Bacterial Infections

Identification and Characterization of the Neisseria gonorrhoeae MscS-Like Mechanosensitive Channel

Zhemin Wang, Xiaomin Wang, Ping Lu, Chunshan Ni, Yuezhou Li, Stijn van der Veen
Shelley M. Payne, Editor
DOI: 10.1128/IAI.00090-18

ABSTRACT

Mechanosensitive channels are ubiquitous in bacteria and provide an essential mechanism to survive sudden exposure to a hypo-osmotic environment by the sensing and release of increased turgor pressure. No mechanosensitive channels have thus far been identified and characterized for the human-specific bacterial pathogen Neisseria gonorrhoeae. In this study, we identified and characterized the N. gonorrhoeae MscS-like mechanosensitive channel (Ng-MscS). Electrophysiological analyses by the patch clamp method showed that Ng-MscS is stretch activated and contains pressure-dependent gating properties. Further mutagenesis studies of critical residues forming the hydrophobic vapor lock showed that gain-of-function mutations in Ng-MscS inhibited bacterial growth. Subsequent analysis of the function of Ng-MscS in N. gonorrhoeae by osmotic down-shock assays revealed that the survival of Ng-mscS deletion mutants was significantly reduced compared with that of wild-type strains, while down-shock survival was restored upon the ectopic complementation of mscS. Finally, to investigate whether Ng-MscS is important for N. gonorrhoeae during infections, competition assays were performed by using a murine vaginal tract infection model. Ng-mscS deletion mutants were outcompeted by N. gonorrhoeae wild-type strains for colonization and survival in this infection model, highlighting that Ng-MscS contributes to in vivo colonization and survival. Therefore, Ng-MscS might be a promising target for the future development of novel antimicrobials.

INTRODUCTION

Bacteria are separated from their environment by a cell membrane consisting of a phospholipid bilayer that is maintained under constant positive turgor pressure. Its integrity is provided by the peptidoglycan layer that constrains the cell membrane and prevents expansion. However, during their normal life cycle, bacteria are exposed to fluctuating osmotic environments that include sudden exposure to hypo-osmotic conditions (1). During this osmotic down-shock, water rapidly moves inside bacterial cells, resulting in increased turgor pressure and membrane expansion. Mechanosensitive (MS) channels are ubiquitous in bacteria and are able to sense increased physical membrane tension and gate in response, which results in the rapid release of cytoplasmic osmolytes and a reduction in turgor pressure (2, 3). The MS channel of small conductance (MscS) is a heptameric transmembrane protein, with each unit containing an N-terminal domain facing the periplasmic space, three transmembrane helices, and a large C-terminal domain extending into the cytoplasm. The pore of the channel is generated by seven copies of transmembrane helix 3 (TM3) (4), which is highly conserved in MscS proteins of different bacterial species (2, 5). When the channel is closed, the most constricted region of the pore is formed by rings of L105 and L109, which are located in TM3 and create a hydrophobic vapor lock (4, 6).

So far, no MS channels have been identified or characterized for Neisseria gonorrhoeae, which is a human-specific bacterial pathogen that causes the sexually transmitted disease gonorrhea. N. gonorrhoeae generally colonizes the mucosal epithelia of the urogenital tract, where it might experience osmotic fluctuations, including hypo-osmotic shock (7). Therefore, MS channels might be important for N. gonorrhoeae to survive in this environment of osmotic fluctuations. According to WHO estimates, the annual global incidence of N. gonorrhoeae is approximately 106 million new cases (8). In men, most gonorrhea cases present as urethritis, while in women, cervical infections are generally asymptomatic. However, when left untreated, complicated or disseminated disease might develop, including ectopic pregnancies, sterility, or pelvic inflammatory disease (9, 10). N. gonorrhoeae is considered a global threat to public health because of the rise of multidrug resistance and dwindling treatment options (11–13). Therefore, N. gonorrhoeae was included in a recent report by the WHO on multidrug-resistant bacteria for which the development of novel antimicrobials is urgently needed (14). Given that bacterial MS channels are structurally distinct from MS channels in animals and that they are highly conserved in bacteria, they have been suggested to be suitable targets for the development of novel antimicrobials (15). Indeed, recently, an in silico approach was used to design antimicrobial compounds targeting the MS channel of large conductance (MscL) from multidrug-resistant Staphylococcus aureus (16). The most potent compound in that study was able to clear infection in a nematode model of infection.

Here, we identified and functionally characterized an MscS-like MS channel in N. gonorrhoeae. Electrophysiological analyses and osmotic down-shock assays showed that the channel was activated by membrane pressure and essential for survival after sudden exposure to hypo-osmotic conditions. Furthermore, the channel appeared to be important for colonization and survival in a mouse model, while gain-of-function mutations inhibited bacterial growth. Therefore, the N. gonorrhoeae MscS-like channel might be an interesting target for the future development of novel antimicrobial treatments against this bacterium.

RESULTS

Identification of NGO2057 as the N. gonorrhoeae MscS-like channel.To identify putative MS channels in N. gonorrhoeae, the amino acid sequences of all seven MS channels present in Escherichia coli were compared with all predicted proteins of the fully sequenced N. gonorrhoeae strain FA1090 (17). Only one predicted N. gonorrhoeae protein (NGO2057) showed significant similarity to any of the seven E. coli MS channels. The amino acid sequence of E. coli MscS (Ec-MscS) showed 33% identity to NGO2057 (Ng-MscS), and the DNA sequence showed 49% identity. Ng-MscS consists of 282 amino acids with a predicted mass of 31 kDa and contains a MS channel motif spanning residues 71 to 270. Further alignment of Ng-MscS with Ec-MscS and the characterized or annotated MscS proteins from 14 other bacteria belonging to both Gram-negative and Gram-positive species using Clustal X (18) showed a high level of identity (see Fig. S1 in the supplemental material). Particularly, the hydrophobic lock in the pore-forming transmembrane helix 3 was well preserved. To further characterize Ng-MscS, a homology model was created based on the open-channel crystal structure of Ec-MscS (PDB accession number 2VV5) (19). Besides the hydrophobic lock residues (L105 and L109) (4), the residues involved in mechanosensing (A34, I37, A85, and L86) (20) and the residues important for gating interactions (F68 and L111) (21) were fully conserved in Ng-MscS (Fig. 1). Interestingly, the residue at position 113, which is involved in the inactivation of the channel (22), was an aspartic acid in Ng-MscS instead of a glycine as observed for Ec-MscS.

FIG 1
FIG 1

Structural comparison of Ec-MscS and the Ng-MscS homology model shows that important residues are conserved. The structures are cartoon representations of the three transmembrane helices of four subunits of the heptameric channel. Important residues for mechanosensing (orange), gating interactions (purple), inactivation (red), and the hydrophobic lock (green and blue) are indicated.

Ng-MscS shows typical MscS-like electrophysiological properties.To characterize the activity of Ng-MscS, the electrophysiological properties of Ng-MscS were determined and compared with those of Ec-MscS by patch clamp analyses of E. coli spheroplasts (23, 24). Membrane patches containing Ng-MscS or Ec-MscS were exposed to a gradual increase in negative pressure. The Ng-MscS channels could be activated at a pressure of 30 to 70 mm Hg, while for Ec-MscS, a higher pressure of 60 to 120 mm Hg was required (Fig. 2A). Interestingly, when the pressure was kept constant at the level required for channel opening, a rapid inactivation of Ec-MscS channels was observed, while for Ng-MscS, channel inactivation did not occur when pressure was maintained. Subsequently, traces at 20 mV were investigated for single-channel properties. It appeared that at a positive voltage, the conductance of single Ec-MscS channels was higher than that of single Ng-MscS channels (Fig. 2B), while at a negative voltage, the conductance of single Ng-MscS channels was higher. Similarly, current-voltage plots for Ec-MscS and Ng-MscS showed that the conductance was higher for Ec-MscS at positive pipette voltages (1.22 ± 0.02 nS for Ec-MscS versus 0.88 ± 0.02 nS for Ng-MscS), while at negative pipette voltages, the conductance was higher for Ng-MscS (0.77 ± 0.01 nS for Ec-MscS versus 1.16 ± 0.04 nS for Ng-MscS) (Fig. 2C). Therefore, Ng-MscS showed outward rectification behavior. Finally, to confirm the observed differences in pressure sensitivity, the pressure threshold ratios of Ng-MscS and Ec-MscS were determined in comparison with MscL. After pressure was applied to open the MscS channels, additional pressure was applied until the opening of MscL was observed. Ng-MscS showed a significantly higher pressure threshold ratio than did Ec-MscS (Fig. 2D), indicating that Ng-MscS is activated at a lower membrane tension than Ec-MscS. Overall, Ng-MscS showed typical MscS-like stretch-activated and pressure-dependent gating properties, although the specific properties, such as conductance, pressure sensitivity, and inactivation, were different from those observed for Ec-MscS.

FIG 2
FIG 2

Ng-MscS shows typical MscS-like channel properties in electrophysiological analyses by the patch clamp method. All analyses were performed on inside-out patches from at least two independent E. coli spheroplast preparations. (A) Representative traces at +20 mV showing the kinetic phenotypes for Ng-MscS and Ec-MscS channels. (B) Representative current recordings for single channels of Ng-MscS and Ec-MscS at pipette voltages of both +20 mV and −20 mV. (C) Current-voltage plots of single channels of Ng-MscS and Ec-MscS. (D) Mechanosensitivity of Ng-MscS and Ec-MscS. Mechanosensitivity was measured for Ng-MscS and Ec-MscS by determining their pressure threshold ratios with MscL (PL/PS). E. coli strain MJF431, containing MscL, was used, and the ratio was calculated by dividing the pressure at which the first MscL opens by the pressure at which the first Ng-MscS or Ec-MscS opens. The graph represents the means and standard deviations of the pressure threshold ratios, and significant differences were identified by Student's two-tailed unpaired t test (GraphPad Prism). ***, P < 0.001.

Ng-MscS is essential for osmotic down-shock survival.Given that the biological role of MS channels is to provide a rapid release of turgor pressure upon sudden exposure to a hypo-osmotic environment, osmotic down-shock assays were performed to investigate whether Ng-MscS functions as a MS channel involved in the release of turgor pressure. Indeed, a ΔmscS deletion mutant generated in the international N. gonorrhoeae reference strain ATCC 49226 (25) showed a major reduction in osmotic down-shock survival compared with the wild-type strain (Fig. 3A), indicating that Ng-MscS truly functions as a MS channel that releases high turgor pressure. The ectopic expression of Ng-mscS under the control of its native promoter only partly complemented the observed down-shock sensitivity of the ATCC 49226-ΔmscS mutant, indicating that ectopic expression might have reduced the promoter activity. Therefore, complementation strains were generated, in which Ng-MscS or Ec-MscS was expressed by a strong isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter. Indeed, down-shock survival was completely restored to the wild-type level for both of these complementation strains, further indicating that Ec-MscS is fully functional in N. gonorrhoeae and able to replace Ng-MscS. These results were not strain dependent and were fully reproducible in N. gonorrhoeae clinical isolate ZJXSH86 (26). The ZJXSH86-ΔmscS deletion mutant similarly showed a major reduction in osmotic down-shock survival compared with the wild-type strain (Fig. 3B). Survival was again restored when the ΔmscS mutant was complemented ectopically with Ng-mscS or Ec-mscS under the control of the IPTG-inducible promoter. Given that Ec-MscS was able to replace the function of Ng-MscS in N. gonorrhoeae down-shock survival, the ability of Ng-MscS to complement Ec-MscS in E. coli down-shock survival was subsequently investigated. Indeed, when either Ng-MscS or Ec-MscS was expressed in E. coli MJF465 (2), which lacks the MS channels MscS, MscL, and MscK, down-shock survival was restored (Fig. 3C). Gonococcal survival in the osmotic down-shock assays was verified by live/dead staining and fluorescence microscopy. Microscopic images of wild-type and ΔmscS mutant strains after down-shock predominantly showed green cells (live) for the wild-type strains and predominantly red cells (dead) for the ΔmscS mutant strains (Fig. 3D). Quantification of the microscopic images for live and dead cells further showed that 64 to 70% of the wild-type bacteria survived osmotic down-shock, while only 11 to 21% of the ΔmscS mutant bacteria survived this treatment (Fig. 3E). These results show that Ng-MscS is indeed a MS channel that is essential for N. gonorrhoeae to survive sudden exposure to a hypo-osmotic environment.

FIG 3
FIG 3

Ng-MscS and Ec-MscS are exchangeable and important for survival during hypo-osmotic shock. The survival of N. gonorrhoeae ATCC 49226 and ZJXSH86 wild-type and mutant strains and E. coli MJF465 strains expressing Ng-MscS or Ec-MscS was determined upon exposure for 20 min to hypo-osmotic medium (N. gonorrhoeae) or deionized water (E. coli) and compared with survival upon exposure to isosmotic medium. Graphs represent the means and standard deviations of data from at least three independent experiments. (A) Survival of N. gonorrhoeae ATCC 49226 (wild type [WT]), ATCC 49226-ΔmscS::kanR, ATCC 49226-Ng-mscS-C, ATCC 49226-Ng-mscS-C(i), and ATCC 49226-Ec-mscS-C(i) upon osmotic down-shock. (B) Survival of N. gonorrhoeae ZJXSH86 (wild type), ZJXSH86-ΔmscS::kanR, ZJXSH86-Ng-mscS-C(i), and ZJXSH86-Ec-mscS-C(i) upon osmotic down-shock. (C) Survival of E. coli MJF465 expressing Ng-MscS or Ec-MscS and the empty vector control upon osmotic down-shock. Significant differences were identified by analysis of variance (GraphPad Prism). *, P < 0.05; ***, P < 0.001. (D) The viability of N. gonorrhoeae wild-type and ΔmscS strains was visualized by live/dead staining after osmotic down-shock. Representative images are shown. (E) Quantification of viable bacteria after live/dead staining. The graph presents the percentages of viable bacteria after osmotic down-shock quantified in three independent experiments analyzing multiple images with a total number of >100 bacteria per condition per experiment. Significant differences were identified by Student's two-tailed unpaired t test (GraphPad Prism). *, P < 0.05; ***, P < 0.001.

Ng-MscS is very sensitive to gain-of-function mutations.Residues L105 and L109 are pivotal for channel gating and function as a hydrophobic lock under closed conditions (4). Therefore, modifications of these hydrophobic residues to polar or less-hydrophobic residues would allow the channel to become more open or leaky in its closed state. One of these so-called “gain-of-function” mutations, L109S, has been studied extensively in E. coli and causes growth defects when the expression of the channel is induced (6). To investigate whether L105 and L109 are similarly important for channel gating in Ng-MscS, L109S, L109A, L105S, and L105A mutations were introduced into Ng-MscS. However, no viable colonies were obtained after many attempts to transform E. coli with pB10b-Ng-mscS containing any of the L109S, L109A, or L105S mutations. Only L105A was successfully introduced in Ng-mscS, but Ng-mscS containing this mutation was not transformable in N. gonorrhoeae. Growth experiments with E. coli MJF465 expressing Ng-MscS and Ng-MscS(L105A) showed that the induction of Ng-MscS(L105A) expression but not of Ng-MscS expression resulted in growth arrest (Fig. 4A and B), similar to what was observed for Ec-MscS and Ec-MscS(L109S) (Fig. 4C and D) (6). These results show that Ng-MscS is very sensitive to gain-of-function mutations.

FIG 4
FIG 4

Induction of Ng-MscS containing a gain-of-function mutation inhibits growth of E. coli. Growth curves of E. coli strain MJF465 expressing wild-type Ng-MscS or Ec-MscS or channels containing gain-of-function mutations in the hydrophobic gas vapor lock were generated with or without induction with IPTG at 2 h postinoculation. The graphs represent the means and standard deviations of data from three independent experiments. (A) Ng-MscS; (B) Ng-MscS(L105A); (C) Ec-MscS; (D) Ec-MscS(L09S). Significant differences for each time point between induced and uninduced conditions were identified by Student's two-tailed unpaired t test (GraphPad Prism). ***, P < 0.001.

Given the sensitivity of Ng-MscS to gain-of-function mutations, this channel might be an interesting target for the development of novel antimicrobials against N. gonorrhoeae that induce channel opening without the requirement of membrane tension. Several compounds were previously shown to activate MS channels without the presence of membrane tension (27–29). Two of these compounds, brilliant green and deoxycholic acid, were used to investigate their MICs against both ATCC 49226 and ZJXSH86 and their ΔmscS mutant strains. For both compounds, the MIC against the ZJXSH86-ΔmscS mutant was 2-fold higher than that against ZJXSH86 (see Table S1 in the supplemental material), indicating that the opening of Ng-MscS might have a small effect on its viability. However, for ATCC 49226, no differences in MICs between the wild-type and mutant strains were observed, indicating that these compounds might be limited in their ability to cause channel gating.

Ng-MscS is important for colonization and survival in a murine vaginal tract model of infection.During an infection of the urogenital tract, N. gonorrhoeae might experience osmotic fluctuations, including rapid hypo-osmotic conditions. Therefore, to investigate whether Ng-MscS contributes to colonization and survival in vivo, competition assays were performed in two independent studies using a murine vaginal tract infection model (30, 31). In the first study, the mouse vaginal tract was colonized with a bacterial suspension containing equal numbers of bacteria of ATCC 49226-catA2, which is chloramphenicol resistant, and ATCC 49226-ΔmscS::kanR, which is kanamycin resistant. After infection, CFU counts recovered on agar plates containing chloramphenicol were significantly higher than those on agar plates containing kanamycin. For each mouse, a competition index (CI) was calculated, which consistently resulted in values below 1.0 (mean CI = 0.33) (Fig. 5), indicating that higher numbers of CFU were recovered for the wild-type strain than for the ΔmscS deletion mutant. To ensure that this difference was not attributed to a general growth defect, competition assays were also performed for in vitro growth in optimal liquid growth medium. The CIs for in vitro growth gave values of ∼1, indicating that under optimal growth conditions, the ΔmscS deletion mutant does not show a growth defect. Further statistical analysis showed that the difference in CIs between in vivo and in vitro growth conditions was significant, indicating that Ng-MscS is important for in vivo colonization and survival. To verify these results and to ensure that the specific antibiotic selection markers did not contribute to the observed differences, a second study was performed in which the selection markers were swapped. A bacterial suspension containing equal numbers of ATCC 49226-kanR and ATCC 49226-ΔmscS::catA2 bacteria was used to inoculate the mice. Again, higher CFU counts of the wild-type strain were recovered from the mice, and significant differences in CIs were observed between in vivo and in vitro growth conditions (mean CI = 0.46) (Fig. 5). As a control, the mscS complementation strain ATCC 49226-Ng-mscS-C, which is resistant to both kanamycin and chloramphenicol, was used in both in vivo and vitro assays with the ATCC 49226-ermC strain, which is resistant to erythromycin. No significant differences in CIs were observed for these strains under in vivo and in vitro growth conditions. Furthermore, in vivo CIs for the deletion mutant in both studies were significantly lower than those for the complemented strain, indicating that Ng-MscS is truly important for colonization and survival in the murine vaginal tract infection model.

FIG 5
FIG 5

N. gonorrhoeae wild-type strains outcompete ΔmscS mutant strains in a murine vaginal tract infection model. Bacterial mixtures containing equal amounts of ATCC 49226-catA2 and ATCC 49226-ΔmscS::kanR (study 1), ATCC 49226-kanR and ATCC 49226-ΔmscS::catA2 (study 2), and ATCC 49226-ermC and ATCC 49226-Ng-mscS-C (study 2) were used to inoculate the vaginal tracts of mice. After 24 h, mice were sampled, and growth of the bacteria on agar plates containing selective antibiotics was investigated. The competition index (CI) was calculated as (mutant/wild type)output/(mutant/wild type)input. As a control, in vitro competition indices of liquid cultures were determined after growth for 6 h. Significant differences were identified by analysis of variance (GraphPad Prism). *, P < 0.05; **, P < 0.01.

Ng-MscS does not contribute to epithelial cell adhesion, invasion, or intracellular survival.Given that Ng-MscS contributes to colonization and survival in vivo, the role of Ng-MscS in in vitro colonization and the intracellular survival of epithelial cells was next investigated by using gentamicin protection assays with HeLa human cervix epithelial carcinoma cells. The ATCC 49226 wild-type strain, the ΔmscS deletion mutant, and complementation strains were used in single-strain assays. However, no significant differences in adhesion, invasion, or intracellular survival were observed (see Fig. S2A in the supplemental material). Subsequently, competition assays between the wild-type and deletion mutant or the complemented strain were performed. Bacterial suspensions containing equal numbers of ATCC 49226-catA2 and ATCC 49226-ΔmscS::kanR or ATCC 49226-ermC and ATCC 49226-Ng-mscS-C bacteria were added to HeLa cells, and adhesion, invasion, and intracellular survival were quantified by using agar plates containing the selective antibiotics. However, no significant differences were observed between the wild-type strain and the ΔmscS deletion mutant (Fig. S2B) or between the wild-type and complemented strains (Fig. S2C). Based on these data, it became apparent that Ng-MscS does not contribute to the colonization of epithelial cells in in vitro gentamicin protection assays.

DISCUSSION

To cope with changing osmotic environments, bacteria have evolved numerous adaptation mechanisms, including the accumulation of compatible solutes, like glycine, betaine, carnitine, and proline, in response to hyperosmotic shock and aquaporins for water efflux or MS channels in response to hypo-osmotic shock (32). Bacterial MS channels are sensor transducers that are able sense increased cytoplasmic membrane tension as a result of hypo-osmotic shock and respond by opening the central pore inside the MS channel complex, which allows the release of cytoplasmic osmolytes and reduces membrane tension. In this study, the MscS-like MS channel of N. gonorrhoeae was characterized both for its electrophysiological properties and for its role in N. gonorrhoeae physiology and host colonization. Ng-MscS showed typical MscS-like electrophysiological properties, but the details of the specific parameters were different from those for Ec-MscS. In our patch clamp analyses, Ng-MscS displayed higher conductance at negative pipette voltages than at positive voltages, indicating that Ng-MscS has outward rectification behavior, meaning that positive ions preferentially move out of the bacterial cell. In contrast, in both our analyses and previous studies, Ec-MscS showed inward rectification (33–35), similar to what is observed for MscS-like channels for most bacterial species, including Silicibacter pomeroyi (36), Corynebacterium glutamicum (37), Vibrio cholerae (38), and Methanococcus jannaschii (39). Interestingly, the archaeon Haloferax volcanii contains two MscS-like channels, and one of these channels showed outward rectification, similar to Ng-MscS, while the other channel showed inward rectification (40). Differences in rectification might be explained by different steric effects in these channels that result in the obstruction of osmolytes and by different charge distributions in the cytoplasmic vestibule of the pore (34, 35, 41, 42). However, differences in channel substate activity and the orientation of channels in the spheroplast patches have also been suggested as explanations for rectification behavior (33, 36, 42). Thus, while the difference in conductance between Ng-MscS and Ec-MscS was dependent on whether positive or negative pipette voltages were applied, for a number of other bacteria, it has already been shown that conductance was lower at all pipette voltages than in Ec-MscS (36, 37, 43). Therefore, Ng-MscS appears to be unusual in its conductance and rectification behaviors compared with those of most of the other members of the MscS family.

Besides rectification and conductance, a major difference between Ng-MscS and Ec-MscS in inactivation was observed as well. Whereas Ec-MscS showed rapid inactivation, Ng-MscS did not show any inactivation during the time measured. A similar lack of inactivation was observed for MscS-like channels belonging to C. glutamicum (37), M. jannaschii (39), S. pomeroyi (36), and H. volcanii (40). Previously, it was shown that the residue at position 113 is pivotal for the straightening and buckling of TM3 during transitions between the open and closed states of the channel, and therefore, it is a critical residue for inactivation (22, 36, 44). Modification of this residue from a glycine, as is observed in E. coli, to an alanine or aspartate prevented the inactivation of the channel (22, 36, 41). Interestingly, both Ng-MscS and the MscS-like channel of M. jannaschii contain an aspartate at position 113, which might explain the observed lack of inactivation. Finally, Ng-MscS showed higher mechanosensitivity than did Ec-MscS, and therefore, gating was observed at a lower membrane tension. Similar observations were made for Pseudomonas aeruginosa (43), while in contrast, MscS from S. pomeroyi showed lower mechanosensitivity than did Ec-MscS (36). Interestingly, one of the two MscS-like channels of H. volcanii shows very low pressure sensitivity, resembling what is observed for MscL (40). Therefore, although the overall modes of action of MscS-like channels belonging to different bacteria are similar, species-specific fine-tuning of the different electrophysiological properties in order to adapt to their respective environments has likely occurred.

Although the specific electrophysiological properties of Ng-MscS and Ec-MscS were different, both channels were able to fully complement each other for survival in osmotic down-shock assays. This in contrast to what was observed for one of the MscS-like channels from P. aeruginosa. In this opportunistic pathogen, two MscS-like channels, PaMscS-1 and PaMscS-2, have been identified. The conductance of PaMscS-2 was lower than that of PaMscS-1, and therefore, only PaMscS-1 was able to provide resistance to osmotic down-shock in E. coli that lacked other MS channels, while PaMscS-2 was unable to do this (43). Similarly, the MscS-like channel of the Gram-positive bacterium C. glutamicum, which showed a lower conductance than that of Ec-MscS, was not functional in response to abrupt osmotic down-shock (37, 45). Given its fairly unusual electrophysiological parameters and its apparent role in glutamate and betaine transport, it has been considered a sustainable metabolic valve instead of an osmoregulator (37, 45).

Since Ng-MscS appears to be essential for survival during sudden exposure to hypo-osmotic conditions, it was interesting to speculate that Ng-MscS might also contribute to the ability of N. gonorrhoeae to survive in vivo. For Campylobacter jejuni, it was previously shown that MscS-like channels contributed to the colonization of chick ceca. C. jejuni contains two MscS-like channels, and a double-deletion mutant was severely attenuated in this infection model (46). Since N. gonorrhoeae colonizes the urogenital tract, it is exposed to various stresses related to mucosal immunity factors, acidification, and osmotic fluctuations (7, 47–49). Based on our study, it is apparent that Ng-MscS was important for colonization and survival in the murine vaginal tract infection model. In in vivo competition assays, Ng-mscS-deficient strains were unable to compete with wild-type strains, indicating that Ng-MscS was important to cope with the osmotic stress encountered in the murine vaginal tract. Although MS channels have not been studied previously in N. gonorrhoeae, another pump has already been identified for its role in host survival. The MtrCDE efflux pump, which spans both the inner and outer membranes and confers resistance to hydrophobic and amphipathic compounds (50, 51), has been reported to contribute to the colonization and persistence of gonococci in the urogenital tract (30). Given that Ng-MscS appears to be important for host colonization, it might be considered a new virulence factor, similarly to MtrCDE (52, 53).

Multidrug-resistant gonococcal infections are spreading globally, and future treatment is therefore insecure. Therefore, the development of novel antimicrobials against N. gonorrhoeae is urgently needed (54–56). Since Ng-MscS is important for host colonization and structurally different from MS channels in mammals, it might be an interesting new target for the development of antimicrobial treatments against N. gonorrhoeae. However, compounds that inhibit the activity of Ng-MscS in the host still require host factors for clearing infection. Another option for targeting Ng-MscS is aiming for compounds that constitutively activate the channel, which results in the constitutive leakage of cytoplasmic contents and, ultimately, bacterial cell death. This is a particularly attractive option against N. gonorrhoeae, since Ng-MscS appeared particularly sensitive to gain-of-function mutations that affect the hydrophobic gas vapor lock. The replacement of the leucines at position 105 or 109 with a hydrophilic serine was shown to cause growth inhibition in E. coli when expression was induced (6). However, these mutations appear to be detrimental in Ng-MscS, since no viable bacteria were obtained after many attempts to make them. When replacement with alanine, which is less hydrophobic and “bulky” than leucine (19), was attempted, only the L105A mutant was successfully made. This mutated Ng-MscS was unable to transform into N. gonorrhoeae, but in E. coli, it showed a phenotype similar to that of the Ec-MscS(L109S) mutant. Based on these results, Ng-MscS appears to be more sensitive to gain-of-function mutations than Ec-MscS. Several compounds were previously shown to activate MscS channels without stimulation by increased membrane tension, including parabens, brilliant green, eriochrome cyanine R, and deoxycholic acid (27–29). However, in our analyses, the modes of action of brilliant green and deoxycholic acid did not seem specific for Ng-MscS. Therefore, further studies are required to identify specific and potent antimicrobial compounds that are able to stimulate Ng-MscS gating in the absence of increased membrane tension.

In conclusion, our study provides a foundation for further structural and functional analyses of the MscS-like channel in N. gonorrhoeae and enhances our understanding of gonococcal physiology and survival during an infection. In addition, Ng-MscS appears to be an interesting novel target for the future development of antimicrobials against N. gonorrhoeae.

MATERIALS AND METHODS

Ethics statement.The animal studies and procedures were approved by the Zhejiang University Animal Care and Use Committee under project license number ZJU2015-032-01. The animal procedures were performed according to the guidelines of the Administration of Affairs Concerning Experimental Animals of the People's Republic of China and adhered to the principles of the Declaration of Helsinki. Clinical N. gonorrhoeae isolate ZJXSH86 was an anonymized strain obtained from the Zhejiang Xiaoshan Hospital research strain collection. Ethical approval for its use in research was not required.

Bacterial strains and culture conditions.N. gonorrhoeae international reference strain ATCC 49226 (25), a recent clinical isolate (ZJXSH86) (26), and their derivatives (Table 1) were stored at −80°C in brain hearth infusion broth (Oxoid) containing 15% glycerol (Biosharp). For all experiments, strains were grown overnight at 37°C in the presence of 5% CO2 on GC agar (Oxoid) supplemented with 1% (vol/vol) Vitox (Oxoid). The antibiotic kanamycin (100 μg/ml; Inalco), chloramphenicol (7.5 μg/ml; Inalco), or erythromycin (5 μg/ml; BBI) was added when appropriate. Escherichia coli strains MJF465 (ΔmcsL::catA2 ΔmscS ΔmscK::kanR) (2) and MJF431 (ΔmscS ΔmscK::kanR ΔyjeP) (2) and their derivatives (Table 1) were stored in Luria-Bertani (LB) broth (BD) containing 15% glycerol at −80°C. To initiate experiments, E. coli strains were grown on LB agar overnight at 37°C. Antibiotics were added when appropriate at concentrations of 100 μg/ml for kanamycin and ampicillin (Inalco) and 30 μg/ml for chloramphenicol.

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TABLE 1

Bacterial strains used in this study

Construction of vectors and mutant strains.To perform a comparative analysis of the functional properties of N. gonorrhoeae MscS (Ng-MscS) and Escherichia coli MscS (Ec-MscS), Ng-MscS and Ec-MscS were ectopically expressed in E. coli strains lacking MS channels (MJF465 and MJF431) (2). The N. gonorrhoeae mscS gene (Ng-mscS) was amplified by using primers mscS-F and mscS-R (Table 2) and cloned into pB10b-Ec-mscS (57), thereby replacing Ec-mscS and giving rise to vector pB10b-Ng-mscS (Table 3). These vectors were subsequently transformed into E. coli strains MJF465 (MJF465-Ec-mscS and MJF465-Ng-mscS) and MJF431 (MJF431-Ec-mscS and MJF431-Ng-mscS). For additional studies on gain-of-function mutations, pB10b-Ng-mscS and pB10b-Ec-mscS were used as the templates for site-directed mutagenesis PCR with primers Ng-mscS(L105A)-F and Ng-mscS(L105A)-R and primers Ec-mscS(L109S)-F and Ec-mscS(L109S)-R to generate vectors pB10b-Ng-mscS(L105A) and pB10b-Ec-mscS(L109S), respectively. To generate mscS deletion mutants in ATCC 49226 and ZJXSH86, the up- and downstream flanking regions of Ng-mscS were amplified by using primers mscS-A to mscS-D and cloned into pUC57-kanR (58) and pUC57-catA2 (58) adjacent to the kanamycin and chloramphenicol resistance genes, resulting in vectors pUC57-ΔmscS::kanR and pUC57-ΔmscS::catA2. Purified vectors were linearized by restriction digestions and used for transformation and recombination into the genomes of ATCC 49226 and/or ZJXSH86. Gonococci grown overnight were harvested and spotted onto supplemented GC agar plates in 10-μl droplets. After drying, 10 μl DNA (0.5 to 1.5 μg) was added, and plates were incubated for 4 to 6 h at 37°C in the presence of 5% CO2. Bacteria were transferred to supplemented GC agar plates containing antibiotics for the selection of transformants in which Ng-mscS was deleted (ATCC 49226-ΔmscS::kanR, ATCC 49226-ΔmscS::catA2, and ZJXSH86-ΔmscS::kanR). Derivatives of ATCC 49226 containing antibiotic resistance selection markers for kanamycin, chloramphenicol, and erythromycin in a locus unrelated to mscS were generated by cloning the antibiotic resistance genes into the convergent locus between lctP and aspC. lctP and aspC were amplified by using primers lctP-F, lctP-R, aspc-F, and aspC-R and cloned into pUC57-kanR, pUC57-catA2, and pUC57-ermC (58) flanking the resistance genes, giving vectors pUC57-lctP-kanR-aspC, pUC57-lctP-catA2-aspC, and pUC57-lctP-ermC-aspC. These vectors were subsequently used to transform ATCC 49226 to give the derivatives ATCC 49226-kanR, ATCC 49226-catA2, and ATCC 49226-ermC. The ATCC 49226-ΔmscS::kanR and ZJXSH86-ΔmscS::kanR deletion mutants were complemented for both Ng-mscS and Ec-mscS in the ectopic lctP-aspC locus. Ng-mscS and Ec-mscS were amplified from vectors pB10b-Ng-mscS and pB10b-Ec-mscS by using primers pB10b-F and pB10b-R. The resulting PCR product included the IPTG-inducible promoter and lacI. The PCR products were cloned into pUC57-lctP-catA2-aspC between lctP and catA2, and the resulting vectors, pUC57-lctP-Ng-mscS-lacI-catA2-aspC and pUC57-lctP-Ec-mscS-lacI-catA2-aspC, were transformed into ATCC 49226-ΔmscS::kanR and ZJXSH86-ΔmscS::kanR to give the complemented strains ATCC 49226-Ng-mscS-C(i), ATCC 49226-Ec-mscS-C(i), ZJXSH86-Ng-mscS-C(i), and ZJXSH86-Ec-mscS-C(i). In addition, ATCC 49226-ΔmscS::kanR was complemented for Ng-mscS expressed from its native promoter. Ng-mscS was amplified by using primers mscS(np)-F and mscS-R and cloned into vector pUC57-lctP-catA2-aspC between lctP and catA2, and the resulting vector, pUC57-lctP-Ng-mscS(np)-catA2-aspC, was transformed into ATCC 49226-ΔmscS::kanR to give the complemented strain ATCC 49226-Ng-mscS-C.

View this table:
TABLE 2

PCR primers used in this study

View this table:
TABLE 3

Vectors used in this study

Preparation of spheroplasts.Preparation of E. coli spheroplasts of MJF465 and MJF431 containing vectors pB10b-Ng-mscS and pB10b-Ec-mscS was performed according to previously described procedures (23). Single colonies were used to inoculate modified LB broth containing 0.5% (wt/vol) NaCl (Vetec) and ampicillin and grown overnight at 37°C with shaking. Cultures grown overnight were subsequently used to inoculate (1:100) fresh modified LB broth, and cultures were incubated at 37°C with shaking until an optical density at 600 nm (OD600) of 0.2 was reached. The cultures were diluted (1:10) in fresh modified LB broth containing 0.06 mg/ml cephalexin (MedChem Express) and incubated until filamentous bacteria with a length of 50 to 150 μm became apparent. Subsequently, the expression of mscS was induced by the addition of 1 mM IPTG (Sangon Biotech), and bacteria were incubated for 30 min. Afterwards, bacteria were centrifuged at 2,000 × g for 10 min, and pellets were resuspended in 2.5 ml of 0.8 M sucrose (Sigma). In the following order, 125 μl 1 M Tris-HCl (pH 8.0) (Sigma), 30 μl 5 mg/ml lysozyme (Amresco), 30 μl 5 mg/ml DNase (BBI), and 30 μl 125 mM EDTA (pH 7.8) (Amresco) were added. After a 5-min incubation, the reaction was terminated by the addition of 1 ml stop solution (19.4 mM MgCl2 [Sigma], 9.7 mM Tris-HCl [pH 8.0], 0.67 M sucrose). The mixture was subsequently diluted in 10 ml of a dilution solution (10 mM MgCl2, 10 mM Tris-HCl [pH 8.0], 0.8 M sucrose) in 15-ml tubes kept on ice. Spheroplasts were centrifuged for 5 min at 1,800 × g at 4°C, and pellets were gently resuspended in some of the remaining dilution solution. Spheroplasts were stored in 50-μl aliquots at −20°C.

Electrophysiological analysis of MscS.Inside-out patches excised from E. coli giant spheroplasts were studied according to previously described procedures (23, 24). All recordings were made by using symmetrical solutions for the bath and pipette (5 mM HEPES buffer [pH 6.0] [Sigma] containing 0.3 M sucrose, 200 mM KCl [Sigma], 90 mM MgCl2, and 10 mM CaCl2 [Sigma]). The negative pressure resulting from suction applied to the patch was monitored with a piezoelectric pressure transducer (World Precision Instruments). The presence of channels in a patch was tested by applying suction and voltage to the patch pipette. The channel currents on E. coli MJF465 patches were recorded with an AxoPatch 200B amplifier in conjunction with Axoscope software (Axon). Conductivity was tested by using E. coli MJF465 giant spheroplasts. Conductance was estimated based on the current-voltage plots generated by single-channel analyses from at least three patches from two independent spheroplast preparations. Mechanosensitivity was tested by using E. coli MJF431 giant spheroplasts. To normalize for differences in the pressure required to open channels due to patch-to-patch variations, MscL was used as an internal control for determining gating thresholds. MscL and MscS can sense tension in the membrane and will be affected equally by variations in patch geometry (59). Pressure thresholds were obtained by dividing the pressure at which the first MscL opens by the pressure at which the first MscS opens (PL/PS). PL/PS ratios were calculated from at least three patches from at least two independent spheroplast preparations. Measurements were analyzed by using Clampfit 10.2 (Molecular Devices).

Osmotic down-shock assays.Gonococcal wild-type and mutant strains were grown overnight and suspended at a concentration of approximately 2.5 × 107 CFU/ml in 20 ml GC broth (1.5% [wt/vol] proteose peptone [Oxoid], 0.1% [wt/vol] starch [Vetec], 0.5% [wt/vol] NaCl, 0.4% [wt/vol] K2HPO4 [Vetec], 0.1% [wt/vol] KH2PO4 [Vetec], 0.043% [wt/vol] NaHCO3 [Vetec]) containing 1% (vol/vol) Vitox and 234 mM sucrose. For the induction of the inducible promoter, 1 mM IPTG was added when appropriate. Cultures were incubated at 37°C with shaking at 250 rpm until the OD600 reached 0.5 to 0.6. Duplicate samples of 5 ml were withdrawn and centrifuged at 3,214 × g for 5 min. Pellets were resuspended in prewarmed isosmotic medium (GC broth containing sucrose) or hypo-osmotic medium (GC broth without sucrose and NaCl). After a 20-min incubation at 37°C, bacteria were serially diluted in isosmotic or hypo-osmotic medium and plated onto GC agar plates. Plates were incubated for 24 to 48 h, and colonies were enumerated. Down-shock survival was determined by dividing the number of colonies of bacteria in hypo-osmotic medium with the number of colonies of bacteria in isosmotic medium. To verify down-shock survival of the wild-type and ΔmscS mutant strains, viability was determined by using the Live/Dead BacLight bacterial viability kit (Thermo) according to the manufacturer's recommendations. Fluorescent images were quantified by counting at least 100 bacteria under each condition. All down-shock assays with N. gonorrhoeae were performed in at least three independent biological repeats.

For E. coli down-shock experiments, single colonies of MJF465 containing pB10b-Ng-mscS and pB10b-Ec-mscS were grown overnight in modified LB broth containing 0.5% (wt/vol) NaCl at 37°C with shaking at 250 rpm. Cultures grown overnight were used to inoculate (1% [vol/vol]) fresh modified LB broth and grown at 37°C to an OD600 of approximately 0.15. The cultures were subsequently mixed (1:1) with fresh LB broth containing 0.91 M NaCl and grown until an OD600 of 0.2 was reached. A total of 1 mM IPTG was added, and the cultures were further incubated for 1 h. Aliquots of 20 μl were withdrawn and diluted into 180 μl prewarmed isosmotic LB broth containing 0.5 M NaCl or deionized water. After a 20-min incubation at 37°C, samples were serially diluted in isosmotic LB broth or deionized water and plated onto modified LB agar plates containing 0.5% (wt/vol) NaCl. Plates were incubated overnight at 37°C, and colonies were enumerated. All assays were performed in at least three independent biological repeats.

Growth phenotypes of gain-of-function mutants.Single colonies of E. coli MJF465 containing the different pB10b derivatives were used to inoculate LB broth containing ampicillin. Cultures were grown overnight at 37°C with shaking at 200 rpm and diluted 1:200 in fresh LB broth containing ampicillin. Cultures were incubated at 37°C with shaking at 200 rpm for 2 h. The expression of mscS was subsequently induced by the addition of 1 mM IPTG, and cultures were incubated further. Throughout the time course of the growth experiments, samples were taken every hour for OD600 measurements. All growth experiments were performed in three independent biological repeats.

Mouse vaginal tract competition assays.Competition assays using the mouse vaginal tract infection model were performed as described previously (30, 31). Six- to eight-week-old female BALB/c mice (Shanghai SLAC Laboratory Animal Company) in their diestrus stage of the estrus cycle were administered β-estradiol (Aladdin) subcutaneously on days −2, 0, and 2. To reduce the commensal microflora during this period, trimethoprim (0.4 g/liter; Meilunbio) was provided in the drinking water, and vancomycin (0.6 mg; Meilunbio) and streptomycin (1.2 mg; BBI) were administered intraperitoneally twice daily. For all mouse vaginal tract competition assays, derivatives of N. gonorrhoeae strain ATCC 49226 were used, which naturally contain an rpsL allele that confers streptomycin resistance (identical to rpsL of strain FA1090) (60). Cultures of N. gonorrhoeae wild-type and mutant strains grown overnight were suspended in phosphate-buffered saline (PBS) containing 0.5 mM CaCl2, 1 mM MgCl2, and 1% (wt/vol) gelatin (Aladdin) and mixed to equal numbers. Mice were inoculated intravaginally with the mixed suspensions at a dose of 5 × 107 CFU for each strain. Bacterial loads were monitored and sampled by using vaginal swabs. Undiluted and serially diluted samples were plated onto GC agar plates supplemented with 1% (vol/vol) Vitox, vancomycin, colistin (Meilunbio), nystatin (Meilunbio), trimethoprim, streptomycin, and specific selective antibiotics to distinguish the wild-type and mutant strains. After 48 h of incubation, colonies were enumerated. To ensure reliability in the competition assay, total CFU counts below 150 were excluded from data analyses. The competition index (CI) was calculated as the ratio of CFU counts from the mutant strains to those from wild-type strains in the inoculum and vaginal swabs [(mutant/wild type)output/(mutant/wild type)input]. A CI of <1.0 implicates a fitness advantage for the mutant (61).

Gentamicin protection assays.HeLa human cervix carcinoma cells (ATCC CCL-2) were seeded into 12-well plates at 3 × 105 cells/well in RPMI 1640 cell culture medium (Biological Industries) supplemented with 10% fetal bovine serum (FBS; Bovogen) and grown overnight at 37°C in the presence of 5% CO2. Cells were washed twice with PBS and challenged with N. gonorrhoeae wild-type and mutant strains grown overnight at a multiplicity of infection of 100 in prewarmed RPMI 1640 cell culture medium. After 1 h of infection, cells were washed three times with PBS, and fresh RPMI 1640 medium containing 200 μg/ml gentamicin (Songon Biotech) was added. Samples were taken before and after the addition of gentamicin in a time series up to 6 h. Samples were incubated with 1% saponin (Sigma) to lyse the HeLa cells, serially diluted, and plated onto GC agar plates containing 1% Vitox. Colonies were enumerated after 48 h of incubation at 37°C in the presence of 5% CO2. Experiments were performed in three independent biological repeats.

ACKNOWLEDGMENTS

We thank Dante Neculai from the Zhejiang University School of Medicine for generating the Ng-MscS homology model.

This research was supported by startup funding from Zhejiang University to S.V.D.V., by the Ministry of Science and Technology of China under grant 2014CB910302 to Y.L., and by the Chinese Ministry of Education Project 111 Program under grant B13026 to Y.L. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We declare that we have no conflicts of interest.

FOOTNOTES

    • Received 1 February 2018.
    • Returned for modification 9 March 2018.
    • Accepted 15 March 2018.
    • Accepted manuscript posted online 26 March 2018.

  • Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00090-18.

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KEYWORDS

gonococcus
electrophysiology
patch clamp
hypo-osmotic shock
host colonization
MscS
Neisseria gonorrhoeae

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