Helicobacter pylori, a gram-negative bacterium capable of chronic colonization of the human stomach, modifies its lipid A by removal of phosphate groups from the 1- and 4′-positions of the lipid A backbone. In this study, we identify the enzyme responsible for dephosphorylation of the lipid A 4′-phosphate group in H. pylori, Jhp1487 (LpxF). To ascertain the role these modifications play in the pathogenesis of H. pylori, we created mutants in lpxE (1-phosphatase), lpxF (4′-phosphatase) and a double lpxE/F mutant. Analysis of lipid A isolated from lpxE and lpxF mutants revealed lipid A species with a 1 or 4′-phosphate group, respectively while the double lpxE/F mutant revealed a bis-phosphorylated lipid A. Mutants lacking lpxE, lpxF, or lpxE/F show a 16, 360 and 1020 fold increase in sensitivity to the cationic antimicrobial peptide polymyxin B, respectively. Moreover, a similar loss of resistance is seen against a variety of CAMPs found in the human body including LL37, β-defensin 2, and P-113. Using a fluorescent derivative of polymyxin we demonstrate that, unlike wild type bacteria, polymyxin readily associates with the lpxE/F mutant. Presumably, the increase in the negative charge of H. pylori LPS allows for binding of the peptide to the bacterial surface. Interestingly, the action of LpxE and LpxF was shown to decrease recognition of Helicobacter LPS by the innate immune receptor, Toll-like Receptor 4. Furthermore, lpxE/F mutants were unable to colonize the gastric mucosa of C57BL/6J and C57BL/6J tlr4 -/- mice when compared to wild type H. pylori. Our results demonstrate that dephosphorylation of the lipid A domain of H. pylori LPS by LpxE and LpxF is key to its ability to colonize a mammalian host.Modification of bacterial surface structures, such as the lipid A portion of lipopolysaccharide (LPS), is used by many pathogenic bacteria to help evade the host innate immune response.  Helicobacter pylori has been identified as the leading cause of gastritis and peptic ulcer disease, infecting around 50% of the world's population. Infected patients are at increased risk for gastric cancers, allowing for classification of H. pylori as a class I carcinogen by the World Health Organization. H. pylori has only one well defined niche, the human stomach. Since no other reservoirs exist, a unique balance must be established during infection permitting long-term survival of both the bacterium and its human host. Here, we show that H. pylori modifies its primary surface component, lipopolysaccharide (LPS), making the bacterium undetectable by components of the innate immune system and highly resistant to antimicrobial compounds secreted by host cells. Mutant strains of H. pylori unable to modify their surface show increased sensitivity to antimicrobial peptides (∼1000 fold) and increased recognition by components of the innate immune system. H. pylori mutants were unable to colonize mouse models, suggesting that remodeling of LPS is essential for survival within the gastric mucosa. Understanding the adaptations used by H. pylori to survive and persist within the human host is key towards unraveling how this unique organism impacts gastric disease.Since its discovery in 1982  Helicobacter pylori, a gram-negative bacterium with only one well-defined niche, the human stomach, can persist for several years without manifestation of symptoms. However, over time serious complications may appear including peptic ulcer disease and gastric cancer, allowing for classification of H. pylori as a class I carcinogen by the World Health Organization H. pylori is composed of lipopolysaccharide (LPS), a surface exposed glycolipid.manno-octulosonic acid), serves as a bridge to link the lipid anchor to the carbohydrate domains of LPS. The core polysaccharide is well conserved within a bacterial species; however, this is not the case with O-antigen. H. pylori shows great diversity in expression of its O-antigen LPS is anchored to the bacterial membrane by its hydrophobic lipid A domain. Extended from lipid A is the core oligosaccharide followed by the O-antigen creating a uniform hydrophilic surface layer that interphases with the surrounding environment. The first sugar of the core, Kdo  complex  lipid A , easily H. pylori produce a minimal Kdo-lipid A structure that is tetra-acylated with a phosphoethanolamine residue attached at the 1 position of the disaccharide  of H. pylori was shown to be important for CAMP resistance, its role in hTLR4-MD2 activation remains unclear H. pylori and demonstrate that the 1 and 4′-phosphatases act synergistically to produce a bacterial surface that is highly resistant to CAMP attack. We also show that dephosphorylation of H. pylori lipid A at the 1 and/or 4′ position results in LPS with attenuated hTLR4-MD2 activation. Most exciting, our results suggest that modification of H. pylori lipid A phosphate groups are important for colonization of a host.tructure  by a fivtructure  and a siH. pylori is Kdo-lipid A that is tetra-acylated with a phosphoethanolamine attached at the 1-position , EptA (phosphoethanolamine transferase), KdoH1/2 (Kdo Hydrolase) and LpxR (3′-O-deacylase), which all act in an ordered efficient manner to produce a single lipid A species found on the bacterial surface  using the Blastp algorithm E. coli PgpB. PgpB is a membrane-bound phosphatidylglycerol phosphatase involved in phospholipid biosynthesis. A ClustalW2 alignment of Jhp0324, Jhp0787 and Jhp1487 against LpxE revealed a score of 8%, 7% and 20%, respectively. Score is defined as percent identities divided by number of residues compared. Given that Jhp1487 showed the highest score (20%) when aligned with LpxE, it became the likely target. A knockout in jhp1487 was created by interruption of the coding sequence with an antibiotic resistance cassette in H. pylori strain J99 and named J99 lpxF. Complementation of J99 lpxF was achieved by re-introduction of lpxF into the chromosome and named J99 +lpxF, lpxF.Previous attempts to identify in vitro assays were performed using radiolabelled Kdo2-[4′32P]lipid A as the substrate (H. pylori J99 membranes show robust LpxF activity (lane 2) as indicated by the appearance of free inorganic phosphate (32Pi), while membranes isolated from J99 lpxF were completely free of 4′-phosphatase activity (lane 3). LpxF activity was restored in membranes isolated from the complemented strain J99 +lpxF, lpxF (lane 4), indicating that the H. pylori lipid A 4′-phosphatase is encoded by jhp1487. Reaction products from the previously characterized 1-phosphatase (LpxE) jhp1487 had no effect on 1-phosphatase or Kdo hydrolase activity. It was demonstrated previously H. pylori LpxF as a lipid A 1-phosphatase.To screen for LpxF activity in membrane fractions, ubstrate . FollowiH. pylori, a series of mutants were generated, including a single lpxE, lpxF and a double lpxE/F mutant. Single lpxE and double lpxE/F mutants were created by introduction of a previously described lpxE mutation lpxF background by natural transformation, creating J99 lpxE and J99 lpxE/F, respectively. Complementation of J99 lpxE was achieved by re-introduction of lpxE into the chromosome and named J99 +lpxE, lpxE. Additionally, all mutations were moved into the mouse adapted H. pylori strains, B128 and X47 To clearly ascertain the role LpxE and LpxF play in the pathogenesis of 32Pi, the lipid A purified, separated by TLC and visualized by phosphorimaging. H. pylori wild type strains J99, B128 and X47 all revealed a single identical species of lipid A indicating no variation between backgrounds , which is tetra-acylated without a phosphate group at the 4′-position and a phosphoethanolamine residue at the 1-position (lpxF mutant spectrum showed a predominant peak at m/z 2091.0 consistent with the [M–H]- ion of a lipid A species that is hexa-acylated with a phosphate group at the 4′-position and a phosphoethanolamine residue at the 1-position (predicted [M–H]- at m/z 2091.5), confirmation that the H. pylori LpxF is encoded by jhp1487. The presence of hexa-acylated lipid A species in the lpxF mutant suggest an ordered modification system in which H. pylori LpxR (3′-O-deacylase) activity is dependent on removal of the lipid A 4′-phosphate group by LpxF. The lpxE mutant spectrum (m/z 1504.8 consistent with the [M–H]- ion of a lipid A species that is tetra-acylated bearing a single phosphate group (predicted [M–H]- at m/z 1505.1), in agreement with the published lipid A structure of lpxE deficient H. pylori strains lpxE/F mutant spectrum showed a predominant peak at m/z 2048.2 consistent with the [M–H]- ion of a lipid A species that is hexa-acylated with a phosphate group at the 1- and 4′-positions (predicted [M–H]- at m/z 2048.5)  mass spectrometry in the negative ion mode. The wild type J99 spectrum showed a predominant peak at position . The lpxspectrum  showed a 2048.5) . Complem. pylori . Moreove. pylori  and X47 . pylori  backgrouH. pylori whose only known reservoir is the human gastric mucosal surface. H. pylori is highly resistant to the CAMP polymyxin B (PMB) with the minimal inhibitory concentration (MIC) of wild type strains ranging from 250–1000 µg/ml peptide H. pylori wild type strain J99 and all complemented strains exhibited a PMB MIC (469.3±73.9 µg/ml) comparable to published determinations lpxE, lpxF and double lpxE/F mutants in background J99 showed a 16, 360 and 1020 fold decrease in resistance to PMB, with MICs of 29.3±4.6, 1.3±0.3 and 0.46±0.03 µg/ml, respectively  to that shown by wild type H. pylori  the human cathelicidin LL-37 produced by both leukocytes and epithelial cells, (ii) human β-defensin 2 (HBD-2) found throughout the gastrointestinal tract, (iii) P-113, a fragment of histatin 5 found within the oral cavity, (iv) and HNP-2, an α–defensin produced by neutrophils The lifecycle and transmission of lpxE, lpxF and double lpxE/F mutants all showed a decrease in resistance against LL37, P-113, and HBD-2, similar to what is seen against PMB , related to the insect cecropins that bind to the phosphate groups of lipid A H. pylori is naturally resistant to Hp (2.20), and it has been suggested that release of this peptide into the stomach may contribute to clearance of other gastrointestinal pathogens providing some benefit to H. pylori infected individuals lpxE/F mutant shows a large decrease (≥ 60 fold) in resistance against Hp (2.20)  . The levH. pylori completely lack unsubstituted phosphate moieties on its LPS H. pylori strains produce an LPS anchored to the membrane by bis-phosphorylated lipid A   lipid A  providinH. pylori wild type and double lpxE/F mutant in strain J99 were incubated for 10 minutes in the presence of 0, 1, 25, or 250 µg/ml PMB-OG and visualized by phase contrast and fluorescence microscopy . Briefly, croscopy . OverlayH. pylori strains, stained cells were re-suspended in PBS and fluorescent intensity measured  producing a mixture of lipid A species, complicating the findings H. pylori, P. gingivalis, and Leptospira interrogans elicit an immune response through hTLR2 H. pylori and the mutants generated herein produce an abundant single species of lipid A  increases in activation of hTLR4-MD2 at 10000 ng/ml when compared to wild type, indicating that LpxF imparts greater influence on attenuated hTLR4-MD2 when compared to LpxE. This agrees with the published crystal structure of the hTLR4-MD2-LPS complex showing that the 4′ phosphate group is involved for ligand recognition lpxR deficient mutant activated hTLR4-MD2 similar to wild type LPS indicating that lipid A dephosphorylation plays a larger role in lowering endotoxicity compared to deacylation of Helicobacter lipid A , a known TLR4 antagonist E. coli (10 and 1000 ng/ml) were used as a negative and positive controls for activation of hTLR4-MD2, respectively , even at LPS concentrations as high as 10,000 ng/ml when compared to wild type LPS (E. coli (1000 ng/ml) and Pam3CSK4 (10 and 1000 ng/ml), a synthetic lipopeptide, were used as a negative and positive control for activation of hTLR2, respectively  increases in activation of mTLR4-MD2 at 10000 ng/ml when compared to wild type (lpxR deficient background is not surprising considering the promiscuity of this receptor for lipid A isoforms (i.e. tetra- and penta-acylated lipid A) E. coli (10 and 1000 ng/ml) were used as negative and positive controls for activation of mTLR4-MD2, respectively , even at LPS concentrations as high as 10,000 ng/ml when compared to wild type LPS (data not shown).Previous research has demonstrated that hTLR4-MD2 and murine TLR4-MD2 (mTLR4-MD2) display differential recognition of LPS ild type , suggestectively . LPS froH. pylori LPS modifications in host colonization. Presumably, dephosphorylation by LpxE and LpxF provides an advantage through resistance to CAMPs at the gastric mucosal surface and attenuated hTLR-MD2 activation. Francisella mutants lacking lpxF were greatly attenuated in animal studies and considered avirulent F. tularemia does not establish a long-term colonization of its human host but rather, results in quick progression to severe illness and/or death in vivo animal study to emphasize the role played by LpxE and LpxF in host colonization.To date, nothing is known regarding participation of Campylobacter jejuni and H. pylori have demonstrated that lipid A modification enzymes can modulate motility and O-antigen expression, respectively H. pylori background J99 were used to illustrate these findings. However, identical results were found in both mouse-adapted strains, B128 and X47 (data not shown).For these experiments, all mutations were transferred into the mouse-adapted strains B128 and X47 tlr4-/-, were colonized. The TLR4 deficient mice were used in conjunction with wild type mice to help determine which plays a larger role in host colonization: CAMP resistance or attenuated hTLR4-MD2 activation. Mice were infected orogastrically with 2×108 bacteria per mouse. Colonization rates were determined by enumeration of colony forming units per gram (CFU/g) of stomach. Two independent colonization experiments were performed. The first was a 15-day time point using both B128 and X47 strains in C57BL/6J. The second was a time course colonization experiment at days 3, 15, and 45 using only strain B128 in both C57BL/6J and C57BL/6J tlr4-/- mice.Two mice populations, C57BL/6J and C57BL/6J lpxE, lpxF and double lpxE/F mutants, in background B128 and X47, showed a statistically significant colonization defect (p<0.0001) . In X47, complementation of lpxE and lpxF deficient strains resulted in partial recovery of the colonization defect but still significantly different from wild type (p<0.05). Data from both cohorts of mice at day 15 were combined to increase significance and robustness of our analysis. The double lpxE/F mutant in strain X47 was not included because the addition of a chloramphenicol cassette shows a strain specific decrease in the fitness of the bacteria in colonization experiments (unpublished observation). Overall, lpxF deficient strains in both B128 and X47 background showed the largest defect in colonization, suggesting LpxF activity is essential for host colonization.Results from the first experiments revealed that in wild type C57BL/6J mice the single <0.0001) . In B128tlr4-/- mice. A 3, 15 and 45 day time course was included in this study to determine if H. pylori strains were cleared soon after infection or if it was a gradual loss of colonization. Wild type strain B128 colonized C57BL/6J and C57BL/6J tlr4-/- mice equally well (p>0.05) throughout the time course . However, the LpxE deficient strain colonized well until day 45 (tlr4-/- mice (p<0.01). This suggests that resistance to CAMPs, resulting from the dephosphorylation of lipid A by LpxF, is important for survival within a host. The role LpxF dependent attenuated TLR4-MD2 activation plays in the colonization of a host cannot be determined because they are so quickly cleared from the mouse stomach, presumably due to CAMP sensitivity. However, LpxE deficient strains retain partial resistance to CAMPs  found on H. pylori lipid A. Additionally, our data suggest that H. pylori LPS does not activate hTLR2 even at high concentrations of LPS, helping to clarify some conflicting reports in the literature LPS serves as one of the most powerful activators of the innate immune system. Presumably, 2 stains , requiriH. pylori lipid A in host colonization. This work clearly demonstrates that dephosphoryation of H. pylori lipid A by LpxE and LpxF is essential for efficient host colonization, with LpxF activity being the largest determinant of bacterial survival within a host. LpxF deficient strains are unable to colonize host and are quickly cleared within 3 days of inoculation  in This study was carried out in strict accordance with the European Union Directive 2010/63/EU (and its revision 86/609/EEC) on the protection of animals used for scientific purposes. Our laboratory has the administrative authorization for animal experimentation (Permit Number 75–1250) and the protocol was approved by the Institut Pasteur Review Board that is part of in the Regional Committee of Ethics of Animal Experiments of Paris region (Permit Number: 99–174).H. pylori were started from methyl-cellulose stocks routinely stored at −80°C, plated onto 5% sheep blood agar medium (BAP) and incubated at 37°C for 36 to 60 h in a microaerobic atmosphere . The methyl-cellulose storage media is composed of cellulose-methyl ether 4000 CPS grade (1.0% w/v) and MgSO4 (0.1 M) and the BAP is composed of Tryptic soy agar (acumedia Cat. 7100A) and defibrinated sheep blood (Remel Cat. R54020). Liquid cultures of H. pylori were started from overnight culture on BAP and inoculated into brucella broth supplemented with 7% fetal bovine serum (Hyclone) and vancomycin (10 µg/ml). Liquid cultures of H. pylori were grown to an A600 of ∼1.0 at 37°C under microaerobic conditions for 24 to 36 h with shaking. E. coli was routinely grown at 37°C in LB broth with appropriate antibiotics using standard conditions.The bacterial strains and plasmids used in the study are summarized in Plasmids were isolated using a QIAprep Spin Miniprep Kit (Qiagen). Custom primers (Table S1 in hp1580 (lpxF), from H. pylori 26695 was obtained from a previously published library hp1580 using standard cloning techniques. The generated plasmid, pILLhp1580:kan, was used to create 4′-phosphatase defective mutants in H. pylori J99, B128 and X47 by natural transformation hp1580 corresponds to jhp1487 in H. pylori J99. Mutation of jhp0019 (lpxE) and jhp0634 (lpxR) in H. pylori B128 and X47 was achieved by introduction of a chloramphenicol or kanamycin cassette into the coding sequence of each gene resulting in the interruption and removal of a large portion of its sequence, using previously described suicide vectors lpxE/F double mutant was created by introduction of the lpxE mutation into a lpxF defective mutant background using natural transformation as described above.To construct the lipid A 4′-phosphatase mutant, plasmid pILL570 containing hp0954 (rdxA) renders H. pylori resistant to metronidazole making it an ideal candidate for chromosomal complementation lpxE and lpxF defective mutants were complemented by insertion and consequent disruption of rdxA. Briefly, the lpxE and lpxF genes plus 500 bps upstream were amplified by PCR , Table S1 in H. pylori J99 genomic DNA using Pfu Turbo (Stratagene) according to the manufacturer's instructions. The lpxE and lpxF amplicons were digested with BamH1 and EcoR1, gel purified, and subsequently ligated into the previously described H. pylori rdxA complementation plasmid pET634comp H. pylori J99 lpxE and lpxF deficient mutants by natural transformation The interruption of H. pylori (25 ml) and E. coli (5 ml) cultures were grown in the presence of 5.0 µCi/ml 32Pi (PerkinElmer) to an A600 of ∼1.0 using standard liquid growing conditions described above and the cells harvested by centrifugation. 32P-labelled lipid A and phospholipids were isolated using published protocols H. pylori cultures (25 ml) were grown to an A600 of ∼1.0 using standard liquid growing conditions described above and the cells harvested by centrifugation. Lipid A was purified from bacteria as described previously 2 laser (337 nm) using a 20 Hz firing rate. The spectra were acquired in negative ion reflectron mode and each spectrum represented the average of a minimum of 4000 shots. The matrix used was a saturated solution of 6-aza-2-thiothymine in 50% acetonitrile and 10% tribasic ammonium citrate . The samples were dissolved in chloroform-methanol  and deposited on the sample plate, followed by an equal portion of matrix solution (0.3 µl).H. pylori cultures (200 ml) were harvested at A600 of ∼1.0 and the cells harvested by centrifugation. Cell free extracts, membrane-free cytosol, and washed membranes were prepared as previously described 2-[4′-32P]lipid A (at ∼3000–5000 cpm/nmol) as the substrate. Kdo2-[4′-32P]lipid A was prepared as previously described The 4′-phosphatase activity of Jhp1487 (LpxF) was assayed under optimized conditions in a 10- µl reaction mixture containing 50 mM MES, pH 6.0, 0.2% Triton X-100, and KdoH. pylori were grown to an A600 of ∼0.8 to 1.0 at 37°C under microaerobic conditions for 24 to 36 h. The cells were washed with PBS (3X) followed by dilution to A600 of 0.05 in PBS. Polymyxin B Oregon Green 514 conjugate (Invitrogen) was added to 50 µl diluted cells at select concentrations and incubated at 37°C for 10 minutes. Following the incubation cells were washed with PBS (3X), resuspended in 50 µl of PBS, 15 µl placed onto poly-L-lysine coated slides (Electron Microscopy Sciences), and coverslips added. Bacteria were viewed at a magnification of 1000X with a Nikon Eclipse 80i microscope equipped with a 100x 1.4NA PLAN APO lens, a GFP band pass emission filter set with a 480±15 nm excitation range, a 535±20 nm emission range and a Photometrics Cool SNAP HQ2 camera. NIS-Elements AR 3.0 software was used to capture and create the merged images. To quantify PMB-OG binding, strains were cultured and stained as described above but in a volume of 200 µl. After washing, the cells were resuspended in PBS and placed into 96-well plates for analysis. Fluorescence (480 nm excitation and 535 nm emission) and A600 of each well was determined using a Synergy Mx multi-mode microplate reader (BioTek). Each experiment was repeated in triplicate and data reported as a ratio of Fluorescence intensity to A600. The results of three experiments were pooled and a one tailed Mann–Whitney test was used to determine statistical significance of observed differences .Liquid cultures of H. pylori was isolated from a 500 ml liquid culture grown under standard conditions to an A600 of ∼1.0 using the hot water-phenol method LPS from H. pylori strains were grown overnight in brucella broth supplemented with 7% fetal bovine serum. The overnight cultures were inoculated into a 96-well microtiter plates at a starting A600 of 0.05, in a total volume of 100 µl standard H. pylori growing medium, supplemented with select concentration of CAMPs. The 96-well plates were incubated at 37°C in a microaerobic atmosphere  with constant shaking and A600 of each well determined using a Synergy-Mx monochromator based multi-mode microplate reader at 24 and 48 hours. Each experiment was repeated in triplicate. A positive growth control containing no CAMP and a negative control containing no bacteria was performed with every replicate. The MIC was taken as the lowest concentration of CAMP that reduced growth (A600) by more than 50% when compared to the positive growth control. The following CAMPs were purchased and used in the MIC experiment: polymyxin B (Sigma), human β defensin-2 (Phoenix Pharm), human cathelicidin LL-37 (Anaspec), histatin-5 P-113 (Sigma), human neutrophil peptide-2 (Bachem), and H. pylori peptide HP2-20 (Invitrogen). All peptides were stored according to manufacturers' instructions. Because of cost, MICs were only determined for mutants in the J99 background. Complemented strains were also excluded.MIC determination using Polymyxin B Etest strips (Biomérieux) was determined as previously described 2 atmosphere at 37°C.For TLR4 and TLR2, human epithelial kidney (HEK) 293 cells stably co-transfected with m or hTLR4, m or hMD2, and m or hCD14 (denoted as HEK-m/hTLR4) or m/hTLR2 and m/hCD14 (denoted as HEK-m/hTLR2) were purchased from InvivoGen . All cell lines stably express secreted embryonic alkaline phosphatase (SEAP) under the control of a promoter inducible by NF-κB and activator protein 1 (AP-1). Thus, stimulation of m/hTLR4-MD2 or m/hTLR2 will result in an amount of extracellular SEAP in the supernatant that is proportional to the level of NF-κB induction. The cell lines were maintained in standard Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) (Gibco) supplemented with 4.5 g/L glucose, 2 mM L-glutamine, 50 U/mL penicillin, 50 ug/ml streptomycin and 1X HEK-Blue selection (InvivoGen) in a 5% saturated COTM colorimetric assay (InvivoGen). The assay was performed according to manufacturer's protocols. Briefly, cells were seeded in a 96-well plate in triplicate (2.5×104 cells/well for HEK-m/hTLR4 and 5×104 cells/well for HEK-m/hTLR2) in the presence or absence of 10-fold dilutions of purified LPS. Controls included Rhodobacter sphaeroides LPS (TLR4 antagonist) (InvivoGen), E. coli W3110 LPS (TLR4 agonist) and the synthetic triacylated lipoprotein Pam3CSK4 (TLR2 agonist) (InvivoGen). After 20–24 h incubation, supernatants (20 ul) were transferred to a 96-well plate and incubated at 37°C with QUANTI-Blue (180 ul) for 1-2. SEAP activity was measured by reading optical density at 655 nm with a Synergy Mx multi-mode microplate reader (BioTek). The results of several (at least three) experiments were pooled and a one tailed Mann–Whitney test was used to determine statistical significance of observed differences .The induction of TLR signaling in HEK-m/hTLR4 and HEK-m/hTLR2 cell lines was assessed by measuring SEAP activity using QUANTI-Bluetlr4 -/- female mice from Pasteur Institute (kindly provided by Shizuo Akira) aged 4 to 5 weeks were infected orogastrically with feeding needles with either X47 or B128 strains (2×108 bacteria per mouse). Colonization rates were determined after 3, 15 or 45 days by enumeration of colony forming units (CFU) per gram of stomach. Mice were euthanized with CO2 and the stomachs were ground and homogenized in peptone broth. The samples were then diluted and spread on blood agar plates supplemented with 200 µg/ml of bacitracin, and 10 µg/ml of nalidixic acid, to inhibit the growth of resident bacteria from the mouse forestomach. The CFUs were enumerated after 8 days of incubation under microaerobic conditions. The results of several (at least two) colonization experiments were pooled and a one tailed Mann–Whitney test was used to determine statistical significance of observed differences .C57BL/6J female mice purchased from Charles River Laboratories and C57BL/6J Text S1H. pylori LPS remodeling supporting information.(PDF)Click here for additional data file.


N-glycosidic activity of ricin toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a toxin (RTAHDEL) showed a similar cytotoxic effect on yeast as a corresponding RTAKDEL variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in toxin-treated spheroplast cultures of S. cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (∆end3) and/or in RTA depurination activity on 28S rRNA (∆rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B toxin transport from the plasma membrane through the endosomal compartment to the ER.The plant A/B toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the  ERendoplasmic reticulumRTAricin toxin A chainRTBricin toxin B chainPIpropidiumiodideRicinus communis produces a heterodimeric protein toxin (ricin) which represents one of the most powerful A/B toxins of biological heritage [N-glycosidase by cleaving a specific adenine residue at position 4324 within the sarcin/ricin loop of 28S rRNA [in vivo binding of elongation factor 2 (EF2) which is required to initiate protein translation on the eukaryotic 80S ribosome [N-acetylglucosamine residues on the surface of mammalian cells, the toxin is taken up by endocytosis and transported to early endosomes [E. coli heat labile toxin HLT [The development of effective protecting drugs and antidotes against various human diseases and biological toxins is still a major goal in ongoing biomedical research. The plant heritage . The matheritage ,3. Ricin28S rRNA . Loss ofribosome . Thus, rribosome . After tndosomes ,8. Whilendosomes ,10,11, ondosomes . Synthaxndosomes . In contoxin HLT , ricin ioxin HLT . Therefooxin HLT ,17,18. Aoxin HLT ,19.in vivo toxicity, comparatively little is known about the intracellular toxin transport and the cellular components involved in this process. A deeper mechanistic understanding of toxin trafficking could not only help to design more effective antidotes and immunotoxins, it would also foster development of novel therapeutic strategies for the treatment of various human diseases including cancer [Despite our detailed knowledge on RTA g cancer ,21,22. in vivo analyses of toxin uptake and transport in more detail. Previous studies already demonstrated that yeast ribosomes are highly sensitive to, and depurinated by, RTA [in vivo toxicity of ricin up to 250 fold [in vivo toxicity against HeLa cells. As we observed similar cytotoxicity for both toxin variants, RTAHDEL and RTAKDEl, we built-up a yeast-based bioassay for the analysis of RTA uptake and intracellular transport. Further studies with this novel test system should shed more light on ricin trafficking in vivo, thereby helping to identify host cell proteins that are mechanistically involved in the intoxification by microbial and plant A/B toxins.The focus of our present study was to develop a yeast-based bioassay which would allow  by, RTA ; however by, RTA . Based o250 fold , we askeE. coli TOP10 (F’mcrA Δ (mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ (ara-leu) 7697 galU galKrpsL (StrR) endA1 nupG) was used for cloning. Using pRS316-RTA as template, RTA(His)6, RTA(His)6HDEL and RTA(His)6KDELwere PCR amplified in the presence of HiFi polymerase (Roche) and the following primer pairs: 5'-RTA (5'-GGATCCATGATAATTCCCAAACAATACCCAATTATAAACTTTAC) and 3'-RTA(His)6 (3'-AAGCTTGTCGACTTAATGATGATGATGATGATGAAACTGTGACGATGGTGGAGGTGC) for RTA(His)6, 5'-RTA and 3'-RTA(His)6HDEL (3'-AAGCTTGTCGACTTACAGTTCATCATGATGATGATGATGATGATGAAACTGTGACGATGGTGGAGGTGC) for RTA(His)6HDEL, and 5'-RTA and 3-RTA(His)6KDEL (3'-AAGCTTGTCGACTTACAGTTCATCTTTATGATGATGATGATGATGAAACTGTGACGATGGTGGAGGTGC) for RTA(His)6KDEL. Each primer introduced either a 5'BamHI or 3'HindIII/SalI cleavage site (underlined). After amplification, the corresponding DNA fragment was cloned into pSTBlue-1 (Novagen), sequenced and finally cloned as BamHI/HindIII fragment into pET24a(+) (Novagen) to obtain the expression vectors pET-RTA, pET-RTAHDEL and pET-RTAKDEL, respectively. All constructs were subsequently transformed into E. coli BL21 (DE3)  λ  and after cells had reached an optimal density (OD600≈0.8–1), expression of each RTA variant was induced in the presence of 1 mM IPTG for 2 h at 28 °C. Cells were harvested, washed twice with sterile water and resuspended in binding buffer  for subsequent RTA purification. Cell debris was removed and the supernatant was collected for Ni2+-NTA affinity chromatography. Sonicated supernatants were immediately used for SDS–PAGE, western analysis and/or Coomassie blue staining.Standard molecular manipulations were performed as described by . E. coliS. cerevisiae wild-type strain BY4742  and its isogenic knock-out mutants YNL084C (∆end3) and YDR418W (∆rpl12B) were obtained from Open Biosystems. Yeast cells were grown in YEPD  at 30 °C. For spheroplast preparation, each strain was grown in YEPD to late exponential phase (4–6 ×107 cells per mL), harvested at 8000 rpm and washed twice with sterile water. Subsequently, 1 × 109 cells were resuspended in 100 mL heroplast buffer , 10 mM CaCl2, 2 mM DTT and 200 µg/mL zymolyase 20 T), incubated at 30 °C for 75 min, harvested at 4 °C and 2000 rpm and washed twice with incubation buffer , 10 mM CaCl2,10 mM glucose).The E. coli clones expressing the (His)6-tagged RTA variants were applied onto a 5 mL HisTrap FF column  and eluted in a single step by the addition of imidazol . Eluted protein fractions were desalted and equilibrated either in PBS (pH 7.4) for studies on mammalian cells or in incubation buffer for yeast experiments. Ni2+-NTA purified supernatants of E. coli expressing the empty vector pET24a(+) without RTA served as negative control. After concentration through 10 kDa cut-off spin columns , purified proteins were stored at 4 °C. Coomassie staining was employed to analyze protein purity and the level of protein expression was verified by western blot analysis. Total protein content was determined by using a BCA protein assay kit (Pierce).Sonicated supernatants of E. coli supernatants and Ni2+-NTA purified fractions were analyzed by SDS-PAGE by separating protein samples in 15% Tris-tricine SDS polyacrylamide gels [After RTA expression, ide gels . After eide gels .5  cells per well in DMEM medium containing 10% FCS and 1% penicillin-streptomycin and incubated at 37 °C in the presence of 5% CO2 for 18 h. After an additional incubation in the presence of purified RTA and/or control samples for 24 or 48 h, phase contrast microscopy was performed using an Olympus IX70 microscope under standard settings. For trypan blue staining, DMEM medium was removed, cells were stained in 0.4% trypan blue (Sigma) for 5 min, washed with PBS and analyzed under the microscope and counted in an automated cell counter (Invitrogen). For counting, cells were harvested 24 or 48 h after RTA treatment. Sample supernatants were collected, adherent growing cells trypsinated and pooled. Subsequently, pooled supernatants were harvested at 2000 rpm, washed once with PBS, resuspended in 100 µL PBS, and 100 µL 0.4% Trypan Blue were added. Cells were counted after 3 min of incubation.HeLa S3 cells were seeded in 24 well plates at a density of 1 × 107 cells) were seeded into 96 well oyxgen-sensor microtiter plates . Various concentrations of RTA variants were adjusted to a final volume of 200 µL incubation buffer per well. In each measurement, a two point calibration was conducted. The k100 value was measured with air saturated water and the k0 value with water containing 0.1% Na2SO3 solution. Oyxgen concentration was measured every 20 min over a time window of 16 h. Each sample measurement was performed in triplicate at 30 °C, 120 rpm and with a shaking diameter of 1 mm. Measurements were carried out in a fluorescence reader equipped with an integrated shaker  [pO2 values were calculated according to the equations shown in (1) and (2).Wild-type yeast cells and selected knock-out mutants were grown in YEPD to exponential phase and spheroplasted by treatment with zymolyase 20 T as described above. Yeast spheroplasts  in the presence of each RTA variant in incubation buffer at 30 °C and low shaking (115 rpm) for 24 h. Measurements were performed using a PARADIGM . Subsequently, cells were treated with 2.5 µL propidium iodide  for 3 min and fluorescence was measured at an extinction wavelength of 535 ± 25 nm and an emission wavelength of 635 ± 25 nm for 140 ms. Fluorescence was expressed as shown in (3).Yeast wild-type cells and knock-out mutants were cultivated and spheroplasted as described above. At a density of 1.5 × 10n = 5) and the corresponding standard deviations are indicated for each experiment.All experiments and measurements were repeated five times  at a wavelength of 450 nm. Cytotoxicity was expressed as shown in (4).HeLa S3 cells were obtained from ATCC and maintained in DMEM medium supplemented with 10% FCS and 1% penicillin-streptomycin. For cell vitality assays , HeLa cells were seeded in 24 well plates at a density of 1 × 10n = 3) and standard deviation is displayed in each figure. Statistical significance of the values was calculated by using the t-test method. Each experiment was performed in triplicate  to obtain the expression vectors pET-RTA, pET-RTAKDEL and pET-RTAKDEL , RTAHDEL (31.5 kDa) and RTAKDEL (31.5 kDa)  B. As expmg/L RTA B.E. coli are biologically active against mammalian cells, XTT cell viability assays were performed in which mitochondrial dehydrogenase activity of HeLa S3 cells was measured as an indirect means of cell viability. To ensure that the cells are not negatively affected by endogenous E. coli proteins, a negative control was included corresponding to NTA-purified proteins from E. coli clones expressing the empty vector. As shown in KDEL or RTAHDEL  (data not shown); after toxin treatment for 48 h, loss in cell viability further increased to 71.4% for RTAKDEL and 68% for RTAHDEL . We thus assume that cells treated with low doses of RTA are being inhibited in protein biosynthesis; however this inhibition is not sufficient to induce in vivo cell death. This might explain the observed lack in RTA toxicity under low toxin concentrations in XTT viability assays. To our knowledge, we showed here for the first time that the addition of a yeast specific ER retention signal (HDEL) to RTA results in a comparably strong cytotoxic effect to what can be observed for RTAKDEL on HeLa cells. Dose response curves and p-value calculations of both constructs did not show any significant difference in the presence of 0.1 to 80 µg toxin (data not shown). One possible explanation for the increased cytotoxicity of RTAHDEL could be that mammalian cells—in contrast to yeast—possess three different ER retention signal recognizing KDEL receptors. One of them (Erd23) has been shown to recognize HDEL carrying proteins and to catalyze subsequent retrograde transport back to the ER [C-terminal HDEL motif; our present cell viability data are consistent with these findings. To fortify the cell viability data, we looked at the morphology of HeLa cells before and after RTA treatment  or RTAKDEL (50.5%); both variants—RTA and RTAKDEL—showed similar kinetics at a low toxin concentration of 3 µg . Taken together, these results indicate that the yeast-specific ER retention motif HDEL is more efficient in ensuring retrograde RTA transport than the mammalian-specific retention signal KDEL. This might be explained by the fact that yeast—in contrast to mammals—only possess a single cellular HDEL receptor (Erd2p) which preferentially recognizes and binds HDEL-carrying proteins within the secretory pathway [In further experiments we wanted to analyze whether or not RTA, RTA surface . Therefo surface ,24,31. B surface . If RTA 0 µg RTA B, while 0 µg RTA B which ir 20 min D. As no  of 3 µg C. p-valu pathway ,36.in vivo cell killing [end3 mutant which is blocked in early steps of both, fluid-phase and receptor endocytosis [end3 cells were effectively protected against all three toxin variants RTA, RTAKDEL and RTAHDEL. The same resistant phenotype was also seen in spheroplasts of a Δrpl12B mutant  staining of yeast spheroplasts and determined PI fluorescence intensity after RTA treatment. As summarized in C-terminus of RTA significantly increases its in vivo toxicity against mammalian cells and yeast cell spheroplasts. Furthermore, we established an oxygen sensor based bioassay which now opens the possibility to use yeast as model to study A/B toxin uptake and intracellular trafficking in more detail. We believe that this bioassay will be helpful to dissect and mechanistically understand how microbial, plant and viral A/B toxins can efficiently intoxicate mammalian cells. Such comprehensive screening in yeast might also bring up novel targets and therapeutic strategies for the treatment of various human diseases, including cancer.In the present study we demonstrate that both the addition of a mammalian- and/or a yeast-specific ER retention signal  to the SP-RTA. Special thanks to Klaus Witte for technical assistance in the affinity purification of recombinant RTA variants, and to the members of the Schmitt lab for helpful discussions, especially to Nina Müller, Thorsten Hoffmann, Esther Gießelmann, Björn Diehl and Frank Breinig. BB was kindly supported by a PhD fellowship from Saarland University. This study was in part supported by a grant from the Deutsche Forschungsgemeinschaft to MJS.We are grateful to Lynne Roberts for kindly providing the ricin A antibody and the plasmid pRS316-K2


Geobacillus stearothermophilus. The E2 scaffold self-assembles into a 60-mer core that is 24 nm in diameter, with a molecular weight of 1.5 MDa, similar to a virus like particle with up to 60 copies of a heterologous protein accessible on the surface. Env(V3)-E2 multimers were tested alone and in combination with Env(gp160) DNA in mice and rabbits. Following two or more co-immunizations with Env(V3)-E2 and Env gp160 DNA, all 18 rabbits developed potent autologous neutralizing antibodies specific for V3 in six weeks. These neutralizing antibodies were sustained for 16 weeks without boosting, and comparable responses were obtained when lipopolysaccharide, a contaminant from expression in E. coli, was removed. Co-immunizations of Env(V3)-E2 and DNA expressing gp160 elicited moderate CD8-specific responses and Env-specific antibodies in mice. Co-immunization with DNA and E2 was superior to individual or sequential vaccination with these components in eliciting both neutralizing antibodies in rabbits and CD8+ T cell responses in mice. Co-immunization with DNA and multimeric E2 scaffolds appears to offer a highly effective means of eliciting rapid, specific, and sustained immune responses that may be a useful approach for other vaccine targets.To obtain proof of concept for HIV vaccines, we generated recombinant multimeric particles displaying the HIV-1 Envelope (Env) third hypervariable region (V3) as an N-terminal fusion protein on the E2 subunit of the pyruvate dehydrogenase complex of  CD8-specific cellular immune responses contribute to early resolution of primary viremia and the maintenance of viral load Despite the fact that HIV-1 utilizes highly effective mechanisms of immune evasion Geobacillus stearothermophilus. E2 oligomers form 1.5 MDa 60-mer particles and are capable of displaying heterologous peptides and proteins in vitro without the help of chaperonins + T cells. Surprisingly, this strategy requires only two immunizations to derive sustained, potent responses.We have been exploring the potential of the antigen display system E2DISP based on the acyltransferase component (E2) of the pyruvate dehydrogenase complex from d in mice, and the C332G and K305R mutations. Env(V3)-E2 and Env(minV3)-E2 monomers were expressed in E. coli as inclusion bodies (IB), purified, and refolded with equimolar amounts of E2 wild type (E2wt) monomers in stepdown dialysis. The resulting 60-mer particles were purified by size exclusion chromatography -E2 with and without (<0.05 EU/ml) LPS were prepared. Resulting E2 preps were >90% pure as determined by quantitative analysis of purified protein gels . Identity was assessed by quantitative western blot . We tested the multimeric particles, separately and together, alone or in combination with HIV-1 SF162 Env (gp160) DNA, in rabbits and mice. Effects of LPS and adjuvant on the resulting immune responses were determined. Vaccine compositions and regimens tested are shown in A DNA fragment encoding the HIV-SF162 V3 region was fused to the E2 gene in the E2DISP expression plasmid. One Cys residue (position 332) was mutated to Gly in an effort to produce homogeneous peptides with no disulfide-bonded loops (C332G) Fig. 1A.4–106) of E2 antibodies were elicited in all groups; and despite the strong E2 response, we observed the priming and boosting of Env- and V3-specific antibodies. Kinetics of V3 antibody development were similar to those of the E2 antibodies  were immunized with both Env(minV3 and V3)-E2 VLPs in combination with Env(gp160) plasmid DNA (DNA) using three different immunization regimens described in detail in tibodies Fig. 2B.w12 = 32) . To increase responses in the DNA primed rabbits We measured NAbs against the autologous HIV-1 SF162 pseudovirus using the TZM-bl assay w6 = 466 and Max NAbw6 = 714). Moreover, the level of NAbs in all animals from this group remained fairly constant with boosts over a period of 28 weeks (Mean NAbw28 = 609 and Max NAbw28 = 1058). To determine which Env-E2 VLP was more effective in eliciting NAbs, each construct was tested separately and co-administered with DNA , after two and three immunizations, better than either of these components delivered alone or sequentially. Area under the NAb titer curve (AUC) for the co-immunization regimen  was significantly higher compared to that for either sequential regimen  DNA (Group 3) rapidly developed a potent autologous NAb response Fig. 3C.Neutralization of HIV-SF162 in sera from rabbits from all of the groups was specifically inhibited when samples were pre-incubated with the SF162 V3 peptide, but not with a V3 scrambled peptide Table 2.w12 = 1679), they were not significantly different than those observed in rabbits co-inoculated with LPS-contaminated Env(V3)-E2 , but did not reach levels observed in rabbits receiving IFA. NAbs in Group 7 without addition of adjuvant were significantly lower than in Groups 5 and 6 . Boosting at week 20 restored the NAbs to the prior level. These results confirmed the rapid development of high titer NAbs in all rabbits co-immunized with Env-E2 and Env(gp160) DNA, with a duration of at least three months from the last immunization.To examine the effect of LPS on the immune responses, we co-immunized a group of rabbits with LPS-free Env(V3)-E2 particles and Env(gp160) DNA with and without IFA (Groups 6 and 7). Binding antibodies directed to E2wt, V3 peptide, and gp140 were similar in both timing and magnitude using LPS-free E2 proteins Fig. 5 cA subgroup of samples was selected from weeks 6 and 28 in Groups 3 and 5 to measure heterologous neutralization. The only significant responder from Group 2 (rabbit 6869) was included. Sera were assayed in the TZM-bl assay against a panel of 12 Clade B pseudoviruses with differing degrees of sensitivity  in vitro stimulation, effector cells were tested for V3 peptide-specific responses by dextramer T cell staining analysis and intracellular cytokine staining (ICS). A representative set of dextramer T cell staining obtained by flow cytometry for splenocytes isolated from each group of immunized mice is illustrated in + splenocytes from co-immunized mice (Group 11) stained positive for the V3 dextramer, compared to mice immunized with plasmid DNA or VLP alone. Likewise, 7.43% of splenocytes  specifically stained positive for the V3 dextramer in mice that were co-immunized with LPS-free VLPs (Group 12). These results suggest that co-administration of DNA and Env-E2 VLPs was required to induce in vivo peptide-specific CD8+ T-cells able to recognize the class I MHC IGPGRAFYA peptide. Splenocyte-derived CD8+ T cells were assessed for IFN-γ production by intracellular cytokine staining, as shown in a representative example  displayed a significantly higher fraction of CD8+ T cells producing IFN-γ  expression plasmid. Previously, E2 particles displaying HIV-1 Gag were shown to bear helper T cell epitopes and to elicit CTL and antibody responses directed to HIV-1 Gag-p17 in HLA-A1 transgenic mice Antibody titers directed to the E2 core were rapidly induced, as was the development of antibodies targeting to the HIV-1 Env epitope presented on the scaffold. All nine rabbits receiving both Env(V3)-E2 scaffolds together with Env(gp160) DNA following any of the three different immunization regimens (Groups 1–3) developed specific antibodies targeting Env. Our results show that Env(gp160) DNA is more effective in eliciting specific antibodies targeting the whole gp140 protein, whereas Env(V3)-E2 proteins are more effective at focusing the response to the V3 peptide presented on the E2 surface. The individual contributions of each of these components can be complemented by the other following sequential administration as shown for Groups 1 and 2. The effect is clearly potentiated when both components are administered together as is the case of Groups 3–8, since all 18 animals from those groups developed the highest responses to both V3 and gp140, and these responses were sustained during the entire immunization schedule.+ weeks and many more immunizations P<0.05). Following as few as three immunizations, this combination protein plus DNA approach elicited a stronger NAb response than a more standard DNA-prime/protein-boost regimen. The V3-E2 constructs, not surprisingly, focused the immune responses to the V3 epitope displayed on the surface. The V3 constructs tested were not effective in generating broadly NAbs; however, we observed a modest level of neutralization of some Tier 1 viruses. It is possible that constrained V3 peptides might be more effective in this context in eliciting broader responses than we observed, as seen with recombinant CTB displaying V3 Intramuscular immunization of rabbits with Env(minV3)-E2, Env(V3)-E2, or both, co-administered with HIV-1 Env(gp160) DNA delivered ID consistently elicited strong NAb responses after only two immunizations (by week 6), levels typically not seen until after 20+ cellular immune responses in mice. As was seen in rabbits with antibodies, the CD8 response was significantly higher compared to that elicited by E2 or DNA when they were administered individually, where responses were only rarely distinguishable from the non-immunized control group. These results suggest that both the E2 and DNA components are important in inducing both cellular and humoral immune responses. This observation is in agreement with previous observations + T-cell responses Env-E2 particles delivered IM co-administered with Env(gp160) DNA delivered IM at a different sites elicited V3-specific CD8in vivo, preserving conformation-sensitive epitopes in the context of cellular antigens as would take place during virion production. Immunogens that are capable of preserving native HIV-1 Env trimeric structure are thought to elicit better NAb responses One of the theoretical advantages of DNA vaccines is their ability to express the native proteins and particles It has been proposed that CpG motifs present in DNA trigger toll-like receptors (TLRs) and thereby stimulate immunity. In this study we demonstrated the intrinsic immunogenicity of the E2 particles, evidenced in the rapid anti-E2 antibody responses after a single immunization. Removing LPS from Env-E2 formulations did not diminish immunity, indicating that immunogenicity was neither enhanced nor diminished in the presence of LPS. Taken together these properties of both DNA and E2-VLPs could, in part, explain the potentiation of the immune response that we observed in the present study. Further experiments are needed to understand the mechanisms of the co-immunization methods that we have used. Combining the properties of these two antigen delivery systems may have the effect of maximizing the efficiency of antigen presentation and more effectively engaging both arms of the adaptive immune response. Repetitive presentation of an epitope, as with E2 particles, can induce a stronger immune response by triggering oligomerization of B cell receptors recognizing the epitope Altogether, these results suggest that this antigen presentation system, coupled with the simultaneous DNA and E2 particle immunization regimen, may hold significant hope for effective immunogenicity of HIV and other vaccines in the clinic.d MHC) were obtained from the Charles River Laboratory . Animals were housed under specific pathogen free conditions at the Animal Facility of the CNR, Naples, Italy.All rabbit studies were performed in accordance with the standards outlined by the National Institutes of Health Guide for the Care and Use of Laboratory animals. The ONPRC is an AAALAC-accredited institution. Rabbit studies were performed according to the rules approved by the Institutional Animal Care and Use Committee (IACUC) at the Oregon Health & Science University, protocol no. 0825. All mouse experiments were carried out in accordance with European Union Laws and Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Review Board and performed according to rules approved by the Animal Ethics Committee (permission no. 137/2006-A). Female New Zealand White rabbits  were housed at the Oregon National Primate Research Center (ONPRC) at Oregon Health & Science University. Eight-week-old female BALB/c mice   lipopolysaccaride-blasts (1.25×106 cells/ml LPS-blasts) produced from non-immunized BALB/c mouse. Ag-pulsed LPS blast cells consisted of splenocytes that were cultured in RPMI 1640, in the presence of 25 µg/ml LPS (Sigma), supplemented with 10% FCS, 5×10−5 M 2-ME, 1 mM glutamine, 1 mM sodium pyruvate, and 7 µg/ml dextran sulfate (Sigma) for 3 days and pulsed for 3 h with 10 µg/ml of IGPGRAFYA311–318 peptide. After 6 days of co-culture, effector cells were harvested and assayed for dextramer staining and Intracellular cytokine IFNγ staining (IFNγ-ICS).Four different groups of three BALB/c mice were intra-muscularly immunized twice following four different immunization regimens : at day GGCGGCGGCCCATGGCCTCTGTAGAAATTAATTCTAC and GGCGGCGGCCCCGGGTTCTCCACTAATGTTACAATG containing the restriction sites NcoI and XmaI (New England Biolabs). Cycling conditions for the PCR were as follows: denature at 94°C for 2 min, 10× , 20× , and a final elongation of 72°C for 7 min. The PCR product and the pETE2DISP vector were double digested with NcoI and XmaI and ligated together with T4 DNA ligase (New England Biolabs) before transformation into BL21 (DE3) CodonPlus-RIPL cells (Stratagene). To decrease proteolytic degradation of Env(V3)-E2, a K305R mutation was introduced using primer CTAACAATAATACAAGAAGAAGTATAACTATAGGACCGG. Likewise, to decrease intramolecular bonding and increase solubility of Env(V3)-E2, a C332G mutation was introduced using the primer GAGATATAAGACAAGCACATGGCAACATTTAGTGGAGAACC. Both mutations were generated using the QuikChange Multi Site-Directed Mutagenesis kit (Stratagene), per manufacturer's instructions. The minimized V3 construct Env(minV3C332G/K305R)-E2 was generated from the corresponding full-length Env(V3C332G/K305R)-E2 construct via deletion of amino acids 291–299 using the QuikChange II Site-Directed Mutagenesis (Stratagene) and primer CCATGGCCTCTGTAGAAATTAATTGTACCATGGCTAACAATAATACAAGAAGAAGTATAAC, which contains an NcoI site corresponding to residue 299. In-frame ligation of all constructs was confirmed by sequencing. For simplicity, these constructs Env(V3C332G/K305R)-E2 and Env(minV3C332G/K305R)-E2 are annotated in this document as Env(V3)-E2 and Env(minV3)-E2, respectively.The Env(V3)-E2 and Env(minV3)-E2 expression vectors were constructed from the previously described pETE2DISP plasmid Escherichia coli strain BL21 (DE3) CodonPlus-RIPL cells (Stratagene). The soluble E2wt-containing fraction was recovered from E. coli lysates after centrifugation at 10,000× g for 10 min at 4°C and was purified using a Sephadex G-25 column for buffer exchange , a Detoxi-Gel for LPS removal (Pierce), and a Q-Sepharose anion exchange column . Peak fractions containing E2wt were pooled and concentrated with a 10 kD molecular weight cut off (MWCO) using Amicon Ultra Centrifugal Filter (Millipore). The retentate was loaded onto a Superdex200 gel filtration column  using Solubility Buffer 2.2 , 50 mM NaCl, 250 mM L-Arginine (Sigma)) and fractions containing the 1.5 MDa E2 60mer particles were concentrated to 1 mg/ml using the Ultra Centrifugal devices and then stored in Solubility Buffer 2.2 at −80°C.Plasmids encoding the E2wt and HIV-1 Env-E2 fusion proteins were maintained and expressed after induction with Isopropyl b-D-1 thiogalactopyranoside (IPTG) in E.coli and were purified from the inclusion bodies, which were washed three times with Inclusion Body Wash Buffer (1 M guanidine hydrochloride (GuHCl), 50 mM NaCl, 1 mM DTT, 1× PBS, 10% glycerol, 0.5 M arginine, pH 7.4) before being dissolved in Unfolding Buffer .Env(V3)-E2 and Env(min)-V3 proteins formed inclusion bodies in To produce the HIV-1 Env-E2 virus-like particles, E2wt was combined with the Env-E2 inclusion bodies at a 1∶1 molar ratio in Unfolding Buffer  rocking at 4°C for a minimum of 3 h. The proteins were transferred to SnakeSkinT Dialysis Tubing, 10 K MWCO (Thermo Fisher Scientific) and subjected to step-down dialysis against 4 M, 2 M, 0 M GuHCl Refolding Buffers . A final dialysis was performed in Solubility buffer 2.2 . Refolded soluble 60-mer virus-like particles were confirmed by gel filtration using the Superdex200 gel filtration column , and purity was assessed by SDS-PAGE and Western blot analysis. The purified VLPs were either stored directly at −80°C or subjected to LPS removal before storing at −80°C. LPS removal from protein samples utilized TritonX-114 (Sigma), as previously described Expression, refolding, and identity of recombinant proteins were assessed by SDS-PAGE, western blot analysis, and ELISA. Samples were prepared as described above and resolved on Invitrogen NuPAGE 4–12% Bis-Tris mini-gels  under reducing conditions. For SDS-PAGE, gels were stained with SimplyBlue™ SafeStain (Invitrogen). For western blot analysis, proteins were transferred onto nitrocellulose paper (Invitrogen), blocked with Odessey blocking buffer (LI-COR Biosciences) overnight at 4°C. The following day, the blot was probed simultaneously with a 1∶8,000 dilution of rabbit sera specific for E2 and a 1∶20,000 dilution of the monoclonal human antibody 447-52D for 1 h at room temperature. After washing 5 times, secondary antibodies IRDye680 Goat anti-Rabbit and IRDye800 Goat anti-human (LI-COR Biosciences) were used at 1∶15,000. Membranes were scanned using the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences) to allow simultaneous two-color detection of E2 and the HIV-1 Envelope V3 region. Integrated intensities were used in conjunction with protein concentrations determined by Nanodrop to calculate protein purity and concentration. Purified particle preparations [wild type E2 and Env(V3)-E2] were tested for binding to antibody 447-52D after coating to ELISA plates at 200 ng/well using standard methods described previously Plasmid DNA was precipitated onto 1 mm diameter gold beads, and bullets were prepared according to the manufacturer's instructions (Bio-Rad) and loaded with a total of 2 µg of DNA. To verify that the bullets were functional, COS-7 cells were transfected via Gene gun with the DNA carried by the gold beads. Cells were incubated at 37°C for 48 h and then fixed and stained for immunofluorescence using 0.5 µg/mL polyclonal primary antibody chimpanzee IgG derived from and HIV-infected animal (CHIVIG) and 1∶50 dilution of the secondary antibody FITC-conjugated goat anti-human IgG (Zymed). The presence of envelope-transfected cells was visualized by fluorescent microscopy.Binding antibody responses in immunized mice and rabbits to E2-wt protein, V3 peptide and HIV-1 envelope antigen gp140 were measured by indirect ELISA as described previously luc reporter gene expression compared to that obtained in virus control wells containing virus and cells only {[(virus+cell)−(tested serum+virus+cell)]/virus+cell}×100 = % neutralization. Background control wells contained cells only. A pre-immune sera pool and the murine leukemia virus (MLV) were used as negative controls. A well-characterized immune serum was used as a positive control. Neutralization dose-response curves were fitted by non-linear regression and a final titer was informed as the reciprocal of the dilution of serum necessary to achieve 50% neutralization. Experiments were performed in parallel at ONPRC and at Duke University on key samples to compare results.Neutralization assays were performed using the single-cycle TZM-bl neutralization assay as described previously 50 of SF162 pV. Percent reduction in neutralization was calculated as: [1−(titer with V3 peptide)/(titer with V3 scrambled peptide)]×100.Detection of NAbs specifically directed to V3 peptide was performed as previously described + T cells specific for IGPGRAFYA311–318 peptide were determined by MHC H-2Dd class I dextramer staining. Briefly, cells were transferred in 96-well round-bottom plates at density of 1.5×106 cells/well and washed with PBS containing 5% FCS. Aliquots (10 µl) of PE-conjugated IGPGRAFYA311–318 H-2Dd dextramers or PE-conjugated H-2Dd control dextramers  were then added to the cells. The plates were gently vortexed and incubated in the dark at room temperature for 10 min. Control dextramer that does not recognize mouse CD8+ T cells was used to assess non-specific staining. Cells were then stained with FITC-conjugated anti mouse-CD8 mAb for 20 min in the dark at 2–8°C. After two washes with buffer, stained cells were resuspended in PBS and a minimum of 50,000 live, CD8-positive, gated events were acquired and analyzed by flow cytometry using a FACSCanto (BD Biosciences). Results are expressed as the percentage of CD8+ cells that are positive for the MHC I/peptide dextramer.CD8g-secreting CD8+ T cells, we performed intracellular cytokine IFNγ staining with Cytofix/Cytoperm Leucoperm™ kit , as previously described by Caivano et al 6 splenocytes per well were stimulated with 10 µg/ml of synthetic IGPGRAFYA311–318 peptide in U-bottom 96-well plates. The cultures were incubated at 37°C in a 5% CO2 incubator for 5 h with 10 µg/ml of the secretion inhibitor Brefeldin-A (Sigma-Aldrich). The IFNγ release induced by 30 ng/ml of Phorbol 12-myristate 13-acetate  plus 1 µg/ml of ionomycin (Sigma) was used as a positive control. After the stimulation period, cells were washed with PBS containing 1% FCS, subsequently incubated for 15 min at 4°C with FITC-conjugated anti-mouse CD8 mAb for surface staining, followed by fixation with Cytofix/Cytoperm™ solution for 15 min at 4°C. The surface-stained cells were then permeabilized with 1X Perm/Wash™ solution and stained intracellularly by incubation with PE-conjugated anti-mouse IFNγ mAb for 30 min at room temperature. The cells were finally washed twice with PBS and acquired on FACSCanto (BD Biosciences).To identify antigen-specific IFN311–318 peptide, a 9-mer peptide corresponding to residues 311–318 of the V3 loop of HIV-1 Envelope glycoprotein, was purchased from PRIMM srl . For peptide competition assays, the HIV-1 SF162 V3 peptide (PNNNTRKSITIGPGRAFYATGD) and the V3 scrambled peptide (PNNNTRKSIFYRGAPGITATGD) were purchased from Invitrogen  and Genscript , respectively.The following monoclonal antibodies (mAb) were used for FACS analysis: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD8 ; phycoerythrin (PE)-conjugated anti-mouse IFNγ . MAb 447-52D to HIV-1 V3 was obtained through the NIH AIDS Research and Reference Program, Division of AIDS, NIAID, NIH from Susan Zolla-Pazner. Polyclonal antibody CHIVIG was described previously Statistical analyses were performed using either the unpaired Student t-test, the Mann-Whitney test (comparison between two groups) and Analysis of Variance . To compare NAb levels along the different immunization regimens, the area under the NAb titer curve (AUC) minus baseline was calculated and divided by the duration of each regimen, and significance was determined using the Mann-Whitney test. In all cases differences were considered statistically significant when P<0.05 .


Nkx2.5 is a cardiac specific homeobox gene critical for normal heart development. We previously identified Nkx2.5 as a target of sumoylation, a posttranslational modification implicated in a variety of cellular activities. Sumoylation enhanced Nkx2.5 activity via covalent attachment to the lysine residue 51, the primary SUMO acceptor site. However, how sumoylation regulates the activity of Nkx2.5 in vivo remains unknown. We generated transgenic mice overexpressing sumoylation deficient mutant K51R (conversion of lysine 51 to arginine) specifically in mouse hearts under the control of cardiac α-myosin heavy chain (α-MHC) promoter (K51R-Tg). Expression of the Nkx2.5 mutant transgene in the wild type murine hearts did not result in any overt cardiac phenotype. However, in the presence of Nkx2.5 haploinsufficiency, cardiomyocyte-specific expression of the Nkx2.5 K51R mutant led to congenital heart diseases (CHDs), accompanied with decreased cardiomyocyte proliferation. Also, a number of human CHDs-associated Nkx2.5 mutants exhibited aberrant sumoylation. Our work demonstrates that altered sumoylation status may underlie the development of human CHDs associated with Nkx2.5 mutants. Nkx2.5, a cardiac specific homeobox gene, is one of the earliest markers for cardiac progenitor cells and is central for normal fetal cardiac structural morphogenesis. Nkx2.5 null mice died from cardiac malformations Nkx2.5 is a moderate activator of its target genes. In addition to its physiological activity achieved via DNA binding through its HD, it functioned through associations with other transfactors such as SRF, GATA4 and TBX proteins +/−/SUMO-1+/− mice exhibited more severe cardiac phenotypes than the mice with single heterozygous knockout of either Nkx2.5 or SUMO-1 +/−) mice led to CHDs.Recent studies pointed to an important role of SUMO conjugation in cardiac structural morphogenesis and dysfunction The animals were handled in accordance with institutional guidelines with appropriate approvals (protocol ID 09018) by IACUC at Texas A&M Health Science Center.CGGCACTCTTAGCAAACCTC; reverse, CACCGCCTTCTGCAGCGC. The expression level of the transgene was evaluated by western blot. K51R-Tg offspring were then crossed back to C57BL/6 for one to four generations. Thus, the animals obtained from this cross were examined under the mixed genetic background of FVB and C57BL/6. The generation of Nkx2.5 knockout (Nkx2.5+/−) mouse line was detailed previously +/− mouse lines was performed as needed. Genomic DNA was isolated from tail biopsies performed on weaned animals (about 3-week old pups) and screened by PCR. The animals were handled in compliance with institutional guidelines at Texas A&M Health Science Center at Houston Campus.The human influenza hemagglutinin (HA) tagged K51R mutant was generated as reported Antibodies against Nkx2.5, HA and GAPDH were purchased from Santa Cruz Biotechnology, Inc., and anti-flag antibody (M2) was from Sigma.6-tagged SUMO-1 cDNA was a gift from Dr. Hay RT  and was subcloned into PCGN vector on XbaI and KpnI sites. All Nkx2.5 point mutations, R25C (conversion of arginine 25 to cysteine), T177M (conversion of threonine 177 to methionine), N187K (conversion of asparagine 187 to lysine), R188G (conversion of arginine 188 to glycine), Y190C (conversion of tyrosine 190 to cysteine), C265Y (conversion of cysteine 265 to tyrosine), V309L (conversion of cysteine 309 to leucine), were generated using Stratagene mutagenesis kit based on manufacturer's protocol. Mutations were confirmed by sequencing. For cell culture, Hela cells were maintained in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum. Transfections were performed on Hela cells cultured on either 6 cm plates for western blot assay or 12 well plates for luciferase activity assay using Lipofectamine 2000 based on the protocol provided by the supplier. For western blot analysis, 100 µg of protein extracts obtained from mouse hearts was subjected to NuPage SDS gel (Invitrogen) and transferred to PVDF membrane, which was labeled with desired antibodies and revealed by chemiluminescence. Luciferase activity assays were performed as previously described The cardiac α-actin promoter driven luciferase reporter construct (Ca-actin-Luc), the expression vector encoding HA-tagged wild type Nkx2.5 and flag-tagged SUMO-1 was detailed previously 2+-NTA chromatography were detailed previously 2+-NTA chromatography, Hela cell lysates containing expressed Nkx2.5 wild type or one its mutants in the presence of His6-tagged SUMO-1 were precipitated by Ni2+-NTA beads, and subsequently subjected to NuPage SDS gel. The blot was probed with anti-Nkx2.5 antibody and revealed by chemiluminescence. The relative level of sumoylated Nkx2.5 wt or its mutants was determined by quantification followed by normalization of the level of SUMO-conjugated Nkx2.5 to the level of free Nkx2.5 protein. The level of sumoylated wt Nkx2.5 was taken as 100%. The data from two independent assays were analyzed using software ImageJ .In vivo sumoylation assay and NiMouse neonatal hearts were dissected and fixed in 4% paraformaldehyde (PFA) at 4°C for overnight. Hearts were sectioned transversely at 10 µm thickness, which were stained with Hematoxylin and Eosin (H&E) according to the standard protocol.The mouse hearts at P7 were fixed in 10% formalin prepared in 1× phosphate-buffered saline (PBS), dehydrated and embedded in paraffin for histological sections. For antigen retrieval, 5 µM sections were boiled in sodium citrate buffer  for 10 minutes, and then blocked in 2% bovine serum albumin /PBST (1XPBS/0.05% Tween20), followed by incubation with the primary antibodies monoclonal anti-cardiac troponin T  and rabbit anti-Ki67  in blocking solution at 4°C overnight. Sections were then washed, followed by incubation with 1∶500 Alexa fluor® 594 goat anti-rabbit secondary antibody  and Alexa fluor® 488 donkey anti-mouse secondary antibody  in blocking solution at RT for 30 minutes. Sections were mounted with Vectashield® with DAPI (Vector Laboratory) and photographed under a Leica fluorescent microscope. TUNEL staining was conducted according to manufacturer's protocol (Promega). The number of double Ki67 positive or TUNEL positive and cTnT positive cells (Ki67+/cTnT+ or TUNEL/cTnT+) was scored in at least three randomly selected fields in the left ventricle or atrioventricular septum and expressed as mean ± SEM.Two dimensional M-mode of Vevo 770  was used to measure the cardiac function on mice of interest with various ages as designed. Briefly, mice were anesthetized by inhalation of 1% isofluorane and fixed to a warm pad. Chest hair was removed and contacted with the probe to record cardiac function indices. Evaluations of cardiac function and heart dimensions were performed by an expert analyst blinded to the genotype of animals.gtgtccaacacagatctgatggat; reverse, gcctagtccactctgggctccaat; BNF: forward, gaaggtgctgcccagatgattct; reverse, gacggatccgatccggtctatctt; cardiac α-actin cctctttcattggtatggaatct; reverse, gtacaatgactgatgagagatgg; cardiac α-MHC ggaagagtgagcggcgcatcaagga; reverse, tctgctggagaggttattcctcgt; cardiac β-MHC: forward, ggccaacaccaacctgtccaagtt; reverse, tgcaaaggctccaggtctgagggc; MLC-2v gccaagaagcggatagaagg; reverse, ctgtggttcagggctcagtc; skeletal α-actin agacaccatgtgcgacgaaga; reverse, ccgtccccagaatccaacacga; SERCA forward, gagtcgaccagtcaattcttac; reverse, tcagtattaactccagttgcca; connexin 40: forward, atctcccacattcgttattg; reverse, aggaagatcccatagaggag; conexin 43 taccacgccaccaccggccca; reverse, cattttggctgtcgtcagggaa. GAPDH: forward, atgttccagtatgactccactcac; reverse, gaagacaccagtagactccacga.RNAs were extracted from K51R-Tg and age-matched control mouse hearts using Trizol based on manufacturer's protocol. 1 or 5 µg total RNAs were used for reverse transcription reaction using cloned reverse transcriptase (Invitrogen), followed by quantitative PCR on machine MX3000 (Strategene) using specifically designed probes for the particular genes of our interest. The sequences (5′ to 3′) of oligos used in this study were shown below: ANF: forward, t test, Chi-square test was applied to determine statistical significance between groups when applicable and shown in each Figure legend.The unpaired Student's 2+-NTA chromatography was performed on Hela cell lysates expressing His6-tagged SUMO-1 and Nkx2.5 wild type (wt) or one of seven point mutants created on murine Nkx2.5 protein, R25C, T177M, N187K, R188G, Y190C, C265Y and V309L . In addition, the cardiac index LV mass/body weight ratio also showed no significant difference between the two age-matched groups (data not shown). Despite no discernable cardiac phenotype(s) in the K51R-Tg mice as evaluated by cardiac function, transcripts of cardiac genes such as cardiac brain natriuretic peptide (BNP) and myosin light chain 2v (MLC-2v) were elevated in the K51R-Tg hearts at P50 compared with those in the age-matched control hearts . However+/− mice were obtained after cross-breeding between K51R-Tg and Nkx2.5+/− mice. The compound K51R-Tg/Nkx2.5+/− mice were born in an expected Mendelian ratio (26.15%), however, the mortality rate in the first 14 days after birth was significantly higher than those of three other genotype groups , indicative of severe cardiac dysfunctions. Global heart morphology showed enlarged hearts from K51R-Tg/Nkx2.5+/− mice  in 10 out of 11 demised K51R-Tg/Nkx2.5+/− mice examined, and a single ASD in 1 mouse. Dead pups from other genotype groups showed either normal structure  or a single ASD (wt)  . Thus, e+/− mice, cardiomyocyte proliferation in the heart sections from age-matched wt, K51R-Tg, Nkx2.5+/− and compound K51R-Tg/Nkx2.5+/− mice (n = 3 for each group) at P7 was first assessed using antibodies against Ki67, a cell proliferating maker +/− mice was substantially lower than those in other three genotype animals . Next, the same tissue sections mentioned above were stained with TUNEL reagent. The rates of double TUNEL+/cTnT+ positive cardiomyocytes were similar between these four genotypes , and during diastole . Also, compound K51R-Tg and Nkx2.5+/− mice showed a decrease in cardiac contraction capacity as indicated by both percent ejection fraction  and percent fractional shortening K51R-Tg/Nkx2.5+/− 26.59±4.96 vs. wt 41.75±7.26, Nkx2.5+/− 36.84±7.13, and K51R-Tg 42.45±2.14, respectively). However, the difference of the above-mentioned indexes between these groups was not statistically significant due to variations among individual K51R-Tg/Nkx2.5+/− mice examined. Further elaborated interrogation revealed enlarged left ventricular chamber with impaired cardiac functions in two of four compound K51R-Tg/Nkx2.5+/− mice examined  have been observed in other SUMO targets How these mutations affected Nkx2.5 sumoylation in the presence of entire principle SUMO site remains unclear. It appears that the relative distance from a mutation site to the SUMO site had no any predictable impact on Nkx2.5 sumoylation. For instance, four HD mutations and V309L, the former closer to K51 than C265Y, and the latter farther away from K51 than C265, all showed repressive effects on Nkx2.5 sumoylation. R25C, on the other hand, which is the most adjacent to K51, increased the sensitivity of Nkx2.5 to sumoylation. Possibly, these mutations caused changes of three dimensional structure of Nkx2.5, which mask the SUMO site and/or make it less accessible to SUMO pathway components (HD and C309L mutations), or expose the SUMO site better for sumoylation (R25C mutation). Alternatively, these sites are required for a direct physical contact of Nkx2.5 with SUMO pathway factors, and mutations either disrupt or enhance this contact. The above findings indicate that all HD mutations of Nkx2.5, which negatively affect its DNA binding activity, also severely affect its sumoylation. These observations point to the likelihood that various cardiac phenotypes associated with different Nkx2.5 mutants are in part attributable to the different degrees of altered sumoylation activity of Nkx2.5 mutants.+/− mice might not be attributable only to the defective sumoylation of K51R; the certain level of dominant negative activity of K51R mutant should also be taken into consideration Previous studies demonstrated that expressing wild type Nkx2.5 or one HD mutant I183P caused embryonic lethality or cardiac anomalies +/− mice indicated a positive role of Nkx2.5 in cardiomyocyte division. Whether this diminished cardiomyocyte proliferation was solely responsible for the development of CHDs present in the compound K51R-Tg/Nkx2.5+/− mice requires further interrogation.The exact mechanism(s) by which K51R mutant caused CHDs in the presence of one wild type Nkx2.5 allele remains unclear. Apparently, apoptosis was not involved, despite it is a critical process for cardiac development and function. Although normal cardiomyocyte proliferation was observed in the hearts of Nkx.2.5 null embryos at embryonic day (E) 10.5 One of the limitations of the approach used in the present study is that cardiac α-MHC controlled K51R expression was restricted to myocardium, therefore the anomalies involving great vessels such as double outlet right ventricle (DORV) and Tetralogy of Fallot (TOF) that were seen in human patients associated with heterozygous Nkx2.5 mutants were not expected/observed in our model. Thus, it will be interesting to see if K51R knockin in murine model would lead to the development of great vessel defects that resemble those observed in human patients.cis-regulatory sequence of the SUMO-1 gene in a patient with both cleft lip/palate and ASD Involvement of the SUMO pathway in a number of pathogenic processes such as the development of craniofacial defects, neurodegenerative disease progression and cancer development has been considerably investigated


As a consequence of acute kidney injury (AKI), proximal tubular cells hyperrespond to endotoxin  by exaggerated renal Tnf-α Production. This LPS hyperresponsiveness is transcriptionally mediated. The epigenetic pathways that control these responses are unknown.We applied multiplex chromatin immunoprecipitation platform (Matrix ChIP) to explore epigenetic pathways that underlie endotoxin hyperresponsiveness in the setting of preceding unilateral renal ischemia/reperfusion (I/R) in mouse AKI model. Endotoxin exposure after I/R resulted in enhanced transcription, manifested by hyperresponsive recruitment of RNA polymerase II (Pol II) at the Tnf-α gene. At this locus, LPS but not I/R increased levels of Pol II C-terminal domain (CTD) phosho-serine2 &5 and induced dephosphorylation of the transcription-repressive histone H4 phospho-serine-1. In contrast, I/R but not LPS increased the transcription-permissive histone phosphorylation  at the Tnf-α gene. In agreement with these observations, I/R but not LPS increased activity of cognate kinases  at the Tnf-α locus. Cross-talk of histone phosphorylation and acetylation synergize to active gene expression. I/R and LPS increased histone acetylation. . Levels of some histone acetyltransferases at this gene (PCAF and MOF) were increased by I/R but not by LPS, while others were induced by either I/R or LPS and exhibited endotoxin hyperresponsive patterns . The adaptor protein 14-3-3 couples histone phosphorylation with acetylation, and tethers chromatin modifiers/transcription elongation factors to target genes. Both I/R and LPS increased levels of 14-3-3 and several chromatin/transcription modifiers  at the Tnf-α gene, all exhibiting endotoxin hyperresponsive recruitment patterns similar to Pol II.Our results suggest that I/R and LPS differentially trigger phosphorylation (Pol II and histone) and acetylation (histone) epigenetic pathways that interact at the Tnf-α gene to generate endotoxin hyperresponse in AKI. Classical studies from several laboratories, including Nath et al Epigenetic processes play a critical role in transcriptional regulation. These processes are driven by changes in covalent modifications of DNA and associated proteins, alterations in chromatin structure, and recruitment of a diversity of signal responsive transcription factors and enzymes. The molecular mechanisms controlling chromatin and transcription are remarkably conserved in organisms as diverse as yeast, flies and mammals Guided by other model systems To study mechanisms of renal endotoxin hyperresponsiveness, we chose an I/R model RT-PCR was used to assess Tnf-α mRNA levels. Cyclophilin A (CypA) gene was used for comparison http://genome.ucsc.edu/), thus it is appropriate to use this gene site for comparison with the Tnf-α gene. Injury-induced increases in Pol II levels were seen at both the 5′ and 3′ ends of the Tnf-α locus, indicating that transcriptional initiation plays a role in the transcriptional hyperresponse to endotoxin in this AKI model.Measurement of Pol II levels along a locus provides means to assess rates of transcription. Analysis of Pol II binding at the Tnf-α gene, estimated by antibodies to the CTD and N-terminal domains, revealed increased levels of the polymerase following either I/R or LPS treatment . Importa1S2P3T4S5P6S7), which are sites for modifications that define the state of the polymerase Mammalian Pol II CTD contains 52 heptapeptide repeats . I/R, but not LPS, also increased H3pSer10 at the CypA gene. The histone variant H3.3 is enriched at actively transcribed genes row 3). There was little or no change in H4pSer1 at the CypA site.Phosphorylation of H3 serine 10 (H3pSer10) is thought to act synergistically with histone acetylation in bringing p-TEFb elongation complex to transcribed genes Higher levels of H3K36m3 have been correlated with enhanced transcription elongation rates Analysis of H3K9m2, H3K9m3, H3K27m3 and H4K20m3 repressive marks The above results  togetherGiven that Pol II and histones are phosphorylated in our model, protein kinase cascades may play a prominent role in AKI- and LPS-induced transcription changes. Different classes of kinases are being discovered bound to genes in situMsk1/2, activated by Erk1/2, is one of the enzymes that phosphorylate serine 10 of histone H3 Next we tested binding of two other H3pSer10/H3.3pS31 kinases, Aurora and IκB kinase α (IKKα). While the Aurora A pattern was similar to H3pSer10 and H3.3pSer31 , the IKKI/R and LPS increased histone acetylation of all four histones at the Tnf-α gene . These PHistone H3 acetylation and phosphorylation were both induced at Tnf-α by renal injury &4, consiThe 14-3-3 family of proteins brings several chromatin factors to transcribed loci, including bromodomain containing factors involved in transcriptional elongation We have previously shown that BRG1 binds to the Tnf-α gene and activates its expression in renal ischemia BRG1 interacts with heterochromatin proteins (HP) that regulate chromatin structure We set out to define transcription and chromatin bases and factors to explain augmented expression of endotoxin-inducible Tnf-α gene in AKI. Herein, we uncovered previously unknown genomic events associated with renal Tnf-α gene transcription induced by endotoxin and I/R. PTMs, Pol II and chromatin modifiers that bind to Tnf-α gene can be approximately grouped into five synchronous recruitment patterns , and bacterial lipopeptides signal through TLR2 Ramesh et.al. has previously shown that treatment with nephrotoxic cisplatin but not LPS activated MAPK pathways in cultured mouse proximal tubule cells I/R but not LPS increased H3pSer10 and H3.3pSer31 . In agrerow 2 and row2). Different TLR4 agonists can differentially activate Tnf-α expression in the same cell We also found changes that were induced more strongly by LPS than by I/R including Pol II phosphorylation of CTD serine 2 and 5 . HistoneIn AKI, proximal tubules are a major source of cytokine production contributing to renal injury row 3) could also be also contributing to the endotoxin hyperresponse, We suggest that combination of histone phosphorylation and acetylation changes induced by I/R (histone/CTD phosphorylation and acetylation) and LPS (CTD phosphorylation histone acetylation and H4 serine-1 dephosphorylation)  and cause recruitment of Pol II to the Tnf-α promoter ylation) , 5–7 couTnf-α mRNA stability is tightly regulated Endotoxin hyperresponsiveness of cytokine production in AKI has been known for nearly a decade 3VO4, Na2MoO4 and β-glycerophosphate were from Sigma. The antibodies were commercially available and are listed in Bovine serum albumin (BSA), phosphate buffered saline (PBS), salmon sperm DNA, and protein A were from Sigma, and proteinase K was from Invitrogen. Matrix ChIP-MeDIP 96-well polypropylene plates were from Bioexpress. Formaldehyde, ethanol, NaCl, EDTA, Triton X-100, NP-40, Tris, leupeptin, PMSF, p-nitrophenyl phosphate, NaF, NaThe experimental protocol is shown in RNA was extracted from tissue fragments using Trizol reagent as per the manufacturer’s protocol. To synthesize cDNA, 400 ng of Trizol-extracted total RNA was reverse transcribed with 200 units MMLV reverse transcriptase (Invitrogen) and random hexamer primers in 20 µl reactions. RT reactions were diluted 100-fold prior to running qPCR The multiplex microplate Matrix ChIP method was previously described To acquire, store and analyze large data sets generated by the high throughput Matrix ChIP platform, we developed a novel graphical method (GraphGrid). Pair-wise statistically significant differences are represented by the size of a circle for each comparison made; p<0.05 by small circle, p<0.01 by large circle and no circle indicating non-significance. The tool automatically adjusts the computed pairwise p-value (significance t-test) using a Bonferroni correction factor –C.Additional information about the methods and data are available upon request.Figure S1Ischemia-reperfusion and endotoxin acute renal injury model and data analysis. A. Model. Mice were anesthetized and subjected to a midline abdominal incision under sterile conditions and, after 30 min of unilateral renal artery occlusion, the clamp was released . Twenty-four hours later, I/R injury mice received a tail vein injection of either lipopolysaccharide (LPS) or saline B.GraphGrid analysis. Results of statistical analysis of endotoxin and I/R responses are shown. Graph bars represent mean values±SEM. Solid yellow circles positioned at line intersections below the graph designate significant differences between given pairs of means. Bar above the circle represents one of the paired means. The second of the paired means is located above the very left end of horizontal line crossing the yellow circle. Five different paired statistical comparisons are done as shown with the numbered circles (right panel). C. Statistical analysis is done using Bonferroni correction. Statistical differences between two means  are shown by the size of the solid yellow circle. : p<0.05 by small circle, p<0.01 by large circle, and no circle indicating the differences are not statistically significant.(TIF)Click here for additional data file.Figure S2Matrix ChIP analysis of repressive histone lysine methylated marks at the TNF-α genes following unilateral kidney I/R and LPS injection. Sheared cross-linked renal cortex chromatin from mice were assayed using antibodies to histone H3 and H4 lysine methylated residues. ChIP DNA were analyzed at the Tnf-α first and last exon and CypA first exon in real-time PCR. Data represent mean ± SEM , expressed as fraction of input.(TIF)Click here for additional data file.Figure S3Synchronized epigenetic changes grouped into different types of responses to I/R and LPS treatment in AKI.Type I, I/R and LPS co-responsive/hyperresponsive; Type II, I/R only responsive; Type III, I/R and LPS co-responsive at saturation; and Type IV and V, LPS only responsive.(TIF)Click here for additional data file.Table S1Sequences of primers used in ChIP-qPCR primers.(DOCX)Click here for additional data file.Table S2Antibodies used in Matrix ChIP assays.(DOCX)Click here for additional data file.