Biological characteristics of mechanosensitive channels MscS and MscL in Actinobacillus pleuropneumoniae

ABSTRACT Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance. IMPORTANCE Bacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

and cellular osmotic regulation (4).To meet these physiological functions, individual bacteria possess multiple ion-transporting systems.
Mechanosensitive channels (MS) are conserved ion channels that sense tension changes on the surface of the cell membrane and can convert the physical signals on the surface of the cell membrane into biochemical signals within the cell, thereby regulating the physiological functions of the cell (5,6).During hypotonic stress, cells can rapidly open MS channels and release histocompatibility substances to reduce osmotic pressure in cells, preventing cell lysis due to excessive pressure (7).
Depending on the open pore, mechanosensitive channels can be divided into four categories: MscM (mini, M), MscS (small, S), MscL (large, L), and MscK (potassium, K) (8).So far, MscL and MscS are the most important and well-studied channels.MscL and MscS channels serve primarily to regulate cellar pressure in response to changes in environmental osmotic pressure (9).When Escherichia coli was exposed to hypotonic shock, MscS channels are activated first, and then MscL channels open when membrane tension approaches lysis tension to prevent cell lysis.Abnormal opening of MscL and MscS channels may lead to abnormal discharge of cell contents, resulting in slow cell growth and even death (10,11).
In addition to osmotic pressure, MscL is also involved in regulating bacterial antibiotic sensitivity.Studies have shown that streptomycin and especially dihydrostreptomycin can enter E. coli cells through MscL channels (12).In addition, the novel compound K05, discovered by Wray et al., can bind directly to the MscL to regulate its gating, increasing the efficacy of common antibiotics (13).The compounds SCH-79797 and IRS-16 can directly bind and activate MscL, making the cell membrane permeable (14).Adding MscL activators to antibiotic compound can reduce the toxicity of antibiotics while preventing the development of antibiotic resistance (14).
PCP, caused by infection with A. pleuropneumoniae, can result in significant economic losses annually.Furthermore, it remains unclear whether and what kind of function MscL performs in A. pleuropneumoniae.In this study, we identified and characterized the function of MscL and MscS in A. pleuropneumoniae.We constructed the deletion mutants of mscL and mscS and investigated the role of MscL and MscS in osmotic stress adapta tion, antibiotic resistance, biofilm formation, and virulence of A. pleuropneumoniae.

Bioinformatics analysis of MscS and MscL
To investigate the role of MscL and MscS in A. pleuropneumoniae, we compared the amino acid sequence of MscL and MscS with those of A. pleuropneumoniae and E. coli.BlastP analysis revealed that the MscL sequence of A. pleuropneumoniae and E. coli had 76% amino acid sequence identity (Fig. 1A), while the MscS sequence of both species had 78% amino acid sequence identity (Fig. 1B).According to the consensus motif mentioned in the literature (5), two conserved motifs, TM1 and TM2, were found in the amino acid sequence of MscL, and the homology with E. coli was 90% and 84% (Fig. 1A).Additionally, three conserved motifs, TM1, TM2, and TM3, which respectively share 55%, 57%, and 89% homology with E. coli, were also found in MscS (Fig. 1B).SWISS-MODEL results suggested that MscL and MscS in A. pleuropneumoniae were polymeric porous structures composed of 5 and 7 monomers, respectively (Fig. 1C and D), which were similar to those found in E. coli.In order to gain a comprehensive understanding of the role of MscL and MscS in A. pleuropneumoniae, we used A. pleuropneumoniae S4074 [serovar 1 str, the wild-type (WT) strain] to construct the mscL mutant strain (ΔmscL), mscS mutant strain (ΔmscS), and both mscL and mscS mutant strains (ΔmscL-ΔmscS) (Fig. S1).The growth curve results showed that deleting mscL or mscS did not affect the growth rate of A. pleuropneumoniae (Fig. S2).The scanning electron microscopy (SEM) of ΔmscL, ΔmscS, and ΔmscL-ΔmscS cells revealed that mscL and mscS did not affect cell morphology (Fig. 1E).

MscL is required for sodium hyperosmotic tolerance of A. pleuropneumoniae, but MscS is not
In order to investigate the function of MscS, MscL channels in A. pleuropneumoniae, we measured the activity of the WT, ∆mscL, complemented (C)∆mscL, ∆mscS, complemented (C)∆mscS, and ∆mscL-∆mscS strains in the presence or absence of specific salt ions.In the medium containing 0.3 M Na + , WT, ∆mscS, C∆mscS, and C∆mscL can form colonies efficiently, but ∆mscL and ∆mscL-∆mscS cannot.In addition, the growth of ∆mscL, ∆mscS, and ∆mscL-∆mscS was identical to that of WT, C∆mscS, and C∆mscL in the medium with 0.3 M K + supplemented or Na + removed (Fig. 2A).Furthermore, after overnight incubation in the medium supplemented with 0.3 M Na + , the osmotic survival rates of ∆mscL (47.2%) and ∆mscL-∆mscS (43.3%) were significantly decreased compared to WT, C∆mscS, and C∆mscL, while ∆mscS (100%) had no significant changes in osmotic survival rates (Fig. 2B).To further investigate the reasons for the decreased survival rate of ∆mscL, we used SEM to observe the changes in bacterial morphology and length.The results of SEM showed that the bacterial length of ∆mscL [mean = 2.40 µm; standard deviation (SD) = 1.575] increased significantly (P = 0.0029) compared to that of WT (mean = 1.85 µm; SD = 0.9968) when grown with the supplementation of 0.3 M Na + (Fig. 2C and D).These data demonstrated that MscL plays an essential role in sodium hyperosmolar tolerance.

MscL and MscS are required for some antibiotic resistance in A. pleuropneu moniae
Studies have shown that mechanosensitive channels play a decisive role in antibiotic sensitivity (12,15).In order to investigate the role of MscS and MscL in antibiotic resistance of A. pleuropneumoniae, we performed antibiotic susceptibility tests on six strains.Compared with WT, C∆mscS, and C∆mscL, the sensitivity of ∆mscL and ∆mscL-∆mscS to antibiotics such as chloramphenicol, erythromycin, and penicillin has declined.The sensitivity of ∆mscL to oxacillin has also declined, while the sensitivity of ∆mscS and ∆mscL-∆mscS remains unchanged.Additionally, the sensitivity of ∆mscS to penicillin increased, but the sensitivity of ∆mscL and ∆mscL-∆mscS decreased (Table 1).This indicated that MscL and MscS are required for some antibiotic resistance in A. pleuro pneumoniae.

MscL is required for the biofilm formation in A. pleuropneumoniae, but MscS is not
To evaluate the effects of MscS and MscL on A. pleuropneumoniae, we measured biofilm formation of six strains at 42°C as previously reported.In crystal violet coloration, it can be seen that WT, ∆mscS, C∆mscS, and C∆mscL visibly form biofilms, while ∆mscL and ∆mscL-∆mscS showed almost no biofilm formation (Fig. 3A).Quantitative analysis also showed that, compared to WT, C∆mscS, C∆mscL, and ∆mscS, biofilm formation of ∆mscL and ∆mscL-∆mscS decreased significantly (Fig. 3B).These suggested that MscL is required for A. pleuropneumoniae biofilm formation.

MscL or MscS does not affect A. pleuropneumoniae virulence in mice
To further investigate the impact of mscL and mscS on the virulence of A. pleuropneumo niae, survival and colonization assays were performed in vivo in KM mice using WT, ∆mscL, C∆mscL, ∆mscS, C∆mscS, and ∆mscL-∆mscS strains.As shown in Fig. S2, the survival rates for WT, ∆mscL, C∆mscL, ∆mscS, C∆mscS, and ∆mscL-∆mscS groups were 20%, 30%, 20%, 20%, 20%, and 20%, respectively.There was no significant difference in survival rates between the six groups (Fig. S3A).The bacterial loads in vivo among the six groups also showed no significance difference (Fig. S3B).In summary, these results indicated that MscL and MscS do not affect the virulence of A. pleuropneumoniae in mice.

DISCUSSION
All living organisms need to detect and respond to both external stimuli and inter nal environmental changes, such as gravity and membrane deformation caused by osmotic pressure, to ensure proper growth, development, and reproduction, especially for microorganisms.Different proteins and their complexes in microbial cells can rapidly sense and respond to various mechanical forces to efficiently regulate life processes.Mechanosensitive channels play a crucial role in facilitating this process.As common mechanosensitive channels in bacteria, MscL and MscS participate in various life activities of bacteria.
In the present study, multiple sequence alignments showed that MscL and MscS possess homology with counterparts in E. coli.During the opening of MscL gated channels, the TM1 and TM2 domains tilt and rotate, resulting in thinning of membranes in the membrane plane (16)(17)(18).According to the consensus motif mentioned in the literature (5), two conserved motifs, TM1 and TM2, were found in the amino acid sequence of MscL (Fig. 1A).Additionally, three conserved motifs, TM1, TM2, and TM3, were also found in MscS (Fig. 1B).All of these motifs were similar to sequences found in E. coli.These data indicated that MscL and MscS are highly conserved.
As mechanosensitive channels, MscL and MscS primarily serve to protect cells from lysis when exposed to hypotonic environments, but few studies have been conducted on their role in hypertonic environments.In this study, the protective effect of MscL was demonstrated in the adaptation of A. pleuropneumoniae to hypertonic stress, whereas MscS did not appear to have this effect.In E. coli, the expressions of MscS and MscL were found to be upregulated in response to hypertonic stress.This upregulation is mediated by the stress sigma factor RpoS (19).Notably, A. pleuropneumoniae does not possess  rpoS gene, and the deletion of mscS does not influence the survival of A. pleuropneumo niae in hypotonic environments.Consequently, the role and mechanism of MscL in A. pleuropneumoniae may not be entirely analogous to that in E. coli, which may be limited by its environmental niche.Furthermore, the growth of mscL or mscS deletion mutant strains was not affected on TSA solid medium containing 0 M NaCl (Fig. 2A), probably due to the insufficient downward osmotic pressure applied.Regardless of the initial and final osmotic pressure, lysis of mscL or mscS deletion mutant strains only occurs when the osmolarity drops equivalent to >0.2 M NaCl (19).Commonly used TSA media have an osmolarity of ≈278 mOsm (equivalent to ≈0.14 M NaCl), and removing all NaCl of TSA (equivalent to ≈0.085 M NaCl) cannot cause lysis of mscL or mscS, or both mscL and mscS deletion mutant strains.
As the abuse of antibiotics, resistance as a self-defense mechanism against antibiot ics has been popular around the world.To date, many bacterial antibiotic resistance mechanisms have been elucidated, including antibiotic target modification, envelope permeability reduction, efflux system activation, and enzymatic inactivation (20).MscL channels is considered as a potential drug target, and adding MscL-activating compo nent to antibiotic compounds may help to evade antibiotic resistance (13,14,21).However, little information is available about MscL or MscS in A. pleuropneumoniae, which has hindered the development of antibacterial drug for this bacterium.
In this study, the deletion of mscL has declined the sensitivity to chloramphenicol, erythromycin, oxacillin, and penicillin.MscL functions as an emergency release valve, excreting cytoplasm solutes under hypotonic stress.Inappropriate opening of MscL allows solutes, even drugs, to pass into cytoplasm (22,23).Therefore, MscL may allow chloramphenicol, erythromycin, and oxacillin to enter the cell and play a role.The deletion of mscS has increased the sensitivity to penicillin.Our findings indicate that MscS also plays a role in antibiotic resistance mechanisms.Moller et al. mentioned that MscL and MscS must act sequentially (24), and the presence of mscL may mitigate the deficiencies caused by the deletion of mscS.Consequently, we constructed strains that are deficient in both mscL and mscS.Compared to the deletion of mscL, deleting both mscL and mscS has increased the sensitivity to chloramphenicol, oxacillin, and penicillin.This seems to indicate that MscL and MscS play an antagonistic role, and MscL plays a dominant role.This could explain the differential effects of MscL and MscS on penicillin resistance.Additionally, in osmotic adaptation and biofilm formation, deleting mscL alone or deleting both mscL and mscS resulted in similar defects, while deleting mscS did not cause any defects, further confirming that mscS does not play a role in these physiological activities.
At present, no studies have shown that MscL is involved in biofilm formation.However, in the present study, we showed that MscL is required for the synthesis of biofilm in A. pleuropneumoniae.Berrier et al. suggested that MscL affects the secretion of thioredoxin, elongation factor Tu, and DnaK during osmotic shock (25).Morra et al. showed that proteins are released in a manner dependent on MscL.These findings suggested that MscL influences the secretion of cytoplasmic proteins (26).Biofilms are aggregated bacterial groups that are embedded in a matrix of extracellular polymeric substances (27).Biofilm contains extracellular polymeric substances (EPS), whose main components are extracellular DNA, polysaccharide, and proteins (28).The formation of biofilm involves a series of steps, and the secretion of exopolysaccharide matrix and proteins is an essential part of this process (29).In addition, biofilms served as a growth mode to help bacteria resist external stimuli, and the ability to form biofilm reflects the virulence of bacteria to a certain extent (28,30).MscL, as a protein secretion system, should have an impact on bacterial virulence.In this study, the deletion of mscL resulted in decreased biofilm production, but it did not affect bacterial virulence.Detailed analysis of the molecular mechanism by which MscL affects A. pleuropneumoniae biofilm formation will be the focus of further research.
In conclusion, our research has provided valuable insights into the role of MscL and MscS in A. pleuropneumoniae, showing that MscL and MscS are involved in various physiological functions of A. pleuropneumoniae.Given the potential of MscL and MscS as targets for antibiotic development, our findings are expected to contribute to the advancement of antibiotic therapies targeting MscL and MscS.Subsequent research will aim to elucidate the mechanism by which MscL and MscS affects A. pleuropneumoniae resistance, thereby enhancing the understanding of MscL and MscS.

Construction of mutant and complementary strains
To obtain upstream and downstream of the target gene by PCR amplification, the genome of A. pleuropneumoniae was used as template.The upstream and downstream fusion fragments from fusion amplification were ligated to the suicide plasmid pEMOC2 to create a recombinant plasmid, which was sequenced to ensure its precision.Then, the plasmid was transferred to E. coli β2155.After homologous recombination, the mutant strains of the target gene were selected by chloramphenicol screening.
The gene was ligated to the shuttle plasmid pJF224-XN with corresponding restriction enzymes and transferred into the mutant strain by electroporation (2.5 KV; 25 μ FD; 800 Ω) to generate complementary strains.The mutant and complementary strains were verified by PCR and DNA sequencing (data not shown).

SEM
The SEM was performed to investigate the effect of mscL and mscS on bacterial cell morphology.Wild-type S4074 and its mutant derivatives were cultured on TSB medium (supplemented with 10% FBS and 10% NAD) with or without 0.3 M NaCl.Bacterial cells were harvested after centrifugation, fixed with 2.5% glutaraldehyde, and then deposited onto copper grids (200 mesh; Zhongjingkeyi, China).The air-dried copper grids were mounted on the sample stage using carbon conductive tape (Zhongjingkeyi, China) and then coated with a sputter-coater.Subsequently, samples were observed using the SEM (VEGA3; TESCAN, Czech Republic).

Osmotic shock
Survival rate after osmotic shock was examined according to the method previously described (9,31).A. pleuropneumoniae (WT, ΔmscL, CΔmscL, ΔmscS, CΔmscS, and ΔmscL-ΔmscS) cultured overnight were transferred to TSB medium, shaken in 37°C, serially 10-fold diluted at OD 600 nm of 0.6, and spotted or plated onto different TSB plates.The survival rate was calculated (nshock/ncontrol) from the number of colonyforming units of cells that experienced osmotic shock (nshock) and those that did not experience the shock (ncontrol).

Biofilm assay
The biofilm experiment was carried out following the procedure outline in the literature (32).The overnight cultured A. pleuropneumoniae (WT, ΔmscL, CΔmscL, ΔmscS, CΔmscS, and ΔmscL-ΔmscS) were diluted 1:100 in fresh TSB medium, transferred to 200 µL of diluent to each well in a 96-well microplate, and four replicated wells were used for each strain.Then, we incubated the microplate at 42°C for 48 h.After culture, we removed the bacterial solution, washed each well three times with sterile saline (0.9% NaCl, wt/vol), and stained it with 100 µL per well of crystal violet (0.1%, wt/vol) for 10 min at room temperature.We removed crystal violet solution and washed the wells under running tap water to remove the excess dye.The microplate was then inverted and allowed to dry at 37°C for 30 min.Once the microplate was completely dry, it was photographed.For quantitative analysis, 100 µL of glacial acetic acid solution (33%, vol/vol) was added to each well to dissolve the crystal violet.Subsequently, the dissolved solution was measured at OD 590 nm using Multi-Detection Microplate Reader.

Determination of minimum inhibitory concentrations (MIC)
MICs were measured on 96-well microplates using 2-fold broth dilution.To prepare the bacterial standard solution, A. pleuropneumoniae (WT, ΔmscL, CΔmscL, ΔmscS, CΔmscS, and ΔmscL-ΔmscS) were cultivated at 37°C to OD 600 nm of 0.6, adjusted to 0.5 McFarland standard with sterile saline (0.9% NaCl, wt/vol), and further diluted 1:100 with TSB.To each well, 100 µL of standard bacterial solution and 100 µL of antibiotics were added.A blank control was prepared by adding 100 µL of TSB and 100 µL of antibiotic to corresponding wells.A positive control was prepared by adding 200 µL of WT stand ard solution to the corresponding wells.The antibiotics used in the experiment were chloramphenicol (0.25-100 mg/mL), erythromycin (0.01-15 mg/mL), oxacillin (5-50 mg/ mL), and penicillin (0.08-5 mg/mL).The 96-well plates were incubated at 37°C for 24 h.The minimum concentration that inhibits the visible growth was confirmed as MIC.All experiments were performed in triplicate.

Animal experiments
A total of 36 four-week-old female Kunming (KM) mice (CTGU University Laboratory Animal Center, Yichang, China) were randomly divided into six groups of six mice each.Each group of mice received an intraperitoneal injection of 1 × 10 7 CFU A. pleuropneu moniae (WT, ΔmscL, CΔmscL, ΔmscS, CΔmscS, and ΔmscL-ΔmscS) per mouse, and the remaining four mice were injected intraperitoneally with the same dose of normal saline (0.9% NaCl, wt/vol) as a blank control.Survival for each group was recorded once a day for 1 week following the injection.
Four-week-old female Kunming (KM) mice (n = 6 in each group) were used to assess colonization ability of A. pleuropneumoniae ΔmscL, ΔmscS, and ΔmscL-ΔmscS.The mice were administered intraperitoneally A. pleuropneumoniae WT, ΔmscL, CΔmscL, ΔmscS, CΔmscS, and ΔmscL-ΔmscS (1 × 10 7 CFU/mouse).At 6 h after injection, the mice were euthanized, and lungs were collected aseptically.Half of the lung was homogenized in normal saline (0.9% NaCl, wt/vol) and serially diluted.Appropriate diluents of lung tissue were plated on TSA plates for determination of bacterial load.The remaining lung was immersed in tissue fixative solution (Biosharp, Beijing, China) for histopathological analysis as previously described (33).

Bioinformatic and statistical analysis
The amino acid sequences of mscS and mscL were downloaded from National Center for Biotechnology Information.MEGA 11 (Mega Limited, New Zealand) was used to compare their homology, prediction of transmembrane domains (TM) was preformed using DTU/ DeepTMHMM -BioLib, and the results were processed using GeneDoc 2.7 (FSF Inc., USA).The three-dimensional structures of MscL and MscS were predicted using SWISS-MODEL.The statistical analyses of experimental data were performed using two-tailed Student's t-tests through GraphPad (GraphPad Inc., USA).All data were presented as means ± SD.Statistical significance was determined if the P value is <0.05.

TABLE 1
Susceptibility of Actinobacillus pleuropneumoniae strains to various antibiotics a

Antibiotics MIC (µg/mL) of six A. pleuropneumoniae strains
a MIC, minimal inhibitory concentration.