s2fieldsofstudy: Biology Medicine Chemistry

text:
Shikonin exerts antitumor activity in Burkitt’s lymphoma by inhibiting C-MYC and PI3K/AKT/mTOR pathway and acts synergistically with doxorubicin

Burkitt’s lymphoma (BL) is a highly aggressive malignancy molecularly characterized by deregulation of the C-MYC proto-oncogene. Recently, it has been confirmed that phosphatidylinositol-3-kinase (PI3K) pathway activation is a crucial element in the malignant transformation of the B cells in BL. Despite the better outcome of adults with BL treated with high-intensity chemotherapy regimens, the overall survival rate for patients older than 60 years remains dismal. Shikonin, a natural naphthoquinone derived from Chinese herbal medicine plant, has the potential to induce cell death in a series of human cancer. In the present study, we investigated the effect and molecular mechanisms of Shikonin in treatment with BL. Shikonin suppressed cellular proliferation and induced caspase-dependent apoptosis in BL cells. Inhibition of C-MYC and suppression of PI3K/AKT/mTOR pathway played critical roles in SHK-induced apoptosis in BL both in vitro and in vivo. Besides, Shikonin potentiated doxorubicin-induced growth inhibition and apoptosis in vitro. Furthermore, the growth of a subcutaneous xenograft tumor model of BL was significantly inhibited by shikonin. Importantly, we did not find the effect of shikonin on liver function in mice. In summary, these data suggest that shikonin may be an encouraging chemotherapeutic agent in the clinical treatment of BL.

constitutively activated transcription factor also involves the activation of PI3K through augmenting the BCR signaling. Additionally, C-MYC contributes to the activation of the PI3K pathway by promoting expression of mir-17-92 cluster, one of which decreases expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) 9 . Overall, these results demonstrate that C-MYC and PI3K pathway have emerged as promising therapeutic targets in BL.
Natural herbs play a vital role in cancer treatment owing to their pharmacological effects and low toxicity 10,11 . Shikonin (SHK) is an active naphthoquinone derivative compound extracted from root tissues of the traditional Chinese medical herb Lithospermum erythrorhizon which had been broadly applied by ancient Chinese doctors for thousands of years to treat burns and to promote wound healing. Recent emerging researches confirmed that SHK had the potential to induce apoptosis in a variety of human tumor cell lines including leukemia cell lines in vitro and in vivo with minimal or no toxicity to healthy human cells [12][13][14][15] . The anti-tumor activity of SHK may involve in its ability to generate reactive oxygen species 12 , activate pro-apoptotic caspase family members, inhibit the expression of C-MYC 13 and suppress PI3K phosphorylation 14 . Thus much attention has been focused on the potential value of SHK in the therapy of kinds of cancer. However, the effect of SHK on the growth of BL and the possible mechanism had never been reported.
In this study, we investigated whether SHK alone could have the anti-tumor effect on BL cells both in vitro and in a xenograft mouse model, and whether SHK could have the potential to act as a chemosensitizing agent to improve the therapeutic index of doxorubicin (DOX) in vitro. The results showed that SHK suppressed cellular proliferation and induced caspase-dependent apoptosis through inhibition of the expression of C-MYC and the modulation of PI3K signaling in BL cells. Besides, SHK strongly potentiated DOX-induced growth inhibition and apoptosis. The present data suggest that SHK may serve as a novel agent for the treatment of BL because of its likely targets.

Shikonin suppresses cellular proliferation and induces caspase-dependent apoptosis in Burkitt's lymphoma cells. For investigating the effect of SHK on the proliferation of human BL cells, Raji
and Namalwa cells were treated with different concentration of SHK for various durations. Cell viability measured by a MTT assay showed that SHK suppresses cellular proliferation in dose-dependent and time-dependent manners ( Fig. 1A-C). The two cell lines responded differently to the SHK treatment and Namalwa cells were apparently more sensitive to SHK than Raji cells. The differences noted between two cell lines were in agreement Previous researches demonstrated that induction of apoptosis is the main way for SHK-induced cell death in vitro 12,15 . So we detected whether apoptosis also took part in this process in BL after SHK treatment, and the apoptosis rate of Namalwa cells was examined using Annexin V and 7-AAD staining assay followed by flow cytometry analysis. As shown in Fig. 1D, the percentage of apoptotic cells dramatically increased with growing concentration of SHK from 250 to 1000 nM after incubation for 24 h. Furthermore, the pan-caspase inhibitor ZVAD-FMK (20 μM) was administered in advance for 2 h to determine whether the apoptosis induced by SHK was caspase-dependent. And we found that the apoptosis rate was significantly decreased, especially for the late apoptosis compared to the early apoptosis (Fig. 1D,E).
To observe the nuclear condensation or fragmentation which signifies apoptosis, we used Hoechst 33342 and PI staining in Namalwa cells after 6 h incubation with SHK. As shown in Fig. 2A, on the contrast with untreated cells presenting round nuclei with dark blue color, part of the cells treated by SHK had shrunken nuclei in light blue color without or with red color indicating they were early apoptotic cells or late apoptotic cells, respectively. Furthermore, the numbers of apoptotic cells were also positively related to the concentration of SHK treatment. On western blot analysis (Fig. 2B), the same doses of SHK dose-dependently activated caspase pathway as evidenced by splicing events of caspase-9, -8, -3 and PARP, which indicate the initiation of apoptosis. All these data suggest that SHK activates caspase cascade and induces caspase-dependent apoptosis in BL cells.

Shikonin inhibits both C-MYC and PI3K/AKT/mTOR activity in vitro.
Numerous studies have established that C-MYC deregulation is one of the most important events for BL malignant transformation 16,17 . The previous study showed that C-MYC is a potential target of SHK in U937 cells 13 . Therefore, we evaluated the effect of SHK on C-MYC in Namalwa and Raji cells. As showed in Fig. 3A, SHK significantly inhibited the protein expression of C-MYC in a dose-dependent manner in both BL cells.
C-MYC regulates a large number of micro-RNAs (miRs) that function as oncogenes apart from inducing cell proliferation and growth 18 . MiR-19a is one of the oncogenic miR-17-92-cluster members which is up-regulated by C-MYC and can cause the activation of the PI3K/AKT pathway through down-regulation of PTEN 9 . In this study, we found that SHK also induced a dose-dependent reduction of miR-19a for Namalwa cells (Fig. 3B). Similar results were obtained with Raji cells (Fig. 3C). A known C-MYC inhibitor 10058-F4 was used as a positive control drug. As shown in Fig. 3E,F, 10058-F4 inhibited the expression of both C-MYC and miR-19a.
It is known that PI3K activity plays a central role in the development and ongoing maintenance of BL 19,20 . And several studies found that SHK could inhibit PI3K activity in different tumor cells 14,21 . So we first investigated the role of SHK on PI3K and its downstream AKT expression by western blot analysis (Fig. 3D), SHK decreased p110α which is one of the PI3K subunits and phosphorylation of AKT at Ser473 in a dose-dependent fashion in Namalwa and Raji cells. As expected, SHK also strongly inhibited phosphorylation of mTOR at Ser2448, a marker for mTORC1 activity 22 , as well as phosphorylation of p70S6K, the best-characterized target of mTORC1. Moreover, SHK successfully inhibited phosphorylation of mTOR at Ser2481, a marker for the presence of mTORC2 complexes 22 , which was consistent with the decreased level of phosphorylation of AKT at Ser473. The activity of mTORC1 and mTORC2 in Namalwa and Raji cells was completely inhibited by the treatment with 1000 nM SHK accompanied by slight degradation of protein expression of mTOR.
The synergistic anti-proliferation activity of shikonin and doxorubicin is that Shikonin potentiates doxorubicin-induced apoptosis in Burkitt's lymphoma cells. Doxorubicin is one of the most commonly used chemotherapeutic drugs in patients with BL. Given the fact that the acquisition of doxorubicin resistance is a primary cause of chemotherapy failure and high doses of doxorubicin easily lead to toxic side effects, we tested the effect of combination therapy of doxorubicin and SHK by MTT assay. Therefore, Namalwa and Raji were treated with a series of doses of SHK or/and doxorubicin. As shown in Fig. 4A, SHK (200 nM) or doxorubicin (400 nM) decreased the cell viability of Namalwa cells to 49.33% or 73.63%, respectively, whereas the viability of Namalwa cells treated with the combination therapy decreased to 38.93% and the similar result was also observed in Raji cells. The combination index analysis showed that CI range values in Namalwa cells were 1.73 to 0.68 for fractional effect corresponding to 0.06 to 0.61 (Fig. 4B), indicating that SHK in combination with doxorubicin has synergistic anti-proliferation activity.
Next, we examined whether this synergy effect of combination treatment of SHK and doxorubicin was due to induction of apoptosis. As shown in Fig. 4C-E, the results of the flow cytometric analysis revealed a marked increase in the proportion of apoptotic cells after combined treatment with SHK and doxorubicin for 24 h compared with that of SHK or doxorubicin alone. And SHK distinctly enhanced doxorubicin-induced activation of caspase-3 and PARP indicating the activation of apoptosis pathway (Fig. 4F). These data are consistent with MTT assay, suggesting that synergistic cell growth inhibition resulting from combination treatment with SHK and doxorubicin may owe to, at least partly, the induction of increased apoptosis in BL cells.

Shikonin inhibits xenograft tumor growth.
Given the potent inhibitory activity of SHK on BL growth in vitro, it is believed that SHK has potent anti-tumor effects in treating BL in vivo. Thus, we established NOD/SCID mice xenografts bearing Namalwa BL cells. As shown in Fig. 5A, there was a significant decrease in mean tumor volume for mice that received the intraperitoneal injection of SHK compared to which were treated with fat emulsion. The last average tumor volume of vehicle group and SHK group reached to 3356.61 mm 3 and 1842.63 mm 3 , respectively (P < 0.01). At the end of the experiment, tumors were isolated from mice and weighted. The mean tumor weight was significantly more substantial in control group compared with SHK-treated mice (P < 0.01) (Fig. 5B). Importantly, there was no significant difference between the vehicle group and SHK group in mice mean body weight (Fig. 5C). Moreover, there was also no significant difference in serum aspartate transaminase and alanine transaminase levels between SHK-treated mice and control mice (Fig. 5D).
Additionally, consistent with our previous findings in vitro, the miR-19a expression of the SHK group was remarkably lower than the control group (Fig. 5E). To evaluate the histopathology changes in the tumor tissues, we performed hematoxylin and eosin (HE) and immunohistochemical staining after the tissues being extracted. As presented in Fig. 5F, HE staining demonstrated that SHK caused severe pathological changes including cell death, tumor necrosis and angiogenesis inhibition compared with control tissues. Furthermore, treatment with SHK obviously decreased the number of Ki-67-positive cells in tumors compared with controls. Similar results   ). The volume of tumor was measured at the indicated times. Data are presented as mean ± SD (n = 5 per group). * and ** represents P < 0.05 and P < 0.01, respectively. (B) The scatter plot represents mean of the tumor weight from SHK-treated and control mice. Data are presented as mean ± SD (n = 5 per group). ** represents P < 0.01. (C) The body weight of mice was measured at the indicated times. Data are presented as mean ± SD (n = 5 per group). (D) Serum activity of alanine transaminase (ALT) and aspartate transaminase (AST) was measured in control and SHK group by spectrophotometric methods. Data are presented as mean ± SD (n = 5 per group). (E) RT-PCR was performed to evaluate the relative expression of miR-19a in Namalwa xenografts. Data are presented as mean ± SD (n = 3 per group). (F) Hematoxylin and eosin hematoxylin and eosin (HE) staining was performed to observe pathological changes in tumor tissues. Immunohistochemistry was performed to measure cell proliferation by Ki-67 staining, and the expression of C-MYC, P-AKT (Ser473) and P-mTOR (Ser2448). Original magnification: 400x. Samples from three animals in each group were analyzed, and representative data are shown. (G) Western blot analysis monitors the expression of C-MYC, PI3K and P-mTOR (Ser2448). Samples from two animals in each group were analyzed. GAPDH was used as a loading control. Cropped blots/gels were used in the figure and the gels had been run under the same experimental conditions; the fulllength blots/gels are presented in Supplementary Figure S4.

Discussion
With the development of intensive chemotherapy regimens, pediatric BL treatment is very successful, most of which can be cured 23 . However, the elderly patients often can not tolerate these rigorous BL regimens. Moreover, the bone marrow and the immune responses are consequently suppressed by these regimens, placing a premium on supportive care, which is to detect and treat infections promptly and efficiently. Hence, it is urgent to exploit new treatment regimens which are less immunosuppressive and better tolerated for BL.
SHK is a small molecule natural compound derived from Chinese herbal medicine plant. Since its long history used in Chinese traditional medicine, it is considered to be much safe for human beings. Recent experiments found out that SHK had anti-tumor effects on glioma 24 , gastric cancer 25 , hepatocellular carcinoma 26 and leukemia 12,15 . In this study, we confirmed that SHK suppressed cellular proliferation and induced death in Raji and Namalwa BL cells in a dose and time-dependent manner. Apoptosis induced by chemotherapy, is one of the essential patterns participating in eliminating cancer cells. It is a consequence of a series of precisely controlled events involving cell death 27 . Two related pathways are resulting in apoptosis: the extrinsic pathway and the intrinsic pathway 28 . Generally speaking, the former involves activating caspase-8, and the latter involves activating caspase-9. Then activated caspase-8 or caspase-9 could trigger downstream proteins, such as caspase-3 and poly ADP-ribose polymerase (PARP). Our data presented that SHK induced apoptosis in BL cells via caspase-dependent pathways, which was validated by nuclear fragmentation, apoptotic cell morphology and pan-caspase inhibitor ZVAD-FMK.
C-MYC deregulation plays a critical role in the pathogenesis of BL. Indeed, BET Bromodomain inhibitors that can inhibit C-MYC indirectly have been examined in mouse models of BL and been undergoing clinical trials in patients with hematologic malignancies [29][30][31] . Besides chromosome translocation, some other mechanisms are accounting for C-MYC deregulation in malignancy, for instance, gene amplification and insertional viral mutagenesis 18 . The C-MYC protein usually acts as a transcription factor that binds to DNA and regulates transcription. It can make the cell phase transition from the G0/1 phase to the S phase, which ultimately induces DNA replication, protein biosynthesis and cell proliferation and growth 32 . In our study, we found that SHK could significantly decrease the expression of C-MYC in protein level even at a low dose as well as its specific inhibi-tor10058-F4 did at a relatively high dose in BL cells. Apart from association with cell proliferation and growth, C-MYC ties up with a great number of micro-RNAs that function as oncogenes such as miR-19a 9 or tumor suppressor genes such as miR-34a 33 . Similar to the previous results, SHK also down-regulated the expression of miR-19a in a dose-manner in BL cells, which negatively regulated the expression of PTEN. It will be interesting to investigate the relationship between SHK and PTEN in future.
It has proved that C-MYC dysregulation alone does not lead to lymphoma 34 and the t(8;14) is also detected in blood cells and bone marrow of healthy individuals 35 . Until recently an engineered mouse model expressing deregulated C-MYC and constitutively-active PI3K specifically in germinal center B cells identified that PI3K signaling could cooperate with C-MYC in the development of BL 7 . In the meantime, by comprehensive high-throughput RNA sequencing, Schmitz et al. further revealed that mutations of TCF3/ID3 genes resulted in continuous PI3K activation in a large panel of BL cases 8 . Therefore, more and more attention is paid to PI3K inhibitors and several pan-PI3K or isoform-selective PI3K inhibitors have been undergoing clinical trials in non-Hodgkin lymphoma 36,37 . In this study, we found that SHK inhibited protein expression of p110α, which is one of the catalytic subunits of Class IA PI3Ks. Moreover, SHK decreased the activity of mTORC1 and mTORC2, as evidenced by dephosphorylation of mTOR on Ser2448 and Ser2481. Paralleled with these results, it elicited potent suppression of phosphorylation of p70S6K and phosphorylation of AKT at Ser473 in BL cells, standing for one of the downstream targets of mTORC1 and a substrate of mTORC2, respectively. The data that both dephosphorylation of mTOR on Ser2448 and Ser2481 by SHK is exciting and inspiring because there is a negative feedback loop between mTORC1 and mTORC2 38 . Once mTORC1 was inhibited, mTORC2 could be further activated through PI3K signaling. It is the reason that single-agent rapamycin analogues have limited efficacy in cancer as a result of incomplete mTOR inhibition 39 . Although this dual inhibition of mTORC by SHK still needs more investigation, these data at least partly suggest SHK might directly target mTORC1/mTORC2.
Long-term exposure of the tumor to certain drugs can lead to drug resistance. Multi-drug combination is a promising approach to overcome drug resistance 40,41 . The effect of SHK combined with DOX was evaluated and we found that low dose SHK potentiated anti-proliferation activity of DOX in BL cells. The molecular mechanisms of this synergistic effect may be involved with enhancement of apoptosis. Furthermore, our findings in vitro have been recapitulated in vivo in a subcutaneous BL model, indicating that the modulation of C-MYC and PI3K/AKT/mTOR is, at least, one of the molecular mechanisms by which SHK may manifest its effect against BL.
In summary, our results indicate that cytotoxic effect mediated by SHK against BL attributes to caspase-dependent apoptosis. Inhibition of C-MYC and suppression of PI3K/AKT/mTOR activity play critical roles in SHK-induced apoptosis in BL both in vitro and in vivo. We also demonstrated that SHK has property to enhance chemotherapeutic sensitivity in BL cells. Altogether, these data suggest that SHK may be an encouraging chemotherapeutic agent in the clinical treatment of BL. MTT. Cell viability was monitored by 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay following the manufacturer's instructions. Briefly, cells (2 × 10 4 cells/well) were seeded in 96-well plates and treated with drugs for the indicated times. After incubation, 20 μl of MTT solution (5 mg/ ml) was added to each well and then the plates were incubated at 4 °C for 3 h. The absorbance of the reaction was measured by a 96-well plate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. IC50 values (half maximal inhibitory concentration) were calculated. . Aliquots containing 30 μg proteins were separated on sodium dodecyl sulfate (SDS) -polyacrylamide gels containing 6-12% acrylamide gradients and then transferred to PVDF membranes (Millipore, Billerica, USA). The membranes were blocked for 2 h in Tris-buffered saline containing 0.1% Tween and 5% skim milk and then incubated with primary antibodies overnight at 4 °C. After washing three times, the membranes were incubated with anti-rabbit/mouse IgG antibody conjugated to horseradish peroxidase (Multi Sciences) for 2 h at room temperature. The results were visualized with the ECL detecting kit (Biological Industries, Cromwell, CT, USA). All primary antibodies were purchased from Cell Signaling Technology except C-MYC and p-mTOR (Ser2481) (Abcam, Cambridge, UK).

Materials and Methods
Hoechst 33342 and propidium iodide (PI) staining. Hoechst 33342 (Beyotime Biotechnologies, Shanghai, China) and PI (Multi Sciences) staining were used to analyze the nuclear morphology. Cells (2 × 10 5 cells) were treated with either DMSO (control) or shikonin at the indicated concentration for 6 h at 37 °C. After washed twice with PBS, the cells were incubated with Hoechst 33342 (1 mg/mL) and PI (5 mg/mL) at room temperature for 15 min and observed under fluorescence microscope (DFC450, Leica, Germany). Apoptotic cells were identified by morphologic changes in their nuclear assembly by observing chromatin condensation and fragment staining by Hoechst 33342 or both Hoechst 33342 and PI.
Real-time PCR. Total RNA of BL cells were extracted using the TRIZOL Reagent (Invitrogen, CA, Carlsbad, USA) according to manufacturer's instructions. Reverse-transcribed complementary DNA was synthesized with the Prime-Script ® RT Reagent Kit (Takara, Tokyo, Japan). Amplification reactions were performed using SYBRP remix Ex Taq (Takara) in a 20 μL volume on a 96-well optical reaction plate in CFX96 Real-Time PCR Detection System (Bio-Rad). RT-PCR was performed with an initial denaturation at 95 °C for 5 min followed by 40 cycles including denaturing at 95 °C for 10 s, annealing at 60 °C for 20 s and extension at 70 °C for 10 min. Melting curves were computed after PCR product amplification. MiR-19a was normalized to U6. Comparative RT-PCR was conducted in triplicate, including no-template controls. The comparative cross-threshold (Ct) method was used to calculate relative expression. The primers for miR-19a and U6 were synthesized by RiboBio (Guangzhou, Guangdong, China).

Combination index analysis.
For evaluating the synergistic efficacies of shikonin and doxorubicin, the combination index (CI) isobologram method of Chou and Talalay was used. This commonly used analysis involves plotting concentration-effect curves for each agent and multiple diluted fixed-ratio combinations by using the median effect equation and the combination index equation. A value of CI less than, equal to or greater than 1 indicates synergism, additivity and antagonism, respectively. The synergy of Doxorubicin with shikonin was analyzed with the use of CalcuSyn software (Biosoft, Cambridge, UK). Establishment and treatment of Namalwa xenografts. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee. All experiments in vivo were performed in accordance with relevant guidelines and regulations. Male non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (4 weeks old) were obtained from Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). For establishment of the xenograft, a mixture of 1 × 10 7 Namalwa cells and PBS was injected subcutaneously in the right oxter of each mouse. The tumor volumes were determined by measuring length (L) and width (W), and calculating volume (V = 0.5 × L × W 2 ) at the indicated time points. When each tumor volume reached about 100 mm 3 , the mice were randomly assigned to two groups (five mice per group): Vehicle, SHK (4 mg/kg). Mice were treated by intraperitoneal (i.p.) injection of 4 mg/kg SHK in fat emulsion every other day, or by i.p. injection of the same volume of the fat emulsion according to the same schedule. At the end of treatment, the mice were sacrificed, and the tumors were removed and weighed.
Scientific RepoRtS | (2018) 8:3317 | DOI:10.1038/s41598-018-21570-z Immunohistochemistry. The extracted tumor tissues were fixed in 10% formalin at room temperature, processed and embedded in paraffin. Paraffin-embedded tissues were sectioned (5 mm thick). For histopathology analysis, the paraffin sections of tumors were stained with HE. Tissue sections were primarily stained with indicated antibodies for immunohistochemical analysis. Then biotinylated secondary antibodies detected the signal with DAB. Images were acquired using a Nikon E300 fluorescence microscope equipped with a Nikon digital camera (400x). Statistical analysis. All data are presented as the mean ± SD for at least three independent experiments.
Comparisons between two groups were performed using Student's t-test and P < 0.05 was considered statistically significant.
Data availability. Original data files are available upon a reasonable request.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Protein kinase A inhibition induces EPAC-dependent acrosomal exocytosis in human sperm

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l−1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02–0.1 μmol l−1), whereas higher concentrations (>5 μmol l−1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.

INTRODUCTION
40%-80% gradient (PureCeption Lower and Upper Phase Gradient) and centrifuged (5810R, Eppendorf, Hamburg, Germany) for 10 min at 3000 g at room temperature. The lower layer containing the sperm was collected and resuspended twice in Ham's F-10 medium containing 21 mmol l −1 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 25 mmol l −1 sodium bicarbonate (Cat No. 144-55-8), 0.6% human serum albumin, 7.6 mmol l −1 sodium lactate  washed in Ham's F-10, then centrifuged again, and the sperm allowed to "swim up" after the last wash at 37°C. The motile cells (over 80% motile cells) were collected without the pellet and resuspended in capacitation medium. This procedure allowed motile sperm to be obtained without leukocyte contamination. All experimental protocols were approved and performed according to the relevant guidelines and regulations of the Helsinki Committee of Sheba Hospital, Ramat-Gan, Israel, and informed consent was obtained from all participants.

Assessment of sperm acrosomal exocytosis
Human sperm (1 × 10 7 cells per ml) were incubated under capacitation conditions for 160 min, and then various compounds as described for each experiment in the figure legends were added for an additional 20 min. The AE inducers described for each experiment in the figure legends were added for 1 h. The percentage of acrosome-reacted sperm was determined microscopically (Axio imager Z1, Zeiss, Jena, Germany) using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA). An aliquot of spermatozoa (10 6 cells per 10 µl) was smeared on a glass slide and allowed to air-dry. The sperm were then fixed with methanol for 15 min at room temperature and washed three times at 5-min intervals. The first and third washes were performed with distilled water (dH 2 O), and the second wash with Tris-buffered saline (TBS) (137 mmol l −1 NaCl [Cat No. 7647-14-5], 2.7 mmol l −1  and 20 mmol l −1 Tris-HCl, pH 7.6). The slides were air-dried and then incubated in a moist environment with PSA-FITC (50 mg ml −1 in TBS) for 35 min, then washed twice with dH 2 O at 5-min intervals and sealed with ProLong Gold antifade reagent (Thermo Fisher Scientific, Waltham, MA, USA). For each treatment, at least 100 cells per slide were evaluated on triplicate slides, using × 400 magnification under an Axio imagerZ1 fluorescence microscope. Cells with green staining over the acrosomal cap were considered acrosome intact; those with equatorial green staining or no staining were considered acrosome-reacted. The percent acrosome-reacted cells (5%-10%) at time zero was subtracted from each measurement.

Intracellular cAMP determination
The total cAMP concentration was determined by an enzyme-linked immunosorbent assay (ELISA) using a commercial kit from Arbor Assay (Cyclic AMP Direct EIA Kit) according to the manufacturer's instructions. Briefly, sperm (10 7 cells per ml) were incubated under capacitation conditions. At the end of the incubation, the sperm were lysed, and ELISA was performed according to the manufacturer's protocol. cAMP was determined relative to a standard curve.

Determination of actin polymerization
The method was described by us previously. 16 Sperm cells were spread on microscope slides, air-dried, and fixed in 1.75% formaldehyde in TBS for 10 min, then washed once with dH 2 O for 5 min, placed in 0.2% Triton X-100 in TBS for 30 min, washed three times at 5-min intervals in TBS and air-dried. Afterward, sperms were incubated in a moist chamber with 4 µmol l −1 phalloidin-FITC in TBS for 60 min, washed four times with dH 2 O at 5-min intervals and mounted with ProLong Gold antifade reagent. Images were captured on Axio imagerZ1 fluorescence microscope at ×400 magnification. Sperm pictures were photographed within 24 h to minimize the loss of fluorescence, at the same exposure. The fluorescence intensity, which indicates the F-actin level, was quantified for whole cells using the "MetaMorp Image J" software (National Institutes of Health Universal Imaging Corp., West Chester, PA, USA) and the background intensity was subtracted. All experiments were carried out in duplicate slides, and at least 50 cells per slide were quantified for fluorescence intensity.

Statistical analyses
Data were analyzed for statistical significance by analysis of variance (ANOVA) followed by a Tukey's honestly significant difference (HSD) comparison for at least three replicates. Differences of P < 0.05 were considered statistically significant. Sample size appears in the figure legends.

The effect of PKA inhibition at the end of sperm capacitation on acrosomal exocytosis
In the present study, we investigated the involvement of cAMP/PKA in human sperm AE. Inhibition of PKA (using H89) at the end of the capacitation process revealed a 3-fold increase in the AE rate (Figure 1). Treatment of capacitated human sperm with TG or progesterone also induced the occurrence of AE and the augmentation of AE by H89, TG, or progesterone was significantly reduced when both soluble and the transmembrane adenylyl-cyclase (AC) were inhibited using 2-Hydroxy-2-methylpropiophenone (2-OH) ddAdo (Figure 1). Interestingly, the induction of the AE by H89 was almost completely blocked by adding a relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l −1 ) or by increasing intracellular cAMP using 1-methyl-3-isobutylxanthine (IBMX); this agent inhibits cAMP-phosphodiesterase, the enzyme that degrades cAMP (Figure 1). Similar inhibition was observed when . The values shown represent the mean ± standard deviation of duplicates from three experiments from three different donors. ** P < 0.01 and *** P < 0.001, significant difference compared to the corresponding control. cAMP: cyclic adenosine monophosphate; AE: acrosomal exocytosis; sAC: soluble adenylyl cyclase; tmAC: transmembrane adenylyl cyclase; TG: thapsigargin. the AE was induced by TG or progesterone (Figure 1). TG induces AE by inhibiting the Ca 2+ -ATPase (Ca 2+ pump) localized in the outer acrosomal membrane, resulting in a decrease of intra-acrosomal Ca 2+ leading to the opening of the store-operated-Ca 2+ -channel (SOCC) of the plasma membrane. [17][18][19] Progesterone activates CatSper, which is a sperm-specific Ca 2+ -channel in the plasma membrane. 20 A dose dependence analysis of the effect of 8Br-cAMP on the AE revealed that 5 µmol l −1 and 25 µmol l −1 8Br-cAMP stimulates AE, whereas higher concentrations showed no effect or even inhibition; nevertheless, all concentrations used inhibited the H89induced AE (Supplementary Figure 1). The data in Figure 1 and Supplementary Figure 1 indicate that intracellular cAMP mediates the AE induced by PKA inhibition, while relatively high cAMP inhibits this effect. Thus, the different sensitivity to cAMP levels, and the fact that inhibition of PKA caused cAMP-dependent AE suggest that endogenous cAMP levels are probably enhanced when PKA is inhibited. Indeed, we found a 50% increase (from 2.6 to 3.9 pmol per 10 7 cells) in cAMP levels in cells treated with H89. Treatment with IBMX, a cAMP-phosphodiesterase (PDE) inhibitor, caused a much higher increase in [cAMP]i (up to 5.6 pmol per 10 7 cells), which is inhibitory to the AE process as mentioned above.

The possible involvement of EPAC in AE induced by PKA inhibition
Since inhibition of PKA (Figure 1) or low concentrations of 8Br-cAMP (Supplementary Figure 1) induced AE, we suggested that cAMPdependent-EPAC might mediate this effect. We, therefore, tested the effect of various concentrations of 8-(4-Chlorophenylthio)-2'-Omethyladenosine-3′,5′-cyclic AMP (8pCPT), a selective activator of EPAC 21 on AE. Activation of EPAC by addition of 8pCPT induced AE at relatively low concentrations of 8pCPT (0.02-10 µmol l −1 ), whereas at higher concentrations of 8pCPT (25-50 µmol l −1 ), the induction was much lower (Figure 2). These results indicate that AE is mediated by EPAC, and relatively high concentrations of EPAC substrate inhibit AE. Increasing intracellular cAMP using IBMX also show significant inhibition (75%) in AE induced by 0.1 µmol l −1 8pCPT (Figure 2).
To further characterize the role of EPAC in H89-induced AE, we tested the effect of the EPAC-specific inhibitor, ESI09 22,23 on AE induced by H89 and other inducers. As shown in Figure 3, ESI09 inhibited AE induced by 8pCPT, H89, TG, Angiotensin II, progesterone or the Ca 2+ -ionophore A23187, indicating that the AE induced by these six inducers is largely EPAC-dependent.
Two isoforms of EPAC are present in sperm cells, EPAC1 and EPAC2; 24,25 both are inhibited by ESI09. 22,23 EPAC1 contains a binding site for phosphatidic acid (PA) which is important for its activation. 26 Thus, the reduction of free PA in the cells by treatment with N-butanol will inhibit EPAC1 activity. PA is produced in cells from phosphatidyl-choline hydrolysis catalyzed by phospholipase D (PLD) activity. In the presence of the primary alcohol N-butanol, the free PA is converted back by PLD to phosphatidyl-butanol resulting in reduced levels of free PA in the cells. 27 This system is active in sperm cells, as we showed that actin polymerization during sperm capacitation is inhibited by N-butanol treatment, which can be reversed by the addition of free PA to the cells. 16 Here, we show that treatment with N-butanol inhibited AE induced by 8pCPT, H89, TG, or A23187 (Figure 4). No effect of 0.5% secondary-butanol which is not a PLD substrate, could be on AE or on sperm motility (not shown) indicating the specificity of the primary-butanol as an inhibitor of the PLD pathway. These results suggest that EPAC1 is the dominant isoform involved in AE.
It was shown that EPAC is involved in actin modulation. 32 During sperm capacitation, G-actin monomers are polymerized to F-actin, and before the AE, this F-actin should be dispersed to enable AE. 2,33 F-actin formation during capacitation can occur only if the actin-severing protein gelsolin is inhibited by its binding to PIP 2 and its tyrosine phosphorylation by Src. 15 Before the AE, PIP 2 is hydrolyzed by PLC, causing the release of p-gelsolin which undergoes dephosphorylation/activation resulting in F-actin dispersion. 15 To examine the relationship between EPAC and actin in sperm, we looked for EPAC involvement in AE induced by activation of gelsolin using polyphosphoinositide-binding-peptide (PBP10), a peptide containing the PIP 2 -binding domain of gelsolin. Treatment with PBP10 will release gelsolin from PIP 2 , leading to its activation and F-actin dispersal, resulting in AE. 15,33 It is seen in Supplementary Figure 2 that AE induced by PBP10 is almost completely blocked by 0.1 mmol l −1 8Br-cAMP or 50 µmol l −1 8pCPT or the EPAC inhibitor ES109, suggesting that EPAC mediates F-actin breakdown and/or the AE process downstream of F-actin dispersion. To solve this question, we tested the involvement of EPAC in the process of F-actin breakdown. It is shown in Figure  5 that TG, PBP10, H89, or 8pCPT induce F-actin breakdown and this breakdown is significantly inhibited by the EPAC inhibitor ESI09. These data clearly indicate that EPAC mediates F-actin depolymerization.
Interestingly, the addition of TG to cells treated first with H89 or PBP10, there is 68% or 80% stimulation of AE, respectively (Supplementary Table 1).

DISCUSSION
In the present study, we investigated the involvement of cAMP/PKA in human sperm AE. Treatment of capacitated human sperm with the PKA inhibitor H89, TG, or progesterone induced the occurrence of AE which was significantly reduced when both soluble and the transmembrane AC were inhibited indicating that cAMP-mediated this AE. However, the induction of the AE by H89, TG, or progesterone was significantly inhibited by adding a relatively high concentration of the membrane-permeable 8Br-cAMP (0.1 mmol l −1 ) or by increasing intracellular cAMP using IBMX. In human sperm, progesterone activates Rap1 in the acrosomal region in a cAMP-dependent manner, and Rap1 activates PLC, leading to Ca 2+ release from the acrosome. 35 TG  causes also reduction of intra-acrosomal Ca 2+ leading to the opening of SOCC. Since intracellular concentrations of cAMP are well regulated by its synthesis and degradation systems, it is reasonable to assume that elevated [cAMP]i would inhibit this Ca 2+ mobilization process. Since the activation of AE by the PKA inhibitor H89 is also inhibited by 8Br-cAMP, it is possible that the AE induced by H89 also requires Ca 2+ mobilization. This point is further discussed below.
The reduction in the AE induced by PKA-inhibition by the AC inhibitors indicates that a cAMP-dependent-PKA-independent mechanism mediates this effect. Moreover, the significant reduction of AE induced by PKA inhibition by relatively low concentration of 8Br-cAMP (25 µmol l −1 ), further supports such a cAMP-dependent-PKA-independent mechanism. A dose-dependence analysis of the effect of 8Br-cAMP on the AE revealed that 25 µmol l −1 8Br-cAMP stimulates AE, whereas higher concentrations showed no effect or even inhibition; nevertheless, all concentrations used inhibited the H89induced AE. Thus, the AE induced by PKA inhibition is highly sensitive to increased cAMP concentrations. The data indicate that intracellular cAMP mediates the AE induced by PKA inhibition, while relatively high cAMP inhibits this effect. Thus, the different sensitivity to cAMP levels, and the fact that inhibition of PKA caused cAMP-dependent AE suggest that endogenous cAMP levels are probably enhanced when PKA is inhibited. Indeed, we found a 50% increase in [cAMP]i levels in cells treated with H89. Treatment with IBMX caused a much higher increase in [cAMP]i (up to 5.6 pmol per 10 7 cells), which is inhibitory to the AE process as mentioned above. Thus, 3.9 pmol cAMP per 10 7 cells is the suitable concentration for AE induction; however, 5.6 pmol cAMP per 10 7 cells is inhibitory to the AE process. The strong effects of small changes in cAMP levels emphasize the importance of tight regulation of intracellular cAMP levels by the sperm cell. The enhanced effect of PKA-inhibition on [cAMP]i is probably due to the fact the inhibition of PKA might decrease binding of cAMP to PKA, resulting in an increase in free cAMP in the cells.
Since inhibition of PKA or low concentrations of 8Br-cAMPinduced AE, we suggested that cAMP-dependent-EPAC might mediate this effect. It was shown previously that EPAC mediates AE in human sperm. 14 Activation of EPAC by addition of 8pCPT induced AE at relatively low concentrations of 8pCPT (0.1-10 µmol l −1 ), whereas at higher concentrations (25-50 µmol l −1 8pCPT), the induction was much lower. These results indicate that AE is mediated by EPAC, and relatively high concentrations of EPAC substrate inhibit AE. The effect of 0.1-1.0 µmol l −1 8pCPT on AE is similar to the effect of H89, whereas higher concentrations of 8pCPT (5-50 µmol l −1 ) inhibit the induced effect of H89 on AE. Thus, at relatively high concentrations, 8pCPT inhibits AE induced by low 8pCPT or by H89. Moreover, AE induced by a low concentration of 8pCPT is inhibited by treatment with IBMX, suggesting that EPAC or its downstream effectors are inhibited by relatively high concentrations of intracellular cAMP.
To further prove the role of EPAC in H89-induced AE, we tested the effect of the EPAC-specific inhibitor, ESI09 22,23 on AE. ESI09 inhibited AE induced by 8pCPT, H89, TG, Angiotensin II, progesterone or the Ca 2+ -ionophore A23187, indicating that the AE induced by these six inducers is largely EPAC-dependent. Thus, AE induced by PKA inhibition is mediated by EPAC; however, relatively high concentrations of EPAC substrate inhibit this activity. This finding is further supported by the inhibitory effect of high cAMP concentration on AE induced by PKA inhibition or by low concentrations of 8pCPT using IBMX.
The cAMP-dependent changes in EPAC dynamics are induced by allosteric rather than simple binding effects. 36 These authors also show that EPAC contains multiple clusters of residues for which dynamics is either quenched or enhanced by cAMP. Thus, it is possible that at relatively high concentrations of cAMP, EPAC activity is reduced by allosteric effects, and when EPAC binds ESI09, this inhibitory effect is partially reversed, and its activity is rescued. Das et al. 36 also showed that Gly-238 in EPAC is highly sensitive to cAMP although not to the antagonist Rp-cAMP, suggesting an allosteric role at Gly-238. These findings suggest differences in the affinities of cAMP and ESI09 to different regions of EPAC.
It has been suggested that the regulatory unit of EPAC2 contains high (B-site) and low (A-site) affinity sites for cAMP. 37 Binding of cAMP to the B-site activates EPAC, whereas it is possible that binding to the A-site inhibits EPAC activity. This idea might explain why a relatively high level of cellular cAMP is inhibitory to EPAC-dependent AE. ESI09 is a competitive inhibitor of EPAC, which competes with cAMP binding. 23,38 When low concentrations of 8pCPT (0.1 µmol l −1 ) were used to activate EPAC-dependent AE, ESI09 caused almost complete inhibition of AE. However, when 50 µmol l −1 8pCPT was used, which itself caused very slight stimulation of AE, a two-fold increase in AE was observed in the presence of ESI09, and an even greater increase in AE induced by ESI09 was seen when both 50 µmol l −1 8pCPT and H89 were present in the reaction mixture. We suggest that in the presence of 50 µmol l −1 8pCPT, the A-site of EPAC (the inhibitory site) is occupied by the cAMP analogue, and therefore no stimulation of AE is observed. The addition of ESI09 competes with cAMP for binding to this site, inducing its release and resulting in EPAC reactivation. These dual effects of ESI09, support our suggestion of high affinity activating, and low-affinity inhibitory sites for cAMP binding in EPAC.
In conclusion, to achieve optimal EPAC-dependent AE, intracellular cAMP levels must be well regulated by its production and degradation systems in the cell.
Two isoforms of EPAC are present in sperm cells, EPAC1 and EPAC2; 24,25 both are inhibited by ESI09. 23,27 EPAC1 contains a binding site for PA which is important for its activation. 26 Here, we show that treatment with N-butanol inhibited AE induced by 8pCPT, H89, TG, or A23187 indicating that EPAC1 is the dominant isoform involved in AE. The fact that AE induced by TG is only slightly inhibited by N-butanol (31%) suggests that AE-induced by TG is probably , or antibiotic A23187 calcium ionophore (A23187; 10 μmol l −1 ) were added. The values represent the mean ± standard deviation of duplicates from three experiments from three different donors. * P < 0.05, ** P < 0.01, and *** mediated by EPAC2 as well. Although N-butanol inhibits actin polymerization and increases spontaneous-AE when present during sperm capacitation, 39 it does not cause any increase in the AE when added at the end of the capacitation, probably because of its inhibitory effect on EPAC1.
PLC is an important factor involved in the AE mechanism. 29,30 TG-induced AE, which occurs through SOCC activation, was over 90% inhibited by PLC inhibition using U73122. AE induced by 8pCPT, H89, progesterone, or A23187 were also almost completely blocked by PLC inhibition. These data demonstrated that AE, whether induced by activation of EPAC using 8pCPT, or by inhibition of PKA, activation of progesterone-R, or Ca 2+ -ionophore is mediated by PLC. Thus, under these various conditions by which EPAC mediates AE, we see that PLC is also involved. The inhibition of 8pCPT-induced AE by the PLC inhibitor U73122 suggests that EPAC might activate PLC. Indeed, it was shown that the human sperm PLC ε isoform is activated by EPAC-activated Rap1 and is required for Ca 2+ mobilization from the acrosome. 14 The fact that AE induced by the Ca 2+ -ionophore A23187 depends on EPAC and PLC indicates that high influx of Ca 2+ is not sufficient to induce AE and that Ca 2+ mobilization from the acrosome is necessary for AE to occur. This conclusion was supported by other studies working with permeabilized human sperm. 13,14 To examine the relationship between EPAC and actin in sperm, we looked for EPAC involvement in AE induced by activation of gelsolin using PBP10, a peptide containing the PIP 2 -binding domain of gelsolin. Treatment with PBP10 release gelsolin from PIP 2 , leading to its activation and F-actin dispersal, resulting in AE. 15,33 It was seen that AE induced by PBP10 is almost completely blocked by 0.1 mmol l −1 8Br-cAMP or 50 µmol l −1 8pCPT or the EPAC inhibitor ES109, suggesting that EPAC mediates F-actin breakdown and/or the AE process downstream of F-actin dispersion. It was shown that TG, PBP10, H89, or 8pCPT induce F-actin breakdown and this breakdown is significantly inhibited by the EPAC inhibitor ESI09. These data clearly indicate that EPAC mediates F-actin depolymerization.
We showed that AE induced by TG, H89, or 8pCPT is mediated by PLC activity. In our previous study in human sperm, we showed that hydrolysis of PIP 2 by PLC leads to gelsolin activation and F-actin breakdown however F-actin breakdown induced by PBP10 is independent of PLC activity. 15 It was suggested elsewhere that EPAC mediates PLC activation, 14 which might be the mechanism by which EPAC leads to gelsolin activation. The fact that EPAC mediates PLCindependent F-actin breakdown induced by PBP10 indicate that EPAC has a direct effect on F-actin depolymerization in addition to its indirect effect due to PLC activation.
It is possible that other mechanisms not involving PLC are likely to mediate the effect of EPAC on the AE. It has been suggested that EPAC can activate the fusion component soluble-NSF-attachment protein receptor (SNARE) 40 known to mediate the AE. 40,41 SNARE is physically associated with F-actin leading to granule exocytosis in platelets. 42 In MIN6 cells, F-actin negatively regulates insulin exocytosis via binding to the SNARE protein, syntaxin 4. 43 Furthermore, in this insulin secretion system, gelsolin forms a complex with syntaxin 4 under basal conditions, which is relieved on stimulus-induced Ca 2+ influx to activate gelsolin and induce its dissociation from syntaxin 4 to facilitate insulin exocytosis. 44,45 Thus, it seems that syntaxin 4 activity is regulated by its binding to F-actin and gelsolin. To allow the outer acrosomal and the plasma membranes to fuse, the F-actin network produced between the two membranes during capacitation should be dispersed. 46 Activation of gelsolin in sperm causes F-actin dispersion which would activate SNARE and other fusion proteins leading to AE. 47,48 A working model suggested for the mechanism of AE indicates that EPAC might affect the AE through two mechanisms: one is EPAC-RAP-PLC-IP 3 leading to Ca 2+ release from the acrosome, and the second is EPAC-Rab3A-SNARE-AE. 14,42 Since F-actin dispersion and AE induced by PBP10 is independent of PLC activity, we suggest that the inhibition of PBP10-induced AE by the EPAC inhibitor ESI09 is due to inhibition of the EPAC-Rab3-SNARE -AE cascade.
TG treatment further stimulates the AE in the presence of PBP10 or H89. These suggest that intracellular Ca 2+ mobilization induced by TG is an important step for achieving AE, even under conditions whereby F-actin is already dispersed. The fact that AE induced by TG is inhibited by ESI09 and by U73122 suggest that EPAC involve in PLC-dependent Ca 2+ mobilization which is supported by previous study. 31 The sperm-specific Ca 2+ -channel CatSper is essential for the fertilizing ability of sperm. 34 Since AE depends upon Ca 2+ influx into the sperm, we tested whether CatSper mediates AE induced by EPAC activation and by other inducers using the CatSper inhibitor, NNC 55-0396. AE induced by TG was almost completely inhibited by NNC, while the AE induced by H89, 8pCPT, or A23187 was only slightly reduced. We suggested before that AE induced by H89 is mediated by EPAC. Here we showed that AE induced by H89 or by direct activation of EPAC using 8pCPT are not affected by CatSper inhibition. These data further support our model that inhibition of PKA activates EPAC leading to AE. Moreover, these data suggest that AE induced by EPAC is probably not mediated by Ca 2+ influx into the cell via CatSper but rather by Ca 2+ mobilization from the acrosome as a result of PLC activation by EPAC-activated GTPase, as described above. This conclusion is supported by other studies in human sperm, which show that EPAC activation induces PLCdependent Ca 2+ mobilization. 14 The fact that AE-induced by progesterone is mediated by EPAC/PLC indicates that Ca 2+ mobilization from the acrosome is a necessary step for AE even under conditions by which CatSper is activated. Moreover, the fact that AE induced by H89, 8pCPT, and A23187 is independent of CatSper activity, suggest that intracellular Ca 2+ mobilization might be sufficient to induce AE, although it is possible that other plasma membrane Ca 2+ channels might be active in Ca 2+ influx.
In mouse sperm, CatSper 34 and PKA 10 are localized to the flagellum. Thus, it is not clear how these two factors are involved in AE. Regarding CatSper, it has been suggested that Ca 2+ influx via this channel starts in the tail excluding initiation via Cav2.3 channel which localize in the head 49 and lead to AE. 50 It was recently shown that CatSper, as well as PKA, are conclusively linked to AE. 51 Moreover, it has been shown that disruption of PKA from AKAP triggers AE in capacitated mouse sperm, 51 which supports our findings.

CONCLUSIONS
In summary, the data presented here indicate that the inhibition of PKA results in an increase in intracellular cAMP and activation of EPACdependent AE. A further increase in cAMP levels is inhibitory for this process. We showed that low concentrations of the selective EPAC substrate added to the cells stimulate AE, whereas high concentrations show no effect on AE, and inhibit AE induced by PKA inhibition. It is well known that cellular levels of cAMP are precisely regulated by its production and decomposing systems. In this study, we provide one of the reasons why such regulation is important. Moreover, this study provides data regarding the involvement of cAMP, PKA, and EPAC in the AE mechanism.
AUTHOR CONTRIBUTIONS DI performed the experiments, helped to write the manuscript, and performed the statistical analysis. YN made critical notes and helped to analyze the data. HB wrote the manuscript, interpreted the data, and supervised DI in her master studies. All authors read and approved the final manuscript.


extfieldsofstudy: Medicine Biology

text:
Global discovery of small RNAs in the fish pathogen Edwardsiella piscicida: key regulator of adversity and pathogenicity

Recently, bacterial small RNA (sRNA) has been shown to be involved as a key regulator in stress responses. sRNAs of Edwardsiella piscicida, an important aquatic pathogen, are not well characterized to date. In this study, using RNA-seq technology, we globally found and identified sRNA candidates expressed from E. piscicida grown in normal LB medium, acid pressure, iron deficiency stress, and oxidation pressure. A total of 148 sRNAs were found, including 19 previously annotated sRNAs and 129 novel sRNA candidates by searching against the Rfam database. Compared in normal condition, the expression of 103 sRNAs (DEsRNA, differentially expressed sRNA) and 1615 mRNAs (DEmRNAs, differentially expressed mRNA) showed significant differences in three stress sample. Based on the prediction by IntaRNA and relational analysis between DEsRNAs and DEmRNAs, 103 DEsRNAs were predicted to regulate 769 target mRNAs. Pleiotropic function of target DEmRNAs indicated that sRNAs extensively participated in a variety of physiological processes, including response to adversity and pathogenicity, the latter was further confirmed by infection experiment. A large number transcription factors appeared in target genes of sRNAs, which suggested that sRNAs likely deeply interlaced within complex gene regulatory networks of E. piscicida. Moreover, 49 Hfq-associated sRNAs were also identified in this study. In summary, we globally discovered sRNAs for the first time in pathogenic bacteria of fish, and our findings indicated that sRNAs in E. piscicida have important roles in adaptation to environmental stress and pathogenicity. These results also provide clues for deciphering regulation mechanism of gene expression related to physiological response and pathogenicity. Electronic supplementary material The online version of this article (10.1186/s13567-018-0613-z) contains supplementary material, which is available to authorized users.

Introduction
Edwardsiella piscicida (formerly included in E. tarda) [1,2], one family member of Enterobacteriaceae, is a Gram-negative, motile, rod-shaped bacterium. It is a serious fish pathogen and infects a wide range of host that includes multiple species of economically important fish such as Japanese flounder (Paralichthys olivaceus), turbot (Scophtalmus maximus), eel (Anguilla japonica), striped bass (Morone saxatilis), red sea bream (Pagrus major), tilapia (Oreochromis niloticus), and channel catfish (Ictalurus punctatus) [3][4][5]. Fish infected by E. piscicida frequently develop a systemic disease called edwardsiellosis, which in Japanese flounder is often manifested in hemorrhage, septicemia, skin lesions, and necrosis of liver, gut, and kidney [4,6]. Heavy economic losses due to E. piscicida-related edwardsiellosis have been reported to occur in the Asia, United States, and Europe. Currently, control of E. piscicida in aquaculture relies chiefly on antibiotics in most countries including China. In recent years, a large number of studies have been carried out to examine the virulence mechanism of E. piscicida in different fish models. Many virulence factors/systems, such as type III (T3SS) and type VI (T6SS) secretion systems, two-component regulatory system, hemolysin, LuxS/ AI-2 quorum sensing system, molecular chaperons and RNA-binding protein Hfq, ferric uptake regulator, lysozyme inhibitors, and so on, are known to be involved in E. piscicida stress resistance or pathogenicity [7][8][9][10][11][12][13][14][15][16]. However, the regulation of stress resistance and pathogenicity in E. piscicida is largely unknown so far.
Bacteria are constantly exposed to stressful and challenging environment. To cope with adverse environment and to survive, bacteria have evolved intricate mechanisms to sense the surrounding milieu and to adequately respond by changing their gene expression patterns and thus phenotypes [17]. Among various regulatory factors, small RNA (sRNA) of bacteria have attracted more and more attention in recent years. The majority of sRNAs function as regulators of gene expression at the post-transcriptional level and play critical regulatory roles in major biological processes, such as adaptation to various environmental stresses, quorum sensing, biofilm formation, motility, and pathogenicity [17][18][19][20][21][22][23][24]. Bacterial sRNAs are usually untranslated transcripts with length ranges from 50 to 500 nucleotides. Almost all of the so far characterized sRNAs regulate their target mRNA's translation and/ or stability by the way of base pairing [25]. Binding of sRNA with ribosome-binding sites (RBSs) blocks initiating ribosomes and then inhibit translation initiation. As positive regulators, they can either stabilize the mRNA by interfering cleavage of RNase or enhance translation by sRNA-binding-induced structure modulation to render translation initiation sites available [17,26,27]. Bacterial sRNA can be divided into two types: cis-encoded and trans-encoded sRNA. The former is expressed from the same locus as their sole target with which they share full complementarity. The latter is expressed from loci elsewhere sharing only partial complementarity with their targets and target multiple mRNAs with specific seed sequences [28,29]. In many bacteria, trans-encoded sRNAs often need the help of Hfq, a close relative of the Sm/Lsm family of proteins involved in splicing and RNA decay. The important homohexameric helper protein has multiple effects on mRNA translation by protection and stabilization of sRNAs from degradation, by facilitating its interaction with the target mRNA, or by increasing the rate of sRNA-mRNA annealing [17,23,30]. On the contrary, cis-encoded sRNAs do not require Hfq for stability and regulation. Deleting Hfq, which has pleiotropic effects on the stability of several sRNAs, results in numerous phenotypes, including resistance to environmental stresses and pathogenicity [31,32]. Our previous study showed that Hfq played an important role in responding to adversity and pathogenicity of E. piscicida [13], but its mechanism remains unknown.
Currently, the study about sRNA in the pathogenic bacteria of teleost fish was very scarce. Only several sRNAs were identified in E. tarda [33]. Information about the number and function of sRNAs in E. piscicida is unknown. In this present study, we discovered and identified sRNAs in E. piscicida globally and analyzed their functions in the pathogenicity of E. piscicida.
Edwardsiella piscicida TX01 was cultured in normal LB medium or stress condition, i.e., in LB medium with pH = 5.0 (acid stress, Ac), in LB medium with 100 μM dipyridyl (iron deficiency, Dp), and in LB medium with 500 μM hydrogen peroxide (oxidation pressure, Pe). Bacteria were cultured to exponential phase and collected. Then the bacteria were used for subsequent RNA sequencing. The experiment was performed three times.

RNA isolation and RNA sequencing
sRNA isolation, library construction, and high-throughput sequencing were carried out by Beijing Genomics Institute (BGI), Shenzhen, China. Total RNA was isolated using TRIzol Reagent according to the manufacturer's instructions (Invitrogen). The RNA samples were treated with DNaseI to remove residual genomic DNA. The quantity and purity of the RNA were monitored using a NanoDrop ™ 1000 spectrophotometer and agarose gel electrophoresis. RNA fragments with length of 50 nt to 500 nt were isolated following gel filtration and purification. A total of four small RNA libraries were constructed and single-end sequencing was performed on an Illumina Hiseq 4000 by BGI. At the same time, the total RNA libraries of four samples were prepared and sequenced.

Analysis of sequencing reads
The reads were processed by filtering low quality reads, removing adapter and impurity sequence, then the clean reads were obtained. After assembling the clean reads, the full-length tags were obtained. The tags were mapped with genome of E. piscicida, which produce bioinformatics of sequence alignment. Among these information, the tags within intergenic or intragenic region of genes were chosen to the candidate sRNAs. Analysis of the follow-up biological information of these candidate sRNAs included: (1) sRNA annotation, (2) prediction of promoter and Rho-independent transcription terminators, (3) prediction of secondary structure, (4) prediction of target genes by IntaRNA, (5) GO annotation of target genes, (6) differential expression analysis of sRNA and mRNA. The relative transcript abundance was measured by reads per kilobase of transcript per million (RPKM) mapped sequence reads. The differential expression analysis of sRNA and mRNA was performed using the DEGseq package. The sequences of sRNAs were extracted and searched against the sRNAMap, Rfam database, sRNATarBase, SIPHI, and BSRD.

Northern blot analysis
Northern blot analysis was carried out using a DIG Northern Starter Kit (Roche) following the manufacturer's protocol as described by Yan et al. [35].

sRNAs knockout
The primers used in this study were listed in Table 1. To construct the sRNA knockout mutant, in-frame deletion of segment of sRNA was performed by overlap extension PCR as follows: the first overlap PCR was performed with the primer pair KOF1 and KOR1, the second overlap PCR was performed with the primer pair KOF2 and KOR2, and the fusion PCR was performed with the primer pair KOF1 and KOR2. The PCR products were inserted into the suicide plasmid pDM4 at the BglII site, resulting in pDMsRNA. S17-1λpir was transformed with pDMsRNA, and the transformants were conjugated with TX01 as described previously [13]. The transconjugants were selected on LB agar plates supplemented with 10% sucrose. One of the colonies that were resistant to sucrose and sensitive to chloramphenicol (marker of pDM4) was analyzed by PCR, and the PCR products were subjected to DNA sequencing to confirm in-frame deletion.

Identification of Hfq-associated sRNAs
To identify the Hfq-associated sRNAs, a markerless hfq in-frame deletion mutant TXhfq [13], was used this study. After culturing to exponential phase, TX01 and TXhfq were collected and RNA isolation were conducted as describe as above. sRNAs expressions were analyzed by RNA sequencing.

Cellular infection and pathogenicity analysis
FG-9307 cells were cultured at 23 °C in 96-well cell culture plates (~10 5 cells/well) with L-15 medium (Gibco, USA) as described previously [36]. The cells were infected with TX01 or sRNA mutants at a MOI of 10:1 for 2 h. After washing with PBS three times, the cells were lysed with 1% Triton X-100, and the lysate was plated on LB agar plates. After incubation at 28 °C for 24 h, the colony number was counted.

Statistical analysis
All statistical analyses were performed with SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). Data were analyzed with analysis of variance (ANOVA), and statistical significance was defined as P < 0.05.

Primer
Sequences

Sequencing of RNA from E. piscicida
To identify globally sRNAs and investigate the sRNA transcriptome profiles of E. piscicida, total RNA was separately isolated from bacteria grown in normal media (Con) and three adverse conditions, i.e., acidic condition (Ac), iron deficiency (Dp), and oxidation pressure (Pe). Small-fragment RNAs with length of 50 to 500 nt were gel-purified and subjected to deep sequencing. Removing reads of poor quality and N > 10%, a total of 26 765 798 reads was obtained. After splicing, a total of 1 622 257 unique tags from four samples were mapped to E. piscicida genome. After removing tRNAs, rRNAs, and repeated tags, 5663 tags as candidate sRNA were produced.

Discovery of the sRNAs in E. piscicida
We extracted sRNA transcripts with sequential bases forming peaks of high-level expression which are distinct from those of its flanking regions. A total of 148 sRNAs with predicted promoter or Rho-independent terminator were finally identified (Additional file 1). Against the Rfam database and sRNAMap, 19 annotated sRNA homologs were detected. The remaining 129 sRNAs appeared to be novel sRNA candidates. The most highly expressed sRNAs (> 10 000 RPKM in four samples) include sR176, sR318, sR100, sR065 (RsmY), sR111, sR214. The expression of 26 sRNAs distribute among 1000-9999 RPKM in four samples.

Validation and characterization of selected sRNA candidates
To check whether the boundaries of sRNAs transcripts were in accordance with those predicted by our methods, the sizes of one annotated sRNA (sR084) and six novel sRNAs (sR042, sR069, sR176, sR205, sR214, and sR274) were determined by Northern blot, and 5S rRNA as a control. The results showed that the transcript lengths of seven sRNAs detected by Northern blot analysis were approximately consistent with the lengths observed by deep sequencing (Figure 1).

sRNAs expression in different conditions
The expression profiles of the 148 sRNAs were normalized with RPKM and then tested for significant difference among different samples with Bonferroni correction. The results showed that 90 sRNAs were expressed in all examined sample (Con, Ac, Dp, and Pe) ( Figure 2). Ten sRNAs were specifically expressed in Pe sample. One, eighteen, four, and four sRNAs were expressed in Con and Ac, Con and Pe, Dp and Pe, and Ac and Pe, respectively. One sRNA was expressed in three sample of Con, Ac, and Dp. Two sRNAs were expressed in Con, Dp, and Pe. Eighteen sRNAs were expressed in Con, Pe, and Ac.
No sRNA was specifically expressed in Con, Ac, or Dp. No sRNA was specifically expressed in Con and Dp, Dp and Ac, Dp and Ac and Pe ( Figure 2).

Differentially expressed sRNAs (DEsRNAs) during stress condition
Compared to Con, 103 sRNAs showed significant differences (> twofold and P < 0.05) in three stress sample (Ac, Dp, and Pe) (Figure 3 and Additional file 2). For convenience, these differentially expressed sRNAs were named DEsRNAs. Facing acid pressure (Ac), 15 sRNAs were significantly upregulated and 26 sRNAs were significantly downregulated. In iron deficiency (Dp), 13 sRNAs were significantly upregulated and 13 sRNAs were significantly downregulated. When bacteria grew in medium with hydrogen peroxide (Pe), 67 sRNAs were significantly upregulated and 15 sRNAs were significantly downregulated. There are 15, 20, 19 sRNAs showed significant differences in both Ac and Dp, both Dp and Pe, and both Ac and Pe, respectively. Ten sRNAs expressions showed significant differences in all three adverse environments, most of them were downregulated. Specifically, four sRNAs (sR162, sR165, sR188, and sR231) were consistently downregulated and two sRNAs (sR230 and sR355) were consistently upregulated, four sRNAs (sR040, sR214, sR281, sR365) were downregulated in Ac and Dp but upregulated in Pe (Figure 3 and Additional file 2).

Differentially expressed mRNAs (DEmRNA) during stress condition
In order to investigate the effect of the sRNA on target gene, global change in gene expressions associated with stress condition was examined. Based on the RNA-Seq data, it was found that the expressions of 1615 genes were significantly (> twofold and P < 0.01) altered during stress condition (Figure 4). Compared to Con, 55 genes expressions were significantly upregulated and 99 genes were significantly downregulated in Ac, 278 genes were significantly upregulated and 408 genes were significantly downregulated in Dp, 916 genes were significantly upregulated and 626 genes were significantly downregulated in Pe (Additional file 3). For convenience, these differentially expressed mRNAs were named DEmRNAs. Of these DEmRNAs, 20 DEmRNAs were existed in all three  sample, 29, 16, and 198 DEmRNAs were found in Ac and Dp, Ac and Pe, and Dp and Pe, respectively (Additional file 3).

Prediction of DEsRNA target genes among DEmRNA
Based on the prediction of DEsRNA target genes by IntaRNA and relational analysis between DEsRNA and

Enrichment analysis of DEsRNA target genes
The DEsRNA target genes identified above were subjected to gene ontology (GO) analysis. As shown in Figure 5, GO annotation of the 769 target genes indicated that they were grouped into three major categories: Biological process, Cellular component, and Molecular function. In the group of "Biological process", the largest number of genes belong to metabolic process term. In the group of "Cellular Component", the term of cell and cell part make up the main part. In the group of "Molecular function", the largest number of genes is catalytic activity term. In particular, some genes involved acid tolerance were observed among the target genes, including genes coding acid shock protein, putrescine transport protein PotE, NADH dehydrogenase, and succinate dehydrogenase. Iron uptake and transport related genes, such as genes coding siderophore biosynthesis protein, hemin transport, hemin uptake protein, hemin receptor, ion transport protein, and ferrous iron transport protein, were among the target genes. Genes participating response to oxidation pressure, such as superoxide dismutase SodB, fumarate reductase, cytochrome D ubiquinol oxidase, methionine-R-sulfoxide reductase, and NADH:ubiquinone oxidoreductase appeared in the target genes. Moreover, there are a large number of genes that play an important role in virulence among these DEsRNA target genes, for example, heat shock protein, molecular chaperone, invasion, adhesin, methyl-accepting chemotaxis protein, regulator of cell autolysis, temperature sensitive hemagglutinin, type III secretion, type

Hfq-associated sRNAs
Since Hfq is an important RNA chaperone protein, many sRNAs are Hfq-dependent [32]. Our previous study showed that Hfq mutation attenuated remarkably bacterial virulence [13], which suggested Hfq-associated sRNAs maybe play an important role in pathogenicity of E. piscicida. To identify the Hfq-associated sRNAs, the different expression of sRNAs between TX01 and TXhfq, a markerless hfq in-frame deletion wild type, was analyzed by RNA-seq. The results showed that 19 sRNAs expression were significantly upregulated and 30 sRNAs expressions were significantly downregulated when Hfq was inactivated ( Figure 6). qRT-PCR was conducted to examine the mRNA levels of 5 upregulated sRNAs and 14 downregulated sRNAs. The results showed that, of 5 upregulated sRNA identified by RNA-seq, 4 sRNAs were significantly upregulated by qRT-PCR, of 14 downregulated sRNAs identified by RNA-seq, 12 sRNAs expression were significantly downregulated or undetectable by qRT-PCR (Additional file 4).

sRNAs potentially involved in E. piscicida pathogenicity
Since sRNAs widely participate in bacterial pathogenicity, we investigated the potential roles of different kinds of sRNAs in E. piscicida pathogenicity. Six Hfq-associated sRNAs (sR012, sR043, sR082, sR084, sR145, and sR355) were chosen to perform the pathogenicity experiment. Meanwhile, three Hfq-nonassociated sRNAs, such as high expression sRNA (sR318, see Additional file 5), moderate expression sRNA (sR274), and low expression sRNA (sR114), were also included. These nine sRNAs mutants were constructed and their pathogenicity were examined. The results showed that the ability of four sRNAs to infect host cell declined significantly, and the ability of two sRNAs to infect host cell enhanced significantly, compared to wild strain TX01 (Figure 7). Among the six Hfq-associated sRNAs, only one sRNA (sR355) was not related to pathogenicity. Among the three Hfqnonassociated sRNAs, only one sRNA (sR274) was related to pathogenicity.

Discussion
During the last decade, sRNAs have emerged as essential post-transcriptional regulators in bacteria. Nearly all important physiological and stress responses are modulated by these sRNAs regulator. Researchers have successfully predicted, identified, and characterized sRNAs in a large number Gram-negative and Gram-positive species. It is assumed that an average bacterial genome encodes about 100-300 sRNAs [20]. Currently, sRNAs in many human pathogen and plant pathogen, such as Yersinia pestis, Yersinia pseudotuberculosis, Streptococcus pneumoniae, and Agrobacterium tumefaciens, have been identified [35,[37][38][39]. In this study, 148 candidate sRNAs in fish pathogen E. piscicida were identified, including 19 annotated sRNA homologs. As far as we know, our study is the first report about systematic identification of sRNA in fish pathogen. Homohexameric RNA chaperone Hfq has been shown to play a critical role in sRNA-mediated gene regulation. In many bacteria, besides playing a role in protection of sRNAs from RNase E-mediated degradation, Hfq has been considered to a key factor in sRNA-mediated gene regulation and efficient base pairing between transencoded sRNA and its target mRNA [40,41]. For example, CyaR and RprA interaction with their targe mRNA required Hfq [42]. Deleting Hfq predictably resulted in numerous phenotypes, mainly consisting of resistance to various environmental stresses [31,43]. In our previous report, we found that deletion of Hfq in E. piscicida exhibited multiple effects, including retarding retarded Figure 6 Identification of Hfq-dependent sRNAs. Edwardsiella piscicida TX01 and TXhfq, a markerless hfq in-frame deletion mutant were collected and RNA isolation were conducted. sRNA expression was analyzed by RNA sequencing. sRNAs showed significant difference expression (> two fold and P < 0.05) were identified to Hfq-dependent sRNA.
planktonic and biofilm growth, decreased resistance against oxidative stress, and attenuated virulence, and Hfq exerted a regulatory effect on a wide range of genes [13]. In this study, deficiency of Hfq influenced the expression of 49 sRNAs, of which 30 sRNAs expressions were downregulated, several sRNAs expression even could not be detected in hfq mutant by qRT-PCR. These results indicated that a great many of sRNAs were Hfqassociated sRNA in E. piscicida, and the regulatory effect of Hfq on target genes were likely achieved by sRNAs.
In their natural habitats, bacteria are constantly exposed to stressful and even challenging conditions. Studies have shown that bacterial sRNAs play important roles as regulators in coping with stress and survival. For example, sRNA DsrA and RprA both confer acid resistance [44]. RyhB is a key actor of iron homeostasis regulation [45]. MicF and OxyS are related to oxidative stress [46,47]. In this study, during acid condition, 41 sRNA expressions were significantly affected, and upregulated sRNAs were more than downregulated sRNA among the DEsRNAs expressions. Especially, sRNA004, sRNA050, sRNA040, and sRNA371 displayed extremely remarkable difference expression. These findings suggested that multiple sRNAs in E. piscicida deeply participated in resisting to acid pressure. sRNA040 is homologous with sraG, which was reported to participates in PNPase homeostasis [48], but there is no report of sraG about acid tolerance to the present. During iron deficiency stress, the expressions of 26 sRNAs changed significantly in E. piscicida, the amount of upregulated sRNAs were basically equal to those of downregulated sRNA among the DEsRNAs expressions. These DEsRNAs include some annotated sRNA such as RyhB (sR355) and CyaR (sR103), and many novel sRNA such as sRNA043 and sRNA300. It is well known that RyhB was involved in regulating iron homeostasis [45]. It was reported that CyaR in E. coli participated in regulating an acid-resistance membrane protein HdeD [42], and its regulation of target genes assisted bacterial survival in the face of envelope stress [49]. However, in E. piscicida, we observed that the expression of CyaR was regulated by iron deficiency stress, but not by acid stress. Unlike the expression of sRNA in acid stress and iron deficiency stress, when facing with the stress of hydrogen peroxide, more sRNAs (82 sRNAs) expression were affected, and the vast majority of sRNAs (about 3/4) exhibited upregulated expression upon oxidative stress. These DEsRNAs included some annotated sRNA such as GlmZ_SraJ, rhyA, csrB, csrC, and a lot novel sRNA such as sR073, sR147, and sR370. Amongst the three different kinds of adversity stress, the sum of DEsRNAs, and the number of upregulated sRNA or downregulated sRNA showed distinct difference, which indicated that sRNAs possessed diversity of function. Facing adversity stress, many significantly upregulated sRNAs have target mRNAs that were downregulated. On the other hand, some downregulated sRNAs have upregulated target genes. These results indicated that sRNAs could potentially negatively regulate target genes, which was similar to other studies [50].
Except as an important regulator of adaptation to adversity, sRNAs also play a key role in bacterial pathogenesis. The role of bacterial sRNAs in virulence has received more and more attention in recent years. It was reported that many of the identified sRNAs in human pathogen S. pneumoniae have important global and niche-specific roles in virulence [38]. In many other pathogens, a large number sRNAs, such as Rli27, LhrC, teg49, RsmY, AsdA, were reported to participate in regulation of virulence [51][52][53]. Lately, several sRNAs were identified in E. tarda and speculated to play a regulatory role of virulence [33]. Consistently, in this study, a large number of target genes of sRNAs were virulence-associated genes, such as temperature sensitive hemagglutinin, invasion, heat shock protein, cell autolysis factor, type III secretion system protein, type VI secretion system protein, and so on. And we also examined the role of several sRNAs in virulence. Of the nine sRNAs used to detect pathogenicity, 6 sRNAs were found to involve in infection of host FG cell. It is known E. piscicida can invade and survive in FG-9307 cells, which often used as an in vitro infection model of E. piscicida [54]. However, in vitro cell infections are different from in vivo animal infection and partially address the pathogenicity. In vivo animal experiments are needed to confirm the role of these sRNAs in the pathogenesis of the infection.
As regulator, the function of sRNA is reflected by its target genes. In E. piscicida, we found that sRNAs could target a large number transcription factors, including LysR family transcriptional regulator, GntR family transcriptional regulator, two-component transcriptional regulator, phage transcriptional regulatory, transcriptional repressor, transcriptional activator, and so on. These discoveries provided obvious evidence that sRNA likely deeply interlaced within complex gene regulatory networks of E. piscicida. Except transcriptional regulator, various types of functional target genes of sRNAs appeared. For example, dps, which was involved in iron limitation and acid stress [55], showed differentially expression in acid and oxidation pressure and was predicted to be regulated by sR031. atoE, which might be required for the resistance to prolonged acid exposure [56], displayed downregulated expression in acid stress and was predicted to be regulated by sR084 and sR177. During iron deficiency, the target genes of sRNAs included many iron uptake and transport genes, such as genes encoding ferrous iron transport protein A, iron transporter, periplasmic ferric iron-binding protein, ABC transporter. In our previous study, we found Fur showed a very close relationship with iron homeostasis in E. piscicida, and regulated a lot of proteins including PotE, SpeF, SpeF2, Ndh, ompF, and napA, which play important role in helping bacterial adapt adverse circumstance [14]. Similarly, in this study, these genes were found to be among targets of sRNAs and showed differently expression in iron deficiency environment. Compared the acid stress and iron deficiency, much more sRNA target genes were found in oxidation pressure condition. A lot of genes directly related to oxidation stress, such as gene encoding alkyl hydroperoxide reductase, thiol peroxidase [57], chaperonin GroEL and GroES [58], superoxide dismutase SodB and SodC [14,59], thioredoxin TrxA and TrxH [60], were among target DEmRNAs. Moreover, many mRNA targets of DEsRNAs code hypothetical proteins, including some very significant differentially expressed hypothetical proteins, which indicate that many unknown things remain to be elucidated in this organism.
In conclusion, we globally discovered candidate sRNAs for the first time in pathogenic bacteria of fish. Many novel sRNAs were identified and expression patterns of DEsRNAs and DEmRNAs in E. piscicida during adversity condition growth were revealed. DEsRNA target genes among DEmRNAs were predicted. The role of sRNAs in E. piscicida pathogenicity was characterized. Hfq-associated sRNAs were also identified. Our findings showed that sRNAs in E. piscicida have important functions in adaptation to environmental stress and pathogenicity. These results also provide clues for deciphering regulation mechanism of gene expression related to physiological response and pathogenicity in E. piscicida.