A cyclohexadepsipeptide from entomogenous fungi Metarhizium
anisopliae inhibits the Helicobacter pylori induced pathogenesis
through attenuation of vacuolating cytotoxin-A activity
Min-Chuan Kao,
a,1Yerra Koteswara Rao,
b,1Yu-Wen Hsieh,
a,1Shih-Hsien
Weng,
aTzu-Li Lu,
cDavid T. W. Tzeng,
dJau-Jin Liu,
aChun-Jung Lin,
a,eChih-Ho Lai,
a,f,* Yew-Min Tzeng
b,*
a Department of Microbiology and Graduate Institute of Basic Medical Science, China
Medical University, Taichung, Taiwan
b Institute of Biochemical Sciences and Technology, Chaoyang University of Technology,
Taichung, Taiwan
c Department of Medical Laboratory Science and Biotechnology, China Medical University,
Taichung, Taiwan
d Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung,
Taiwan
e Graduate Institute of Clinical Medical Science, China Medical University, Taichung,
Taiwan
f Department of Nursing, Asia University, Taichung, Taiwan
*Corresponding authors. Tel.: +886-4-23323000 ext. 4471; fax: +886-4-23395870.
E-mail address: [email protected] (Y.-M. Tzeng) or [email protected] (C.-H. Lai)
1 Equally contributed to this work
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 21 22 23 24
ABSTRAT
Infection with Helicobacter pylori in the gastric mucosa is thought to be increased the risk of peptic ulcer disease and gastric cancer. Vacuolating cytotoxin A (VacA) was an important bacterial virulence factor and was closely associated with H. pylori-induced pathogenesis. Upon H. pylori infection, the VacA can be transferred directly from bacteria into the host cells where can stimulate vacuolar-type ATPase (V-ATPase) proton pump and induce the formation of vacuoles in gastric epithelial cells followed by intoxication of cells. Destruxin B (Dtx-B), a cyclodepsipeptide from the fungus Metarhizium anisopliae bearing insecticidal and anticancer effects. In this study, Dtx-B demonstrated inhibitory effects against H. pylori VacA-induced vacuolization in gastric epithelial cells. Our data further showed that attenuation of H. pylori-induced vacuolization in gastric epithelial cells by Dtx-B is mediated through inhibition of V-ATPase activity rather than VacA expression levels. The results from this study reveal a novel role for Dtx-B in the modulation of H. pylori VacA-induced pathogenesis.
Keywords: Destruxin B; Helicobacter pylori; Vacuolating cytotoxin A; V-ATPase 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
1. Introduction
Gram-negative microaerophilic bacterium Helicobacter pylori is an important pathogen that colonizes in the stomach and infects approximately half of the human population in the world [1, 2]. Persistent infection by H. pylori that is usually caused several types of gastrointestinal diseases, including chronic gastritis, peptic ulcer, and gastric adenocarcinoma [3, 4]. Previous studies reported that a set of virulence factors that was involved in H. pylori-induced pathogenesis and contributed to enable bacterium to survive, multiply, protect from immune attack, and eventually lead to persistent infection in the host [5−7]. H. pylori contained two major bacterial cytotoxins: vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), which were considered to be more virulent and that were closely associated with disease progression [8]. The VacA can be secreted from H. pylori in the culture supernatant, but remained a large amount on the bacterial cell wall [9]. Infection of H. pylori, the VacA can be transferred directly from bacteria into the host cells where can lead to intoxication of cells [9]. Previous studies indicated that H. pylori VacA-induced vacuoles were associated with rab7, which was a small GTPase and localized at the late endosomal compartments [10]. The intracellular compartments endowed with vacuolar-type ATPase (V-ATPase) which is a proton pump can be activated by VacA, resulting in increasing anionic permeability and lead to cell swelling [11, 12]. Following two decades of the extensive research of VacA, this has been recognized as a multifunctional toxin [12, 13]. Therefore, development of an inhibitor against VacA might be an ideal strategy for attenuation of H. pylori-related diseases.
Identification of natural constituents as alternative drugs against H. pylori-induced pathogenesis has been pursued by many pharmaceutical research groups. Destruxins (Dtx) isolated from the fungus Metarhizium anisopliae have been well characterized previously [14]. The broad activities bearing by Dtx have been known as insecticidal effect [15], 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
immunosuppressive [16], and anti-tumoral activity in various types of cancer cells [17−19]. Destruxin B (Dtx-B), a cyclic cyclohexadepsipeptide, which has been shown to contain the inhibitory effect against V-ATPase activity [20]. Therefore, it is reasonable to investigate whether Dtx-B harboring the potent in the inhibition of H. pylori VacA-associated pathogenesis. In this study, the biological functions of Dtx-B against H. pylori VacA-actions were further explored. Our investigation indicates that Dtx-B attenuates H. pylori-induced vacuolization in gastric epithelial cells mediated through inhibition of V-ATPase activity rather than suppression of VacA levels.
2. Materials and methods
2.1. Antibodies and reagents
Antibodies against V-ATPase and H. pylori VacA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor568-conjugated goat anti-rabbit IgG, and 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA). Bafilomycin A1 (Baf A1), neutral red, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents were of the highest grade commercially available and supplied either by Sigma-Aldrich or Merck (Whitehouse Station, NJ).
2.2. Production and purification of destruxin B (Dtx-B)
The compound destruxin B (Dtx-B) used in this study was obtained from the submerged cultivation of Metarhizium anisopliae F061 according to the production and purification processes as we described previously [18, 19]. The purity of the Dtx-B was >98% by HPLC. Its structure (Fig. 1A) was confirmed by comparison of its mass and nuclear magnetic resonance spectral data with those in the literature [18, 19].
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
2.3. H. pylori strains and bacterial culture
The H. pylori reference strain 26695 (ATCC 700392), and vacA isogenic mutant (ΔVacA) strain were obtained as we described previously [21]. H. pylori strains were cultured on Brucella agar plates (Becton Dickinson, Franklin Lakes, NJ), supplemented with 10% sheep blood and contained vancomycin (6 μg/mL), amphotericin B (2 μg/mL). The bacterial strains were incubated under microaerophilic condition at 37°C for 2-3 days prior to infection of epithelial cells [22].
2.4. Cell culture and cytotoxicity assay
The human gastric epithelial cell line, AGS cells, was purchased from the American Type Culture Collection (ATCC CRL 1739, Rockville, MD) and cultured in F12 medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% FBS (Hyclone, Logan, UT) at 37°C in 5% CO2. The MTT assay was employed to determine the cytotoxicity of Dtx-B in AGS cells as
described previously [23]. 2.5. Neutral red uptake assay
H. pylori-induced vacuolization activity was analyzed by using neutral red uptake (NRU) assay [24]. AGS cells (2×104/well) were cultured in 96-well plates for 18 h and then added
Dtx-B (0-1000 nM) or Baf A1 (10 nM) prior to infection with H. pylori at an MOI of 100 for 12 h. The culture medium was removed and the cells were stained with 0.05% (w/v) neutral red (Sigma-Aldrich). After staining for 10 min, the cells were washed and the neutral red was then extracted using acidified alcohol (1% of 12N HCl in 75% alcohol) [25]. The level of NRU was measured at OD 570 nm by a Microplate Photometer (Thermo Scientific, Waltham, MA). The control was a population of AGS cells uninfected with H. pylori and was used to define 100% cellular vacuolization. The results were expressed as the percentage of relative vacuole formation by comparison with the control group.
2.6. Analysis of reversal of H. pylori-induced vacuolization by Dtx-B 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114
AGS cells (1.5×105/well) were seeded in a 6-well plate for 18 h. The cells were then
infected with H. pylori at an MOI of 100 for 8 h followed by treatment with penicillin (100 units/mL) and streptomycin (100 μg/mL) to inhibit the over-growth of bacteria. Dtx-B (1000nM) was added at the indicated time points and evaluated the vacuolating activity continually for an additional 14 h. The cell vacuolization was determined from five-independent microphotographs per sample (×200 magnification with a phase-interference inverted microscope) by counting the number of cells having more than five vacuoles per cell [26].
2.7. Immunofluorescence labeling of cells
AGS cells (1.5×105/well) were seeded on coverslips in 6-well plates for 18 h. Cells were
treated with or without Dtx-B (1000 nM) followed by infection with H. pylori at an MOI of 100 for 12 h. The treated cells were washed and then fixed with 4% paraformaldehyde (Sigma-Aldrich). To label VacA and V-ATPase, cells were incubated with anti-VacA and anti-V-ATPase antibodies (Santa Cruz Biotechnology) at 4°C for 18 h, and then probed with Alexa Fluor 488-conjugated goat mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG (Invitrigen), respectively. Samples were washed and mounted with Fluoromount-G (Southern Biotechnology Assoc., Birmingham, AL) along with DAPI, and then analyzed under a confocal laser scanning microscope (Zeiss LSM 510, Carl Zeiss, Göttingen, Germany) with a 63 oil immersion objective.
2.8. Western blot analysis
AGS cells (1.5×105/well) were cultured in 6-well plates for 18 h and then treated with or
without Dtx-B (1000 nM) prior to infection with H. pylori at an MOI of 100 for 12 h. The cells were washed with phosphate-buffered saline (PBS) and lysed with RIPA (150 mM NaCl, 50 mM Tris base pH 7.4, and 1 mM EDTA, 1% NP-40, 0.25 mM deoxycholate) containing protease inhibitor (Roche, Indianapolis, IN). The samples were boiled in SDS-115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139
PAGE sample buffer for 10 min and then resolved by 12% SDS-PAGE followed by transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The blots were incubated with antibodies against V-ATPase or β-actin at 4°C for 18 h. The membranes were washed and then incubated with horseradish peroxidase-conjugated secondary antibody (Millipore). The expression levels of V-ATPase or β-actin were detected using ECL Western blotting detection reagents (GE Healthcare, Piscataway, NJ) and visualized using X-ray film (Kodak, Rochester, NY).
2.9. Statistical analysis
The data are presented as mean ± standard deviation of triplicate experiments. The Student's t-test was performed to calculate the statistical significance of experimental results between two groups. P < 0.05 was considered significant.
3. Results
3.1. Inhibition of H. pylori-induced vacuolization by Dtx-B
H. pylori vacuolating cytotoxin A (VacA) can stimulate V-ATPase proton pump and induce the formation of vacuoles in gastric epithelial cells [27]. Destruxin B (Dtx-B), one of destruxin constituents which harbor the inhibitory activity of V-ATPase [14]. In this study, we sought to investigate whether Dtx-B can serve as an inhibitor of V-ATPase and therefore prevent the H. pylori VacA-induced vacuolization of gastric epithelial cells. We first tested the influence of Dtx-B on the growth of gastric epithelial cells. AGS cells were untreated or treated with Dtx-B at the concentrations of 100−1000 nM for 24 h and assessed the cell viability using the MTT approach. As shown in supplementary Fig. 1, at the maximum concentration (1000 nM) of the Dtx-B which exhibited barely any effects on AGS cell growth. In parallel, Dtx-B did not show bactericidal activity on the growth of wild-type and ΔVacA isogenic mutant H. pylori (supplementary Fig. 2).
We then explored whether Dtx-B can inhibit H. pylori-induced vacuolization of gastric 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164
epithelial cells. AGS cells were untreated or treated with Dtx-B (1000 nM) followed by infection with H. pylori for 12 h. As shown in Fig. 1B, the cell vacuolization was dramatically inhibited by treatment with Dtx-B compared to that un-treatment control. In this study we used bafilomycin A1 (Baf A1) as a positive control which is reported as a specific inhibitor of V-ATPase, inhibit acidification and prevent the vacuole formation [28]. The neutral red uptake (NRU) assay was then employed for detection of cell vacuolization [25]. Our data showed that AGS cells infected with H. pylori accumulate significantly more dye than un-infected cells (Fig. 1C). In the treatment of cells with Baf A1 which exhibited great inhibition of H. pylori-induced vacuolization when compared to the control group. Moreover, treatment of cells with various concentrations of Dtx-B, the level of H. pylori-induced NRU was inhibited in a concentration-dependent manner. In contrast, the vacuolization was not observed in cells treated with ΔVacA isogenic H. pylori mutant (supplementary Fig. 3).
These results indicate that Dtx-B has comparable inhibitory effect with Baf A1 on H. pylori-induced vacuolization in gastric epithelial cells.
3.2. Attenuation of H. pylori-induced vacuolization by Dtx-B
Since Dtx-B was able to prevent the formation of vacuoles, we therefore investigated whether Dtx-B can rescue H. pylori-induced vacuolization. AGS cells were infected with H. pylori followed by treatment with Dtx-B (1000 nM) at the time points of 8 h and 12 h, respectively. As shown in Fig. 2A, Dtx-B effectively reversed H. pylori-induced vacuolization. Moreover, the cells can be rescued and recovered to be normal appearance by the culture contained Dtx-B up to 24 h.
3.3. Dtx-B inhibits V-ATPase activity rather than VacA levels
To understand the role of Dtx-B played in the inhibition of H. pylori-induced vacuolization, we next attempted to assess the expression levels of V-ATPase and H. pylori 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188
VacA. As shown in Fig. 2B, the expression levels of V-ATPase in AGS cells was increased after infection with wild-type strain rather than ΔVacA H. pylori strain. After treatment of cells with Dtx-B, the expression level of H. pylori-induced V-ATPase was dramatically reduced when compared to Dtx-B untreated group (Fig. 2B and C). However, the protein expression level of H. pylori VacA in cells treated with Dtx-B was similar to that in Dtx-B
untreated group (Fig. 2B). We then directly observed whether Dtx-B inhibited H.
pylori-induced V-ATPase using confocal microscopy. The fluorescence intensity signals showed that equal expression of VacA in the H. pylori-infected cells upon un-treated or treated with Dtx-B (Fig. 3). Without treatment of Dtx-B in H. pylori-infected AGS cells, more apparent V-ATPase was localized around the vacuoles in the cytoplasm. However, remarkably reduced the vacuoles and faint V-ATPase signal was observed in H. pylori-infected AGS cells upon treated with Dtx-B. Our data indicate that attenuation of H. pylori-induced vacuolization in gastric epithelial cells by Dtx-B might mediate via inhibition of V-ATPase activity rather than VacA expression levels.
4. Discussion
VacA, one of the most important virulence factors which secreted from H. pylori, has been extensively studied for over two decades. Infection of H. pylori strains bearing toxigenic vacA alleles that were highly associated with an increased risk of peptic ulcer disease and gastric adenocarcinoma [29]. Previous studies indicate that VacA has various biological functions on host and has been recognized as a multifunctional toxin [12]. Several characteristic activities were exerted by VacA including induction of cell vacuolization [10], inhibition of T cell proliferation [5], and induction of apoptosis [30]. The mechanism for VacA-actions in vacuolization, apoptosis, and inhibition of lymphocyte proliferation is relied on one event so-called as pore-forming activity which eventually leads to osmotic swelling and vacuolization of cells [12]. Considering the critical role of VacA in H. pylori-induced 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213
pathogenesis, development of a new strategy to target VacA was thought to be a potential therapeutic modality.
Dtx, cyclodepsipeptides, showed extensive spectrum of bioactive functions that included antifungal, anticancer, and immunosuppressant effects [14]. Several studies have been demonstrated that Dtx-B harbors insecticidal and antiviral effects [31, 32]. However, the real actions of Dtx-B relying on anti-bacterial infection have never been explored. In the present study, we showed that Dtx-B has no role on H. pylori viability (supplementary Fig. 1). Additionally, our study demonstrated that Dtx-B has inhibition effect on H. pylori-VacA functions. This study firstly demonstrated that Dtx-B attenuates VacA-induced vacuolization in H. pylori-infected cells (Fig. 1B, C). Moreover, presence of Dtx-B in the cultured cells for a longer time effectively reversed H. pylori-induced vacuolization (Fig. 1C). The molecular mechanism for Dtx-B attenuates H. pylori-induced vacuolization in gastric epithelial cells is through inhibition of V-ATPase activity by which the function was similar to Baf A1 [10, 28]. It is worth noting that the expression level of VacA did not influent by Dtx-B treatment (Fig. 3), suggesting that the inhibition of cell vacuolization by Dtx-B neither mediated via prevention of VacA binding on cell surface nor internalization into cells (Fig. 4).
Baf A1, a specific inhibitor of V-ATPase, is able to inhibit the growth of human gastric cancer cells [33]. The molecular actions of Baf A1-induced apoptosis of cancer cells were included increasing lysosomal pH and activation of caspase-3 [33]. Additionally, a very recent study reported that treatment of gastric cancer with Baf A1, decreased the lysosomal chloride and therefore causes dysfunction of autophagy [34]. In the study of Dtx-B which harbored the same activity with Baf A1 that have been demonstrated bearing apoptosis effects on several types of cancers including lung cancer [18], colorectal cancer [35], and lymphoma [36]. Moreover, VacA and other virulence factors of H. pylori that were associated with increasing the risk of gastric cancer [1]. Our present study demonstrated that 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238
Dtx-B, similar to Baf A1, which is a potent inhibitor of V-ATPase and VacA-mediated vacuolization. Therefore, implying that inhibition of VacA-actions by Dtx-B may modulate the development of gastric cancer induced by H. pylori.
In conclusion, we have demonstrated for the first time that a cyclodepsipeptide destruxin B from fungus Metarhizium anisopliae potently reverses H. pylori VacA-induced vacuolization of gastric epithelial cells. Furthermore, destruxin B also found has potent effects in inhibiting pathogenesis of cells through attenuation of VacA-elicited ATPase activity. Therefore, suggesting that destruxin B might serve as a potent compound for developing a complementary therapeutic drug in the prevention of H. pylori-associated diseases.
Conflict of interest statement
The authors declare that there are no potential conflicts of interest.
Acknowledgements
This work was supported by the Ministry of Science and Technology, Taiwan (102-2811-M-324-001, 101-2313-B-039-004-MY3, and 103-2633-B-039-001), China Medical University (CMU102-BC-2), Clinical Trial and Research Center of Excellence Funds (MOHW103-TD-B-111-03) from the Taiwan Ministry of Health and Welfare, and the Tomorrow Medicine Foundation. The authors thank Shu-Chen Shen (Scientific Instrument Center, Academia Sinica) for confocal microscopy analysis.
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Figure legends
Fig. 1. (A) Chemical structures of destruxin B. (B) Attenuation of H. pylori-induced
vacuolization by Dtx-B. AGS cells were treated with Dtx-B (0-1000 nM) or Baf A1 (10 nM) followed by infection with H. pylori at an MOI of 100 for 12 h. The vacuolization activity was observed using reverse optical microscope. Scale bar, 10 μm. (C) The effect of DtxB inhibits H. pylori-induced vacuolization was assessed using neutral red uptake assay. Statistical significance was evaluated using Student t-test (*, P < 0.05).
Fig. 2. (A) Reversion of H. pylori-induced vacuolization by Dtx-B. AGS cells were infected
with H. pylori at an MOI of 100 for 8 h and then treated with penicillin (100 units/mL) and streptomycin (100 μg/mL) to inhibit the over-growth of bacteria. Cells were treated with Dtx-B (1000 nM) at the time points of 8 h and 12 h after H. pylori-infection of cells. The cell vacuolization was determined as described in the materials and methods. Open circle, cells were infected with H. pylori at the indicated time. Closed circle, H. pylori-infected cells were treated with Dtx-B at 8 h and 12 h, respectively (indicated by arrows). The data are the means and standard deviations of three independent experiments. (B) Inhibition of H. pylori VacA-induced V-ATPase activity in AGS cells. AGS cells were treated or untreated with Dtx-B (1000 nM) and infected with wild-type or ΔVacA isogenic mutant H. pylori strains for 12 h. Whole-cell lysates were analyzed the expression levels of V-ATPase, VacA, and β-actin. (C) The level of v-ATPase was evaluated by densitometric analysis. β-actin was detected to ensure equal loading. The values are means and standard deviations from 3-independent experiments. *P < 0.05.
Fig. 3. Dtx-B inhibits H. pylori VacA activity is mediated via down-regulation of V-ATPase.
AGS cells were treated or untreated with Dtx-B (1000 nM) followed by infection or 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390
un-infection with H. pylori for 12 h. The cells were fixed and stained with anti-VacA (green), anti-V-ATPase (red), and with DAPI (blue) to visualize cell nucleus. The samples were then observed by confocal fluorescence microscopy as described in Materials and Methods. Region of VacA and V-ATPase co-localization appeared in yellow in the overlay. Scale bar, 5 μm.
Fig. 4. Model depicting Dtx-B inhibits H. pylori VacA-induced vacuolization in gastric
epithelial cells. (A) VacA secreted by H. pylori binds to receptors followed by delivery into the cytoplasm where can induce V-ATPase activation and enhance vacuole formation. (B) Dtx-B inhibits H. pylori VacA-induced vacuolization is mediated through inhibition of V-ATPase.
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