Anaerobe 28 (2014) 18e23
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Molecular biology, genetics and biotechnology
Anti-stress proteins produced by Bacteroides thetaiotaomicron after nutrient starvation Anne-Cécile Hochart-Behra a, b, c, f,1, Hervé Drobecq a, d, g, Mélissa Tourret a, b, f, Luc Dubreuil a, e, f, Josette Behra-Miellet a, b, f, *,1 a
Univ Lille Nord de France, F-59000 Lille, France UDSL, EA 4481, UFR Pharmacie, F-59000 Lille, France CHArmentières, F-59280, France d Peptide CSB Platform, IBL, 59000, Lille, France e UDSL, U995, 59000 Lille, France f Faculté de Pharmacie, Laboratoire de Bactériologie, 3 rue du Pr Laguesse, BP 83, 59006 Lille Cedex, France g Institut de Biologie de Lille, 1 rue du Pr Calmette, 59000 Lille, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2013 Received in revised form 2 April 2014 Accepted 17 April 2014 Available online 28 April 2014
Bacteroides thetaiotaomicron maybe one of the most adaptable intestinal bacteria due to its complex genome. Known to be an opportunistic pathogenic anaerobe, B. thetaiotaomicron has recently been described as a symbiont with anti-inflammatory properties. In this study, peptide mass finger printing technique was used to identify the stress proteins (maybe anti-stress proteins for the host) extracted from B. thetaiotaomicron grown under nutrient starvation (without heme, blood or bile) prior to be placed in an aerobic solution containing a mild non-ionic detergent derived from cholic acid. We focus here on proteins related to stress, knowing that superoxide dismutase was previously identified in the extract. In parallel, the morphology of the bacterial cells was observed using electronic microscopy before and after the extraction process. The effective antioxidant effect of the extract was evaluated in vitro against hydrogen peroxide. This work highlights the B. thetaiotaomicron ability to produce a large amount of stress proteins and to remain viable during the extraction. Budding vesicles were observed on its cell wall. The extraction process did not exceed 20 h in order to preserve the bacterial viability that decreased significantly after 24 h in preliminary studies. In our experimental conditions, an inhibitory effect of the extract was found against hydrogen peroxide. Animal models of inflammation will later check in vivo if this extract of anti-stress proteins is able to counter the respiratory burst beginning an inflammation process. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Bacteroides thetaiotaomicron Oxidative stress Chaperones Obligate anaerobe Vesicles Antioxidant
1. Introduction Bacteroides thetaiotaomicron (BT) is a bowel commensal anaerobe usually known as a pathogenic opportunist showing an
Abbreviations: BT, Bacteroides thetaiotaomicron; CFU, colony forming unit; E, Bacteroides thetaiotaomicron extract; EM, electronic microscopy; GAD, glutamate decarboxylase; OM, outer membrane; PB, 0.1 M phosphate buffer, pH 7.4. * Corresponding author. Faculté de Pharmacie, 3 rue du Pr Laguesse, BP 83, 59006 Lille Cedex, France. E-mail addresses: [email protected] (A.-C. Hochart-Behra), [email protected] (H. Drobecq), [email protected] (M. Tourret), luc. [email protected] (L. Dubreuil), [email protected] (J. Behra-Miellet). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.anaerobe.2014.04.008 1075-9964/Ó 2014 Elsevier Ltd. All rights reserved.
increasing antibiotic resistance among anaerobes [1e3]. Its genome indicates a high potential for adaptation to environment [4e7]. BT is among the predominant bacteria able to adhere to Caco-2 cells [8] and colonizes artificial mucus gels under conditions of nutrient stress, characteristic of the proximal and distal colon [9]. A new concept has emerged about this symbiont: Kelly et al. have previously shown an anti-inflammatory effect of BT on Caco-2 cells, via the PPAR-gamma pathway [10]. Other studies have illustrated the specific contributions of BT in important aspects of host physiology [11,12]. Recently, a developer of live biotherapeutics has been granted orphan drug designation by the US food and drug administration office for ThetanixÒ (containing BT), a pediatric Crohn’s drug. However, BT is also cited as a probable cause in the deleterious inflammatory process [13]. Therefore, the contradictory facets of BT remain to be investigated. We previously developed an
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innovative BT extract (E) which inhibited superoxide anion in vitro. The method used 3-[(3-cholamidopropyl) dimethylammonio]-1 propanesulfonate (CHAPS), a derivative of cholic acid, that preserved the viability of the strictly anaerobic bacteria during an aerobic extraction, after culture under nutrient starvation on Brucella agar only supplemented by 0.5 mg L1 menadione. Superoxide dismutase (SOD) was identified in the cell-free extract that exhibited a very strong inhibition of the O 2 produced in vitro in an acellular model [14]. This was consistent with Pan and Imlay studies [15]. Conversely, when BT was cultured on the above medium enriched with bile to enhance its growth, the consequent extract exhibited a very different protein pattern, with no antioxidative properties and the viability of BT was altered after extraction. Thus, this abundant [4] symbiont could, under defined hostile conditions, be able to produce very active elements against oxidative stress. In this study, we sought to understand how BT remained viable during the extraction that generated SOD in E. Electronic microscopy (EM) was used to visualize any change of the BT morphology related to any protein export pathway during the extraction. Mass finger printing technique was used to identify other proteins in E. The anti-oxidative effect of E was searched in vitro on H2O2. 2. Materials and methods 2.1. Obtention of the bacterial cell-free extract E under conditions of stress and evaluation of the cell viability Briefly, according to the method previously described [14], the B. thetaiotaomicron strain ATCC 29741 was grown onto Brucella agar medium containing no blood nor hemin nor bile (Becton Dickinson, Le Pont de Claix, France), only supplemented with 0.5 mg L1 menadione. Plates were incubated at 35 C for 5 days in an anaerobic chamber (Don Withley chamber, AES, Combourg, France) containing a gas mixture without O2 (85% N2, 10% H2, CO2 5%). Bacteria were harvested from the surface of the agar plates and transferred into a sterile 7 mM CHAPS, 0.5 mM tris(hydroxymethyl)aminomethane (Tris) hydrochloride (pH 6.8) solution (Serva, Heidelberg, Germany) aereted to create an oxidative stress, kept at 2e4 C under slow mixing for 20 h (a sufficient time laps to allow the protein production, but short enough to avoid protein digestion by proteases). At the end of the extraction period in CHAPS medium, the bacterial suspension was centrifuged at 20,000 g for 1 h at 4 C (5417R centrifuge, Eppendorf, Hamburg, Germany). After evaluating the supernatant protein concentration, it was adjusted to 2.5 mg mL1 with the extraction solution and named “E” [14]. The bacterial viability was compared before, after extraction in the presence of CHAPS and following a transfer into a survival anaerobic quarter-strength Ringer diluent (Merck). This was performed using enumeration of the colony forming units (CFU) obtained after spread plating of 100 mL of five suitable tenfold dilutions of the bacterial suspension in quarter-strength anaerobic Ringer diluent with 0.3 g L1 cysteine (Acros, New Jersey, USA) on surface of Brucella agar complemented with 5 mg L1 hemin, 5% (vol/vol) defibrinated horse blood (Eurobio, Les Ulis, France) and 0.5 mg L1 menadione (Merck). After 48 h incubation under anaerobic conditions at 37 C, the CFU were counted and expressed per g of bacteria harvested (moist and after 15-day drying in an oven) weighed after centrifuging. For the statistical analysis, seven paired series of CFU counts per g of bacteria were tested using the non parametric Wilcoxon’s test (p ¼ 0.05, StatView software, version 4.5, Abacus Concepts, Berkeley, CA, USA) to compare the bacterial viabilities before and after the 20 h extraction in CHAPS and to compare viabilities of BT in the CHAPS solution and the survival diluent.
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2.2. Electronic microscopy Bacteria were prepared for ultrastructure study using a method adapted from Martinez et al. [16] at two different time points: immediately after removal from the agar surface and suspension in a CHAPS solution, and after a 20 h extraction process. Fixation and Epon-embedding procedures were carried out using high osmolarity fixation and washing solutions that optimized the cell structure preservation. Samples were centrifuged at 2500 g and 4 C (5417R centrifuge, Eppendorf). The bacterial pellet was fixed by 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (PB) (Sigma, St Quentin Fallavier, France) for 2 h at room temperature. The bacteria were washed four times in PB and by centrifuging for 3 min at 10,000 g after each bath. The bacteria (pellet) were post-fixed in 1% OsO4 (Electron Microscopy Sciences, Hatfield, PA, USA) in PB for 1 h at room temperature. After centrifuging, the bacteria pellet was dehydrated by successive baths in ethanol (Fisher Scientific, Illkirch, France) 70 , 95 and absolute ethanol and by two baths in propylene oxide (ACROS, Geel, Belgium). The conical tubes containing BT were centrifuged for 5 min at 15,000 g between each bath. A mixture (EPON) of 46% (vol/vol) Epon (epikote) 812, 29% (vol/vol) dodecenyl succinic anhydride, 25% (vol/vol) nadic methyl anhydride (Electron Microscopy Sciences, Fort Washington, PA, USA) and 0.06% (vol/vol) 2,4,6-(trimethylaminoethyl) phenol (Electron Microscopy Sciences) was used for embedding. The bacteria were first settled in an EPON/propylene oxide mixture (vol/vol), overnight at room temperature. Two other settlings were performed at 37 C after removing supernatants and pouring 1 mL of pure EPON instead. Supernatant was finally removed and pure EPON was poured onto the bacteria and a label. After drying for 72 h at 60 C, a pyramid inclusion could be unmolded. Semi-thin and then thin sections (50e70 nm) were made by ultramicrotomy (using a Leica Ultracut R Ultramicrotome, Leica microsystems, Wien, Autria) and collected onto form war/ carbon-coated nickel grids (300 mesh, Euromedex, Souffelwehersheim, France). Sections were stained with saturated uranyl acetate for 20 min and lead citrate at high pH (Euromedex) for 2 min [16]. After staining the semi-thin sections, the ultrastructure of bacteria was examined by EM using a TEM Hitachi 7500 coupled to an AMT CCD camera (Hitachi High-Technologies Europe GmbH, Krefeld, Germany). 2.3. Proteomic analysis BT extracts (500 mg protein aliquots) were prepared (desalted, re-solubilized, and lyophilized) as previously described for twodimensional polyacrylamide gel electrophoresis (PAGE) [14], as well as the 2D-gel realization. Proteins were loaded on precast immobilized pH 3e10 or 4e7 gradient ReadyStripÒ (17 cm, BioRad) in the Bio-Rad Protean IEF cell system for isoelectric focusing (IEF). SDS PAGE was undertaken on vertical gels with 4e 20% acrylamide gradient (non-denaturing gels). Colloidal Coomassie blue staining was then performed after fixing and washing proteins in the gels. Gel images were acquired (400-dpi resolution, blue and red filters) using a GS800 densitometer (Bio-Rad). The protein profile analysis of 5 2D-gels of pH 4e7 gradient was obtained using PDQuest software (Bio-Rad) as previously reported for the “Master cybergels” created [14] from IEF strips of pH 3e10 gradient. Interesting protein spots were excised and prepared as previously described [14] to perform protein identification by peptide mass finger printing technique using matrix-assisted laser desorption ionization time of flight mass spectrometry. Protein sequence database (National Center for Biotechnology Information) searching was performed with “ProFound” and “Mascot” algorithms.
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2.4. In vitro evaluation of the BT effect on hydrogen peroxide Concentration of H2O2 was estimated using an acellular method, adapted from that reported by Leavey et al. [17] and optimized in order to avoid interferences with peroxidases present in E. Briefly, about 9 nmol/mL of H2O2 were incubated with the extract corresponding to protein concentrations ranging from 0 to 250 mg mL1, for 15 min at 37 C in a final volume of 1 mL of HH buffer (Hank’s HEPES e pH 7.4, Sigma). Controls without H2O2 (replaced by HH) were also incubated for each protein concentration of the bacterial extract. After adding 40 mL of 1 N HNO3, 200 mL of 10 M Iron (II) ammonium sulfate and 100 mL of 2.5 M KSCN, the tubes were centrifuged at 1125 g for 5 min. The absorbance in each supernatant, reflecting the ferrithiocyanate red complexes, was measured by spectrophotometry at 480 nm. Concentrations of H2O2 in the samples were determined by using a standard curve ranging from 2.5 to 20 nmol/mL. The specimens used for calibration underwent the same process as the samples containing the protein extract E. Data normality and variance homogeneity were checked (QeQ plot graph and Bartlett’s test at the 0.05 level) using the Openstat free software for window’s XP (Dr William G. Miller, Iowa State University). A statistical analysis was performed using oneway (protein concentration) ANOVA and the Fisher’s Protected least significant difference a posteriori test (Statview 4.5 and Superanova softwares for Macintosh, Abacus Concepts; p ¼ 0.05). 3. Results 3.1. The viability of BT after treatment No statistically significant difference was observed between the BT viabilities observed at the beginning and the end of the extraction process (p ¼ 0.6121; n ¼ 7). CHAPS did not alter the BT
viability compared with the Ringer solution (p ¼ 0.8658 and p > 0.9999 when the moist and dried bacteria weights were considered for calculation, respectively; n ¼ 7). 3.2. Electronic microscopy (EM)-observation of the bacteria EM-observation of the bacteria fixed just before extraction showed bacilli of approximately 1.5e2 mm in length and 0.2e 0.4 mm in width. Pilus- or capsule-like structures were observed on the bacterial surface which appeared fringed (Fig. 1aec). Overall, the bacterial content appeared dense. It was possible to observe the bacterial cell wall and more specifically the probable outer membrane (OM) (Fig. 1c). After the 20 h extraction, the intact bacterial bodies seemed to have dilated to 0.6 mm in width and their content was less dense. At that time, BT showed the formation of several neat protrusions of about 100 nm in diameter on the cell surface. Dense material was present inside the vesicles and the pilus- or capsule-like structures on the cell surface had disappeared (Fig. 1def). 3.3. Stress proteins identified in the extract The major group of spots containing a stress protein in 2D gels was identified (Fig. 2 and on-line supplementary materials 1, 4e5) as glutamate decarboxylase (GAD). Six major peaks of GAD were found using the restricted pH 4e7 gradient for IEF. Chaperone proteins were also detected as GroEL, GroES, molecular chaperone DnaK, FKBP-type peptidylprolyl cis-transisomerase, endopeptidase Clp ATP-binding chain b, peptidylprolyl isomerase, a putative outer membrane protein OmpH. Another rich group of stress proteins was widely represented: it contained antioxidant compounds and enzymes: superoxide dismutase (SOD), thioredoxin C-2, thiol peroxidase, C22 alkyl hydroperoxide reductase, flavodoxin, a
Fig. 1. Scanning electron micrographs showing B. thetaiotaomicron ATCC 29741 just before and after extraction. After collection from agar medium the bacteria were suspended in the extraction solution; bacilli with dense content and a fringed surface can be observed (aec). After extraction for 20 h in the solution bacteria are producing buds or vesicles with a dense content (def). Arrows pointing at portions of the bacterium surface showing initiation (e) and the subsequent vesicle formation (f). Scale bars, 500 nm (a) 100 nm (bef).
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Fig. 2. “Master cybergel” showing proteins of the E extract after migration during isoelectrofocusing over a range of pI from 4 to 7. Construction of the non-linear grid of molecular weights (kDa) and pI (a). Three dimensional view of the “a”e“h” peaks that was identified as glutamate decarboxylase (b).
putative oxidoreductase of pyridinic nucleotide-disulfide bounds and ferritin A. Sigma 54 protein was also identified among the proteins released by BT. Other proteins were found in E: ATP-binding ABC transporter; magnesium chelatase; choloylglycine hydrolase and proteins related to the EmbdeneMeyerhofeParnas pathway or to the synthesis of aminoacids and of purines. Major OM structural proteins of BT such as OmpA, SusC or SusD were not found in the gels. 3.4. In vitro antioxidative effect of the extract against hydrogen peroxide The extract showed an inhibitory effect against hydrogen peroxide in a concentration-dependent manner (Fig. 3). H2O2 was incubated with 6 different extracts at the mean concentration of 9.5 0.4 nmol of H2O2 mL1 of reaction mixture. The H2O2 inhibition by E was statistically significant from 37.5 mg of proteins mL1 (p ¼ 0.05). This inhibition was still significant at the 1% level from the protein concentration of 62 mg mL1.
Fig. 3. Hydrogen peroxide inhibition by the extract E in an acellular model. The extremities of the lowest and the highest bars represent the tenth and 90th percentiles of hydrogen peroxide concentrations (nmol/mL) for each protein concentration, respectively. The 25th, 50th (median) and 75 percentiles correspond to the inferior, interior and superior horizontal bars of the boxes constructed for each protein concentration. The circles represent values over the tenth and the 90th percentiles. a, b and c represent the homogenous mean groups that are statistically different (Overall Fisher’s test, p ¼ 0.0001; Fisher’s PLSD test, p ¼ 0.05, n ¼ 6 independent experiments).
4. Discussion Before extraction, the viability of BT was not more altered by the aerobic solution containing CHAPS than by the anaerobic survival solution. More surprising was the fact that BT could escape to the hostile environment (detergent, oxygenated and hypo-osmolar medium) during the 20 h extraction. Scanning electron micrographs showing B. thetaiotaomicron ATCC 29741 just before and after extraction let suggest the BT mechanism of survival through an active release of vesicles of about 100 nm in diameter. The fringed bacterial surface disappeared after the 20 h extraction. This could be due to the loss of any bacterial capsule in the presence of detergent. E seemed to correspond to the release of targeted compounds, maybe including proteins inside the vesicles. These protrusions have already been reported as a secretion and delivery system enabling the Gram-negative bacteria to survive stressful conditions [18] and for Porphyromonas gingivalis, vesicles appeared to contain membrane proteins somewhat different from the OM fraction [19]. BT is capable of becoming one of the prominent intestinal bacterial human symbionts but it can be also one of the Bacteroides the most frequently isolated from anaerobic infections. The expression levels of four C10 proteases genes, maybe related to BT virulence, has been shown modulated in response to environmental stimuli such as an increased oxygen level by Thornton et al. but they grew bacteria in/on media supplemented with 50 mg mL1 hemin or on with 5% (vol/vol) sheep blood porcine bile [20]. Preliminary assays led us to use culture media without hemin or blood or bile generating no antioxidant extracts. Performing the extraction process at 2e4 C could also prevent the proteins extracted from any action of proteases. Preserving the viability of BT during extraction aimed to avoid any release of lipopolysaccharide into E. The absence of structural OM proteins such as BT OmpA, or SusC and SusD in E is consistent with the preservation of BT during extraction. Apart from a few enzymes of metabolism, chaperone proteins were largely found in E. The main protein found in gels was the glutamate decarboxylase (GAD). Did BT ensure the pH homeostasis when massively excreting GAD from cytosol to the extraction medium? Non-denaturing electrophoresis could explain the molecular weight (Mw) difference for a spot series of GAD at around 70 kDa (higher Mw than the theoretical Mw given by data banks of about 15 kDa) (Fig. 2 and SM 1e5). No other protein could be co-identified with GAD in the 2D-gel spots. Successive phosphorylations or associations with small cofactors e such as pyridoxal phosphate could explain the pattern displaying spots with pI
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regularly spaced at a similar Mw (Fig. 2 and SM 4e5). For GAD (Fig. 2B). Five phosphorylation sites were predicted with the NetPhosBac algorithm (http://www.cbs.dtu.dk/services/NetPhosBac-1. 0/), developed by Miller et al. for protein amino-acid sequences with good specificity for bacteria. This supports the statement that stress proteins are highly phosphorylated under stressful conditions [21]. Protein GroE was detected in E as chaperonins 60 (GroEL) and 10 (Gro ES) (spots c4 and c1 in supplementary materials SM 1e 3). DnaK and ATPase Beta chain of ClpB endopeptidase (found in the respective peak c6 and c7 in SM 1e3), are part of a multichaperone system that reactivates highly aggregated proteins after stress [22]. Peptidylprolyl isomerase and F kappa BP-type peptidylprolyl cistransisomerase were also found (spots c3 and c5 in SM 1e3). The latter or “trigger factor” accelerates the rehabilitation of aggregated proteins [23]. GroEL and DnaK are over-expressed when bacteria are exposed to biliary salts and detergents [24] and could be due to CHAPS, structurally close to bile salts. Another way for BT to adapt the oxidative conditions of the medium relies also on the two component systems [5,7,25]. Sigma 54 protein was identified (spots p1 and 2 in SM 1e3) and might play an important role in the BT defense [26]. BT produced antioxidant compounds in the extraction liquid. In this study, acid-stress (pH 6.8) could lead to the production of antioxidant enzymes by BT to counter reactive oxygen species [27]. SOD was found, once again, in our gels at Mw of 20e 21 kDa compatible with the monomeric form of the enzyme and around 120e126 kDa, suggesting a hexamer [14]. But this enzyme alone, catalyzing the dismutation of O 2 into H2O2, would not be sufficient to protect BT from the oxidative stress imposed during extraction. Thus, a good system of ROS detoxification would be able to eliminate H2O2 before its transformation into HO or HOCl. This is precisely the case of E according the emerging view that this anaerobic opportunistic pathogen is highly evolved to deal with oxidative stress when trauma releases it from the intestine [28]. To limit H2O2 accumulation, C-2 thioredoxin was released in E (spot o1 in SM 1e3). Our findings support the theory that this thioredoxin would be produced by Bacteroides in response to O2 exposure after its growth and during extraction, to maintain the redox balance for cell survival [29e31]. Thioredoxins seemed also interesting in that they have anti-inflammatory effects [32,33]. The periplasmic thiol peroxidase (Tpx, spot o4 in SM 1e3), leading to detoxification of H2O2 and other peroxides in the absence of catalase/peroxidase and C22 alkyl hydroperoxidase AhpC were also found in E 2D-gels (spots o7 and o8 in SM 1e3). Hence, simple aeration of the bacterial environment was already shown to lead to OxyR regulon induction and consequently to the AhpC expression [28]. Herein, AhpC proteins displayed a Mw consistent with a dimer (preserved in our native gels). Flavodoxin (spots o3 in SM 1e3) catalyzing the AhpC reduction or that of the thioredoxin reductase [34] and the monomers of ferritin and bacterioferritin (spots o2 in SM 1e3) were also found. Anaerobes can counter oxidative stress by trapping iron with both proteins and avoiding the formation of HO generated by the Fenton reaction [35,36]. For Bacteroides fragilis and P. gingivalis, the transcription of the ferritin gene may depend, at least indirectly, on OxyR [35,37]. Finally, the cell-free extract E showed an in vitro inhibitory effect against H2O2 that was expected in the presence of functional enzymes such as thioredoxins, Tpx, AhpC . 5. Conclusion Our study highlighted the protein equipment expressed by BT to counteract unfavorable environmental conditions maybe those encountered throughout establishing an opportunistic infection when the integrity of the intestinal barrier fails. However, provided it is placed in an appropriate environment similar to that used in
our study, especially as far as nutrient starvation is concerned (absence of hemin, bile or blood), BT remains viable and might be exploited for its release of functional antioxidant proteins, those found in E. Lipid and glycoside compositions of E are still to be determined, as well as the anti-oxidative/anti-inflammatory effect of this extract in pharmacological models using cellules and animals. Acknowledgments The authors wish to acknowledge the Nord-Pas-de-Calais region (France) as this work was supported by the funds “Fond Régional pour l’Innovation” (Oseo Innovation, number A0802003N). They thank Julie Sam and Stéphane Vernel for their technical assistance, Professor M. Aliouat and Muriel Pottier from Lille 2 University for their contribution to the preparation of the bacteria for electron microscopy (fixation and Epon-embedding), Martine and Paul Mayes for their help with English and the reviewers of Anaerobe for their relevant suggestions. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.anaerobe.2014.04.008. References [1] Behra-Miellet J, Calvet L, Mory F, Muller C, Chomarat M, Bezian MC, et al. Antibiotic resistance among anaerobic Gram-negative bacilli: lessons from a French multicentric survey. Anaerobe 2003;9:105e11. [2] Behra-Miellet J, Calvet L, Dubreuil L. A Bacteroides thetaiotamicron porin that could take part in resistance to beta-lactams. Int J Antimicrob Agents 2004;24: 135e43. [3] Fang H, Edlund C, Nord CE, Hedberg M. Selection of cefoxitin-resistant Bacteroides thetaiotaomicron mutants and mechanisms involved in beta-lactam resistance. Clin Infect Dis 2002;35:S47e53. [4] Comstock LE, Coyne MJ. Bacteroides thetaiotaomicron: a dynamic, nicheadapted human symbiont. Bioessays 2003;25:926e9. [5] Xu J, Bjursell MK, Himrod J, Deng S, Carmichael LK, Chiang HC, et al. A genomic view of the human-Bacteroides thetaiotaomicron symbiosis. Science 2003;299: 2074e6. [6] Xu J, Gordon JI. Inaugural article: honor the symbiont. Proc Natl Acad Sci U S A 2003;100:10452e9. [7] Xu J, Chiang HC, Bjursell MK, Gordon JI. Message from a human gut symbiont: sensitivity is a prerequisite for sharing. Trends Microbiol 2004;12:21e8. [8] Bahrami B, Child MW, Macfarlane S, Macfarlane GT. Adherence and cytokine induction in Caco-2 cells by bacterial populations from a three-stage continuous-culture model of the large intestine. Appl Environ Microbiol 2011;77: 2934e42. [9] Macfarlane S, Woodmansey EJ, Macfarlane GT. Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a twostage continuous culture system. Appl Environ Microbiol 2005;71:7483e92. [10] Kelly D, Campbell JI, King TP, Grant G, Jansson EA, Coutts AG, et al. Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclearcytoplasmic shuttling of PPAR-gamma and RelA. Nat Immunol 2004;5:104e 12. [11] Zocco MA, Ainora ME, Gasbarrini G, Gasbarrini A. Bacteroides thetaiotaomicron in the gut: molecular aspects of their interaction. Dig Liver Dis 2007;39:707e 12. [12] Kelly D, King T, Aminov R. Importance of microbial colonization of the gut in early life to the development of immunity. Mutat Res 2007;622:58e69. [13] Bloom SM, Bijanki VN, Nava GM, Sun L, Malvin NP, Donemeyer DL, et al. Commensal Bacteroides species induce colitis in host-genotype-specific fashion in a mouse model of inflammatory bowel diseases. Cell Host Microbe 2011;9:390e403. [14] Hochart-Behra AC, Behra-Miellet J, Sam J, Drobecq H, Gressier B, Luyckx M, et al. Antioxidative effect of Bacteroides thetaiotaomicron extracts: superoxide dismutase identification. Anal Bioanal Chem 2008;391:415e23. [15] Pan N, Imlay JA. How does oxygen inhibit central metabolism in the obligate anaerobe Bacteroides thetaiotaomicron. Mol Microbiol 2001;39:1562e71. [16] Martinez A, Aliouat E, Standaert-Vitse A, Werkmeister EM, Pottier M, Claire Pinçon C, et al. Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii. PLoS One 2011;6:1e12. [17] Leavey PJ, Gonzalez-Aller C, Thurman G, Kleinberg M, Rinckel L, Ambruso DW, et al. A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion
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[29] Reott MA, Parker AC, Rocha ER, Smith CJ. Thioredoxins in redox maintenance and survival during oxidative stress of Bacteroides fragilis. J Bacteriol 2009;191:3384e91. [30] Rocha ER, Tzianabos AO, Smith CJ. Thioredoxin reductase is essential for thiol/ disulfide redox control and oxidative stress survival of the anaerobe Bacteroides fragilis. J Bacteriol 2007;189:8015e23. [31] Rocha ER, Herren CD, Smalley DJ, Smith CJ. The complex oxidative stress response of Bacteroides fragilis: the role of OxyR in control of gene expression. Anaerobe 2003;9:165e73. [32] Takeuchi J, Hirota K, Itoh T, Shinkura R, Kitada K, Yodoi J, et al. Thioredoxin inhibits tumor necrosis factor- or interleukin-1-induced NF-kappaB activation at a level upstream of NF-kappaB-inducing kinase. Antioxid Redox Signal 2000;2:83e92. [33] Tamaki H, Nakamura H, Nishio A, Nakase H, Ueno S, Uza N, et al. Human thioredoxin-1 ameliorates experimental murine colitis in association with suppressed macrophage inhibitory factor production. Gastroenterology 2006;131:1110e21. [34] Argyrou A, Blanchard JS. Flavoprotein disulfide reductases: advances in chemistry and function. Prog Nucleic Acid Res Mol Biol 2004;78:89e142. [35] Rocha ER, Smith CJ. Transcriptional regulation of the Bacteroides fragilis ferritin gene (ftnA) by redox stress. Microbiology 2004;150:2125e34. [36] Smith JL. The physiological role of ferritin-like compounds in bacteria. Crit Rev Microbiol 2004;30:173e85. [37] Meuric V, Gracieux P, Tamanai-Shacoori Z, Perez-Chaparro J, BonnaureMallet M. Expression patterns of genes induced by oxidative stress in Porphyromonas gingivalis. Oral Microbiol Immunol 2008;23:308e14.
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Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Molecular biology, genetics and biotechnology
Anti-stress proteins produced by Bacteroides thetaiotaomicron after nutrient starvation Anne-Cécile Hochart-Behra a, b, c, f,1, Hervé Drobecq a, d, g, Mélissa Tourret a, b, f, Luc Dubreuil a, e, f, Josette Behra-Miellet a, b, f, *,1 a
Univ Lille Nord de France, F-59000 Lille, France UDSL, EA 4481, UFR Pharmacie, F-59000 Lille, France CHArmentières, F-59280, France d Peptide CSB Platform, IBL, 59000, Lille, France e UDSL, U995, 59000 Lille, France f Faculté de Pharmacie, Laboratoire de Bactériologie, 3 rue du Pr Laguesse, BP 83, 59006 Lille Cedex, France g Institut de Biologie de Lille, 1 rue du Pr Calmette, 59000 Lille, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2013 Received in revised form 2 April 2014 Accepted 17 April 2014 Available online 28 April 2014
Bacteroides thetaiotaomicron maybe one of the most adaptable intestinal bacteria due to its complex genome. Known to be an opportunistic pathogenic anaerobe, B. thetaiotaomicron has recently been described as a symbiont with anti-inflammatory properties. In this study, peptide mass finger printing technique was used to identify the stress proteins (maybe anti-stress proteins for the host) extracted from B. thetaiotaomicron grown under nutrient starvation (without heme, blood or bile) prior to be placed in an aerobic solution containing a mild non-ionic detergent derived from cholic acid. We focus here on proteins related to stress, knowing that superoxide dismutase was previously identified in the extract. In parallel, the morphology of the bacterial cells was observed using electronic microscopy before and after the extraction process. The effective antioxidant effect of the extract was evaluated in vitro against hydrogen peroxide. This work highlights the B. thetaiotaomicron ability to produce a large amount of stress proteins and to remain viable during the extraction. Budding vesicles were observed on its cell wall. The extraction process did not exceed 20 h in order to preserve the bacterial viability that decreased significantly after 24 h in preliminary studies. In our experimental conditions, an inhibitory effect of the extract was found against hydrogen peroxide. Animal models of inflammation will later check in vivo if this extract of anti-stress proteins is able to counter the respiratory burst beginning an inflammation process. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Bacteroides thetaiotaomicron Oxidative stress Chaperones Obligate anaerobe Vesicles Antioxidant
1. Introduction Bacteroides thetaiotaomicron (BT) is a bowel commensal anaerobe usually known as a pathogenic opportunist showing an
Abbreviations: BT, Bacteroides thetaiotaomicron; CFU, colony forming unit; E, Bacteroides thetaiotaomicron extract; EM, electronic microscopy; GAD, glutamate decarboxylase; OM, outer membrane; PB, 0.1 M phosphate buffer, pH 7.4. * Corresponding author. Faculté de Pharmacie, 3 rue du Pr Laguesse, BP 83, 59006 Lille Cedex, France. E-mail addresses: [email protected] (A.-C. Hochart-Behra), [email protected] (H. Drobecq), [email protected] (M. Tourret), luc. [email protected] (L. Dubreuil), [email protected] (J. Behra-Miellet). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.anaerobe.2014.04.008 1075-9964/Ó 2014 Elsevier Ltd. All rights reserved.
increasing antibiotic resistance among anaerobes [1e3]. Its genome indicates a high potential for adaptation to environment [4e7]. BT is among the predominant bacteria able to adhere to Caco-2 cells [8] and colonizes artificial mucus gels under conditions of nutrient stress, characteristic of the proximal and distal colon [9]. A new concept has emerged about this symbiont: Kelly et al. have previously shown an anti-inflammatory effect of BT on Caco-2 cells, via the PPAR-gamma pathway [10]. Other studies have illustrated the specific contributions of BT in important aspects of host physiology [11,12]. Recently, a developer of live biotherapeutics has been granted orphan drug designation by the US food and drug administration office for ThetanixÒ (containing BT), a pediatric Crohn’s drug. However, BT is also cited as a probable cause in the deleterious inflammatory process [13]. Therefore, the contradictory facets of BT remain to be investigated. We previously developed an
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innovative BT extract (E) which inhibited superoxide anion in vitro. The method used 3-[(3-cholamidopropyl) dimethylammonio]-1 propanesulfonate (CHAPS), a derivative of cholic acid, that preserved the viability of the strictly anaerobic bacteria during an aerobic extraction, after culture under nutrient starvation on Brucella agar only supplemented by 0.5 mg L1 menadione. Superoxide dismutase (SOD) was identified in the cell-free extract that exhibited a very strong inhibition of the O 2 produced in vitro in an acellular model [14]. This was consistent with Pan and Imlay studies [15]. Conversely, when BT was cultured on the above medium enriched with bile to enhance its growth, the consequent extract exhibited a very different protein pattern, with no antioxidative properties and the viability of BT was altered after extraction. Thus, this abundant [4] symbiont could, under defined hostile conditions, be able to produce very active elements against oxidative stress. In this study, we sought to understand how BT remained viable during the extraction that generated SOD in E. Electronic microscopy (EM) was used to visualize any change of the BT morphology related to any protein export pathway during the extraction. Mass finger printing technique was used to identify other proteins in E. The anti-oxidative effect of E was searched in vitro on H2O2. 2. Materials and methods 2.1. Obtention of the bacterial cell-free extract E under conditions of stress and evaluation of the cell viability Briefly, according to the method previously described [14], the B. thetaiotaomicron strain ATCC 29741 was grown onto Brucella agar medium containing no blood nor hemin nor bile (Becton Dickinson, Le Pont de Claix, France), only supplemented with 0.5 mg L1 menadione. Plates were incubated at 35 C for 5 days in an anaerobic chamber (Don Withley chamber, AES, Combourg, France) containing a gas mixture without O2 (85% N2, 10% H2, CO2 5%). Bacteria were harvested from the surface of the agar plates and transferred into a sterile 7 mM CHAPS, 0.5 mM tris(hydroxymethyl)aminomethane (Tris) hydrochloride (pH 6.8) solution (Serva, Heidelberg, Germany) aereted to create an oxidative stress, kept at 2e4 C under slow mixing for 20 h (a sufficient time laps to allow the protein production, but short enough to avoid protein digestion by proteases). At the end of the extraction period in CHAPS medium, the bacterial suspension was centrifuged at 20,000 g for 1 h at 4 C (5417R centrifuge, Eppendorf, Hamburg, Germany). After evaluating the supernatant protein concentration, it was adjusted to 2.5 mg mL1 with the extraction solution and named “E” [14]. The bacterial viability was compared before, after extraction in the presence of CHAPS and following a transfer into a survival anaerobic quarter-strength Ringer diluent (Merck). This was performed using enumeration of the colony forming units (CFU) obtained after spread plating of 100 mL of five suitable tenfold dilutions of the bacterial suspension in quarter-strength anaerobic Ringer diluent with 0.3 g L1 cysteine (Acros, New Jersey, USA) on surface of Brucella agar complemented with 5 mg L1 hemin, 5% (vol/vol) defibrinated horse blood (Eurobio, Les Ulis, France) and 0.5 mg L1 menadione (Merck). After 48 h incubation under anaerobic conditions at 37 C, the CFU were counted and expressed per g of bacteria harvested (moist and after 15-day drying in an oven) weighed after centrifuging. For the statistical analysis, seven paired series of CFU counts per g of bacteria were tested using the non parametric Wilcoxon’s test (p ¼ 0.05, StatView software, version 4.5, Abacus Concepts, Berkeley, CA, USA) to compare the bacterial viabilities before and after the 20 h extraction in CHAPS and to compare viabilities of BT in the CHAPS solution and the survival diluent.
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2.2. Electronic microscopy Bacteria were prepared for ultrastructure study using a method adapted from Martinez et al. [16] at two different time points: immediately after removal from the agar surface and suspension in a CHAPS solution, and after a 20 h extraction process. Fixation and Epon-embedding procedures were carried out using high osmolarity fixation and washing solutions that optimized the cell structure preservation. Samples were centrifuged at 2500 g and 4 C (5417R centrifuge, Eppendorf). The bacterial pellet was fixed by 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (PB) (Sigma, St Quentin Fallavier, France) for 2 h at room temperature. The bacteria were washed four times in PB and by centrifuging for 3 min at 10,000 g after each bath. The bacteria (pellet) were post-fixed in 1% OsO4 (Electron Microscopy Sciences, Hatfield, PA, USA) in PB for 1 h at room temperature. After centrifuging, the bacteria pellet was dehydrated by successive baths in ethanol (Fisher Scientific, Illkirch, France) 70 , 95 and absolute ethanol and by two baths in propylene oxide (ACROS, Geel, Belgium). The conical tubes containing BT were centrifuged for 5 min at 15,000 g between each bath. A mixture (EPON) of 46% (vol/vol) Epon (epikote) 812, 29% (vol/vol) dodecenyl succinic anhydride, 25% (vol/vol) nadic methyl anhydride (Electron Microscopy Sciences, Fort Washington, PA, USA) and 0.06% (vol/vol) 2,4,6-(trimethylaminoethyl) phenol (Electron Microscopy Sciences) was used for embedding. The bacteria were first settled in an EPON/propylene oxide mixture (vol/vol), overnight at room temperature. Two other settlings were performed at 37 C after removing supernatants and pouring 1 mL of pure EPON instead. Supernatant was finally removed and pure EPON was poured onto the bacteria and a label. After drying for 72 h at 60 C, a pyramid inclusion could be unmolded. Semi-thin and then thin sections (50e70 nm) were made by ultramicrotomy (using a Leica Ultracut R Ultramicrotome, Leica microsystems, Wien, Autria) and collected onto form war/ carbon-coated nickel grids (300 mesh, Euromedex, Souffelwehersheim, France). Sections were stained with saturated uranyl acetate for 20 min and lead citrate at high pH (Euromedex) for 2 min [16]. After staining the semi-thin sections, the ultrastructure of bacteria was examined by EM using a TEM Hitachi 7500 coupled to an AMT CCD camera (Hitachi High-Technologies Europe GmbH, Krefeld, Germany). 2.3. Proteomic analysis BT extracts (500 mg protein aliquots) were prepared (desalted, re-solubilized, and lyophilized) as previously described for twodimensional polyacrylamide gel electrophoresis (PAGE) [14], as well as the 2D-gel realization. Proteins were loaded on precast immobilized pH 3e10 or 4e7 gradient ReadyStripÒ (17 cm, BioRad) in the Bio-Rad Protean IEF cell system for isoelectric focusing (IEF). SDS PAGE was undertaken on vertical gels with 4e 20% acrylamide gradient (non-denaturing gels). Colloidal Coomassie blue staining was then performed after fixing and washing proteins in the gels. Gel images were acquired (400-dpi resolution, blue and red filters) using a GS800 densitometer (Bio-Rad). The protein profile analysis of 5 2D-gels of pH 4e7 gradient was obtained using PDQuest software (Bio-Rad) as previously reported for the “Master cybergels” created [14] from IEF strips of pH 3e10 gradient. Interesting protein spots were excised and prepared as previously described [14] to perform protein identification by peptide mass finger printing technique using matrix-assisted laser desorption ionization time of flight mass spectrometry. Protein sequence database (National Center for Biotechnology Information) searching was performed with “ProFound” and “Mascot” algorithms.
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2.4. In vitro evaluation of the BT effect on hydrogen peroxide Concentration of H2O2 was estimated using an acellular method, adapted from that reported by Leavey et al. [17] and optimized in order to avoid interferences with peroxidases present in E. Briefly, about 9 nmol/mL of H2O2 were incubated with the extract corresponding to protein concentrations ranging from 0 to 250 mg mL1, for 15 min at 37 C in a final volume of 1 mL of HH buffer (Hank’s HEPES e pH 7.4, Sigma). Controls without H2O2 (replaced by HH) were also incubated for each protein concentration of the bacterial extract. After adding 40 mL of 1 N HNO3, 200 mL of 10 M Iron (II) ammonium sulfate and 100 mL of 2.5 M KSCN, the tubes were centrifuged at 1125 g for 5 min. The absorbance in each supernatant, reflecting the ferrithiocyanate red complexes, was measured by spectrophotometry at 480 nm. Concentrations of H2O2 in the samples were determined by using a standard curve ranging from 2.5 to 20 nmol/mL. The specimens used for calibration underwent the same process as the samples containing the protein extract E. Data normality and variance homogeneity were checked (QeQ plot graph and Bartlett’s test at the 0.05 level) using the Openstat free software for window’s XP (Dr William G. Miller, Iowa State University). A statistical analysis was performed using oneway (protein concentration) ANOVA and the Fisher’s Protected least significant difference a posteriori test (Statview 4.5 and Superanova softwares for Macintosh, Abacus Concepts; p ¼ 0.05). 3. Results 3.1. The viability of BT after treatment No statistically significant difference was observed between the BT viabilities observed at the beginning and the end of the extraction process (p ¼ 0.6121; n ¼ 7). CHAPS did not alter the BT
viability compared with the Ringer solution (p ¼ 0.8658 and p > 0.9999 when the moist and dried bacteria weights were considered for calculation, respectively; n ¼ 7). 3.2. Electronic microscopy (EM)-observation of the bacteria EM-observation of the bacteria fixed just before extraction showed bacilli of approximately 1.5e2 mm in length and 0.2e 0.4 mm in width. Pilus- or capsule-like structures were observed on the bacterial surface which appeared fringed (Fig. 1aec). Overall, the bacterial content appeared dense. It was possible to observe the bacterial cell wall and more specifically the probable outer membrane (OM) (Fig. 1c). After the 20 h extraction, the intact bacterial bodies seemed to have dilated to 0.6 mm in width and their content was less dense. At that time, BT showed the formation of several neat protrusions of about 100 nm in diameter on the cell surface. Dense material was present inside the vesicles and the pilus- or capsule-like structures on the cell surface had disappeared (Fig. 1def). 3.3. Stress proteins identified in the extract The major group of spots containing a stress protein in 2D gels was identified (Fig. 2 and on-line supplementary materials 1, 4e5) as glutamate decarboxylase (GAD). Six major peaks of GAD were found using the restricted pH 4e7 gradient for IEF. Chaperone proteins were also detected as GroEL, GroES, molecular chaperone DnaK, FKBP-type peptidylprolyl cis-transisomerase, endopeptidase Clp ATP-binding chain b, peptidylprolyl isomerase, a putative outer membrane protein OmpH. Another rich group of stress proteins was widely represented: it contained antioxidant compounds and enzymes: superoxide dismutase (SOD), thioredoxin C-2, thiol peroxidase, C22 alkyl hydroperoxide reductase, flavodoxin, a
Fig. 1. Scanning electron micrographs showing B. thetaiotaomicron ATCC 29741 just before and after extraction. After collection from agar medium the bacteria were suspended in the extraction solution; bacilli with dense content and a fringed surface can be observed (aec). After extraction for 20 h in the solution bacteria are producing buds or vesicles with a dense content (def). Arrows pointing at portions of the bacterium surface showing initiation (e) and the subsequent vesicle formation (f). Scale bars, 500 nm (a) 100 nm (bef).
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Fig. 2. “Master cybergel” showing proteins of the E extract after migration during isoelectrofocusing over a range of pI from 4 to 7. Construction of the non-linear grid of molecular weights (kDa) and pI (a). Three dimensional view of the “a”e“h” peaks that was identified as glutamate decarboxylase (b).
putative oxidoreductase of pyridinic nucleotide-disulfide bounds and ferritin A. Sigma 54 protein was also identified among the proteins released by BT. Other proteins were found in E: ATP-binding ABC transporter; magnesium chelatase; choloylglycine hydrolase and proteins related to the EmbdeneMeyerhofeParnas pathway or to the synthesis of aminoacids and of purines. Major OM structural proteins of BT such as OmpA, SusC or SusD were not found in the gels. 3.4. In vitro antioxidative effect of the extract against hydrogen peroxide The extract showed an inhibitory effect against hydrogen peroxide in a concentration-dependent manner (Fig. 3). H2O2 was incubated with 6 different extracts at the mean concentration of 9.5 0.4 nmol of H2O2 mL1 of reaction mixture. The H2O2 inhibition by E was statistically significant from 37.5 mg of proteins mL1 (p ¼ 0.05). This inhibition was still significant at the 1% level from the protein concentration of 62 mg mL1.
Fig. 3. Hydrogen peroxide inhibition by the extract E in an acellular model. The extremities of the lowest and the highest bars represent the tenth and 90th percentiles of hydrogen peroxide concentrations (nmol/mL) for each protein concentration, respectively. The 25th, 50th (median) and 75 percentiles correspond to the inferior, interior and superior horizontal bars of the boxes constructed for each protein concentration. The circles represent values over the tenth and the 90th percentiles. a, b and c represent the homogenous mean groups that are statistically different (Overall Fisher’s test, p ¼ 0.0001; Fisher’s PLSD test, p ¼ 0.05, n ¼ 6 independent experiments).
4. Discussion Before extraction, the viability of BT was not more altered by the aerobic solution containing CHAPS than by the anaerobic survival solution. More surprising was the fact that BT could escape to the hostile environment (detergent, oxygenated and hypo-osmolar medium) during the 20 h extraction. Scanning electron micrographs showing B. thetaiotaomicron ATCC 29741 just before and after extraction let suggest the BT mechanism of survival through an active release of vesicles of about 100 nm in diameter. The fringed bacterial surface disappeared after the 20 h extraction. This could be due to the loss of any bacterial capsule in the presence of detergent. E seemed to correspond to the release of targeted compounds, maybe including proteins inside the vesicles. These protrusions have already been reported as a secretion and delivery system enabling the Gram-negative bacteria to survive stressful conditions [18] and for Porphyromonas gingivalis, vesicles appeared to contain membrane proteins somewhat different from the OM fraction [19]. BT is capable of becoming one of the prominent intestinal bacterial human symbionts but it can be also one of the Bacteroides the most frequently isolated from anaerobic infections. The expression levels of four C10 proteases genes, maybe related to BT virulence, has been shown modulated in response to environmental stimuli such as an increased oxygen level by Thornton et al. but they grew bacteria in/on media supplemented with 50 mg mL1 hemin or on with 5% (vol/vol) sheep blood porcine bile [20]. Preliminary assays led us to use culture media without hemin or blood or bile generating no antioxidant extracts. Performing the extraction process at 2e4 C could also prevent the proteins extracted from any action of proteases. Preserving the viability of BT during extraction aimed to avoid any release of lipopolysaccharide into E. The absence of structural OM proteins such as BT OmpA, or SusC and SusD in E is consistent with the preservation of BT during extraction. Apart from a few enzymes of metabolism, chaperone proteins were largely found in E. The main protein found in gels was the glutamate decarboxylase (GAD). Did BT ensure the pH homeostasis when massively excreting GAD from cytosol to the extraction medium? Non-denaturing electrophoresis could explain the molecular weight (Mw) difference for a spot series of GAD at around 70 kDa (higher Mw than the theoretical Mw given by data banks of about 15 kDa) (Fig. 2 and SM 1e5). No other protein could be co-identified with GAD in the 2D-gel spots. Successive phosphorylations or associations with small cofactors e such as pyridoxal phosphate could explain the pattern displaying spots with pI
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regularly spaced at a similar Mw (Fig. 2 and SM 4e5). For GAD (Fig. 2B). Five phosphorylation sites were predicted with the NetPhosBac algorithm (http://www.cbs.dtu.dk/services/NetPhosBac-1. 0/), developed by Miller et al. for protein amino-acid sequences with good specificity for bacteria. This supports the statement that stress proteins are highly phosphorylated under stressful conditions [21]. Protein GroE was detected in E as chaperonins 60 (GroEL) and 10 (Gro ES) (spots c4 and c1 in supplementary materials SM 1e 3). DnaK and ATPase Beta chain of ClpB endopeptidase (found in the respective peak c6 and c7 in SM 1e3), are part of a multichaperone system that reactivates highly aggregated proteins after stress [22]. Peptidylprolyl isomerase and F kappa BP-type peptidylprolyl cistransisomerase were also found (spots c3 and c5 in SM 1e3). The latter or “trigger factor” accelerates the rehabilitation of aggregated proteins [23]. GroEL and DnaK are over-expressed when bacteria are exposed to biliary salts and detergents [24] and could be due to CHAPS, structurally close to bile salts. Another way for BT to adapt the oxidative conditions of the medium relies also on the two component systems [5,7,25]. Sigma 54 protein was identified (spots p1 and 2 in SM 1e3) and might play an important role in the BT defense [26]. BT produced antioxidant compounds in the extraction liquid. In this study, acid-stress (pH 6.8) could lead to the production of antioxidant enzymes by BT to counter reactive oxygen species [27]. SOD was found, once again, in our gels at Mw of 20e 21 kDa compatible with the monomeric form of the enzyme and around 120e126 kDa, suggesting a hexamer [14]. But this enzyme alone, catalyzing the dismutation of O 2 into H2O2, would not be sufficient to protect BT from the oxidative stress imposed during extraction. Thus, a good system of ROS detoxification would be able to eliminate H2O2 before its transformation into HO or HOCl. This is precisely the case of E according the emerging view that this anaerobic opportunistic pathogen is highly evolved to deal with oxidative stress when trauma releases it from the intestine [28]. To limit H2O2 accumulation, C-2 thioredoxin was released in E (spot o1 in SM 1e3). Our findings support the theory that this thioredoxin would be produced by Bacteroides in response to O2 exposure after its growth and during extraction, to maintain the redox balance for cell survival [29e31]. Thioredoxins seemed also interesting in that they have anti-inflammatory effects [32,33]. The periplasmic thiol peroxidase (Tpx, spot o4 in SM 1e3), leading to detoxification of H2O2 and other peroxides in the absence of catalase/peroxidase and C22 alkyl hydroperoxidase AhpC were also found in E 2D-gels (spots o7 and o8 in SM 1e3). Hence, simple aeration of the bacterial environment was already shown to lead to OxyR regulon induction and consequently to the AhpC expression [28]. Herein, AhpC proteins displayed a Mw consistent with a dimer (preserved in our native gels). Flavodoxin (spots o3 in SM 1e3) catalyzing the AhpC reduction or that of the thioredoxin reductase [34] and the monomers of ferritin and bacterioferritin (spots o2 in SM 1e3) were also found. Anaerobes can counter oxidative stress by trapping iron with both proteins and avoiding the formation of HO generated by the Fenton reaction [35,36]. For Bacteroides fragilis and P. gingivalis, the transcription of the ferritin gene may depend, at least indirectly, on OxyR [35,37]. Finally, the cell-free extract E showed an in vitro inhibitory effect against H2O2 that was expected in the presence of functional enzymes such as thioredoxins, Tpx, AhpC . 5. Conclusion Our study highlighted the protein equipment expressed by BT to counteract unfavorable environmental conditions maybe those encountered throughout establishing an opportunistic infection when the integrity of the intestinal barrier fails. However, provided it is placed in an appropriate environment similar to that used in
our study, especially as far as nutrient starvation is concerned (absence of hemin, bile or blood), BT remains viable and might be exploited for its release of functional antioxidant proteins, those found in E. Lipid and glycoside compositions of E are still to be determined, as well as the anti-oxidative/anti-inflammatory effect of this extract in pharmacological models using cellules and animals. Acknowledgments The authors wish to acknowledge the Nord-Pas-de-Calais region (France) as this work was supported by the funds “Fond Régional pour l’Innovation” (Oseo Innovation, number A0802003N). They thank Julie Sam and Stéphane Vernel for their technical assistance, Professor M. Aliouat and Muriel Pottier from Lille 2 University for their contribution to the preparation of the bacteria for electron microscopy (fixation and Epon-embedding), Martine and Paul Mayes for their help with English and the reviewers of Anaerobe for their relevant suggestions. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.anaerobe.2014.04.008. References [1] Behra-Miellet J, Calvet L, Mory F, Muller C, Chomarat M, Bezian MC, et al. Antibiotic resistance among anaerobic Gram-negative bacilli: lessons from a French multicentric survey. Anaerobe 2003;9:105e11. [2] Behra-Miellet J, Calvet L, Dubreuil L. A Bacteroides thetaiotamicron porin that could take part in resistance to beta-lactams. Int J Antimicrob Agents 2004;24: 135e43. [3] Fang H, Edlund C, Nord CE, Hedberg M. Selection of cefoxitin-resistant Bacteroides thetaiotaomicron mutants and mechanisms involved in beta-lactam resistance. Clin Infect Dis 2002;35:S47e53. [4] Comstock LE, Coyne MJ. Bacteroides thetaiotaomicron: a dynamic, nicheadapted human symbiont. Bioessays 2003;25:926e9. [5] Xu J, Bjursell MK, Himrod J, Deng S, Carmichael LK, Chiang HC, et al. A genomic view of the human-Bacteroides thetaiotaomicron symbiosis. Science 2003;299: 2074e6. [6] Xu J, Gordon JI. Inaugural article: honor the symbiont. Proc Natl Acad Sci U S A 2003;100:10452e9. [7] Xu J, Chiang HC, Bjursell MK, Gordon JI. Message from a human gut symbiont: sensitivity is a prerequisite for sharing. Trends Microbiol 2004;12:21e8. [8] Bahrami B, Child MW, Macfarlane S, Macfarlane GT. Adherence and cytokine induction in Caco-2 cells by bacterial populations from a three-stage continuous-culture model of the large intestine. Appl Environ Microbiol 2011;77: 2934e42. [9] Macfarlane S, Woodmansey EJ, Macfarlane GT. Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a twostage continuous culture system. Appl Environ Microbiol 2005;71:7483e92. [10] Kelly D, Campbell JI, King TP, Grant G, Jansson EA, Coutts AG, et al. Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclearcytoplasmic shuttling of PPAR-gamma and RelA. Nat Immunol 2004;5:104e 12. [11] Zocco MA, Ainora ME, Gasbarrini G, Gasbarrini A. Bacteroides thetaiotaomicron in the gut: molecular aspects of their interaction. Dig Liver Dis 2007;39:707e 12. [12] Kelly D, King T, Aminov R. Importance of microbial colonization of the gut in early life to the development of immunity. Mutat Res 2007;622:58e69. [13] Bloom SM, Bijanki VN, Nava GM, Sun L, Malvin NP, Donemeyer DL, et al. Commensal Bacteroides species induce colitis in host-genotype-specific fashion in a mouse model of inflammatory bowel diseases. Cell Host Microbe 2011;9:390e403. [14] Hochart-Behra AC, Behra-Miellet J, Sam J, Drobecq H, Gressier B, Luyckx M, et al. Antioxidative effect of Bacteroides thetaiotaomicron extracts: superoxide dismutase identification. Anal Bioanal Chem 2008;391:415e23. [15] Pan N, Imlay JA. How does oxygen inhibit central metabolism in the obligate anaerobe Bacteroides thetaiotaomicron. Mol Microbiol 2001;39:1562e71. [16] Martinez A, Aliouat E, Standaert-Vitse A, Werkmeister EM, Pottier M, Claire Pinçon C, et al. Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii. PLoS One 2011;6:1e12. [17] Leavey PJ, Gonzalez-Aller C, Thurman G, Kleinberg M, Rinckel L, Ambruso DW, et al. A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion
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