MtrR Regulates a Major Lytic Transglycosylase (ltgA) Responsible for Peptidoglycan-Derived Cytotoxin Release and Autolysis in Neisseria gonorrhoeae

TL;DR

This study shows that MtrR regulates a key enzyme in Neisseria gonorrhoeae that affects cell wall breakdown and toxin release.

Contribution

The study identifies MtrR as a regulator of ltgA, linking it to peptidoglycan-derived cytotoxin release and autolysis in Neisseria gonorrhoeae.

Findings

MtrR binds to the ltgA promoter and increases its expression during exponential growth.

Deleting mtrR reduces peptidoglycan monomer release and increases bacterial autolysis.

MtrR regulation of ltgA impacts cytotoxin release and contributes to gonococcal pathogenesis.

Abstract

The multiple-transferable resistance protein (MtrR) is a transcriptional repressor of the mtrCDE-encoded drug efflux pump and Type IV pilus biosynthesis (pilM), and an activator of penicillin-binding protein 1 (ponA) expression in Neisseria gonorrhoeae. Previously published microarray data suggested that MtrR is also an activator of ltgA expression in the gonococcus. LtgA is a lytic transglycosylase responsible for approximately half of recycled peptidoglycan fragments and released peptidoglycan-derived cytotoxins, which cause ciliary damage and induce specific inflammatory responses. The fragments generated by LtgA during peptidoglycan remodeling can either be recognized by the permease AmpG for uptake into the bacterial cytoplasm and recycled for new cell wall growth and general metabolism or released into the external milieu. Therefore, we sought to define the capacity of MtrR to regulate LtgA expression in gonococci. We show that MtrR binds to the ltgA promoter region in a concentration-dependent manner, and that this binding results both in increased ltgA mRNA transcription and LtgA protein levels during exponential growth. Deletion of mtrR in N. gonorrhoeae decreased peptidoglycan monomer release from growing cells and increased autolysis. These results suggest that MtrR regulation of ltgA impacts peptidoglycan-derived cytotoxin release and autolysis in the gonococcus. This study suggests a central role of MtrR in coordinating aspects of the cellular envelope that may contribute to gonococcal pathogenesis.

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1. Importance

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection, gonorrhea, which affects millions of people worldwide because of its ability to survive and suppress both the innate and adaptive immune response, respectively. Thus, the gonococcus has the capacity to stimulate a local inflammatory response by releasing peptidoglycan-derived cytotoxins (PGCT) and concomitantly resist neutrophil killing. We have demonstrated that PGCT release and autolysis via LtgA activity are regulated in Neisseria gonorrhoeae by the same cytoplasmic protein, MtrR, that also regulates antibiotic resistance. Modulation of specific immune responses via MtrR regulation of LtgA activity may provide the gonococcus with a selective advantage over other bacteria at the site(s) of infection.

2. Introduction

Neisseria gonorrhoeae is a strict Gram-negative human pathogen that causes the sexually transmitted infection, gonorrhea. Infections usually initiate on the surfaces of mucosal epithelia located in the genitourinary tract [1], but can also infect the rectum, pharynx, and conjunctiva of the eye [2,3]. The incidence of gonococcal infections remains high despite public health efforts to promote awareness and prevention. It has been estimated by the WHO that over 78 million people are infected with gonorrhea annually [3], which has made it a global concern [4,5]. The Centers for Disease Control and Prevention currently recommends a single dose treatment with ceftriaxone for treatment of gonorrhea [6]; however, recent reports have identified resistance to ceftriaxone and previously recommended antibiotics [7,8]. Recently, the FDA approved two new oral antimicrobials, zoliflodacin (Nuzolvence) and gepotidacin (Blujepa), demonstrating clinical efficacy comparable to standard ceftriaxone-based therapy for uncomplicated urogenital gonorrhea [9]. The global spread of multidrug-resistant strains of N. gonorrhoeae (gonococcus, GC) could lead to increased incidences of pelvic inflammatory disease, infertility, ectopic pregnancy and disseminated gonococcal infections. Hence, early detection and new treatment strategies are essential to reducing the disease burden associated with a gonococcal infection.

Peptidoglycan (PG) is an essential cell wall component, and its synthesis is a major target of several antibiotics. PG comprises parallel glycan strands of alternating aminosugars, N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG), linked by a β-1,4-glycosidic bond, with peptides connected to each NAM residue that are cross-linked to peptides from adjacent glycan strands [10]. Mature PG confers cell shape, rigidity, and resistance to osmotic pressure, and this layer is constantly remodeled during cell growth. PG fragments liberated during this process can be released or recycled. Lytic transglycosylases (LTGs) are a class of PG lyases that facilitate cell wall growth and cell separation [11,12,13], release of cytotoxic muropeptides [14,15], and insertion of secretion systems and surface appendages [16,17,18,19]. These enzymes cleave the β-1,4-glycosidic bond linking NAM and NAG residues, with subsequent formation of a 1,6-anhydro-N-acetylmuramic acid residue. Although N. gonorrhoeae recycles PG fragments, it also releases sufficient immunologically active PG monomers to cause cell damage and inflammatory cytokine responses [10,20,21]. Gonococci release two types of soluble PG monomers: one form is structurally identical to tracheal cytotoxin produced by Bordetella pertussis [14], while the other is the NOD1 agonist 1,6-anhydro-disaccharide-tripeptide PG monomer [22,23]. These gonococcal PG-derived cytotoxins (PGCT) can induce inflammatory cytokines [24,25], NOD1 signaling and mucosal damage [21,23]. Damage at mucosal surfaces reduces ciliary propulsion, can result in tubal-factor infertility, and may affect bacterial attachment and tissue invasion [26,27,28].

Most of the recycled PG and released PG-derived cytotoxins in GC are produced by the activity of two enzymes, LtgA and LtgD [15]. In PG recycling, the cytoplasmic membrane protein AmpG acts as a permease for PG fragments and is specific for molecules containing the GlcNAc-anhydro-MurNAc disaccharide [29]. Thus, PG monomers and free disaccharides are taken up into GC for recycling. Since only LTGs can create the 1,6-anhydro bond on PG, LTGs must liberate the PG fragments if they are to be recycled via the AmpG pathway. Consequently, a double-knockout strain (ltgA and ltgD) cannot recycle PG fragments or release PG monomers [15] and these enzymes also limit TLR2 and NOD2 recognition by generating monomers that weakly activate NOD2 compared to those produced by host lysozyme [30].

Although PG recycling and release via LTG activity has been well studied in Neisseria [10] and other bacteria [31,32], how Neisseria LTGs are regulated during growth, cell separation, and/or infection is unclear. One of the major transcriptional regulators in N. gonorrhoeae is the multiple-transferable resistance repressor protein, MtrR, which affects the cell envelope by altering the expression of the MtrC-MtrD-MtrE efflux pump [33,34,35], penicillin-binding protein 1 (PBP1), and pilus biosynthesis genes in GC [36]. A microarray study performed by Shafer et al. determined that one of the 69 genes regulated by MtrR was ltgA, the only lytic transglycosylase identified in the study [37]. To further define MtrR regulation of ltgA, we examined ltgA mRNA transcription and LtgA levels in N. gonorrhoeae FA19 wild type and isogenic mtrR mutant strains. In this study, we report that MtrR positively regulates ltgA mRNA transcription and LtgA levels through direct binding to the ltgA promoter. Consistent with this observation, metabolic labeling of gonococcal PG and chromatographic separation of released PG fragments showed that the ΔmtrR mutant releases fewer PG monomers and shows increased autolysis at a pH conducive for autolysin activity. These data suggest that MtrR regulates ltgA expression and allows the gonococcus to regulate release of PG-derived cytotoxins and cell lysis during growth.

3. Materials and Methods

Bacterial stains and culture conditions. The bacterial strains used in this study are described in Table 1. All the gonococcal strains were constructed using N. gonorrhoeae FA19 as the parental strain. Gonococcal strains were grown on gonococcal medium base (GCB) agar containing Kellogg’s supplements under 5% (vol/vol) CO_2_ at 37 °C or in GCB liquid medium (GCBL) (Difco/Becton Dickinson, Franklin Lakes, NJ, USA) containing Kellogg’s supplements [38] and 0.042% NaHCO_3_ at 37 °C with aeration. FA19 and its isogenic strains (GC3mtrR) were kindly provided by Dr. William Shafer (Emory University). Strain HL9 (FA19 mtrR::Kan) was generated by transforming genomic DNA from strain KH9 (FA19 mtrR::Kan). The mutation was confirmed by PCR and sequencing, and the resulting transformed strain was then designated HL9. Erythromycin, chloramphenicol, and kanamycin were used at 1 μg/mL, 2 μg/mL, and 50 μg/mL, respectively.

RNA Isolation. GCBL cultures of strains FA19, HL9 (FA19 ΔmtrR), and GC3mtrR (ΔmtrR complemented with wild type mtrR) [36] were grown under normal growth conditions and harvested at the exponential phase. Total RNA was isolated using Trizol (Invitrogen, ThermoFisher, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA integrity was examined by gel electrophoresis and quantified using a Nanodrop1000 (NanodropTechnologies, Wilmington, DE, USA). Samples were checked for genomic DNA (gDNA) contamination using ribonuclease P RNA (rnpB) primers (Table 2) without reverse transcriptase in quantitative real-time PCR reactions as described below.

Quantitative Real-time PCR (qPCR). Total RNA was reverse-transcribed into complementary DNA (cDNA) using the high-capacity cDNA Reverse-Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s protocol. The PCR reaction contained cDNA (300 ng), 5 μM each of gene-specific qPCR primers (Table 2), and Sybr Green (Applied Biosystems, Grand Island, NY, USA) in a final volume of 25 µL. All transcripts were assayed in triplicate on three independent biological samples with appropriate negative controls. Changes in gene expression between wild type and mutant strains were converted to fold differences using the constitutively expressed rnpB gene as the endogenous control [41].

EMSAs. Electrophoretic mobility shift assays (EMSAs) were carried out using purified MtrR fused to maltose-binding protein (MBP-MtrR) [42] (kindly provided by Dr. William Shafer, Emory University, Department of Microbiology and Immunology). All promoter probes used were amplified from FA19 gDNA and labeled using a PCR digoxigenin (DIG) labeling kit (Roche, Indianapolis, IN, USA). The promoter region located upstream of the translational start site for ltgA was amplified using specific primer sets (Table 2). Therefore, a 300 and 120 bp DIG-labeled promoter fragment was used in titration and competitive EMSAs, respectively, while four fragments ranging from 37 to 300 bp were used to localize MtrR binding sites within the putative ltgA promoter region. DIG-labeled promoter fragments were purified using a QIAquick purification kit (Qiagen, Germantown, MD, USA). Increasing amounts of MBP-MtrR (8.7–261 pmol) in 15 µL reactions containing from 0.5 to 4 pmol DIG-labeled ltgA fragments of varying sizes (assay dependent) in binding buffer [5 mM Tris-HCL (pH 7.5), 4% (vol/vol) glycerol, 0.5 mM dithiothreitol, 0.5 mM EDTA, 50 mM NaCl, and 1 µg poly(dI-dC)] were incubated at 4 °C for 30 min. Unlabeled fragments were present at 50-fold excess of DIG-labeled fragments for all competition assays performed. Samples were electrophoresed on a 6% (wt/vol) polyacrylamide gel and then transferred to nitrocellulose membranes. Anti-digoxigenin-AP antibody (diluted 1:5000; Roche) was incubated with membranes overnight at 4 °C and then incubated with goat anti-mouse IgG-HRP conjugate (diluted 1:3000; Bio-Rad, Hercules, CA, USA) for 2 h at room temperature. Bands on the nitrocellulose membranes were detected using 5-bromo-4-chloro-3-indolylphosphate (BCIP) and Nitro Blue Tetrazolium (NBT) and then scanned and analyzed using ImageJ v1.51a [43].

Preparation of whole cell extracts. N. gonorrhoeae strains FA19, HL9, GC3mtrR, and LW9279 (FA19 ΔltgA) were grown overnight on GCB agar plates at 37 °C in 5% CO_2_. Colonies were resuspended to an OD_560_ of 0.08 in 1 L of GCBL medium containing Kellogg’s supplements [38] and 0.042% NaHCO_3_. Cultures were grown at 37 °C with shaking until they reached an optical density OD_560nm_ of 0.4 to 0.6, and the cells were harvested by centrifugation at 3000× g for 15 min. The media was discarded and the cell pellet was resuspended in 5 mL of B-Per Bacterial Protein extraction reagent (Thermo Scientific/Pierce, Pittsburgh, PA, USA). The cell resuspension was vortexed and then gently mixed at room temperature for 10 min. Soluble and insoluble proteins were separated by centrifugation at 27,000× g. The soluble proteins were analyzed by Western blots.

Western Blot Analysis. Whole cell lysates (3 μL) were mixed with 3 µL 2× SDS-PAGE loading buffer, heated for 5 min, and then electrophoresed on an SDS-PAGE (10%) gel. Proteins in the gel were transferred using a Mini Trans-Blot Electrophoretic transfer cell to nitrocellulose membranes in buffer (25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3) and blocked in 5% non-fat dried milk in 1× Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 30 min. Membranes were incubated overnight at 4 °C with a 1:1000 dilution (in 1× TBS) of LtgA-pC, a polycolonal antibody (Genscript Inc., Piscataway, NJ, USA) raised against purified His-tagged LtgA of N. gonorrhoeae. The membranes were washed three times for 10 min each and then incubated with a 1:3000 dilution of goat anti-rabbit IgG-HRP conjugate in 1× TBS (Bio-Rad, Hercules, CA, USA) for 2–3 h at room temperature. After three 5 min washes in TBST, the membranes were developed using 5-bromo-4-chloro-3-indolylphosphate (BCIP) and Nitro Blue Tetrazolium (NBT) as described above.

PG labeling and gel filtration chromatography. Pulse-chase labeling for quantitative comparisons of PG fragment release was performed as previously described [23]. Gonococci were grown to mid-log phase and then resuspended at an OD_560_ of 0.2 in GCBL (containing 0.4% pyruvate, 0.1% glutamine, 0.0002% thiamine pyrophosphate, 0.0005% ferric nitrate, and 0.042% NaHCO_3_) supplemented with 10 µCi/mL [6-^3^H] glucosamine and allowed to grow for 30 min. A sample of each culture was analyzed for ^3^H in the cells, and cultures were diluted to equalize the amounts of labeled material in each culture prior to the chase period. This method allows for quantitative comparisons between strains for peptidoglycan turnover and amounts of different peptidoglycan fragments released during growth [44,45]. Cells were harvested, washed in GCBL medium containing glucose, and then grown in glucose-containing GCBL for an additional 2 h in the same medium. The cells were pelleted, culture supernatants were passed through a 0.22 µm filter and applied to size exclusion columns (tandem 350 mL Bio-Gel P6 and Bio-Gel P30 columns), and ^3^H-labeled PG fragments were eluted with 0.1 M LiCl. Fractions were collected and radioactivity was determined by liquid scintillation counting.

4. Results

MtrR regulates transcription of ltgA in gonococci. Results from a previous microarray study suggested that ltgA transcription is regulated by MtrR [37]. To confirm this result, total RNA was extracted from a set of gonococcal strains during exponential phase growth and the levels of ltgA mRNA transcripts were examined by qPCR. In HL9, which is FA19 containing an insertionally inactivated mtrR gene (Table 1), ltgA mRNA was decreased by 2.5-fold relative to FA19 wild type, but these levels were restored to near wild type in strain GC3mtrR (HL9 complemented with a wild type copy of mtrR in trans) (Figure 1). These data suggest that MtrR is a modulator of ltgA transcription in the gonococcus.

MtrR regulates LtgA protein expression. To ascertain whether the increase in ltgA transcription results in an increase in LtgA protein, we determined the levels of LtgA in FA19, HL9, and GC3mtrR by Western blotting using an anti-LtgA polyclonal antibody (LtgA-pC). The ltgA gene was codon-optimized for E. coli, synthesized, and cloned by GenScript into pUC57 for propagation. An N-terminal 6× His-tag was incorporated during synthesis, as confirmed in the recombinant protein sequence. The construct was subsequently subcloned into a bacterial expression vector, expressed in E. coli, and the resulting LtgA-His_6_ protein was purified by His-affinity chromatography to ~85% purity for antibody production (GenScript, Piscataway, NJ, USA). LtgA-His_6_ produced a single band at approximately 68 kDa (Figure 2A and Figure S1), and a band at the same size as native LtgA was identified at the same MW for the three strains. In contrast, no band was detected with the anti-LtgA polyclonal antibody in lysates of LW9279 (FA19 ΔltgA) (Figure 2A). Consistent with the qPCR results, we observed a decrease in LtgA of 2.5-fold (±0.28; n = 3) in HL9 lysates relative to LtgA in cell lysates of FA19 and GC3mtrR (Figure 2B). These data indicate that in the absence of MtrR, LtgA levels decrease significantly (p value of <0.0001).

MtrR binds to the putative promoter region of ltgA. We next examined whether the transcriptional regulation of ltgA by MtrR was direct or indirect. Analysis of the sequence upstream of the ATG-translational start site of LtgA revealed a putative promoter sequence, −10 and −35 recognition motifs, within 150 bp of the start of the coding sequence (see below). To determine whether MtrR binds to this putative ltgA promoter sequence, increasing amounts of purified MBP-MtrR protein was incubated with a 300 bp PCR-amplified DIG-labeled fragment encompassing both the putative ltgA promoter and the translational start site of ltgA from N. gonorrhoeae FA19 gDNA, and the protein–DNA mixtures were separated on native polyacrylamide gels (Figure 3). These data revealed that MtrR binds to the upstream putative promoter region of ltgA in a concentration-dependent manner (Figure 3). Maximal binding was observed at 174 pmol of purified MBP-MtrR, which is nearly identical to that observed for MBP-MtrR binding to the promoter region of genes involved in the efflux of antibiotics [42], glutamine synthesis [46], and general stress response [37].

Determination of specificity of MtrR binding to the ltgA promoter. To further establish the specificity of binding, competitive EMSAs were performed using unlabeled ltgA promoter fragments in 50-fold molar excess over the DIG-labeled ltgA promoter fragment. The results demonstrated that an excess of unlabeled ltgA promoter fragment competed for MtrR binding to the ltgA DIG-labeled promoter fragment, whereas a 50-fold molar excess of a non-specific fragment (pilT) had no effect on MBP-MtrR binding (Figure 4). The positive control shows a decrease in the electrophoretic migration of the DIG-labeled ltgA probe incubated with purified MBP-MtrR protein. Taken together, these results demonstrate that MtrR binds to the putative promoter of ltgA in both a concentration-dependent and sequence-specific manner, suggesting that MtrR directly regulates ltgA mRNA transcription.

Localization of MtrR binding sites within the ltgA promoter region. Based on the capacity of MtrR to bind the 300 bp putative promoter of ltgA, we further localized the binding site within this region. DIG-labeled PCR fragments of increasing size (37, 75, 120 and 300 bp) from the putative ltgA promoter, each ending at the translational start site, were generated and used in separate EMSA reactions to identify the region that binds MtrR. As shown in Figure 5A, MBP-MtrR binds to the 120 and 300 bp ltgA promoter fragments but not to the 37 and 75 bp fragments. These results suggest that the MtrR binding site(s) is (are) located between 120 and 75 bp upstream of the translational start site of ltgA. After localizing the MtrR binding site to this region, sequence comparisons to additional MtrR-regulated promoter sequences suggested that a putative MtrR binding may be located between the −10/−35 sites in the promoter of ltgA at −104 to −85 bp relative to the translational start site. The putative MtrR binding site in ltgA showed a strong nucleotide identity to MtrR binding sites in promoter sequences of genes involved in the general stress response (rpoH; 52%), drug efflux (mtrCDE; 38%), and glutamine synthesis (glnA; 40%) (Figure 5B). Taken together, these data suggest that the MtrR binding site in the putative promoter sequence of ltgA is located between 85–104 bp upstream of the translational start site.

MtrR regulation of ltgA affects cell viability and autolysis of gonococci. Insertional inactivation of ltgA in strain MS11 has been shown to significantly reduce autolysis at a pH (8.0) that is optimum for autolysin activity [48]; therefore, we examined whether MtrR regulation of ltgA would alter the autolytic potential of N. gonorrhoeae. Lysis was measured in buffers either at an optimal pH (pH 8.0) or at a non-optimal pH (pH = 6.0) for autolysin activity. FA19 and HL9 (ΔmtrR) were grown to mid-log phase, diluted to an OD_560_ of 0.2 in the two buffers at room temperature, and monitored for a decrease in optical density. The percent autolysis was calculated using the following formula: %Autolysis = (OD_0,strain_ − OD_t,strain_/OD_0,strain_) × 100. There was no significant difference in cell lysis for the strains at pH 6 (Supplemental Table S1), but at pH 8, HL9 (ΔmtrR) was more autolytic compared to FA19 and its isogenic HL9 complement strain (Table 3). These data suggest that the absence of MtrR results in increased autolysis.

MtrR regulates PG-derived cytotoxin release. It was shown previously that loss of LtgA decreases the levels of PG monomer released into the culture medium during growth [14]. To determine whether loss of MtrR affects PG fragment release, PG was pulse labeled with [6-^3^H] glucosamine in growing cells, and size exclusion chromatography was used to analyze PG fragments released into the supernatant during the chase period. Gonococcal cultures were diluted following the labeling such that each culture contained the same amount of ^3^H-label in the cell wall. Thus, different gonococcal strains can be directly compared for the amounts of the different types of PG fragments released during the chase period [44,45]. HL9 (ΔmtrR) showed significantly reduced PG monomer release compared to FA19, and monomer release was increased (albeit only to an intermediate level) in the GC3mtrR complement strain (Figure 6). These results show that MtrR regulation of ltgA transcription affects PG monomer release during growth and possibly during infection.

5. Discussion

Based on data from a microarray profile by Folster et al. [37], which suggested that MtrR activated ltgA transcription, the focus of this study was to further define the capacity of MtrR to regulate ltgA and to determine if regulation affected PG monomer release and autolysis. We show here that deletion of mtrR decreases both the transcription of ltgA and expression of LtgA protein, with levels of ltgA transcripts and LtgA protein restored to wild type levels in the complemented strain (GC3mtrR). Our EMSA results indicate that MtrR can bind to a region upstream of the ltgA start codon, which includes predicted promoter elements and shares similarity with known MtrR binding sequences. These findings suggest a potential role for MtrR in regulating ltgA transcription. Additionally, changes observed in PG monomer release and autolysis in the mtrR mutant correlate with altered ltgA expression; however, further studies are needed to confirm whether these effects are directly attributable to MtrR-mediated regulation.

MtrR was first identified as a repressor of the MtrC-MtrD-MtrE multi-drug efflux system in gonococcus, which exports structurally diverse antibiotics and host-derived antimicrobial agents. Lucas et al. [42] showed that MtrR represses expression of the MtrC-MtrD-MtrE multi-drug efflux pump via binding of a promoter sequence between mtrR and the divergently transcribed mtrCDE operon. Thus, a mutation in the MtrR-coding region or promoter sequence (single base pair deletion in a 13 bp inverted repeat sequence) enhances the expression of the MtrC-MtrD-MtrE multi-drug efflux pump, leading to increased resistance to both antibiotics and host-derived antimicrobials. Although MtrR was once considered primarily a regulator of the MtrC-MtrD-MtrE multi-drug efflux pump [33,42], subsequent studies have shown that it is involved in positively or negatively regulating over 70 other gonococcal genes [36,37,46], some of which are also involved in PG synthesis. For example, MtrR was shown to positively regulate ponA, which encodes penicillin-binding protein 1 (PBP1), the major bifunctional transglycosylase/transpeptidase involved in the synthesis of PG during cell growth, and to negatively regulate an operon (pilMNOPQ) encoding Type IV pilus proteins whose promoter region overlaps with the promoter region of ponA. Thus, MtrR functions as both a repressor and an activator depending on promoter context. While MtrR represses the mtrCDE efflux operon by blocking RNA polymerase access, it activates ltgA by binding upstream of its transcriptional start site (−104 to −85 bp) in a region that enhances promoter recognition. This positioning likely stabilizes RNA polymerase recruitment, leading to increased ltgA transcription and LtgA protein levels. Positive regulation of ltgA, a major lytic transglycosylase, allows MtrR to coordinate peptidoglycan remodeling with synthesis and stress responses, thereby influencing cytotoxin release and autolysis during growth and infection. However, lower ltgA expression weakens the cell wall while dysregulated efflux activity adds membrane stress. These combined defects make the HL9 mtrR mutant highly susceptible to autolysin-mediated lysis, resulting in significantly increased autolysis compared to wild type FA19. MtrR likely coordinates peptidoglycan degradation and synthesis by co-regulating LtgA and PBP1, enabling LtgA to cleave existing strands and PBP1 to insert new ones, ensuring cell wall growth and remodeling.

Studies have suggested that MtrR regulates glutamine biosynthesis genes (glnA and glnE) [46,47,49], the protein products of which are used to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), a precursor of PG, LPS, and other macromolecules. These data are consistent with the idea that MtrR may regulate PG synthesis in several ways, i.e., glutamine synthesis (glnA/glnE), PG synthesis (ponA), and PG degradation (ltgA). Given that MtrR regulates ltgA (this study), we speculate that changes in PG recycling and release via LtgA activity may affect glutamine biosynthesis, which potentially could impact de novo synthesis of PG [40]. For example, decreased PG release in mutant mtrR (Figure 6) could increase PG pools salvaged for glutamine synthesis, which could increase fitness during infection at mucosal surfaces that are normally glutamine-limiting [50]. Supporting this idea is the observation that glutamine synthetase (glnA) activity for de novo synthesis of glutamine does not increase in gonococci grown in GCB broth lacking glutamine [46], which suggests the cell may use other sources, e.g., PG, to salvage for glutamine. Hence, MtrR may coordinately regulate genes involved in glutamine biosynthesis and PG release (via LtgA activity) to increase the survival of gonococci in glutamine-limited environments.

Warner et al. [51] reported that loss of MtrR imparts a fitness advantage over wild type in a female mouse model of a genital-tract gonococcal infection, and this increased fitness was attributed primarily to overexpression of the mtrCDE-encoded efflux pump due to the loss of repression by MtrR. However, the study could not exclude the possibility that other MtrR-regulated genes also may have contributed to gonococcal fitness during infection. Our data suggest that MtrR regulation of PG fragment release via LtgA activity may also contribute to increased gonococcal fitness during growth and possibly infection. PG fragments have been implicated in contributing to pathogenicity and in mediating specific host–pathogen interactions during infection [10,21,52,53]. Moreover, most gonococcal strains are MtrR-positive, suggesting that retention of MtrR provides an overall advantage in natural infections. In support of this, Warner et al. [51] observed that while loss of MtrR conferred a short-term fitness benefit in the lower genital tract of female mice, this advantage diminished at later stages of infection. This suggests that MtrR-mediated regulation, including its activation of ltgA, may favor persistence and long-term survival rather than transient competitive growth. Such regulation could help balance PG fragment release and other stress responses to optimize colonization and avoid detrimental inflammatory damage during prolonged infection.

Comparison of N. meningitidis and N. gonorrhoeae PG fragment profiles showed that N. meningitidis releases lower amounts of PG monomers and dimers that induce inflammatory responses during infection. N. meningitidis is more efficient in recycling PG fragments, which is hypothesized to contribute to its long-term carriage in the nasopharynx [23]. Although gonococci release more proinflammatory PG fragments and are not as efficient at recycling, it is feasible that MtrR could modulate PG fragment release during growth and infection. Hence, this would allow the bacteria to regulate when and to what degree it releases PG fragments. While we do not know whether MtrR regulation of LtgA affects recycling or turnover, the decrease in PG release in mtrR strains suggests that recycling and turnover would also be altered like mutant ltgA [14]. This would support the idea that the gonococcus has the capacity to regulate PG levels, and as a result affect the nutrient (e.g., amino acids and nucleotides), energy levels and the growth state of the cell wall. Another possibility is that PG release via MtrR regulation of LtgA could provide a survival advantage during infection. For example, N. gonorrhoeae induces a local inflammatory response (mostly neutrophils) that is driven by the controlled release of PG (via MtrR), but because the organism ultimately resists phagocytic neutrophil killing [54,55], this strategy could provide a selective advantage over susceptible pathogens at local sites of infection. In addition to PG, the release of other proinflammatory molecules [56,57,58], e.g., blebbing of outer membranes containing high levels of lipooligosaccharide [59,60], suggests that inducing an inflammatory response may be beneficial under certain conditions.

6. Conclusions

In this study, we show that PG fragment release and autolysis via LtgA activity is regulated by the cytoplasmic protein, MtrR, in N. gonorrhoeae during normal growth. PG fragment release in mutant mtrR is comparable to the reduced PG fragment levels of a previously reported LtgA knockout strain [14]. Therefore, MtrR regulation of PG fragment release and autolysis via LtgA activity may impact the overall fitness of gonococcus during normal growth and/or during infection.