Veterinary Microbiology 174 (2014) 496–503
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Andrographolide interferes quorum sensing to reduce cell damage caused by avian pathogenic Escherichia coli Xun Guo a, Li-Yan Zhang a,b, Shuai-Cheng Wu a, Fang Xia a, Yun-Xing Fu a, Yong-Li Wu a, Chun-Qing Leng a, Peng-Fei Yi a, Hai-Qing Shen a, Xu-Bin Wei a, Ben-Dong Fu a,* a
Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, No. 5333, Xi’an Road, Changchun, Jilin 130062, China Beijing Keepyoung Technology Co. Ltd., Shangdi East Road, Haidian District, Beijing 100085, China
b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 July 2014 Received in revised form 30 August 2014 Accepted 24 September 2014
Avian pathogenic Escherichia coli (APEC) induce septicemia in chickens by invading type II pneumocytes to breach the blood–air barrier. The virulence of APEC can be regulated by quorum sensing (QS). Andrographolide is a QS inhibitor of Pseudomonas aeruginosa (P. aeruginosa). Therefore, we investigate whether andrographolide inhibits the injury of chicken type II pneumocytes by avian pathogenic E. coli O78 (APEC-O78) by disrupting the bacterial QS system. The results showed that sub-MIC of andrographolide significantly reduced the release of lactate dehydrogenase (LDH), F-actin cytoskeleton polymerization, and the degree of the adherence to chicken type II pneumocytes induced by APEC-O78. Further, we found that andrographolide significantly decreased the autoinducer-2 (AI-2) activity and the expression of virulence factors of APEC-O78. These results suggest that andrographolide reduce the pathogenicity of APEC-O78 in chicken type II pneumocytes by interfering QS and decreasing virulence. These results provide new evidence for colibacillosis prevention methods in chickens. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Andrographolide Avian pathogenic Escherichia coli Quorum sensing Chicken type II pneumocyte
1. Introduction Avian pathogenic Escherichia coli (APEC) infects chicken, turkeys, and other avian species, and causes great loss to the poultry industry (Arne et al., 2000; Knobl et al., 2012; Schouler et al., 2012). Among the 171 serotypes of APEC, O78 is one of the most frequently detected serogroups (Orskov and Orskov, 1992). APEC contains a number of virulence factors, including iron uptake chelate gene D (iucD), ferric yersiniabactin uptake A (fuyA), iron regulatory protein 2 (irp-2), type 1 pili fimC gene (fimC), pyelonephritis-associated pili papC (papC), temperature-sensitive hemagglutinin gene
* Corresponding author. Tel.: +86 431 87835379. E-mail addresses: [email protected], [email protected] (B.-D. Fu). http://dx.doi.org/10.1016/j.vetmic.2014.09.021 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
(tsh), hemolysin E (hlyE), serum survival gene (iss), outer membrane proteins A (ompA), and vacuolating autotransporter toxin gene (vat), among others. These virulence factors are associated with bacterial iron acquisition, metabolism, adhesion and invasion, and serum survival to attack the host (Johnson et al., 2012; Olsen et al., 2011). APEC infection begins in the upper respiratory tract (Edelman et al., 2003) and breaches the blood–air barrier to induce septicemia (Pourbakhsh et al., 1997a). Type II pneumocytes play an important role in maintaining the function of blood–air barrier (Bjornstad et al., 2014; Maina and King, 1982; Makanya et al., 2006), and can be invaded by APEC (Zhang et al., 2014). It has recently been shown that bacterial infections can be fought through interference of bacterial quorum sensing (QS), a cell-to-cell signaling system, involving
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the production and detection of autoinducers (AIs) (Musk and Hergenrother, 2006). Microbes can utilize AIs of QS to control the expressions of multiple virulence factors, regulate survival (Reading and Sperandio, 2006) and coordinate particular phenotypic features (Fuqua et al., 1994). Autoinducer-2 (AI-2), a furanosyl boronated diester, is a type of signaling molecules whose use between Gramnegative and Gram-positive bacteria for intra-species and interspecies communication has been identified (Bassler, 2002; Nazzaro et al., 2013; Schauder et al., 2001). The gene responsible for the production of AI-2 is LuxS (Schauder et al., 2001). QS in E. coli have been demonstrated to act as a key player in the expression of virulence genes at stationary phase (Lazazzera, 2000). Inhibition of QS of APEC O2 (DE17) significantly reduced adherence and invasion by 50.0% and 40.7%, respectively, and mRNA levels of virulence-related genes were also significantly reduced (Han et al., 2013). Andrographolide (Fig. S1) is a major active constituent of Andrographis paniculata (Burm. F) Nees, and has been used in the treatment of bacillary dysentery, urinary tract infections, enteritis, laryngitis, and influenza. Furthermore, andrographolide exhibits anti-inflammatory (Kumar et al., 2004; Rajagopal et al., 2003), antibacterial (Singha et al., 2003) and antiviral activities (Calabrese et al., 2000). It has been reported that andrographolide possesses QS-inhibiting activity in Pseudomonas aeruginosa (P. aeruginosa). In this study, therefore, we set out to investigate whether andrographolide inhibits the injury of APEC-O78 to chicken type II pneumocytes through regulation of the QS system. 2. Materials and methods
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respectively. Cell suspensions were filtrated by 200 mesh sieve, re-suspended with 10% fetal bovine serum (FBS) in a 100-mm culture plate, and incubated for 1 h. Supernatants with the unattached cells were then collected for three times. The unattached cells were centrifuged at 1200 r/min for 5 min, re-suspended in fresh Dulbecco’s modified Eagle’s medium (DMEM) for three times, and filtrated by 400 mesh sieve. Cells were then incubated for 18 h at 37 8C. The adherent cell cultures were chicken type II pneumocytes. Cell cytoplasm was positively stained purple after being stained with alkaline phosphatase (Fig. S2A). Cell purity of 95% was reached. Scanning electron microscope (SEM) was used to observe abundant microvilli on the cell surface (Fig. S2B), and one or more osmophilic lamellar bodies were observed by transmission electron microscope (TEM) (Fig. S2C). 2.3. Measurement of the minimum inhibitory concentration (MIC) of andrographolide against APEC-O78 In order to determine the effect of andrographolide on the growth of APEC-O78, the MIC was measured. We plated andrographolide (50 mg/ml) into peptone culture media and achieved a final bacteria density of 5 105 CFU/ ml via 2-fold serial dilution. To identify the turbidity of the medium, optical density at 630 nm (OD630) were measured via Bio-Tek microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA) after 24 h of cultivation at 37 8C. To understand whether andrographolide affects the growth of APEC-O78 after co-culture with chicken type II pneumocytes, cells were infected with APEC-O78 (108 CFU/ml) in the presence of andrographolide (0.1– 10 mg/ml) for 6 h, and then the optical density at 600 nm (OD600) of supernatants was measured.
2.1. Bacterial strains and culture conditions 2.4. Lactate dehydrogenase (LDH) activity detection APEC-O78 strain (CVCC1418) was purchased from the China Veterinary Culture Collection Center (CVCC, Beijing, China), which was isolated from the heart of chicken with septicemia signs. The bacteria were grown routinely in peptone culture media agar plates at 37 8C. Vibrio harveyi BB152 (V. harveyi BB152) (sensor1+sensor2+) strain was provided by Dr. Han, Xian-Gan of Shanghai Veterinary Research Institute (CAAS, Shanghai, China), V. harveyi BB170 (V. harveyi BB170) (sensor1 sensor2+) strain was donated by Dr. Ke, Cai-Huan of the College of Ocean & Earth Sciences (Xiamen University, Xiamen, China) and cultivated in modified autoinducer bioassay (AB) medium at 30 8C (Bassler et al., 1997). Andrographolide (purity > 98.0%) was purchased from National Institutes for Food and Drug Control (NIFDC, Beijing, China).
Chicken type II pneumocytes were seeded in 24-well plates and incubated in DMEM with 20% FBS without antibiotics for 18 h to 90% confluence in an atmosphere of 5% CO2 at 37 8C. The cells were infected with APEC-O78 (108 CFU/ml) in the presence of andrographolide (0.1– 10 mg/ml) for 6 h. The supernatants were collected after centrifugation at 300 g for 5 min and 12,000 g for 10 min. The LDH activity was determined according to the manufacturer’s protocol (Jiancheng Technology Co., Nanjing, China). LDH activity was calculated as follows: LDH activity (U/L) = (Aexp Acon)/(Asta Abla) 0.2 1000, where Aexp is the absorbance of test samples, Acon is the absorbance of control samples, Asta is the absorbance of standard hole, and Abla is the absorbance of black wells.
2.2. Culture of chicken type II pneumocytes 2.5. Cell viability assay The assay was performed as described previously (Zhang et al., 2014) with modifications. Briefly, lung tissue samples of 13-day-old chicken embryos were cut into small tissue blocks of about 1 cubic centimeter, followed by addition of a designated amount of 0.25% trypsin and 0.1% IV collagenase (Invitrogen-Gibco, Grand Island, NY, USA) for digestion at 37 8C for 10 min and 15 min,
Cell death of chicken type II pneumocytes induced by APEC-O78 was assessed by trypan blue staining (Zhu et al., 2011). Briefly, chicken type II pneumocytes were mixed with APEC-O78 (108 CFU/ml) without antibiotics for 6 h. Cells were then concentrated to 100 ml and stained with 0.4% trypan blue for 5 min at room temperature. To assess
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cell viability, approximately 100 cells were counted with a hemocytometer for each sample. The numbers of total cells and blue cells (dead cells) were counted. The percentage of cytotoxicity was calculated as follows: Cytotoxicity (%) = (blue cells/total cells counted) 100. 2.6. F-actin cytoskeleton polymerization assay F-actin cytoskeleton polymerization assay was performed as described previously (Zhang et al., 2014), with modifications. Chicken type II pneumocytes were seeded on covers-lips in 24-well plates and incubated in DMEM (20% FBS) for 18 h. Cells were then treated with 10 mg/ml andrographolide for 4 h, washed three times with phosphate buffer saline (PBS), and stained by Hoechst 33342 (Invitrogen-Gibco, Grand Island, NY, USA) for 30 min. APEC-O78 (108 CFU/ml) stained by fluorescein isothiocyanate (FITC) (1 mg/ml) (Sigma, St. Louis, MO, USA) was used to treat cells for 3 h. Untreated cells served as a control group, cells treated with DH5a served as a negative control group, and cells only treated with APEC-O78 served as a positive control group. After 3 h post-infection, cells were washed with PBS three times, then fixed for 5 min in 3.7% formaldehyde solution in PBS, washed extensively with PBS, permeabilized with 0.1% Triton X-100 in 0.1 M PBS for 5 min, and washed again with PBS. F-actin was stained with a 5 mg/ml fluorescent rhodamine (TRITC) phalloidin (Sigma, St. Louis, MO, USA) conjugate solution with PBS (containing 1% dimethyl sulfoxide (DMSO) from the original stock solution) for 40 min at room temperature. Cells were washed three times with PBS to remove unbound phalloidin conjugate and then examined by a confocal laser microscope. 2.7. Bacterial adherence assay Chicken type II pneumocytes were exposed to APECO78 (108 CFU/ml) in the presence of andrographolide (0.1– 10 mg/ml) at 37 8C for 6 h. Nonadherent bacteria were removed with PBS for three times, then 400 ml of 0.1% Triton X-100 (Sigma, St. Louis, MO, USA) were added and co-incubated for 5 min at room temperature. The cell suspension was collected, 10-fold serially diluted with PBS, and smeared onto the peptone agar plates to determine the amount of adherence. All assays were performed in triplicate.
plates (Corning Costar, Fisher Scientific, Canada), and incubated at 30 8C for 4 h. Bioluminescence was measured via BHP9504 microplate luminescence analyzer (Beijing Hamamatsu Photonics Technology Co., Beijing, China). The supernatant from overnight cultures of V. harveyi BB152 was used as a positive control, and the supernatant from overnight cultures of E. coli DH5a as a negative control. The AI-2-mediated bioluminescence was expressed as induction (n-fold) over the negative control. For a single experiment, the V. harveyi bioassay was performed in triplicate for each sample. The experiments were repeated three times. 2.9. Reverse transcription polymerase chain reaction (RT-PCR) analysis Chicken type II pneumocytes were washed twice with pre-warmed DMEM and incubated with 108 CFU/ml of APEC-O78 in with the presence or absence of andrographolide (0.1–10 mg/ml) for 6 h. Total RNA of APEC-O78 was isolated using Trizol reagent (Invitrogen Co., USA) according to the standard procedures. For each RT-PCR reaction, 2 mg of total RNA were subjected to synthesize cDNA using Bio RT cDNA First Strand Synthesis Kit (Bioer Technology, Hangzhou, China). Parameters of PCR conditions were as follows: 94 8C for 3 min for one cycle, then 94 8C for 30 s, 51.8–59.5 8C for 30 s, 72 8C for 45 s for 30–35 cycles, and 72 8C for 10 min for one cycle. RT-PCR was performed in a total volume of 25 ml containing 12.5 ml of 2 Power Tap PCR MasterMix (BioTeke Corporation, Changchun, China), 2 ml of cDNA, 0.5 ml of 10 mM forward primer, 0.5 ml of 10 mM reverse primer, and 8.5 ml of ddH2O water. Primer sets were listed in (Table S1). The amplified PCR products were separated by 2% agarose gel electrophoresis and visualized with ethidium bromide staining and UV irradiation. The dnaE was acted as a house-keeping control gene. The relative change in gene expression was recorded as follows: Relative ratio of target gene = (expression of target gene/expression of dnaE) (Wang et al., 2010). 2.10. Statistical analysis Data are presented as means SD. The data were evaluated using one-way ANOVA in SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). P values of less than 0.05 were considered significant.
2.8. Autoinducer bioassays 3. Results The AI-2 bioluminescence assay was performed as described previously (Han and Lu, 2009), with modifications. Briefly, chicken type II pneumocytes were infected with APEC-O78 (108 CFU/ml) in the presence of andrographolide (0.1–10 mg/ml) for 6 h. The supernatant was collected by centrifuging at 12,000 g for 10 min and further filtered through a 0.22 mm filter (Pall Corporation, Ann Arbor, MI, USA). The reporter strain V. harveyi BB170 was grown in AB medium to OD600 of 2.0, diluted at 1:5000 with fresh AB medium, and cultured at 30 8C for 30 min. The above supernatant was diluted 10-fold with V. harveyi BB170 dilution, then added to black flat-bottomed 96-well
3.1. The MIC of andrographolide against APEC-O78 We used a 2-fold dilution method to assess the antibacterial effect of andrographolide on APEC-O78. As shown in Fig. S3A, andrographolide (0.1–50 mg/ml) had no effect on the growth of APEC-O78. Therefore, the MIC of andrographolide against APEC-O78 was >50 mg/ml. Furthermore, Fig. S3B showed that andrographolide (0.1–10 mg/ml) had no effect on the growth of APECO78 after co-culture with chicken type II pneumocytes for 6 h.
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3.2. Effect of andrographolide on cytotoxicity induced by APEC-O78 To investigate whether sub-MIC andrographolide protects chicken type II pneumocytes from damage caused by APEC-O78, LDH activity and cell viability were examined. Fig. 3 showed that the release of LDH from chicken type II pneumocytes exposed to APEC-O78 (184.7 7.2 U/L) increased significantly compared with that from the untreated group (129.5 1.4 U/L). Additionally, treatment with andrographolide (0.1–10 mg/ml) for 6 h significantly reduced the LDH release induced by APEC-O78 (Fig. 1A). Similar observations were seen in trypan blue exclusion, APEC-O78 (108 CFU/ ml) induced 31.5 3.3% dead cells for 6 h, while andrographolide (1 and 10 mg/ml) significantly reduced the percentage of dead cells to 23.5 3.4% and 12.1 3.0%, respectively (P < 0.01) (Fig. 1B). 3.3. Effect of andrographolide on F-actin cytoskeleton polymerization induced by APEC-O78 F-actin was examined to explore whether andrographolide inhibit cytoskeleton polymerization induced by APEC-O78. In control and DH5a groups, F-actin was arranged in neat rows and cytoskeleton rearrangement phenomenon did not occur. However, APEC-O78 significantly increased F-actin cytoskeleton polymerization of chicken type II pneumocytes. After pretreatment with andrographolide (10 mg/ml) for 4 h, F-actin cytoskeleton polymerization was less than that of APEC-O78 group (Fig. 2). 3.4. Effect of andrographolide on bacterial adherence to chicken type II pneumocytes To test the effect of andrographolide on bacterial adherence, chicken type II pneumocytes were treated with APEC-O78 in the presence or absence of andrographolide (0.1–10 mg/ml). We found that the quantity of APEC-O78 adhering to chicken type II pneumocytes was
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(4.2 0.6) 105 CFU/ml. After treatment with andrographolide (10 mg/ml), the quantity of APEC-O78 adhering to cells was significantly lower than that of APEC-O78 group (P < 0.01) (Fig. 3). 3.5. Effect of andrographolide on AI-2 activity produced by APEC-O78 To determine whether andrographolide regulates the quorum sensing of APEC-O78, we examined AI-2 activity produced by APEC-O78 in the presence or absence of andrographolide. The AI-2 activity of APEC-O78 was increased 173 8 fold, similar to that of positive control. After treatment with andrographolide (10 mg/ml) for 6 h, the AI-2 activity of APEC-O78 was significantly decreased (Fig. 4). 3.6. Effect of andrographolide on the expression of virulence genes of APEC-O78 coculture with chicken type II pneumocytes The RT-PCR results showed that after treatment with andrographolide (10 mg/ml), the mRNA levels of virulencerelated genes (papC, fimC, iucD, irp-2, fuyA, hlyE, tsh, iss, vat, luxS) of APEC-O78 were significantly decreased compared with those of APEC-O78 group (P < 0.05). However, the mRNA levels of ompA and pfs were not affected by andrographolide (Fig. 5 and Table 1). 4. Discussion Our results suggest that andrographolide reduces cell damage of chicken type II pneumocytes induced by APECO78 by inhibiting bacterial quorum sensing systems and decreasing the expression of virulence genes. We showed that andrographolide significantly reduced the release of LDH and F-actin cytoskeleton polymerization in chicken type II pneumocytes induced by APEC-O78, and remarkably decreased the degree of adherence, AI-2 activity, and the virulence gene expressions in APEC-O78.
Fig. 1. (A) The effect of andrographolide on the LDH activity of chicken type II pneumocytes induced by APEC-O78. The values represent mean SD of three independent experiments. Error bars indicate standard deviations. ##P < 0.01 vs. control group, **P < 0.01 vs. APEC-O78 group. (B) Quantitative analysis of the viability of chicken type II pneumocytes by trypan blue exclusion study. The cells were treated with APEC-O78 (108 CFU/ml) in the presence or absence of andrographolide (0.1–10 mg/ml) in DMEM (with 5% FBS). The values represent mean SD of three independent experiments. ##P < 0.01 vs. control group, ** P < 0.01 vs. APEC-O78 group.
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Fig. 2. Effect of Andrographolide on F-actin cytoskeleton polymerization induced by APEC-O78. F-actin was stained with TRITC-phalloidin (red), nuclei were stained with Hoechst 33342 (blue) and bacteria were stained with FITC (green) (magnification 2000 ). (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
Adherence of APEC to epithelial cells is the critical initial step for colonization of the respiratory tract. This infection process was mediated by the F1 and P pili (Pourbakhsh et al., 1997b). F1 pili cause E. coli colonization in trachea and air sacs (Dozois et al., 1995), and P pili were present in the air sacs, lungs and internal organs (Pourbakhsh et al., 1997b). Tsh, an essential virulence factor, may act as an adhesin particularly in the initial stages of respiratory tract colonization (Dozois et al., 2000). In the present study, chicken type II pneumocytes were
used to investigate the effect of andrographolide on the pathogenicity of APEC-O78. We found that sub-MIC of andrographolide significantly decreased the degree of adherence and the mRNA levels of fimA, papC and tsh in APEC-O78. These results indicate that andrographolide inhibits bacterial adhesion to the cell surface by reducing the expression of F1 pili, P pili and Tsh. LDH, a biological enzyme widely distributed in animals and plants, and it is a marker for common injuries. The virulence potential of E. coli strains can be determined by
Fig. 3. Effect of andrographolide on the adherence of APEC-O78 to chicken type II pneumocytes. The values represent mean SD of three independent experiments. Error bars indicate standard deviations. ** P < 0.01 vs. APEC-O78 group.
Fig. 4. The AI-2 activity of cell-free culture supernatants from chicken type II pneumocytes treated with APEC-O78 in the presence or absence of andrographolide. The values represent mean SD of three independent experiments. Error bars indicate standard deviations. **P < 0.01 vs. untreated group.
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Fig. 5. Effect of andrographolide on the mRNA expression of ferric uptake regulator (iucD, fuyA, irp-2), adhesin (fimC, papC), haemolysin (tsh, hlyE), antiserum survival factor (iss, ompA), vacuolating autotransporter toxin gene (vat), and QS (luxS, pfs). The APEC-O78 was co-culture with chicken type II pneumocytes in DMEM with 0.1–10 mg/ml of andrographolide for 6 h.
Table 1 The transcriptional levels of selected genes.a Gene
APEC-O78
papC fimC iucD irp-2 fuyA hlyE tsh ompA iss vat luxS pfs
1.11 0.07 0.50 0.04 1.21 0.08 1.10 0.03 1.00 0.07 0.75 0.05 0.89 0.03 0.90 0.03 1.01 0.05 0.87 0.03 1.39 0.05 0.89 0.03
Andrographolide (mg/ml) (mean SD)b 0.1
1
10
1.09 0.06 0.53 0.05 1.12 0.06 1.01 0.05 0.88 0.05 0.68 0.02 0.86 0.03 0.88 0.03 0.94 0.05 0.85 0.03 1.12 0.04** 0.86 0.02
0.91 0.04* 0.47 0.02 1.13 0.02* 1.00 0.06 0.84 0.04* 0.69 0.03 0.82 0.02 0.89 0.03 0.88 0.03 0.76 0.02* 1.34 0.06 0.89 0.02
0.83 0.06* 0.36 0.03* 1.00 0.04* 0.92 0.04* 0.81 0.04* 0.61 0.03* 0.70 0.03** 0.86 0.02 0.81 0.03* 0.63 0.02** 1.23 0.02* 0.80 0.03
a
The mRNA level of each gene was normalized to that of dnaE. Results are shown as relative expression ratios compared to that of APEC-O78 at 108 CFU/ml. Each value represents the mean SD of three different determinations. * P < 0.05 vs. APEC-O78 group. ** P < 0.01 vs. APEC-O78 group. b
examining LDH release from cells (Maldonado et al., 2005). Hemolysin produced by E. coli can promote pores formation in the membrane of target cells and destroy the structural integrity of cell, causing the release of cytokines and inflammatory reactions (del Castillo et al., 1997; Reingold et al., 1999). Vat, a virulence factor produced by E. coli, similar to the cytotoxin of Helicobacter pylori, can induce vacuolation of the primary chicken embryo fibroblast (Salvadori et al., 2001). In this study, andrographolide reduced the LDH release by chicken type II pneumocytes induced by APEC-O78. This is consistent with the findings that andrographolide relieves pathological changes in the lung tissues and the ultrastructure changes in type II alveolar epithelial cells, and improves the viability of MLE-12 cells (Zhu et al., 2013). We also found that the expressions of hlyE and vat were decreased
significantly by andrographolide. These results reveal that andrographolide powerfully protects against cell damage induced by APEC-O78 by inhibiting the secretion of toxins. F-actin plays an important role in the mobility and contraction of cells during cell division and is a constituent of the cell cytoskeleton (Wooley et al., 1998). We found that APEC-O78 induced actin cytoskeleton polymerization, in accordance with the actin polymerization of chicken fibroblast CEC-32 cell induced by APEC-MT78 (Matter et al., 2011). After treatment with andrographolide, F-actin cytoskeleton polymerization was significantly decreased. Similarly, a-cyperone also inhibits actin cytoskeleton polymerization induced by APEC-O78 (Zhang et al., 2014). This indicates that andrographolide can maintain normal cell morphology by decreasing F-actin cytoskeleton polymerization induced by APEC-O78.
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Why sub-MIC of andrographolide protect chicken type II pneumocytes from damage induced by APEC-O78 by reducing the mRNA expression of bacterial virulence factors? Recently researches indicate that this phenomenon is associated with bacterial QS. Our previous study showed that APEC-O78 produces the highest level of AI-2 activity at late log phase (data not shown), while the expression of the virulence factors also reaches a maximum at this stage. Therefore, we hypothesize that andrographolide inhibits the pathogenicity of APEC-O78 by regulating QS system. Here, we found that andrographolide significantly decreased the AI-2 activity of APECO78, indicating that andrographolide can regulate QS system of APEC-O78. The QS system regulates the production of a variety of virulence factors in many pathogenic bacteria. DNA microarrays hybridization identified 242 genes of E. coli (upregulated 154 genes, repressed 88 genes) that are controlled by QS signaling molecules AI-2 (DeLisa et al., 2001). Type III secretion systems of both enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) have also been shown to be controlled by QS (Sperandio et al., 1999). In E. coli, a number of genes including ferric uptake regulator, adhesion, haemolysin, antiserum survival factor and vacuolating autotransporter toxin, facilitate APEC infection (Johnson et al., 2012; Olsen et al., 2011). In this study, andrographolide also significantly reduced the expressions of luxS and these virulence genes, indicating that andrographolide diminishes the pathogenicity of APEC-O78 by regulating QS. Several natural compounds also decreased the virulence of bacterial through QS regulation. For example, 14-alpha-lipoyl andrographolide (AL-1) has been shown to repress the transcriptional level of QS-regulated genes (lasI, lasR, rhlI, rhlR, pqsA, pqsH, and pqsR) in P. aeruginosa (Ma et al., 2012). Additionally, ellagic acid derivatives disrupted the expression of LasR and RhlR to decrease the production of virulence factor in P. aeruginosa PAO1 (Sarabhai et al., 2013). Andrographolide was selected out as one of several quorum sensing inhibitors of P. aeruginosa using molecule docking methods, and may compete for binding to receptor protein binding sites of RhlR with signaling molecules (Sarabhai et al., 2013). However, the mechanism of andrographolide down-regulates of AI-2 activity in APEC has not been fully elucidated and must be further studied. In conclusion, andrographolide reduced cell damage of chicken type II pneumocytes induced by APEC-O78 by interfering with quorum sensing via molecular AI-2 and down-regulating the expression of virulence genes. These results provide new evidence for colibacillosis prevention methods in chickens.
Conflict of interest The authors have declared no conflict of interest. Acknowledgements We thank Prof. Ward Lynds, Jilin University and Matthew Engel, Cornell University for very carefully editing the manuscript.
This work was supported by the National Natural Science Foundation of China (No. 31372470), and Jilin University Fundamental Research Funds (to Dr. B.D. Fu). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2014.09.021. References Arne, P., Marc, D., Bree, A., Schouler, C., Dho-Moulin, M., 2000. Increased tracheal colonization in chickens without impairing pathogenic properties of avian pathogenic Escherichia coli MT78 with a fimH deletion. Avian Dis. 44, 343–355. Bassler, B.L., 2002. Small talk: cell-to-cell communication in bacteria. Cell 109, 421–424. Bassler, B.L., Greenberg, E.P., Stevens, A.M., 1997. Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J. Bacteriol. 179, 4043–4045. Bjornstad, S., Paulsen, R.E., Erichsen, A., Glover, J.C., Roald, B., 2014. Type I and II pneumocyte differentiation in the developing fetal chicken lung: conservation of pivotal proteins from birds to human in the struggle for life at birth. Neonatology 105, 112–120. Calabrese, C., Berman, S.H., Babish, J.G., Ma, X., Shinto, L., Dorr, M., Wells, K., Wenner, C.A., Standish, L.J., 2000. A phase I trial of andrographolide in HIV positive patients and normal volunteers. Phytother. Res. 14, 333–338. del Castillo, F.J., Leal, S.C., Moreno, F., del Castillo, I., 1997. The Escherichia coli K-12 sheA gene encodes a 34-kDa secreted haemolysin. Mol. Microbiol. 25, 107–115. DeLisa, M.P., Wu, C.F., Wang, L., Valdes, J.J., Bentley, W.E., 2001. DNA microarray-based identification of genes controlled by autoinducer 2stimulated quorum sensing in Escherichia coli. J. Bacteriol. 183, 5239– 5247. Dozois, C.M., Dho-Moulin, M., Bree, A., Fairbrother, J.M., Desautels, C., Curtiss 3rd, R., 2000. Relationship between the Tsh autotransporter and pathogenicity of avian Escherichia coli and localization and analysis of the Tsh genetic region. Infect. Immun. 68, 4145–4154. Dozois, C.M., Pourbakhsh, S.A., Fairbrother, J.M., 1995. Expression of P and type 1 (F1) fimbriae in pathogenic Escherichia coli from poultry. Vet. Microbiol. 45, 297–309. Edelman, S., Leskela, S., Ron, E., Apajalahti, J., Korhonen, T.K., 2003. In vitro adhesion of an avian pathogenic Escherichia coli O78 strain to surfaces of the chicken intestinal tract and to ileal mucus. Vet. Microbiol. 91, 41–56. Fuqua, W.C., Winans, S.C., Greenberg, E.P., 1994. Quorum sensing in bacteria: the LuxR–LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176, 269–275. Han, X., Bai, H., Liu, L., Dong, H., Liu, R., Song, J., Ding, C., Qi, K., Liu, H., Yu, S., 2013. The luxS gene functions in the pathogenesis of avian pathogenic Escherichia coli. Microb. Pathog. 55, 21–27. Han, X.G., Lu, C.P., 2009. Detection of autoinducer-2 and analysis of the profile of luxS and pfs transcription in Streptococcus suis serotype 2. Curr. Microbiol. 58, 146–152. Johnson, T.J., Wannemuehler, Y., Kariyawasam, S., Johnson, J.R., Logue, C.M., Nolan, L.K., 2012. Prevalence of avian-pathogenic Escherichia coli strain O1 genomic islands among extraintestinal and commensal E. coli isolates. J. Bacteriol. 194, 2846–2853. Knobl, T., Moreno, A.M., Paixao, R., Gomes, T.A., Vieira, M.A., da Silva Leite, D., Blanco, J.E., Ferreira, A.J., 2012. Prevalence of avian pathogenic Escherichia coli (APEC) clone harboring sfa gene in Brazil. Sci. World J. 2012, 437342. Kumar, R.A., Sridevi, K., Kumar, N.V., Nanduri, S., Rajagopal, S., 2004. Anticancer and immunostimulatory compounds from Andrographis paniculata. J. Ethnopharmacol. 92, 291–295. Lazazzera, B.A., 2000. Quorum sensing and starvation: signals for entry into stationary phase. Curr. Opin. Microbiol. 3, 177–182. Ma, L., Liu, X., Liang, H., Che, Y., Chen, C., Dai, H., Yu, K., Liu, M., Yang, C.H., Song, F., Wang, Y., Zhang, L., 2012. Effects of 14-alpha-lipoyl andrographolide on quorum sensing in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 56, 6088–6094. Maina, J.N., King, A.S., 1982. The thickness of avian blood-gas barrier: qualitative and quantitative observations. J. Anat. 134, 553–562.
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Andrographolide interferes quorum sensing to reduce cell damage caused by avian pathogenic Escherichia coli Xun Guo a, Li-Yan Zhang a,b, Shuai-Cheng Wu a, Fang Xia a, Yun-Xing Fu a, Yong-Li Wu a, Chun-Qing Leng a, Peng-Fei Yi a, Hai-Qing Shen a, Xu-Bin Wei a, Ben-Dong Fu a,* a
Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, No. 5333, Xi’an Road, Changchun, Jilin 130062, China Beijing Keepyoung Technology Co. Ltd., Shangdi East Road, Haidian District, Beijing 100085, China
b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 July 2014 Received in revised form 30 August 2014 Accepted 24 September 2014
Avian pathogenic Escherichia coli (APEC) induce septicemia in chickens by invading type II pneumocytes to breach the blood–air barrier. The virulence of APEC can be regulated by quorum sensing (QS). Andrographolide is a QS inhibitor of Pseudomonas aeruginosa (P. aeruginosa). Therefore, we investigate whether andrographolide inhibits the injury of chicken type II pneumocytes by avian pathogenic E. coli O78 (APEC-O78) by disrupting the bacterial QS system. The results showed that sub-MIC of andrographolide significantly reduced the release of lactate dehydrogenase (LDH), F-actin cytoskeleton polymerization, and the degree of the adherence to chicken type II pneumocytes induced by APEC-O78. Further, we found that andrographolide significantly decreased the autoinducer-2 (AI-2) activity and the expression of virulence factors of APEC-O78. These results suggest that andrographolide reduce the pathogenicity of APEC-O78 in chicken type II pneumocytes by interfering QS and decreasing virulence. These results provide new evidence for colibacillosis prevention methods in chickens. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Andrographolide Avian pathogenic Escherichia coli Quorum sensing Chicken type II pneumocyte
1. Introduction Avian pathogenic Escherichia coli (APEC) infects chicken, turkeys, and other avian species, and causes great loss to the poultry industry (Arne et al., 2000; Knobl et al., 2012; Schouler et al., 2012). Among the 171 serotypes of APEC, O78 is one of the most frequently detected serogroups (Orskov and Orskov, 1992). APEC contains a number of virulence factors, including iron uptake chelate gene D (iucD), ferric yersiniabactin uptake A (fuyA), iron regulatory protein 2 (irp-2), type 1 pili fimC gene (fimC), pyelonephritis-associated pili papC (papC), temperature-sensitive hemagglutinin gene
* Corresponding author. Tel.: +86 431 87835379. E-mail addresses: [email protected], [email protected] (B.-D. Fu). http://dx.doi.org/10.1016/j.vetmic.2014.09.021 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
(tsh), hemolysin E (hlyE), serum survival gene (iss), outer membrane proteins A (ompA), and vacuolating autotransporter toxin gene (vat), among others. These virulence factors are associated with bacterial iron acquisition, metabolism, adhesion and invasion, and serum survival to attack the host (Johnson et al., 2012; Olsen et al., 2011). APEC infection begins in the upper respiratory tract (Edelman et al., 2003) and breaches the blood–air barrier to induce septicemia (Pourbakhsh et al., 1997a). Type II pneumocytes play an important role in maintaining the function of blood–air barrier (Bjornstad et al., 2014; Maina and King, 1982; Makanya et al., 2006), and can be invaded by APEC (Zhang et al., 2014). It has recently been shown that bacterial infections can be fought through interference of bacterial quorum sensing (QS), a cell-to-cell signaling system, involving
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the production and detection of autoinducers (AIs) (Musk and Hergenrother, 2006). Microbes can utilize AIs of QS to control the expressions of multiple virulence factors, regulate survival (Reading and Sperandio, 2006) and coordinate particular phenotypic features (Fuqua et al., 1994). Autoinducer-2 (AI-2), a furanosyl boronated diester, is a type of signaling molecules whose use between Gramnegative and Gram-positive bacteria for intra-species and interspecies communication has been identified (Bassler, 2002; Nazzaro et al., 2013; Schauder et al., 2001). The gene responsible for the production of AI-2 is LuxS (Schauder et al., 2001). QS in E. coli have been demonstrated to act as a key player in the expression of virulence genes at stationary phase (Lazazzera, 2000). Inhibition of QS of APEC O2 (DE17) significantly reduced adherence and invasion by 50.0% and 40.7%, respectively, and mRNA levels of virulence-related genes were also significantly reduced (Han et al., 2013). Andrographolide (Fig. S1) is a major active constituent of Andrographis paniculata (Burm. F) Nees, and has been used in the treatment of bacillary dysentery, urinary tract infections, enteritis, laryngitis, and influenza. Furthermore, andrographolide exhibits anti-inflammatory (Kumar et al., 2004; Rajagopal et al., 2003), antibacterial (Singha et al., 2003) and antiviral activities (Calabrese et al., 2000). It has been reported that andrographolide possesses QS-inhibiting activity in Pseudomonas aeruginosa (P. aeruginosa). In this study, therefore, we set out to investigate whether andrographolide inhibits the injury of APEC-O78 to chicken type II pneumocytes through regulation of the QS system. 2. Materials and methods
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respectively. Cell suspensions were filtrated by 200 mesh sieve, re-suspended with 10% fetal bovine serum (FBS) in a 100-mm culture plate, and incubated for 1 h. Supernatants with the unattached cells were then collected for three times. The unattached cells were centrifuged at 1200 r/min for 5 min, re-suspended in fresh Dulbecco’s modified Eagle’s medium (DMEM) for three times, and filtrated by 400 mesh sieve. Cells were then incubated for 18 h at 37 8C. The adherent cell cultures were chicken type II pneumocytes. Cell cytoplasm was positively stained purple after being stained with alkaline phosphatase (Fig. S2A). Cell purity of 95% was reached. Scanning electron microscope (SEM) was used to observe abundant microvilli on the cell surface (Fig. S2B), and one or more osmophilic lamellar bodies were observed by transmission electron microscope (TEM) (Fig. S2C). 2.3. Measurement of the minimum inhibitory concentration (MIC) of andrographolide against APEC-O78 In order to determine the effect of andrographolide on the growth of APEC-O78, the MIC was measured. We plated andrographolide (50 mg/ml) into peptone culture media and achieved a final bacteria density of 5 105 CFU/ ml via 2-fold serial dilution. To identify the turbidity of the medium, optical density at 630 nm (OD630) were measured via Bio-Tek microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA) after 24 h of cultivation at 37 8C. To understand whether andrographolide affects the growth of APEC-O78 after co-culture with chicken type II pneumocytes, cells were infected with APEC-O78 (108 CFU/ml) in the presence of andrographolide (0.1– 10 mg/ml) for 6 h, and then the optical density at 600 nm (OD600) of supernatants was measured.
2.1. Bacterial strains and culture conditions 2.4. Lactate dehydrogenase (LDH) activity detection APEC-O78 strain (CVCC1418) was purchased from the China Veterinary Culture Collection Center (CVCC, Beijing, China), which was isolated from the heart of chicken with septicemia signs. The bacteria were grown routinely in peptone culture media agar plates at 37 8C. Vibrio harveyi BB152 (V. harveyi BB152) (sensor1+sensor2+) strain was provided by Dr. Han, Xian-Gan of Shanghai Veterinary Research Institute (CAAS, Shanghai, China), V. harveyi BB170 (V. harveyi BB170) (sensor1 sensor2+) strain was donated by Dr. Ke, Cai-Huan of the College of Ocean & Earth Sciences (Xiamen University, Xiamen, China) and cultivated in modified autoinducer bioassay (AB) medium at 30 8C (Bassler et al., 1997). Andrographolide (purity > 98.0%) was purchased from National Institutes for Food and Drug Control (NIFDC, Beijing, China).
Chicken type II pneumocytes were seeded in 24-well plates and incubated in DMEM with 20% FBS without antibiotics for 18 h to 90% confluence in an atmosphere of 5% CO2 at 37 8C. The cells were infected with APEC-O78 (108 CFU/ml) in the presence of andrographolide (0.1– 10 mg/ml) for 6 h. The supernatants were collected after centrifugation at 300 g for 5 min and 12,000 g for 10 min. The LDH activity was determined according to the manufacturer’s protocol (Jiancheng Technology Co., Nanjing, China). LDH activity was calculated as follows: LDH activity (U/L) = (Aexp Acon)/(Asta Abla) 0.2 1000, where Aexp is the absorbance of test samples, Acon is the absorbance of control samples, Asta is the absorbance of standard hole, and Abla is the absorbance of black wells.
2.2. Culture of chicken type II pneumocytes 2.5. Cell viability assay The assay was performed as described previously (Zhang et al., 2014) with modifications. Briefly, lung tissue samples of 13-day-old chicken embryos were cut into small tissue blocks of about 1 cubic centimeter, followed by addition of a designated amount of 0.25% trypsin and 0.1% IV collagenase (Invitrogen-Gibco, Grand Island, NY, USA) for digestion at 37 8C for 10 min and 15 min,
Cell death of chicken type II pneumocytes induced by APEC-O78 was assessed by trypan blue staining (Zhu et al., 2011). Briefly, chicken type II pneumocytes were mixed with APEC-O78 (108 CFU/ml) without antibiotics for 6 h. Cells were then concentrated to 100 ml and stained with 0.4% trypan blue for 5 min at room temperature. To assess
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cell viability, approximately 100 cells were counted with a hemocytometer for each sample. The numbers of total cells and blue cells (dead cells) were counted. The percentage of cytotoxicity was calculated as follows: Cytotoxicity (%) = (blue cells/total cells counted) 100. 2.6. F-actin cytoskeleton polymerization assay F-actin cytoskeleton polymerization assay was performed as described previously (Zhang et al., 2014), with modifications. Chicken type II pneumocytes were seeded on covers-lips in 24-well plates and incubated in DMEM (20% FBS) for 18 h. Cells were then treated with 10 mg/ml andrographolide for 4 h, washed three times with phosphate buffer saline (PBS), and stained by Hoechst 33342 (Invitrogen-Gibco, Grand Island, NY, USA) for 30 min. APEC-O78 (108 CFU/ml) stained by fluorescein isothiocyanate (FITC) (1 mg/ml) (Sigma, St. Louis, MO, USA) was used to treat cells for 3 h. Untreated cells served as a control group, cells treated with DH5a served as a negative control group, and cells only treated with APEC-O78 served as a positive control group. After 3 h post-infection, cells were washed with PBS three times, then fixed for 5 min in 3.7% formaldehyde solution in PBS, washed extensively with PBS, permeabilized with 0.1% Triton X-100 in 0.1 M PBS for 5 min, and washed again with PBS. F-actin was stained with a 5 mg/ml fluorescent rhodamine (TRITC) phalloidin (Sigma, St. Louis, MO, USA) conjugate solution with PBS (containing 1% dimethyl sulfoxide (DMSO) from the original stock solution) for 40 min at room temperature. Cells were washed three times with PBS to remove unbound phalloidin conjugate and then examined by a confocal laser microscope. 2.7. Bacterial adherence assay Chicken type II pneumocytes were exposed to APECO78 (108 CFU/ml) in the presence of andrographolide (0.1– 10 mg/ml) at 37 8C for 6 h. Nonadherent bacteria were removed with PBS for three times, then 400 ml of 0.1% Triton X-100 (Sigma, St. Louis, MO, USA) were added and co-incubated for 5 min at room temperature. The cell suspension was collected, 10-fold serially diluted with PBS, and smeared onto the peptone agar plates to determine the amount of adherence. All assays were performed in triplicate.
plates (Corning Costar, Fisher Scientific, Canada), and incubated at 30 8C for 4 h. Bioluminescence was measured via BHP9504 microplate luminescence analyzer (Beijing Hamamatsu Photonics Technology Co., Beijing, China). The supernatant from overnight cultures of V. harveyi BB152 was used as a positive control, and the supernatant from overnight cultures of E. coli DH5a as a negative control. The AI-2-mediated bioluminescence was expressed as induction (n-fold) over the negative control. For a single experiment, the V. harveyi bioassay was performed in triplicate for each sample. The experiments were repeated three times. 2.9. Reverse transcription polymerase chain reaction (RT-PCR) analysis Chicken type II pneumocytes were washed twice with pre-warmed DMEM and incubated with 108 CFU/ml of APEC-O78 in with the presence or absence of andrographolide (0.1–10 mg/ml) for 6 h. Total RNA of APEC-O78 was isolated using Trizol reagent (Invitrogen Co., USA) according to the standard procedures. For each RT-PCR reaction, 2 mg of total RNA were subjected to synthesize cDNA using Bio RT cDNA First Strand Synthesis Kit (Bioer Technology, Hangzhou, China). Parameters of PCR conditions were as follows: 94 8C for 3 min for one cycle, then 94 8C for 30 s, 51.8–59.5 8C for 30 s, 72 8C for 45 s for 30–35 cycles, and 72 8C for 10 min for one cycle. RT-PCR was performed in a total volume of 25 ml containing 12.5 ml of 2 Power Tap PCR MasterMix (BioTeke Corporation, Changchun, China), 2 ml of cDNA, 0.5 ml of 10 mM forward primer, 0.5 ml of 10 mM reverse primer, and 8.5 ml of ddH2O water. Primer sets were listed in (Table S1). The amplified PCR products were separated by 2% agarose gel electrophoresis and visualized with ethidium bromide staining and UV irradiation. The dnaE was acted as a house-keeping control gene. The relative change in gene expression was recorded as follows: Relative ratio of target gene = (expression of target gene/expression of dnaE) (Wang et al., 2010). 2.10. Statistical analysis Data are presented as means SD. The data were evaluated using one-way ANOVA in SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). P values of less than 0.05 were considered significant.
2.8. Autoinducer bioassays 3. Results The AI-2 bioluminescence assay was performed as described previously (Han and Lu, 2009), with modifications. Briefly, chicken type II pneumocytes were infected with APEC-O78 (108 CFU/ml) in the presence of andrographolide (0.1–10 mg/ml) for 6 h. The supernatant was collected by centrifuging at 12,000 g for 10 min and further filtered through a 0.22 mm filter (Pall Corporation, Ann Arbor, MI, USA). The reporter strain V. harveyi BB170 was grown in AB medium to OD600 of 2.0, diluted at 1:5000 with fresh AB medium, and cultured at 30 8C for 30 min. The above supernatant was diluted 10-fold with V. harveyi BB170 dilution, then added to black flat-bottomed 96-well
3.1. The MIC of andrographolide against APEC-O78 We used a 2-fold dilution method to assess the antibacterial effect of andrographolide on APEC-O78. As shown in Fig. S3A, andrographolide (0.1–50 mg/ml) had no effect on the growth of APEC-O78. Therefore, the MIC of andrographolide against APEC-O78 was >50 mg/ml. Furthermore, Fig. S3B showed that andrographolide (0.1–10 mg/ml) had no effect on the growth of APECO78 after co-culture with chicken type II pneumocytes for 6 h.
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3.2. Effect of andrographolide on cytotoxicity induced by APEC-O78 To investigate whether sub-MIC andrographolide protects chicken type II pneumocytes from damage caused by APEC-O78, LDH activity and cell viability were examined. Fig. 3 showed that the release of LDH from chicken type II pneumocytes exposed to APEC-O78 (184.7 7.2 U/L) increased significantly compared with that from the untreated group (129.5 1.4 U/L). Additionally, treatment with andrographolide (0.1–10 mg/ml) for 6 h significantly reduced the LDH release induced by APEC-O78 (Fig. 1A). Similar observations were seen in trypan blue exclusion, APEC-O78 (108 CFU/ ml) induced 31.5 3.3% dead cells for 6 h, while andrographolide (1 and 10 mg/ml) significantly reduced the percentage of dead cells to 23.5 3.4% and 12.1 3.0%, respectively (P < 0.01) (Fig. 1B). 3.3. Effect of andrographolide on F-actin cytoskeleton polymerization induced by APEC-O78 F-actin was examined to explore whether andrographolide inhibit cytoskeleton polymerization induced by APEC-O78. In control and DH5a groups, F-actin was arranged in neat rows and cytoskeleton rearrangement phenomenon did not occur. However, APEC-O78 significantly increased F-actin cytoskeleton polymerization of chicken type II pneumocytes. After pretreatment with andrographolide (10 mg/ml) for 4 h, F-actin cytoskeleton polymerization was less than that of APEC-O78 group (Fig. 2). 3.4. Effect of andrographolide on bacterial adherence to chicken type II pneumocytes To test the effect of andrographolide on bacterial adherence, chicken type II pneumocytes were treated with APEC-O78 in the presence or absence of andrographolide (0.1–10 mg/ml). We found that the quantity of APEC-O78 adhering to chicken type II pneumocytes was
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(4.2 0.6) 105 CFU/ml. After treatment with andrographolide (10 mg/ml), the quantity of APEC-O78 adhering to cells was significantly lower than that of APEC-O78 group (P < 0.01) (Fig. 3). 3.5. Effect of andrographolide on AI-2 activity produced by APEC-O78 To determine whether andrographolide regulates the quorum sensing of APEC-O78, we examined AI-2 activity produced by APEC-O78 in the presence or absence of andrographolide. The AI-2 activity of APEC-O78 was increased 173 8 fold, similar to that of positive control. After treatment with andrographolide (10 mg/ml) for 6 h, the AI-2 activity of APEC-O78 was significantly decreased (Fig. 4). 3.6. Effect of andrographolide on the expression of virulence genes of APEC-O78 coculture with chicken type II pneumocytes The RT-PCR results showed that after treatment with andrographolide (10 mg/ml), the mRNA levels of virulencerelated genes (papC, fimC, iucD, irp-2, fuyA, hlyE, tsh, iss, vat, luxS) of APEC-O78 were significantly decreased compared with those of APEC-O78 group (P < 0.05). However, the mRNA levels of ompA and pfs were not affected by andrographolide (Fig. 5 and Table 1). 4. Discussion Our results suggest that andrographolide reduces cell damage of chicken type II pneumocytes induced by APECO78 by inhibiting bacterial quorum sensing systems and decreasing the expression of virulence genes. We showed that andrographolide significantly reduced the release of LDH and F-actin cytoskeleton polymerization in chicken type II pneumocytes induced by APEC-O78, and remarkably decreased the degree of adherence, AI-2 activity, and the virulence gene expressions in APEC-O78.
Fig. 1. (A) The effect of andrographolide on the LDH activity of chicken type II pneumocytes induced by APEC-O78. The values represent mean SD of three independent experiments. Error bars indicate standard deviations. ##P < 0.01 vs. control group, **P < 0.01 vs. APEC-O78 group. (B) Quantitative analysis of the viability of chicken type II pneumocytes by trypan blue exclusion study. The cells were treated with APEC-O78 (108 CFU/ml) in the presence or absence of andrographolide (0.1–10 mg/ml) in DMEM (with 5% FBS). The values represent mean SD of three independent experiments. ##P < 0.01 vs. control group, ** P < 0.01 vs. APEC-O78 group.
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Fig. 2. Effect of Andrographolide on F-actin cytoskeleton polymerization induced by APEC-O78. F-actin was stained with TRITC-phalloidin (red), nuclei were stained with Hoechst 33342 (blue) and bacteria were stained with FITC (green) (magnification 2000 ). (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
Adherence of APEC to epithelial cells is the critical initial step for colonization of the respiratory tract. This infection process was mediated by the F1 and P pili (Pourbakhsh et al., 1997b). F1 pili cause E. coli colonization in trachea and air sacs (Dozois et al., 1995), and P pili were present in the air sacs, lungs and internal organs (Pourbakhsh et al., 1997b). Tsh, an essential virulence factor, may act as an adhesin particularly in the initial stages of respiratory tract colonization (Dozois et al., 2000). In the present study, chicken type II pneumocytes were
used to investigate the effect of andrographolide on the pathogenicity of APEC-O78. We found that sub-MIC of andrographolide significantly decreased the degree of adherence and the mRNA levels of fimA, papC and tsh in APEC-O78. These results indicate that andrographolide inhibits bacterial adhesion to the cell surface by reducing the expression of F1 pili, P pili and Tsh. LDH, a biological enzyme widely distributed in animals and plants, and it is a marker for common injuries. The virulence potential of E. coli strains can be determined by
Fig. 3. Effect of andrographolide on the adherence of APEC-O78 to chicken type II pneumocytes. The values represent mean SD of three independent experiments. Error bars indicate standard deviations. ** P < 0.01 vs. APEC-O78 group.
Fig. 4. The AI-2 activity of cell-free culture supernatants from chicken type II pneumocytes treated with APEC-O78 in the presence or absence of andrographolide. The values represent mean SD of three independent experiments. Error bars indicate standard deviations. **P < 0.01 vs. untreated group.
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Fig. 5. Effect of andrographolide on the mRNA expression of ferric uptake regulator (iucD, fuyA, irp-2), adhesin (fimC, papC), haemolysin (tsh, hlyE), antiserum survival factor (iss, ompA), vacuolating autotransporter toxin gene (vat), and QS (luxS, pfs). The APEC-O78 was co-culture with chicken type II pneumocytes in DMEM with 0.1–10 mg/ml of andrographolide for 6 h.
Table 1 The transcriptional levels of selected genes.a Gene
APEC-O78
papC fimC iucD irp-2 fuyA hlyE tsh ompA iss vat luxS pfs
1.11 0.07 0.50 0.04 1.21 0.08 1.10 0.03 1.00 0.07 0.75 0.05 0.89 0.03 0.90 0.03 1.01 0.05 0.87 0.03 1.39 0.05 0.89 0.03
Andrographolide (mg/ml) (mean SD)b 0.1
1
10
1.09 0.06 0.53 0.05 1.12 0.06 1.01 0.05 0.88 0.05 0.68 0.02 0.86 0.03 0.88 0.03 0.94 0.05 0.85 0.03 1.12 0.04** 0.86 0.02
0.91 0.04* 0.47 0.02 1.13 0.02* 1.00 0.06 0.84 0.04* 0.69 0.03 0.82 0.02 0.89 0.03 0.88 0.03 0.76 0.02* 1.34 0.06 0.89 0.02
0.83 0.06* 0.36 0.03* 1.00 0.04* 0.92 0.04* 0.81 0.04* 0.61 0.03* 0.70 0.03** 0.86 0.02 0.81 0.03* 0.63 0.02** 1.23 0.02* 0.80 0.03
a
The mRNA level of each gene was normalized to that of dnaE. Results are shown as relative expression ratios compared to that of APEC-O78 at 108 CFU/ml. Each value represents the mean SD of three different determinations. * P < 0.05 vs. APEC-O78 group. ** P < 0.01 vs. APEC-O78 group. b
examining LDH release from cells (Maldonado et al., 2005). Hemolysin produced by E. coli can promote pores formation in the membrane of target cells and destroy the structural integrity of cell, causing the release of cytokines and inflammatory reactions (del Castillo et al., 1997; Reingold et al., 1999). Vat, a virulence factor produced by E. coli, similar to the cytotoxin of Helicobacter pylori, can induce vacuolation of the primary chicken embryo fibroblast (Salvadori et al., 2001). In this study, andrographolide reduced the LDH release by chicken type II pneumocytes induced by APEC-O78. This is consistent with the findings that andrographolide relieves pathological changes in the lung tissues and the ultrastructure changes in type II alveolar epithelial cells, and improves the viability of MLE-12 cells (Zhu et al., 2013). We also found that the expressions of hlyE and vat were decreased
significantly by andrographolide. These results reveal that andrographolide powerfully protects against cell damage induced by APEC-O78 by inhibiting the secretion of toxins. F-actin plays an important role in the mobility and contraction of cells during cell division and is a constituent of the cell cytoskeleton (Wooley et al., 1998). We found that APEC-O78 induced actin cytoskeleton polymerization, in accordance with the actin polymerization of chicken fibroblast CEC-32 cell induced by APEC-MT78 (Matter et al., 2011). After treatment with andrographolide, F-actin cytoskeleton polymerization was significantly decreased. Similarly, a-cyperone also inhibits actin cytoskeleton polymerization induced by APEC-O78 (Zhang et al., 2014). This indicates that andrographolide can maintain normal cell morphology by decreasing F-actin cytoskeleton polymerization induced by APEC-O78.
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Why sub-MIC of andrographolide protect chicken type II pneumocytes from damage induced by APEC-O78 by reducing the mRNA expression of bacterial virulence factors? Recently researches indicate that this phenomenon is associated with bacterial QS. Our previous study showed that APEC-O78 produces the highest level of AI-2 activity at late log phase (data not shown), while the expression of the virulence factors also reaches a maximum at this stage. Therefore, we hypothesize that andrographolide inhibits the pathogenicity of APEC-O78 by regulating QS system. Here, we found that andrographolide significantly decreased the AI-2 activity of APECO78, indicating that andrographolide can regulate QS system of APEC-O78. The QS system regulates the production of a variety of virulence factors in many pathogenic bacteria. DNA microarrays hybridization identified 242 genes of E. coli (upregulated 154 genes, repressed 88 genes) that are controlled by QS signaling molecules AI-2 (DeLisa et al., 2001). Type III secretion systems of both enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) have also been shown to be controlled by QS (Sperandio et al., 1999). In E. coli, a number of genes including ferric uptake regulator, adhesion, haemolysin, antiserum survival factor and vacuolating autotransporter toxin, facilitate APEC infection (Johnson et al., 2012; Olsen et al., 2011). In this study, andrographolide also significantly reduced the expressions of luxS and these virulence genes, indicating that andrographolide diminishes the pathogenicity of APEC-O78 by regulating QS. Several natural compounds also decreased the virulence of bacterial through QS regulation. For example, 14-alpha-lipoyl andrographolide (AL-1) has been shown to repress the transcriptional level of QS-regulated genes (lasI, lasR, rhlI, rhlR, pqsA, pqsH, and pqsR) in P. aeruginosa (Ma et al., 2012). Additionally, ellagic acid derivatives disrupted the expression of LasR and RhlR to decrease the production of virulence factor in P. aeruginosa PAO1 (Sarabhai et al., 2013). Andrographolide was selected out as one of several quorum sensing inhibitors of P. aeruginosa using molecule docking methods, and may compete for binding to receptor protein binding sites of RhlR with signaling molecules (Sarabhai et al., 2013). However, the mechanism of andrographolide down-regulates of AI-2 activity in APEC has not been fully elucidated and must be further studied. In conclusion, andrographolide reduced cell damage of chicken type II pneumocytes induced by APEC-O78 by interfering with quorum sensing via molecular AI-2 and down-regulating the expression of virulence genes. These results provide new evidence for colibacillosis prevention methods in chickens.
Conflict of interest The authors have declared no conflict of interest. Acknowledgements We thank Prof. Ward Lynds, Jilin University and Matthew Engel, Cornell University for very carefully editing the manuscript.
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