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Deciphering the impact of MreB on the morphology and pathogenicity of the aquatic pathogen Spiroplasma eriocheiris

Biology Direct volume 19, Article number: 98 (2024) Cite this article

Abstract

Background

Spiroplasma eriocheiris has been proved to be a pathogen causing tremor disease of Eriocheir sinensis, it is also infectious to other aquatic crustaceans, resulting in a serious threat on the sustainable development of the aquaculture industry. S. eriocheiris is a helical-shaped microbe without a cell wall, and its motility is related to the cytoskeleton protein MreB which belongs to the actin superfamily and has five MreB homologs.

Results

In this study, we purified MreB3, MreB4 and MreB5, and successfully prepared monoclonal antibodies. After S. eriocheiris treated with actin stabilizator Phalloidin and inhibitors A22, we found that Phalloidin and A22 affect the S. eriocheiris morphology by altering MreB expression. We confirmed that the ability of S. eriocheiris to invade E. sinensis was increased after treatment with Phalloidin, including that the morphology of E. sinensis blood lymphocytes was deteriorated, blood lymphocytes viability was decreased, peroxidase activity and cell necrosis were increased. On the contrary, the pathogenicity of S. eriocheiris decreased after treatment with A22.

Conclusions

Our findings suggest that the MreB protein in S. eriocheiris plays a crucial role in its morphology and pathogenicity, providing new insights into potential strategies for the prevention and control of S. eriocheiris infections.

Background

Spiroplasma was first discovered in maize with dwarf disease by Robert Davis, an American plant pathologist. It belongs to the taxonomic groups Mollicutes, Entomoplasmatales and Spiroplasmataceae. Like other members of Mollicutes, S. eriocheiris is characterized by its absence of a cell wall [1, 2], has a diameter of about 0.1–0.2 μm, and it can pass through a filter membrane with a pore size of 0.22 μm. The vast majority of Spiroplasma species identified to date have been isolated from insects and terrestrial plants, where they commonly exhibit parasitic behavior within host cells. Over the course of their long-term evolution, these bacteria have established three distinct relationships with their hosts parasitism, symbiosis, and pathogenicity [3]. “Trembling disease” is one of the most serious epidemic diseases of E. sinensis, which causes huge economic losses to the aquaculture industry [4,5,6]. There is a research team isolated and cultivated the pathogen that causes the disease, they designated it as S. eriocheiris [7], this discovery challenged the previous notion that Spiroplasma exclusively infects terrestrial plants and insects, thus expanding our understanding of its host range from land to water for the first time [8]. In recent years, S. eriocheiris has also been found in aquatic crustaceans such as M. rosenbergii, P. clarkii, and M. nipponense [8,9,10]. In addition, researchers have isolated a strain of bacteria that is phylogenetically related to S. eriocheiris from blood cultures of Chinese patients who developed a lung infection after surgery. This result shows that S. eriocheiris not only harms aquaculture industry, but also may pose a threat to human health [11].

The morphology of S. eriocheiris is variable due to the absence of a cell wall, exhibiting different forms during various growth stages. Despite lacking flagella or cilia, S. eriocheiris can still rely on its cytoskeleton for movement. The cytoskeleton not only maintains the helical morphology of S. eriocheiris, but also functions as a motor that directly powers its movement [12, 13]. Previously, it was believed that cytoskeletons were exclusive to eukaryotes. In eukaryotes, the cytoskeleton encompasses the cytoplasmic network composed of three protein components, including microtubules, microfilaments, and intermediate filaments. However, recent studies have revealed their presence in prokaryotes such as bacteria. To date, the bacterial proteins FtsZ, MreB, and CreS have been identified as counterparts of tubulin, actin, and intermediate filament in eukaryotic cell cytoskeleton proteins, respectively. These proteins play crucial roles in cellular processes such as cell division, morphological maintenance and regulation [14]. The cytoskeleton protein MreB, among the three cytoskeleton proteins discovered in bacteria, has been identified in numerous bacterial species [15], including Bacillus subtilis, Thermus thermophiles, Escherichia coli, Myxococcus xanthus and so on. The study showed that the S. eriocheiris genome contains five to seven MreB homologs, each of which is an independent replicate and has a different function [13, 16]. MreB polymerizes into non-helical filaments with juxtaposed subunits, in which two filaments interact and are inverted parallel [17]. It was demonstrated that the structure of MreB and fibril is crucial in the movement of S. citri [18], the MreB polymer structure coordinates fibril to maintain helicity produce kinking motions [19]. Due to its distinctive helical structure and locomotion mechanism, S. eriocheiris is widely recognized as a highly appealing model organism for international research [20]. The helical shape of S. eriocheiris is maintained by the cytoskeleton within its cells, which also serves as the propulsive force driving its movement and preserving its helical morphology [21]. In recent years, numerous scholars have conducted preliminary investigations on the cytoskeleton and motility of S. eriocheiris. The MreB proteins constitute a significant portion of the cell cytoskeleton in S. eriocheiris, with five subtypes identified [22]. Among these proteins, four MreB proteins (MreB1, MreB2, MreB4, and MreB5) have been characterized in the genome of S. eriocheiris, demonstrating their crucial role in maintaining cellular morphology and motility [13]. S. eriocheiris exhibited a typical helical shape during the logarithmic growth period, while during the decay period, it displayed a spherical shape (Unpublished data). The transcription of the five MreB genes differed significantly between these two phenotypes (unpublished data), indicating that MreB is closely associated with the phenotype of S. eriocheiris.

A22(S-(3,4-dichlorobenzoyl) isothiourea) is a derivative of S-benzoyl isothiourea, it was discovered during a randomized screening of E. coli chromosome assignment inhibitors using the nucleophile blue test [23, 24]. The compound A22 is a reversible, competitive inhibitor of the actin-like protein MreB that can induce changes in cell shape, division defects and irregular segregation of chromosome in E. coli and Caulobacter crescentus [14]. The A22 was first shown to induce the formation of spherical cells in E. coli [25]. Several studies have demonstrated that A22 disrupts the rod-like structure of bacterial cells by interacting with MreB, resulting in a transition from a rod-like to a spherical shape [26,27,28]. Phalloidin is a bicyclic heptapeptide originally isolated from the death cap mushroom Amanita phalloides and is well known for its ability to bind stochiometricly to F-actin [29]. It binds to actin filaments much more tightly than to actin monomers and shifts the equilibrium between filaments and monomers toward filaments [30]. The investigation into the cytoskeleton proteins of S. eriocheiris is still in its nascent stage, and the findings have established a robust groundwork for subsequent research on the cytoskeleton of S. eriocheiris.

In this study, we localized MreB in S. eriocheiris and successfully observed the effects of Phalloidin and A22 on the morphology of S. eriocheiris, examined the changes in the expression of MreB in response to treatment with Phalloidin and A22, and investigated the effects of Phalloidin and A22 on the pathogenicity of S. eriocheiris.

Materials and methods

Monoclonal antibody preparation of MreB

Five specific peptides of MreB were sent to the company for chemical synthesis and preparing specific monoclonal antibodies, the specific sequences of the five chemically synthesized MreB are shown in Table 1. After the monoclonal antibody was prepared, the effect of the antibody was detected by Western Blot. The logarithmic phase of S. eriocheiris was crushed by a cell crusher, and then the prepared protein samples were subjected to SDS-PAGE gel electrophoresis. PVDF membranes and four sheets of filter paper of appropriate size were cut and equilibrated in the transfer buffer, and then placed in the order of filter paper, gel, PVDF membrane and filter paper, and the transfer was carried out at 80 V. After the membrane transfer, the PVDF film were blocked with 5% skim milk for 2 h, washed with TBST, and then incubated at 4 °C overnight with MreB3, MreB4, MreB5 and the reference SPE-0313 primary antibody. Finally, HRP-conjugated anti-mouse IgG (H + L) was added, and the mixture was incubated at room temperature for 2 h. The proteins were detected with the high-sensitivity ECL chemiluminescence kit. Finally, MreB3, MreB4 and MreB5 were all detected at the corresponding locations (Figure. S1), while MreB1 and MreB2 had no specific bands and their monoclonal antibodies were unavailable, MreB3, MreB4 and MreB5 antibodies were successfully prepared and had good specificity, which was sufficient to meet the needs of downstream experiments.

Table 1 Sequences of MreB to prepare monoclonal antibodies
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Observation on the morphology and cytoskeleton of S. eriocheiris in buffer and H2O

S. eriocheiris inoculated were observed under a phase contrast microscope to ensure that they were not contaminated with stray bacteria. The S. eriocheiris reached logarithmic stage was centrifuged and then suspended with PBS and H2O respectively, phase contrast microscope was applied to observe and photograph. S. eriocheiris was collected and resuspended with PBS, the resuspended S. eriocheiris was dripped onto a copper mesh and kept at room temperature for 10 min. Finally, 1% ammonium molybdate was used for negative dyeing for 1 min. After the excess dyeing solution was removed, the samples were put into an electronic drying oven overnight for observing morphology under electron microscope. S. eriocheiris was treated with the same manner as described above. Triton X-100 was dropped onto the copper mesh containing the sample, and the excess Triton X-100 immediately was sucked off with filter paper, repeating this process three times. The copper mesh was washed 5 times with PBS after Triton X-100 treatment to remove the residual treatment solution. Finally, 1% ammonium molybdate was used for negative dyeing for 1 min, after absorbing excess dyeing solution, the samples were put into an electronic drying oven overnight for observing the cytoskeleton under electron microscope. S. eriocheiris was cultivated to logarithmic stage and suspended with PBS and H2O. Electron microscope was used to observe the corresponding shape is helical or spherical, S. eriocheiris was broken by cell crusher, and the protein samples were prepared for SDS-PAGE gel electrophoresis, Refer to “Monoclonal antibody preparation of MreB” for the procedure of membrane transfer.

Detection of MreB localization using immunoelectron microscopy

The copper mesh with carbon film was irradiated under ultraviolet lamp for 30 min to increase the adsorption capacity of the copper mesh. Two mL S. eriocheiris in the logarithmic phase were taken, centrifuged, and resuspended with H2O and PBS, respectively. The resuspended S. eriocheiris was dripped onto a copper mesh and kept at room temperature for 10 min. Making a wax disc with a candle, and then adding 3% BSA dropwise. The edge of the copper mesh was clamped with tweezers and placed upside down on the BSA. After being sealed at room temperature for 30 min, it was washed with PBST. The monoclonal antibodies to MreB3, MreB4 and MreB5 prepared by the company were used as primary antibodies. The primary antibody was taken and dripped onto the wax plate, the cleaned copper mesh was upside down on the primary antibody, and incubated at room temperature for 1 h before cleaning. Anti-rabbit IgG labeled with colloidal gold particles was used as the secondary antibody, it was mixed with PBS containing 1% BSA, and then the mixed secondary antibody was dripped onto the wax plate. The copper mesh was upside down on the secondary antibody, and the negative dyeing was performed with 1% ammonium molybdate for 1 min. Finally, the excess dye solution was absorbed with filter paper, and samples were observed under electron microscope after being put in an electronic drying oven overnight.

The observation of the morphology and cytoskeleton of S. eriocheiris

The copper grid is exposed to ultraviolet light to increase its adsorption capacity, S. eriocheiris was cultivated to the logarithmic phase with 2.5 µg/mL phalloidin and 200 µg/mL A22, then collected and re-suspended with PBS. The re-suspended S. eriocheiris was dripped onto a copper grid and kept at room temperature for 10 min. Triton X-100 solution is configured with PBS, it is a derivative of polyethylene glycol and was dropped onto the copper grid containing the sample, and immediately suck off the excess Triton X-100 solution with filter paper, repeat this process three times. The copper grid was washed 5 times with PBS after Triton X-100 treatment to remove the residual treatment solution. Finally, 1% ammonium molybdate was used for negative dyeing for 1 min. After absorbing excess dyeing solution, the samples were put into an electronic drying oven overnight for observation under electron microscope. The changes in MreB expression were detected by Western Blot as described above.

Quantity of S. eriocheiris detected using CCU method

The Phalloidin and A22 were dissolved in DMSO and sterile H2O respectively to prepare the solution, and then the overnight cultivated S. eriocheiris was treated with the prepared solution. The treated S. eriocheiris was put in an incubator at 30 °C for 12 h, and the photos were taken after using microscopy to ensure that it was not contaminated with bacteria. After extending the cultivation time of the above S. eriocheiris to 8 days, three groups of S. eriocheiris incubated for 12 h and 8 days were taken for microscopic examination to ensure that they were not contaminated with stray bacteria. The CCU method, also called the colour change unit method, is based on the principle of detecting the relative concentration of S. eriocheiris by using the metabolic activity of the S. eriocheiris in a liquid medium. This method is usually used for microorganisms that are too small to be counted using normal turbidimetry [31]. In this experiment, a new centrifuge tube was taken from each group with S. eriocheiris medium, and then 100 µL of S. eriocheiris from each group that had been cultured for 12 h and 8 days was taken into the first centrifuge tube and mixed. 100 µL of the mixture from the first centrifuge tube was added to the second centrifuge tube and so on, until the tenth centrifuge tube. The above centrifugal tube was sealed and put into an incubator at 30 °C. The sample was taken out for microscopy examination and imaging after 12 h.

Statistics on S. eriocheiris mortality and E. sinensis survival rate

The S. eriocheiris cultivated with 2.5 µg/mL Phalloidin and 200 µg/mL A22 were examined by microscope to ensure that they were not contaminated with other bacteria. The S. eriocheiris were collected by centrifugation at 4 ℃ and 11,000 rpm for 10 min, and the supernatant was discarded. After two times centrifugation washes with pre-cooled PBS. Binding buffer, AnnexinV-FITC and PI Staining Solution were added and gently blown out respectively. Binding buffer was added after incubation for 10 min at room temperature away from light, and the stained samples were detected by flow cytometry. The S. eriocheiris cultivated with Phalloidin and A22 was collected and resuspended, and then divided into four groups, including R2 medium, S. eriocheiris group, Phalloidin treatment group and A22 treatment group. Each E. sinensis was injected with 100 µL of the corresponding S. eriocheiris, and R2 was used as a control. Finally, the circulating water system where the infected E. sinensis lives was heated to 28 °C, and the survival rate was counted daily.

Morphology observation of blood lymphocytes in E. sinensis

Six healthy E. sinensis were selected and their appendages were disinfected with alcohol. A total of 12 tubes of 400 µL of blood were taken in centrifuge tubes containing anticoagulant. After centrifugation, the supernatant was discarded and resuspended in L15 medium. The blood lymphocytes were added to the six-well plate and cultivated in an incubator at 28 °C for 3 to 6 h until they were adherent to the wall. Then the cells were divided into four groups, including L15, S. eriocheiris, S. eriocheiris + Phalloidin and S. eriocheiris + A22 group. The S. eriocheiris cultivated with Phalloidin and A22 was examined under a microscope to ensure that it was free of foreign bacteria. After collecting the S. eriocheiris, it was resuspended with L15 and added into each well of the six-well plate. The cell morphology was observed at 0, 12 and 24 h after S. eriocheiris infection.

Activity detection of blood lymphocytes in E. sinensis

The detailed procedures for the cultivation of blood lymphocytes and the S. eriocheiris treatment can be found in “Morphology observation of blood lymphocytes in the E. sinensis”. To detect the activity of blood lymphocytes, the old medium was removed from the cells after morphological observation, and new medium was added, then 100 µL CCK-8 Solution was added and incubated in an incubator at 28 °C for 1 h. Finally, the absorbance at 450 nm was detected by the ELISA instrument. The culture medium in the six-well plates was removed after infection for 0, 12 and 24 h. The total DNA of blood lymphocytes was extracted according to the EasyPure Genomic DNA kit. The concentration of DNA and dilute each sample to the same concentration. The copy number of S. eriocheiris was detected by qRT-PCR, and finally statistical analysis was conducted by SPSS software. The blood lymphocytes were cultivated according to the same steps as above and then resuspended by centrifugation. L-dopamine and Tris-HCl were added after incubation with ultrasonic crushing, and the absorbance values were measured at 490 nm. The concentration of the broken liquid was measured by Coomassie brilliant blue protein quantitative test box. Finally, the phenoloxidase (PO) activity of blood lymphocytes was detected according to the formula for calculating PO activity.

Flow cytometry for blood lymphocytes apoptosis detection

The detailed procedures for the cultivation of blood lymphocytes and the S. eriocheiris treatment can be found in “Morphology observation of blood lymphocytes in the E. sinensis”. After the cells were stained with Annexin V-FITC/PI Apoptosis Detection Kit, the apoptosis of the cells was detected by flow cytometry. Refer to “Statistics on S. eriocheiris mortality and E. sinensis survival rate” for specific operation methods.

Statistic method

Data were analyzed using SPSS 13.0 software. A t-test was used for comparison between the two groups, significant difference was determined when the P value was less than 0.05.

Results

Morphology of S. eriocheiris and cytoskeleton in buffer and H2O

To observe the S. eriocheiris in the logarithmic stage, S. eriocheiris not only had a typical helical shape in the medium, but also in the PBS buffer (Fig. 1A), S. eriocheiris at logarithmic phase rapidly changes from helical to spherical when cells were placed in H2O (Fig. 1B). When the PBS buffer is added back, it quickly reverts from a spherical shape to a typical helical shape. To observe the morphology of S. eriocheiris more clearly, S. eriocheiris was negatively stained and then observed by transmission electron microscope. Electron microscopy results revealed the typical helical structure of S. eriocheiris in the PBS buffer, with clear observation of the tip structure and tail (Fig. 1C), but appears spherical in H2O (Fig. 1D). The cytoskeleton of S. eriocheiris plays an important role in its growth and morphology. Under electron microscope observation, the S. eriocheiris cytoskeleton in PBS was composed of dumbbell-shaped structure and a flat ribbon-like structure, (Fig. 1E), which was similar to the previous study [13], but the cytoskeleton of S. eriocheiris cell in the H2O showed the round structure, which did not have dumbbell and flat ribbon structure, and only filament structures were observed (Fig. 1F).

Fig. 1
figure 1

Morphology of S. eriocheiris and cytoskeleton in buffer and H2O. (A) Phase contrast microscope image of S. eriocheiris in PBS. S. eriocheiris in PBS buffer presents a helical shape. (B) Phase contrast microscope image of S. eriocheiris in H2O, where the S. eriocheiris appears spherical. (C) Negative electron microscopy image of S. eriocheiris in PBS, S. eriocheiris in PBS is helical shape. (D) Electron microscope negative staining image of S. eriocheiris in H2O, the S. eriocheiris in H2O appears spherical. (E) The cytoskeleton of S. eriocheiris in PBS was observed by electron microscopy. The cytoskeleton of S. eriocheiris in PBS showed a slender helical shape. (F) The cytoskeleton of S. eriocheiris in H2O was observed under electron microscope, and the cytoskeleton of spherical S. eriocheiris in H2O showed a rounded shape

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Alteration of S. eriocheiris cytoskeleton protein expression in buffer and H2O

According to the above experiments, when the morphology of S. eriocheiris changes, its cytoskeleton also changes accordingly. To further investigate whether the expression of the cytoskeleton protein MreB changed when the S. eriocheiris presented a spherical shape, quantitative western blot analysis was performed (Fig. 2A). The results of the experiment indicate that the MreB5 of helical S. eriocheiris was not significantly different compared with spherical S. eriocheiris at 0, 6 and 12 h (Fig. 2D), there was no significant difference in the expression levels of MreB3 and MreB4 in helical S. eriocheiris at 0 and 6 h, but they were significantly higher than spherical S. eriocheiris at 12 h (Fig. 2B and C).

Fig. 2
figure 2

Alteration of cytoskeleton expression in S. eriocheiris. (A) Western Blot analysis revealed that S. eriocheiris MreB5 in the PBS group did not differ at 0, 6 and 12 h compared to H2O group, and the expression of MreB3 and MreB4 was also not different at 0 and 6 h, but was higher at 12 h than H2O group. ORF313 as the internal parameter. (B, C, D) The gray value analysis of Western Blot result of MreB3, MreB4 and MreB5. (*: p < 0.05, ***: p < 0.001)

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Localization of MreB in S. eriocheiris

To determine whether MreB localization on the surface of S. eriocheiris, we incubated S. eriocheiris with MreB-specific antibody, and then incubated it with secondary antibody containing gold particles. After negative staining, the stained samples were observed by electron microscopy. As shown in Fig. 3, in the control group using primary antibodies labeled with His tags as a negative control, no gold particles were observed on the surface of S. eriocheiris (Fig. 3A). But specific primary antibodies of MreB3, MreB4 and MreB5 were presented on the surface of S. eriocheiris, respectively, because some gold particles were aggregated on one end of S. eriocheiris (Fig. 3B and D), and others were uniformly distributed on the whole surface of S. eriocheiris (Fig. 3C). The results revealed that the three kinds of cytoskeleton proteins were distributed in the outer membrane of S. eriocheiris.

Fig. 3
figure 3

Localization of MreB in S. eriocheiris. (A) Immuno-electron microscopy images. No gold particles were observed on the surface of S. eriocheiris after incubation with His-tagged protein primary antibody as a negative control. (B, C) Immuno-electron microscopy image. The S. eriocheiris was incubated with a primary antibody specific to MreB3 and MreB4, and gold particles were observed in an aggregated state at one end of the organism, as indicated by the red arrow. (D) Immuno-electron microscopy images. The strain was incubated with a primary antibody specific to MreB5, resulting in the even distribution of gold particles on the surface of S. eriocheiris, as indicated by the red arrow

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The effects of Phalloidin and A22 on the morphology and MreB expression of S. eriocheiris

After identifying the cytoskeleton protein MreB in S. eriocheiris, we treated S. eriocheiris with the promoter Phalloidin and the specific inhibitor A22, and the morphology was observed by transmission electron microscopy after negative staining. As shown in Fig. 4, compared to the normal form of the S. eriocheiris (Fig. 4A), the length of S. eriocheiris increased significantly after treatment with Phalloidin (Fig. 4B), while the morphology of S. eriocheiris changed from a typical helix to a nearly spherical shape after treatment with A22 (Fig. 4C). The above results suggest that MreB promoter Phalloidin and inhibitor A22 can change the length of S. eriocheiris by promoting or inhibiting MreB polymerization. Phalloidin and A22 can change the morphology of S. eriocheiris by affecting the polymerization of MreB. To further explore the alteration of S. eriocheiris MreB expression at the protein level after treatment with Phalloidin and A22, the S. eriocheiris treated with Phalloidin and A22 was broken, and the protein was quantitatively analyzed by MreB specific antibody. There was no significant alteration in the expression of MreB3 and MreB5 after treatment with MreB promoter Phalloidin and MreB inhibitor A22 (Fig. 4D and F). There was no significant difference between MreB4 in Phalloidin treated S. eriocheiris and normal S. eriocheiris, but the expression of MreB4 was significantly decreased after treatment with A22 (Fig. 4E). The above results suggest that A22 can reduce the expression of MreB and change the morphology of S. eriocheiris.

Fig. 4
figure 4

Morphology of S. eriocheiris and expression of the cytoskeleton protein MreB after treatment with Phalloidin and A22. (A, B, C) S. eriocheiris's TEM negative staining images. A, using S. eriocheiris as a negative control. B, the length of S. eriocheiris was significantly increased after treatment with Phalloidin. C, the morphology of S. eriocheiris transformed from a spiral shape to an almost spherical shape following treatment with A22. (D, E, F) The gray value of MreB3, MreB4 and MreB5 in S. eriocheiris, following treatment with Phalloidin and A22, D and F did not exhibit significant alterations in expression levels. E, the expression of MreB5 was significantly reduced after A22 treatment of S. eriocheiris. (G) Western blot analysis of the expression of the skeleton protein MreB, SPE-0313 was used as reference. 1: S. eriocheiris without any treatment as control. 2: MreB expression in S. eriocheiris after treatment with Phalloidin. 3: MreB expression in S. eriocheiris after treatment with A22. (*: p < 0.05)

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Effects of Phalloidin and A22 on the growth of S. eriocheiris

To investigate the effect of MreB on the pathogenicity of S. eriocheiris, we compared the growth of S. eriocheiris under different treatments. After S. eriocheiris was treated with 2.5 µg/mL Phalloidin for 12 h, the color of the medium changed from red to yellow, while the color of culture medium only changed slightly after treatment with 200 µg/mL A22 for 12 h (Fig. 5A). The above results showed that the Phalloidin had no significant effect on the growth rate of S. eriocheiris, while A22 significantly delayed the growth of S. eriocheiris. In order to further explore the effect of Phalloidin and A22 on the number of S. eriocheiris, S. eriocheiris was treated with 2.5 µg/mL Phalloidin and 200 µg/mL A22, and the number of S. eriocheiris was detected by the CCU method. The results showed that compared with the control group, the number of S. eriocheiris did not change after treatment with Phalloidin, but decreased to106 after treatment with A22 for 12 h (Fig. 5B). The S. eriocheiris after treated with A22 was cultured for 8 days, the number of S. eriocheiris was as high as 108 in the control group (Fig. 5C). These results showed that there was no change in the growth rate and quantity of S. eriocheiris, but A22 could significantly slow down the growth rate of S. eriocheiris, and had no significant effect on the final number of S. eriocheiris.

Fig. 5
figure 5

The impact of Phalloidin and A22 on the S. eriocheiris growth. (A) The alterations of the medium color of S. eriocheiris after treatment with Phalloidin and A22. I: S. eriocheiris as negative control. II: S. eriocheiris was treated with 2.5 µg/mL Phalloidin for 12 h, the medium color changed from red to yellow. III: S. eriocheiris after treatment with 200 µg/mL A22 for 12 h, the medium color slightly changed. (B) Calculating the number alterations of S. eriocheiris in the first three groups presented in 5A using CCU method. I: In the negative control group, the number of S. eriocheiris can reach 108. II: The number of S. eriocheiris treated with Phalloidin could also reach 108, without alterations. III: The number of S. eriocheiris was reduced after 12 h treatment with A22, and only reached 106. (C) The front of three groups of S. eriocheiris were cultivated for 8 days, and calculated the alterations of number by CCU method. I: Negative control group. II: The number of S. eriocheiris could also reach 108 after treatment with Phalloidin, without alterations. III: The number of S. eriocheiris treated with A22 could also reach 108 after culture for 8 days

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Effects of Phalloidin and A22 on the mortality of S. eriocheiris

To explore whether S. eriocheiris could be broken and killed by Phalloidin and A22, S. eriocheiris was treated with 2.5 µg/mL Phalloidin and 200 µg/mL A22, and the fragmentation and death of S. eriocheiris were detected by flow cytometry. The flow cytometry results showed that compared with the control group (Fig. 6A), there was no significant fragmentation or death of S. eriocheiris in the Phalloidin treatment group (Fig. 6B), but 49.5% of the S. eriocheiris in the treatment group of A22 showed fragmentation and death (Fig. 6C). The results show that A22 has a certain effect on the fragmentation and death of S. eriocheiris. After centrifugation, the S. eriocheiris were resuspended with R2, and each E. sinensis was injected with 100 µL S. eriocheiris, and the number of deaths was counted every day. The result revealed a diminished E. sinensis survival rate in the Phalloidin group compared to the untreated, wild-type S. eriocheiris group at 5, 7, and 9 days post-infection (Fig. 6D), suggesting that Phalloidin may amplify the pathogenicity of S. eriocheiris. Conversely, the A22 treated group displayed an elevated E. sinensis survival rate relative to the untreated wild-type S. eriocheiris group at 7 and 9 days (Fig. 6D), indicating a potential reduction in S. eriocheiris pathogenicity due to A22. Notably, no significant difference in E. sinensis survival rates was observed between the wild-type S. eriocheiris group, the A22 treated group, and the Phalloidin treated group 9 days following the injection.

Fig. 6
figure 6

Effects of Phalloidin and A22 on mortality and virulence of S. eriocheiris. (A) The death of S. eriocheiris was detected by flow cytometry, it is the control group. (B) No significant fragmentation and death of S. eriocheiris treated with Phalloidin compared to the control group. (C) 49.5% of the S. eriocheiris in the A22 treatment group were fragmented and dead. (D) The survival rate of E. sinensis infected with S. eriocheiris treated with Phalloidin and A22. The survival rate of S. eriocheiris in the Phalloidin group was lower than the S. eriocheiris group at 5, 7 and 9 days, the survival rate of A22 treated group was higher than S. eriocheiris group at 7, and 9 days. (* indicates a significant difference between this group and the S. eriocheiris group)

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Effects of S. eriocheiris treated with Phalloidin and A22 on blood lymphocytes

Morphology of blood lymphocytes

To explore the effect of S. eriocheiris treated with Phalloidin and A22 on blood lymphocytes of E. sinensis, S. eriocheiris treated with 2.5 µg/mL Phalloidin and 200 µg/mL A22 were used to infect blood lymphocytes of E. sinensis. Blood lymphocytes were observed by inverted fluorescence microscope at 0, 12 and 24 h after infection. It was found that the blood lymphocytes were basically normal fusiform or ellipsoidal type at 0 h (Fig. 7A). Compared with negative control group, S. eriocheiris treated with Phalloidin and A22 group, and S. eriocheiris in the positive control group infected blood lymphocytes for 12 h, a small number of cell fragments were found in all three groups, but the morphology of blood lymphocytes basically remained normal (Fig. 7B). After 24 h of infection, most of the blood lymphocytes in the Phalloidin group were granular, the number of cells decreased significantly, many cells were broken and died, and the cells were surrounded by contents and impurities. However, the survival rate of blood lymphocytes in the A22 treatment group was higher than that in the positive control group, and the membrane growth halo of the surviving cells was obvious, which maintained the normal growth state (Fig. 7C). The above results showed that S. eriocheiris treated with Phalloidin worsened the morphology of blood lymphocytes of E. sinensis, while S. eriocheiris of A22 group had less effect on the morphology of E. sinensis blood lymphocytes.

Fig. 7
figure 7

Blood lymphocytes morphology of E. sinensis under phase contrast microscope. (A) The column results show that the blood lymphocytes of E. sinensis were infected with wild type S. eriocheiris and S. eriocheiris treated with Phalloidin and A22 after 0 h, the hemolymph cells were basically normal fusiform or ellipsoid shape. (B) Compared with the negative control group, the other three groups of S. eriocheiris showed a small number of cellular debris after the blood lymphocytes were infected for 12 h, but the morphology of the blood lymphocytes was basically maintained in a normal state. (C) After 24 h of infection, the number of blood lymphocytes in the Phalloidin group was significantly reduced. Compared with the positive control group, the survival rate of blood lymphocytes in the A22-treated group was increased, and the surviving cells maintained the morphology of the normal growth state

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The ability of S. eriocheiris to invade blood lymphocytes

Compared with the positive control group, the cell morphology of the Phalloidin treatment group was slightly worse, while the A22 treatment group was slightly better. To further verify this phenomenon, we detected the effects of S. eriocheiris infection treated with Phalloidin and A22 on the viability of blood lymphocytes using Cell Counting Kit-8. As shown in Fig. 8, taking the cell viability of the negative control group as the standard, 12 and 24 h after S. eriocheiris infection, the cell activity in the Phalloidin group was significantly lower than that in the positive control group (Fig. 8A). However, the cell activity in A22 treatment group was significantly increased (Fig. 8B). The results showed that the infection of S. eriocheiris to blood lymphocytes was enhanced and the cell viability was significantly decreased in Phalloidin treatment group, while the infection of blood lymphocytes by S. eriocheiris in A22 group was significantly decreased and the cell viability was significantly increased. To investigate whether the changes of cell viability and morphology are directly related to S. eriocheiris infection, we detected the copies of S. eriocheiris in the blood lymphocytes of the Phalloidin group (S. eriocheiris + 2.5 µg/ml Phalloidin), A22 group (S. eriocheiris + 200 µg/ml A22) and positive control group (S. eriocheiris). It was found that there was no significant change in the copy number of S. eriocheiris in the Phalloidin treatment group compared with the control group at 12 and 24 h after infection (Fig. 8C), while there was no significant change in the copy number of S. eriocheiris infected blood lymphocytes in the A22 group after 12 h, but it was significantly lower than that in the positive control group after 24 h (Fig. 8D). This result showed that the ability of S. eriocheiris treated with A22 to infect blood lymphocytes and replicate in them was decreased. To explore the changes of phenoloxidase system of blood lymphocytes induced by S. eriocheiris treated with Phalloidin and A22, blood lymphocytes were infected with S. eriocheiris treated with Phalloidin and A22, and the activity of PO was detected. PO is an innate immune protein whose increase in blood lymphocytes helps to resist infection by pathogens. The results showed that compared with the positive control group, the activity of PO increased significantly 12 and 24 h after Phalloidin group infection (Fig. 8E). After 12 and 24 h of S. eriocheiris infection, the activity of PO in the A22 treatment group was significantly lower than that in the positive control group (Fig. 8F). The above results show that infection of S. eriocheiris treated with both Phalloidin and A22 can affect the PO activity of blood lymphocytes. PO is a kind of innate immune protein, and its elevation in blood lymphocytes is helpful to resist the infection of pathogens. Because the PO activity of blood lymphocytes infected by S. eriocheiris treated with A22 was significantly lower than that of the positive control group, which indicated that the pathogenicity of S. eriocheiris treated with A22 was weakened.

Fig. 8
figure 8

Effects of S. eriocheiris on E. sinensis blood lymphocytes. (A, B) Activity of blood lymphocytes infected with S. eriocheiris and S. eriocheiris treated with Phalloidin and A22. Using the negative control group as the standard, the cell viability of the Phalloidin group was significantly lower than the positive control group after 12 and 24 h of S. eriocheiris infection, while the cell viability of the A22 group was significantly higher (***: p < 0.001). (C, D) When S. eriocheiris was treated with Phalloidin, the intracellular S. eriocheiris copy number did not change significantly at 12 and 24 h after infection compared with the control group. The copy number of A22 group did not change significantly at 12 h, but it was significantly lower than the normal group at 24 h. (*: p < 0.05). (E, F) When E. sinensis was infected with S. eriocheiris of the Phalloidin group, the PO activity of blood lymphocytes was significantly higher after 12 h and 24 h compared with the positive control group, while the PO activity of blood lymphocytes in the A22-treated group was significantly lower than that in the positive control group. (***: p < 0.001, **: p < 0.01)

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Apoptosis of blood lymphocytes infected by S. eriocheiris

To further explore the effects of S. eriocheiris treated with Phalloidin and A22 on the apoptosis and necrosis of blood lymphocytes, blood lymphocytes infected with S. eriocheiris were collected to detect apoptosis. The flow cytometry results showed that after 24 h of normal S. eriocheiris infection, 17.6% of the cells were necrotic, 77.3% of the cells had late withering, and 3.61% of the cells had early withering (Fig. 9A). After 24 h of S. eriocheiris infection treated with Phalloidin, 22.3% of the cells were necrotic, 72.0% of the cells were late withered, and 3.01% of the cells were withered early (Fig. 9B). After 24 h of the blood lymphocytes of the A22 group infected with S. eriocheiris, only 11.6% of the cells were necrotic, but 82.0% of the cells had late apoptosis and 3.85% of the cells had early apoptosis (Fig. 9C). The above results showed that the number of cell necrosis of blood lymphocytes infected with Phalloidin group increased, while the number of necrotic A22-treated cells decreased.

Fig. 9
figure 9

Flow cytometry detection of blood lymphocytes apoptosis infected with S. eriocheiris treated with Phalloidin and A22. (A) The flow cytometry results showed that blood lymphocytes was infected with normal S. eriocheiris, necrosis occurred in 17.6% of the cells, late apoptosis in 77.3% and early apoptosis in 3.61% of the cells after 24 h. (B) After 24 h infection of blood lymphocytes with S. eriocheiris treated with Phalloidin, necrosis occurred in 22.3% of the cells, late apoptosis in 72.0%, and early apoptosis in 3.01% of the cells. (C) Blood lymphocytes was infected with S. eriocheiris treated with A22, necrosis occurred in 11.6% of the cells, late apoptosis in 82.0%, and early apoptosis in 3.85% of the cells after 24 h

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Discussion

The role of MreB in the morphology of S. eriocheiris

In recent years, the research on the cytoskeleton of Spiroplasma mainly focuses on the Spiroplasma-specific fibril proteins, while the actin-like protein MreB has been less frequently reported. Studies on the Spiroplasma cytoskeleton have also only modeled its distribution in the cytoskeletal structure mathematically and physically, and studies on its biological function are lacking [22]. We focused on MreB to explore its biological function in the cytoskeleton structure, and to understand its role in morphological changes. Because S. eriocheiris has no cell wall, its morphology is variable, and S. eriocheiris has different morphology at different growth stages, it has a more typical helical structure during the logarithmic growth phase, and helix during the plateau phase when nutrient conditions are adequate, but takes on several different morphologies during the nutrient-deficient plateau phase and the decay phase, such as dumbbell-shaped, bean-shaped, spherical, pear-shaped, and tadpole-shaped and other irregular shapes. These morphological variations are closely related to the cytoskeleton. S. eriocheiris is typical helix shape during logarithmic growth and spherical during decay, and the transcripts of all five MreB genes are significantly different between the two phenotypes (unpublished data). The morphology of S. melliferum was observed by dark field microscope, and it was found that most of the S. melliferum bodies were dots in the early stage of hysteresis. Electron microscopy revealed that they were mainly irregular near-spherical or slightly curved rod-shaped with swollen ends, while the morphology of S. eriocheiris in the logarithmic growth phase was mainly typical helical. It was also found that there were significant differences in the transcription of MreB5 gene between helical and non-helical S. melliferum, and the transcription amount of MreB gene in helical was significantly higher than that in non-helical [32]. In the S. poulsonii, the MreB subtype forms a polymeric cytoskeleton that acts on the bacteria morphology in a wall-independent manner, however, the high expression of MreB3 is not sufficient to form the bacteria helicity which needs to interact with MreB5 to perform function, the MreB polymeric structures coordinate fibril cytoskeleton, and membrane to maintain the helicity of Spiroplasma, and in in wall-less bacteria, they control the shape of the bacteria through mechanisms independent of peptidoglycan synthesis [19, 33]. The maintenance of helical morphology of Spiroplasma is also closely related to its motility mechanism. Fibril is a constitutive filament-forming cytoskeletal protein that makes up filaments and is unique to members of the genus Spiroplasma [34].The five homologues of MreB interact with each other and form the ribbon structure of the Spiroplasma with the fibril, the cell rotates its body by transmitting helicity and switching along the cell axis, thus moving forward. The helicity transition and its transmission may be caused by conformational changes in the internal helical band structure along the entire cell axis [35, 36]. The combination of S. eriocheiris MreB4-MreB5 and MreB1-MreB5 produces the helical morphology and swimming ability, but the reason for the presence of up to five MreBs is not known, in different environments, they may favour efficient and robust swimming. Some studies have shown that when MreB is combined with filaments, MreB exerts a force on the filaments while swimming, and the filaments may also effectively obtain high energy efficiency and chemotaxis [12, 18].

Similarly, the role of MreB on Spiroplasma morphology and motility has been demonstrated in S. citri. In the nonhelical strain S. citri ASP-1, the loss of helicity and motility is due to a nonsense mutation within the sequence encoding MreB5 that cannot be functionally compensated by any of the other four MreBs. Thus, MreB5 was identified as a major determinant of cell helicity in S. citri [37]. In addition, the researchers found a lipoprotein on the locus encoding MreB in S. citri, which does not interact functionally with MreB, but may positively affect the survival of Citri clade by swimming [38]. A22 is an antibiotic-like small molecule compound that prevents the assembly of MreB and specifically binds to sites on MreB [39]. Our experiment showed that the S. eriocheiris was approximately spherical after A22 treatment, and the expression level of MreB4 was significantly decreased at this time, which suggested that the deformation effect of A22 on S. eriocheiris may mainly depend on MreB4. In summary, MreB plays an important role in the morphology alteration of S. eriocheiris.

The role of cytoskeleton proteins MreB in the pathogenesis of S. eriocheiris

The invasion of S. eriocheiris into E. sinensis has caused great losses to the aquaculture industry, and studies have shown that S. eriocheiris has been observed in blood lymphocytes as well as in other organs of the E. sinensis, making it clear that blood lymphocytes are the target cells of S. eriocheiris [40]. We used S. eriocheiris treated with MreB promoter Phalloidin and inhibitor A22 to infect the blood lymphocytes of E. sinensis, the ability of S. eriocheiris to destroy blood lymphocytes was significantly weakened after A22 treatment, and the cell activity was significantly increased compared with the S. eriocheiris control group, the flow cytometry results also showed that the amount of cell necrosis was lower than S. eriocheiris control group. Compared with the control group, the destructive ability of S. eriocheiris treated with Phalloidin was enhanced and the cell viability was significantly reduced. Flow cytometry results showed an increase in cell necrosis as well. It has been shown that Drosophila S2 cells infested with S. eriocheiri exhibit cytopathic effects, mainly in the form of a gradual deterioration in the state of the cells. The formerly well adherent cells gradually shrink and become surrounded by exudate or cellular debris. Meanwhile, S. eriocheiris induces apoptosis in S2 cells, and the viability of S2 cells severely decreases with the prolongation of infection [41]. The viability of 3T6 cells was also decreased after S. eriocheiris infection of mouse 3T6 cells [42]. In addition, S. eriocheiris infected the blood lymphocytes of E. sinensis, and the cytopathic effect occurred within 36 h, with the morphology changing from initially radial to irregular mass [43]. In the present study, we found that S. eriocheiris treated with Phalloidin was able to infect blood lymphocytes, destroy the morphology of the cells, significantly reduce cell viability, and induce apoptosis, which is strongly pathogenic. Phalloidin is a polypeptide isolated from mushroom Amanita phalloides [44]. In eukaryotes, Phalloidin mainly causes hepatocyte necrosis [45,46,47], Phalloidin binds to F-actin, stabilizes F-actin filaments, and irreversibly prevents filamentous F-actin from depolymerizing into globular G-actin, the bile ducts are wrapped in actin filaments, reducing bile flow and leading to cholestasis [29]. Since the MreB protein in S. eriocheiris is homologous to eukaryotic actin [15], the Phalloidin may have a stable function on MreB, but did not increase the MreB expression. Previous studies have shown that MreB is associated with the ability of S. eriocheiris to swim [17], the effect of Phalloidin on MreB may allow it to maintain a good motility and thus enhance its pathogenicity, however, due to the differences between eukaryotes and prokaryotes, it is necessary to further explore whether Phalloidin have different pathogenic targets for S. eriocheiris.

MreB is one of the cytoskeleton proteins of S. eriocheiris and has an important role in maintaining the morphology of S. eriocheiris [13]. It has also been found that MreB is a virulence gene for pathogenic bacteria [48]. A study on the cytoskeleton proteins of Salmonella revealed a strong link between the cytoskeleton and pathogenicity, and the expression of virulence genes was directly related to the integrity of the cytoskeleton [49]. E263 bacteria expressing MreB-GFP were treated with A22 for 30 min and then infected with purified GVE2 virus, it was found that A22 could change the bacteria morphology by blocking the polymerization of MreB in the host bacteria, and the blocking of MreB polymerization directly inhibited the infection of GVE2 to the host bacteria [50]. Shigella is a gram-negative rod-shaped bacterium, one of the pathogens of bacillary dysentery, its rod-shaped structure is necessary for invading host cells, and its growth is not inhibited by A22. There was no significant difference in the cell adhesion capacity of Shigella with a spherical shape after A22 induction, but the cell invasion rate of Shigella rapidly decreased when the concentration of A22 was 5 mg/mL [51], indicating that the cell invasion of Shigella MreB was inhibited after the morphology change caused by the A22 obstruction. These experimental results showed that when the polymerization of MreB was inhibited by A22, the pathogenicity of bacteria was also affected, indicating that MreB also plays an important role in the pathogenicity of bacteria. The administration of A22 to S. eriocheiris yielded a notable increase in E. sinensis mortality rates 7 and 9 days post-infection (Fig. 6D). Conversely, the Phalloidin group exhibited decreased mortality at 5, 7, and 9 days relative to the untreated, wild-type S. eriocheiris group (Fig. 6D). These findings suggest that MreB significantly influences the pathogenicity of S. eriocheiris. Notably, no statistically significant difference in E. sinensis survival rates was observed between the wild-type S. eriocheiris group, the A22 treated group, and the Phalloidin treated group after 9 days post-injection. This may be attributed to the specific action of A22 and Phalloidin on MreB protein, as neither agent affects the MreB gene. Consequently, newly formed S. eriocheiris daughter cells possess the structure and function of native MreB, resulting in a pathogenesis comparable to the wild-type S. eriocheiris. Similarly, the growth of S. eriocheiris after treatment with A22 confirmed this explanation. Studies suggested that MreB also plays a crucial role in bacterial division [52], S. eriocheiris treated with A22 had a delayed growth due to the inhibition of MreB, which affected their ability to divide, however, A22 did not affect the MreB gene, S. eriocheiris can grow normally after prolonged the cultivation time. MreB has various functions in S. eriocheiris, which can affect its morphology and swimming ability [16, 53], therefore, inhibition of MreB by A22 leads to the decrease of S. eriocheiris pathogenicity. In addition, after the infection of S. eriocheiris treated with A22 in blood lymphocytes, there were still a small number of blood lymphocytes with normal morphology, and the cell viability was also significantly increased compared with normal S. eriocheiris. Our work illustrates that the pathogenicity of S. eriocheiris was significantly reduced in the presence of the cytoskeletal MreB inhibitor A22, which suggests that MreB has an important role in S. eriocheiris pathogenicity.

Conclusion

In contrast to other members of the wall-less Mollicutes family, the S. eriocheiris genome has retained the MreB gene throughout evolution [16]. In this study, we successfully prepared three MreB recombinant proteins and elucidated the role of this protein in the cytoskeleton of S. eriocheiris as well as its effect on the pathogen’s virulence. Following treatment with the cytoskeletal protein promoter Phalloidin and the cytoskeletal protein inhibitor A22, the length of S. eriocheiris in the treatment group was significantly longer compared to the control group. Conversely, the length of S. eriocheiris in the A22 treatment group was significantly reduced, resulting in nearly spherical cells. Western Blot analysis revealed that after treatment with phalloidin and A22 showed no significant changes in the expression levels of MreB3, MreB4, and MreB5 in the Phalloidin-treated group. However, there was a notable decrease in the expression of MreB4 in the A22-treated group, while the expression of MreB3 and MreB5 remained comparable to the levels in normal S. eriocheiris. Furthermore, observations of the growth status of S. eriocheiris and the characteristics of its blood lymphocytes indicated that the pathogenicity of S. eriocheiris was significantly altered by cytoskeletal MreB stabilizator and inhibitor. Specifically, the pathogenicity of S. eriocheiris treated with Phalloidin was significantly enhanced, whereas it was markedly reduced when treated with the inhibitor A22. Our findings suggest that the MreB protein in S. eriocheiris plays a crucial role in its morphology and pathogenicity, providing new insights into potential strategies for the prevention and control of S. eriocheiris infections.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

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Funding

This work was supported by the National Natural Science Foundation of China (NSFC) [32370209]; Natural Science Foundation of Hunan Province, China [2023JJ30503]; Scientific Research Foundation of Hunan Provincial Education Department, China [22A0297]; and Hunan Provincial College Students’ innovation and Entrepreneurship Training Program [S202310555204, S202312650005, X202410555207, S202410555051, X202410555214].

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Author notes

  1. Rong Li, Xiaohui Cao, Jiaxin Chen and Tingting He contributed equally to this work.

Authors and Affiliations

  1. Institute of Pathogenic Biology, Basic Medical School,University of South China, Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, Hengyang, Hunan, 421001, China

    Rong Li, Jiaxin Chen, Yan Zhang, Yaqi Wang, Yifei Wang, Yanyan Qiu, Mengji Xie, Kailin Shi, Yuhua Xu, Siyuan Zhang & Peng Liu

  2. Jiangsu Marine Fisheries Research Institute, Nantong, Jiangsu, 226007, China

    Xiaohui Cao

  3. Shaoxing Center for Disease Control and Prevention, 276 Century Street, Shaoxing, Zhejiang Province, 312000, China

    Tingting He

  4. Jiangsu Key Laboratory for Biodiversity and Biotechnology, Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Jiangsu, China

    Wen Wang

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  1. Rong Li
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Contributions

Conception and design were done by Rong Li, Xiaohui Cao, Jiaxin Chen, Tingting He and Wen Wang (MreB purification and morphology parts), Yan Zhang, Siyuan Zhang, Yaqi Wang and Yifei Wang (Pathogenicity part). Collection and assembly of data were done by Yanyan Qiu, Mengji Xie and Yuhua Xu (MreB purification and morphology parts) and Kailin Shi (Pathogenicity part). Manuscript writing was done by Rong Li and Peng Liu. Final approval of manuscript was done by Peng Liu.

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Correspondence to Peng Liu.

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Li, R., Cao, X., Chen, J. et al. Deciphering the impact of MreB on the morphology and pathogenicity of the aquatic pathogen Spiroplasma eriocheiris. Biol Direct 19, 98 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13062-024-00537-3

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Biology Direct

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