sacculus and an increase in peptide cross-links. Thus, the increased pentapeptide monomer release and the increased dimer release correlate with the changes in sacculus composition. However, dacB sacculi also showed increased dipeptide side chains and decreased tri- and tetrapeptides, but the release of di-, tri-, and tetrapeptide monomers was relatively unchanged in the dacB mutant. Furthermore, it is not clear why dacB mutation would have increased the release of free tripeptides and free tetrapeptides.

For the dacB pbpG double mutant, most of the PG fragments released were PG dimers and larger PG oligomers. This result is consistent with the compositional analysis showing increased cross-linked peptides. Other changes in the PG fragment release pattern included increased release of dipeptide monomers and pentapeptide monomers by the mutant. It appears that no tripeptide monomer was released by the double mutant, and tetrapeptide monomer release was significantly decreased. The changes in the types of monomer released are consistent with the changes in sacculus composition.

In the double complemented strain, the types of PG monomers released were restored, and the dimers and larger multimers were reduced to WT levels. The amounts of monomers released by the double complemented strain were increased over WT amounts, and this difference is likely due to overexpression of PBP3 and PBP4 from the complementation constructs. Increased production of PBP3 and PBP4 may make more PG strands accessible for degradation by LtgA, LtgD, and AmiC.

Free peptide release in the double mutant was greatly diminished or eliminated. The most abundant peptides released by WT gonococci are tetrapeptides and tripeptides. Since the mutant lacks tripeptide side chains in the sacculus, it is not surprising that no tripeptides were released. However, the dacB mutant and the dacB pbpG mutant have the same amounts of non-cross-linked tetrapeptides and decreased or absent tripeptides. Thus, it is not clear why the dacB mutant releases these free peptides and the dacB pbpG mutant does not. One possibility is that the amidase that removes peptides from the glycan strands (AmiC) cannot access these substrates. Perhaps an endopeptidase (PBP3 or PBP4) must cut the cross-links before AmiC can follow and remove the peptides. Arguing against this hypothesis is the fact that AmiC removes peptides from sacculi in vitro (11). However, in bacteria, the enzyme interacts with NlpD and possibly other proteins, and its ability to bind its substrates may be more constrained (31).

Changes in PG fragment release in the double mutant significantly reduced the NOD1 signaling response. This reduced NOD1 signaling is reminiscent of the reduced response seen in an ldcA mutant of N. gonorrhoeae (12). For both the dacB pbpG mutant and the ldcA mutant, tripeptides were no longer present in the sacculi and tripeptide-containing PG fragments were absent in the supernatant. LdcA has been shown to convert tetrapeptides to tripeptides both in the sacculus and for soluble PG monomers. LdcA also cuts DAP-DAP cross-links (12). The absence of tripeptide fragments from the dacB pbpG mutant sacculus and supernatant suggests that the substrates of LdcA are absent or inaccessible. Since tetrapeptide-containing PG is the substrate both for LdcA and for ld-transpeptidases that create DAP-DAP cross-links (24), the decreased amount of tetrapeptides in the dacB pbpG mutant sacculus would leave little substrate for LdcA to turn into tripeptides.

Our investigation made the unexpected discoveries that gonococcal strain FA19 has a high level of dipeptide chains in the sacculus and that mutation of dacB and pbpG results in increases in dipeptides. Strain FA19 has been used for numerous studies of antibiotic resistance, infection, and physiology (32–35). The unusually large amounts of dipeptide in the FA19 sacculus may result in less cross-linking and a possibly weaker cell wall than those for other strains. The large amounts of dipeptide result in greater NOD2 stimulation by the FA19 sacculi and supernatants than by those of strain MS11. The reason that dacB pbpG mutation results in increased dipeptides in the sacculus and increased dipeptides among the released PG fragments is unclear. It is possible that whatever endopeptidase creates these dipeptides uses pentapeptides or cross-linked peptides as a substrate and that the increased amounts of these substrates in the dacB pbpG mutant result in an increased dipeptide product.

Mutation of dacB and pbpG together resulted in a strain that had poor survival characteristics. Log-phase growth was nearly identical to that of the WT parent and complemented strains in our experiments, although Stefanova et al. were able to quantify a slight reduction in the growth rate for the double mutant (23). We found that the mutant cells died dramatically in stationary phase in liquid culture. Similarly, our examination of agar-grown bacteria showed fewer live CFU per colony for the mutant than for the WT. The amount of lysis in culture was not changed, although the level of lysis in buffer was reduced for the mutants. These results may indicate that the alterations to the cell wall, such as increased peptide cross-linking, make the cell harder to lyse. Thus, while the increased death of the mutant in stationary phase remains a mystery, the increased death without increased lysis may be due simply to a more extensively connected cell wall.

MATERIALS AND METHODS

Bacterial strains and growth conditions.All N. gonorrhoeae strains used in this study are derivatives of strain FA19 or MS11 (23, 36, 37). N. gonorrhoeae strain FA19 and its mutant derivatives were kindly provided by Robert A. Nicholas of the University of North Carolina, Chapel Hill. Insertional mutations in dacB and pbpG contain antibiotic marker insertions using spectinomycin resistance (Ω [38]) or kanamycin resistance (kpt [39]) genes, respectively (13, 23). Nonpiliated transparent variants were used for all assays where cell growth and viability were assessed by optical density readings. Liquid cultures of N. gonorrhoeae were grown with aeration using gonococcal base liquid (GCBL) medium, which consists of 1.5% Proteose Peptone No. 3, 0.4% K2HPO4, 0.1% KH2PO4, and 0.1% NaCl (pH 7.2), to which were added Kellogg’s supplements and 0.042% NaHCO3 (40, 41). Gonococci were also grown on GCB agar plates (Difco) with Kellogg’s supplements and were incubated at 37°C the presence of 5% CO2. Antibiotics for selection and the maintenance of selection were used at the following concentrations: 100 µg per ml for streptomycin, 50 µg per ml for spectinomycin, 50 µg per ml for kanamycin, 2 µg per ml for erythromycin, and 2 µg per ml for chloramphenicol.

Construction of the dacB pbpG double mutant complemented strain.Plasmids for the complementation of mutant genes were cloned into Escherichia coli. The dacB gene was amplified by PCR using oligonucleotides PBP3-SacI-F (5′-TGA GCT CAC AAT CAT TCC TCC TGA ATA TTA AGT TTG TGC G) and PBP3-HindIII-R (5′-TTT AAG CTT TCA GGC GCG GCG TTC TTT GC) and was digested with SacI and HindIII. The insert was ligated into pKH37 (42); the resulting colonies were selected from plates containing 25 µg per ml chloramphenicol; and the construct was named pRS146 following sequencing. The pbpG gene was amplified by PCR using oligonucleotides PBP4-SacI-F (5′- TGA GCT CCA TCC AAA CCG ACA CAC GAC GG) and PBP4-HindIII-R (5′-TTT AAG CTT TCA GGA GCG TTG CTG CAG C) and was digested with SacI and HindIII. The insert was ligated into pMR68 (43); the resulting colonies were selected from plates containing 500 µg per ml erythromycin; and the construct was named pRS149 following sequencing.

Spot transformation of complementation constructs into FA19 ΔdacB ΔpbpG on GCB plates was performed as described previously (44). Plasmid pRS149 transformants were selected on GCB plates containing erythromycin, followed by selection of pRS146 on plates containing chloramphenicol. The insertion of complemented genes into the correct loci was confirmed by PCR, and the strain was named RS623. The complemented strain was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and 5 ng per ml anhydrotetracycline to induce the expression of PBP3 and PBP4, respectively.

Peptidoglycan labeling and characterization of released fragments.Gonococcal PG was characterized as described by Schaub et al. in 2016 (8). For labeling the glycan strand of PG, liquid cultures of gonococci were grown with aeration in GCBL medium containing 0.4% pyruvate and lacking glucose, supplemented with 10 µCi/ml [6-3H]glucosamine. For labeling the peptide chains in PG, gonococci were grown in Dulbecco’s modified Eagle’s medium without cysteine, supplemented with 100 µg/ml methionine and 100 µg/ml threonine and containing [2,6-3H]diaminopimelic acid at 25 µCi/ml. Radioactive counts per minute were measured from 100-µl aliquots taken from each culture in order to normalize the amount of radiolabel incorporated into equivalent numbers of cells per culture. Normalization of the radiolabel allows for direct comparison of radiolabeled fragments shed by each culture. Labeled gonococci were then grown in liquid culture for an additional 2.5 h prior to chromatographic analysis of the PG fragments in the supernatant as described previously (8). Fragment release profiles depict a representative chromatogram from multiple assays (six assays for the WT, eight for the ΔdacB mutant, two for the ΔpbpG mutant, six for the ΔdacB ΔpbpG mutant, and two for the complemented strain).

Peptidoglycan purification and composition analysis.PG sacculi were purified from N. gonorrhoeae strains grown in liquid culture as described previously (26). Purified sacculi were digested with mutanolysin and reduced with sodium borohydride, and PG fragments were separated by reversed-phase HPLC. PG fragments of interest were identified by mass spectrometry as described previously (26). Chromatograms are representative of PG extracted from a minimum of three log-phase cultures.

Measurements of gonococcal growth.N. gonorrhoeae strains were grown on GCB agar plates overnight, and gonococci were transferred to GCBL medium with supplements, diluted to an optical density at 540 nm (OD540) of 0.2, and grown with aeration for 2 h to obtain uniform cells undergoing log-phase growth. Cultures were diluted to an OD540 of 0.2, induced if necessary, and grown with aeration for growth determination. Volumes of 20 µl were removed at each time point and were serially diluted and plated for CFU determinations.

The number of CFU per colony grown on GCB agar was determined as described by Kline and Seifert (45). Colonies were lifted from the agar plate using sterile Whatman paper pieces; bacteria were suspended in GCBL medium; and CFU counts were determined by serial dilution and plating. Each assay was repeated in triplicate on three separate occasions.

Measurements of gonococcal lysis.To measure autolysis in buffer, gonococci were grown in GCBL medium at 37°C with aeration to an OD540 of 1.2, representing late-log phase, from an initial OD540 of 0.2. Cells were centrifuged for 5 min at 1,800 × g, washed, and suspended in 1 ml of 50 mM Tris-HCl (pH 6). Approximately 0.3 ml of cells was transferred to two culture tubes, each containing 6 ml of 50 mM Tris-HCl (pH 8). Cell lysis was monitored by a decrease in turbidity from an initial OD540 of 0.2 at room temperature. Each assay was performed four times on separate occasions.

To measure autolysis during growth, N. gonorrhoeae RNA was metabolically labeled by growth in the presence of 2 µCi/ml [3H]adenine, and RNA released into the medium was measured over a 6-h chase period (42). Because of the increased death of the dacB pbpG mutant at later time points during growth, protein measurements determined by the Bradford assay were used to normalize RNA release to the protein content of the cell pellet. This assay was performed in triplicate on separate occasions.

Microscopy.Thin-section electron micrographs of gonococcal strains from two independent experiments were obtained as described by Mehr et al. (46).

Measurement of NOD1 and NOD2 activation.N. gonorrhoeae strains were inoculated at an OD540 of 0.2 and were grown for 2 h in GCBL medium. The bacteria were removed by centrifugation and filtration, and the supernatants were normalized to total protein content. Supernatants were used to treat HEK293 cells overexpressing NOD1 or NOD2 as described previously (11). Secreted alkaline phosphatase (a reporter for NF-κB activation) was measured as absorbance at 650 nm following incubation of the cells in QUANTI-Blue medium according to the manufacturer’s directions (InvivoGen). Assays were repeated independently three times for NOD1 and five times for NOD2.

ACKNOWLEDGMENTS

This work was supported by NIH grant R01AI097157 to J.P.D. D.L.G. was supported by NRSA F31AI054325, and K.M.P.-M. was supported by NIH National Research Service Award T32 GM007215.

We thank R. A. Nicholas of the University of North Carolina at Chapel Hill for providing dacB and pbpG mutants of N. gonorrhoeae.

FOOTNOTES

    • Received 19 November 2018.
    • Accepted 25 November 2018.
    • Accepted manuscript posted online 3 December 2018.

  • Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00833-18.

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KEYWORDS

NOD1
NOD1 agonist
carboxypeptidase
endopeptidase
penicillin-binding proteins
peptidoglycan

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