Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes  were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc. Burkholderia mallei, the etiologic agent of glanders, is a Gram-negative bacillus that primarily infects horses, mules and donkeys, and occasionally humans. In nature, chronically infected horses are believed to be the only reservoir of this host-adapted pathogen B. mallei. Due to the high risk of aerosol infection and the historical use of this organism as a biological weapon, B. mallei is currently listed as a Tier 1 Select Agent by the Centers for Disease Control and Prevention (CDC) B. mallei is a facultative intracellular pathogen that can survive and replicate within the cytosol of eukaryotic cells. Several studies have shown that the organism persists within murine macrophage cell lines in vitro and can be observed within phagocytic cells in animal models of glanders B. mallei rapidly escapes from endocytic vacuoles, enters into the cytosol and uses actin-based motility to promote intra- and inter-cellular spread B. mallei also possesses the unique ability to induce host cell fusion resulting in multinucleated giant cell (MNGC) formation; a phenomenon that is observed both in vitro and in vivoB. mallei in animals have recently been shown to influence key processes during the interactions of the organism with eukaryotic cells. Included among these are an animal pathogen-like Type III secretion system (T3SSAP), the cluster 1 Type VI secretion system (T6SS-1) and the VirAG two-component regulatory system AP is essential for early vacuolar escape, T6SS-1 is necessary for optimal intracellular growth, robust actin-based motility, and MNGC formation in RAW 264.7 murine macrophages virG is required for actin tail formation by B. mallei in J774.2 cells B. mallei T3SSAP, T6SS-1 and VirAG mutants are unclear.Vibrio cholerae VasX protein, T6SS-secreted, non-VgrG effectors that act directly on eukaryotic targets remain largely unidentified Type VI secretion systems (T6SSs) are contact-dependent protein translocation machines expressed by many Gram-negative bacteria in vivo, but are repressed during routine growth in bacteriological culture media B. mallei T6SS-1 genes are transcribed poorly during the routine culture in rich medium, but are expressed following uptake by murine macrophages prior to vacuolar escape in vitro growth of B. mallei, Hcp1 becomes the major protein detected in culture supernatants T6SS expression is precisely regulated at the genetic level and typically involves two-component regulatory systems, transcriptional activator proteins, posttranscriptional modulators or posttranslational activation mechanisms in vitro while still supporting robust growth of B. mallei. We describe the use of minimal media formulations that facilitate Hcp1 production and demonstrate that virG is required for T6SS-1 gene expression during growth in this media. In addition, we show that divalent metals influence T6SS-1 gene expression and Hcp1 production. Since two closely related species, B. pseudomallei (etiologic agent of melioidosis) and B. thailandensis , also harbor homologous T6SS-1 gene clusters in their genomes In the present study, we sought to identify defined growth conditions that would induce T6SS-1 gene expression Escherichia coli, B. pseudomallei and B. thailandensis strains were routinely cultured at 37°C on Luria Bertani (LB) agar  or in LB broth . B. mallei strains were routinely cultured at 37°C on LB agar or in LB broth supplemented with 4% glycerol (LB4G). For growth of the Burkholderia strains in minimal media, the following formulations were used: Difco M9 Minimal Salts supplemented with 0.4% glucose (M9G); M9G supplemented with 0.5% (w/v) Bacto Casamino Acids (M9CG); or M9G supplemented with 0.5% Bacto Tryptone (M9TG). Metal-deplete M9TG media (M9TG-C) was prepared by supplementing M9G media with 0.5% Bacto Tryptone that had been chelated two times with 2.5% (w/v) Chelex 100. When appropriate, antibiotics were added at the following concentrations: 25 µg/ml kanamycin (Km), 50 µg/ml zeocin (Zeo), 15 µg/ml polymyxin B (Pm), and 100 µg/ml ampicillin (Ap) for E. coli; 5 µg/ml Km, 5 µg/ml Zeo for B. mallei; and 100 µg/ml Km for B. thailandensis. Bacterial stocks were maintained at −80°C as 20% glycerol suspensions. All studies utilizing viable B. mallei and B. pseudomallei were conducted in a CDC select agent-certified biosafety level 3 containment facility.The bacterial strains used in this study are shown in Unless stated otherwise, chemicals were purchased from Sigma-Aldrich. Zeocin was purchased from Invitrogen. Puratronic metals were obtained from Alfa Aesar as follows: Cu as Copper (II) sulfate, 99.999%; Fe as Iron (II) sulfate, 99.999%; Mg as Magnesium sulfate, 99.997%; Mn as Manganese (II) sulfate, 99.999%; Ni as Nickel (II) sulfate, 99.9985%; Zinc sulfate, 99.999%.E. coli TOP10 cells were transformed as per manufacturer’s instructions (Invitrogen). Oligonucleotide primers and dual-labeled probes were obtained from Integrated DNA Technologies or Sigma-Aldrich. DNA sequencing was performed by ACGT Inc. The plasmids and primers used in this study are shown in DNA manipulations were performed using standard methods. Restriction enzymes and T4 DNA Ligase (New England Biolabs) were used according to manufacturer’s instructions. PCR was performed using an Expand High Fidelity PCR System (Roche Applied Science) or GoTaq DNA Polymerase (Promega); 1 M Betaine was included in all polymerase chain reaction (PCR) procedures. PCR was performed using the following conditions: 97°C for 5 min; 30 cycles, each consisting of 97°C for 45 s, 55°C for 45 s, and 72°C for 3 min; a final extension step of 72°C for 10 min was included. PCR and restriction digested products were purified using a QIAquick Gel Extraction Kit (Qiagen). Plasmids were purified using a QIAprep Spin Miniprep Kit (Qiagen). Genomic DNA was purified using a Wizard Genomic DNA Purification kit (Promega). Chemically competent B. mallei SR1A were performed with the sacB-based allelic exchange vectors pEX18Zeo and pMo130 virG gene  from pGRV2-ΔA0746 HindIII and KpnI and cloned into pEX18Zeo digested with the same enzymes resulting in plasmid pEXZΔvirG. E. coli S17-1 was used to mobilize pEXZΔvirG or pMo130-ΔBPSS1498 into B. mallei SR1A via conjugative mating essentially as previously described 4, spotted onto LB4G agar plates and incubated for 8 h at 37°C. To select for transconjugants, mating mixtures were plated onto LB4G-Pm-Zeo or LB-Pm-Km agar and incubated at 37°C for 48 h. To select for sucrose resistant colonies, transconjugants were inoculated into LB4G broth without antibiotics and 10-fold dilutions of the overnight cultures were spread onto M9G plates containing 5% sucrose. Following incubation at 37°C for 48–72 h, sucrose resistant colonies were screened for loss of the Zeo or Km resistance markers by replica plating individual colonies onto LB4G and LB4G-Zeo or LB4G-Km. The resolved co-integrates were screened for the presence of the mutant alleles (ΔvirG or Δhcp1) by PCR using the appropriate primer pairs (virG-FH/virG-RKp or BPSS1498-1/BPSS1498-4).Gene replacement experiments with virAG expression experiments, pBHR2 and pvirAG were mobilized into B. mallei SR1A and its derivatives via conjugative mating as described above and transconjugants were selected for on LB4G-Pm-Km agar. Plasmid pBtvirAG was constructed by PCR amplifying virAG from B. thailandensis E264 genomic DNA using the BtvirAG-FKp/BtvirAG2-RXb primer pair and then cloning the resulting PCR product into the KpnI and XbaI sites of pBHR2. pBtvirAG was mobilized into B. thailandensis DW503 via conjugative mating essentially as described for B. mallei.For B. mallei Hcp1 with a C-terminal 6xHis-Tag (BmHcp1-6xHis), the hcp1 ORF (BMAA0742) was PCR amplified from B. mallei ATCC 23344 genomic DNA using the Bmhcp1-HisF1 and Bmhcp1-HisR1 primer pair. For expression of recombinant B. thailandensis Hcp1 with a C-terminal 6xHis-Tag (BtHcp1-6xHis), the hcp1 ORF (BTH_II0868) was PCR amplified from B. thailandensis E264 genomic DNA using the Bthcp1-HisF1 and Bthcp1-HisR1 primer pair. The resulting DNA fragments were digested with BsaI and cloned into pBAD/HisA digested with NcoI/HindIII resulting in pBmhcp1-His and pBthcp1-His, respectively.For expression of recombinant E. coli TOP10 (pBmhcp1-His) or TOP10 (pBthcp1-His) were grown in 500 ml of LB broth plus Ap at 37°C with aeration (200 rpm). When the cultures reached an OD600 of 0.8, protein expression was induced using 0.02% L-arabinose for 5 h. Bacteria were harvested by centrifugation , resuspended in B-PER (Pierce) plus 1 µl/ml Benzonase (Novagen) and incubated for 10 min at room temperature (RT). The insoluble material was pelleted by centrifugation  and resuspended in B-PER and incubated for 10 min at RT. The insoluble material was again pelleted, then resuspended in Solubilization Buffer (50 mM Tris (pH 8.0), 50 mM NaCl, 10 mM Imidazole and 0.2% Sarkosyl) and gently agitated for 60 min at RT. The remaining insoluble material was removed by centrifugation, the supernatant was filter sterilized and loaded onto a gravity fed Ni-NTA agarose (Qiagen) column. The flow through was collected and applied to the column a second time followed by washing of the column with Wash Buffer (50 mM Tris (pH 8.0), 300 mM NaCl and 40 mM Imidazole). Protein was eluted with Elution Buffer . Fractions were analyzed by SDS-PAGE and those containing BmHcp1-6xHis or BtHcp1-6xHis were pooled, concentrated, dialyzed against saline and stored at −20°C. Protein concentrations were determined using a BCA protein assay kit (Pierce).For purification of BmHcp1-6xHis or BtHcp1-6xHis, Purified BmHcp1-6xHis was used to raise BmHcp1-specific polyclonal antiserum in rats, and purified BtHcp1-6xHis was used to raise BtHcp1-specific polyclonal antiserum in rabbits, at Cocalico Biologicals, Inc.  using a standard protocol.All procedures involving animals were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols were approved by the Cocalico Biologicals, Inc. Animal Care and Use Committee.B. mallei, B. pseudomallei or B. thailandensis strains grown in various media  overnight (16–18 h) at 37°C with shaking (200 rpm). Briefly, 0.5–1 ml of bacterial culture was pelleted by centrifugation, the resulting pellets were resuspended in 0.2 ml of 1X Novex Tris-glycine SDS Sample Buffer (Invitrogen) with β-mercaptoethanol and then boiled for 10 min. For immunoblot analyses, the whole cell lysates (10 µl/lane) were separated on 12% Novex Tris-glycine gels (Invitrogen) and electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with StartingBlock T-20 Blocking Buffer (Thermo Scientific) for 20 min, incubated with a polyclonal rat anti-BmHcp1 or rabbit anti-BtHcp1 serum  for 1 h, and then incubated with either goat anti-rat or anti-rabbit IgG horse radish peroxidase conjugate antibodies (1/5000 dilution in TBS-T) for 1 h. Membranes were washed three times with TBS-T following each incubation step. Blots were visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific) and a ChemiDoc XRS imaging system (BioRad).Whole cell lysates were prepared from B. mallei or B. pseudomallei strains grown in various media  for 8 h at 37°C with shaking (200 rpm). RNA extracted from bacterial pellets using TRIzol Reagent (Invitrogen) was treated with RNase-free DNase I and purified using an RNeasy Miniprep Kit (Qiagen) according to the manufacturer’s instructions. SuperScript III (Invitrogen) was used to synthesize first-strand cDNA from 1 µg of RNA following manufacturer’s instructions. qRT-PCR primers and probes specific for target genes were designed using Primer Express 3.0 and are shown in rpoA. Fold changes were calculated using the ΔΔCT method. The housekeeping gene dnaK was used as a control for comparative purposes since expression of this gene has been shown to remain similar in various growth conditions For quantitation of transcript levels, RNA was purified from B. mallei T6SS-1 gene cluster (BMAA0744-0730) is part of the VirAG regulon and is co-regulated with the Burkholderia intracellular motility (bim) locus and the clan CA cysteine protease encoding gene, tssM  in order to monitor T6SS-1 expression during growth of the organism in various media. Utilizing whole cell lysates prepared from B. mallei SR1A (pvirAG) and BM0742 (pvirAG), Western immunoblot analysis confirmed the specificity of the polyclonal antiserum for Hcp1  or pvirAG (expresses virAG) grown in M9 minimal media formulations. Consistent with previous findings, Western immunoblot analysis confirmed that SR1A (pBHR2) grown in LB4G medium did not produce Hcp1  or tryptone (M9TG). When Hcp1 levels were assessed under these growth conditions, results demonstrated robust Hcp1 production during growth in M9CG, but much lower Hcp1 production during growth in M9TG  that induced Hcp1 production by B. mallei during in vitro growth.To confirm that non-plasmid bearing strains of  in M9TG . As expehcp1 transcript levels increase significantly in both B. mallei and B. pseudomallei when virAG is over-expressed in vitroB. mallei in minimal media conditions, we harvested RNA from SR1A grown in M9G, M9CG, M9TG and LB4G, and used qRT-PCR to determine hcp1 and virG transcript levels. As shown in hcp1 transcript levels. Consistent with the observations in hcp1 transcript when compared to growth in LB4G  in comparison those grown in LB4G  and assessed Hcp1 production. As shown in hcp1 and tssA mRNA levels in BM0746 in comparison to SR1A during growth in M9CG media using qRT-PCR. In agreement with the immunoblot analysis, results indicated that in the absence of VirG, hcp1 and tssA transcripts were undetectable  used in this study. While both are digests/hydrolysates of casein, casamino acids preparations are largely devoid of iron in comparison to tryptone. To determine if the presence of iron or other divalent metals in tryptone suppressed Hcp1 production, B. mallei was grown in minimal medium supplemented with Chelex-treated tryptone (M9TG-C) and Hcp1 production was assessed. Results of Western immunoblots showed that, although not as robust as in M9CG grown B. mallei, Hcp1 was detectable in M9TG-C grown cultures , or if the iron concentration was increased 2-fold (20 µM), Hcp1 was virtually undetectable  during growth of B. mallei in M9CG containing both iron and zinc (bimA transcript levels decreased (∼5-fold), but virG, tssM and dnaK transcript levels remained relatively unchanged in the presence of iron and zinc. Taken together, these findings indicated that divalent metals, particularly iron and zinc, play a role in negatively regulating T6SS-1 and bimA expression in B. mallei.The observation that cultures . Extendioduction . When B.tectable . In suppand zinc . In addiB. pseudomallei and B. thailandensis are closely related to B. mallei, and harbor homologous T6SS-1 gene clusters  virAG and bimBCADE as shown in B. mallei, B. pseudomallei T6SS-1 expression is induced following uptake by host cells and requires VirG B. mallei . To determine if Hcp1 production in B. pseudomallei was influenced by divalent metals, strains K96243 and 1026b were grown in M9CG alone or supplemented with various cations and Hcp1 levels were assessed. Results demonstrated that either iron or zinc negatively regulated Hcp1 production in both strains  and used it to monitor Hcp1 production during growth of the organism in LB, M9G, M9CG and M9TG. In contrast to the pathogenic species, B. thailandensis did not produce Hcp1 under any of the media conditions tested  and VirAG virAG results in transcription of the T6SS-1 gene cluster as well as the production and export of Hcp1 virG is expressed during growth in M9CG media and is required for T6SS-1, bimA and tssM expression. Our results showed that virG transcript levels increased (∼32-fold) during growth of B. mallei in M9CG in comparison to LB4G. Analysis of a B. mallei ΔvirG mutant confirmed a critical role for VirG in the expression of T6SS-1 genes (tssA and hcp1) and tssM transcription, and to a lesser extent bimA transcription. These findings are comparable to those from a previous study reporting that the activation of T6SS-1 in B. pseudomallei was completely dependent on VirAG within the intracellular environment virAG in B. pseudomallei had no effect on T6SS-1 gene expression when bacteria were grown in RPMI medium. This discrepancy may be due to differences in the levels of T6SS-1 gene expression in RPMI in comparison to M9CG. It is conceivable that VirAG regulated genes may be transcribed at much higher levels during growth of the organisms in M9CG than in RPMI. Further studies will be necessary to address this issue. Taken together, however, these findings suggest that growth of B. mallei and B. pseudomallei in M9CG provides a signal(s) sensed by VirAG which induces its expression in vitro.Although the regulation of T6SSs varies widely from species to species, some common mechanisms that govern T6SS gene expression include quorum sensing, changes in temperature and pH, and two-component regulatory systems Edwardsiella species, T3SS and T6SS genes have been shown to be activated under conditions that mimic those of a phagosome  and repressed in the presence of high iron Pseudomonas aeruginosa and enteroaggregative Escherichia coli, expression of the H2-T6SS and sci1 T6SS gene clusters, respectively, is repressed in the presence of iron B. mallei, we made the observation that when M9G media was supplemented with tryptone (iron-replete) rather than casamino acids (iron-deplete), Hcp1 production was repressed. Extending upon this finding, we showed that iron and zinc negatively regulate Hcp1 production in both B. mallei and B. pseudomallei. Analysis of mRNA levels revealed that transcription of tssA, hcp1 and bimA was down-regulated in the presence of iron/zinc, but that tssM transcript was unchanged, indicating that not all VirAG regulated genes are metal regulated. While virG transcript levels remained relatively constant in both organisms in the presence of iron/zinc, virG transcript decreased ∼5-fold B. pseudomallei in the presence of iron alone. The reason for this result is currently unclear. Additional experiments will be necessary to investigate this phenomenon as well as to determine the levels and activation state of VirG under these growth conditions.Several recent studies have identified a variety of environmental cues that influence T6SS gene expression including the extracellular concentrations of iron, phosphate and magnesium B. mallei T6SS-1 is expressed within phagolysosomal compartments and with the findings in the current study demonstrating that iron and zinc limiting conditions induce expression of genes encoding this critical virulence factor Based on the results obtained herein, it appears that while VirAG positively regulates T6SS-1 expression, iron and zinc negatively regulate the expression of this important secretion system. Since vertebrate hosts sequester iron as a protective mechanism against bacterial infections, and pathogens often sense this iron depletion as a signal to express virulence genes, our findings are in keeping with previous studies demonstrating that T6SS-1 is expressed within host environments sci1 T6SS in enteroaggregative E. coli, the H2-T6SS in P. aeruginosa and, a T6SS in Edwardsiella tardaE. coli and P. aeruginosa, Fur negatively regulates T6SS expression in the presence of iron by binding to Fur boxes in the promoter regions of the gene clusters encoding these systems. The situation in E. tarda appears to be more complex, and it has recently been reported that in addition to directly suppressing T6SS, Fur also acts indirectly through unidentified downstream transcriptional regulators that interact with components of the PhoB regulon B. mallei ATCC 23344 and B. pseudomallei K96243 B. mallei/B. pseudomallei Fur proteins play a role in regulating T6SS-1 gene expression.The ferric uptake regulator protein Fur has been shown to be involved in the regulation of expression of the B. thailandensis produces a number of homologs of known virulence determinants that are expressed by B. mallei and B. pseudomallei and is commonly used as a model system to study specific aspects related to the pathogenesis of glanders and melioidosis B. thailandensis can invade and replicate within host cells and exhibits a similar intracellular lifestyle as the pathogens B. thailandensis T6SS-1 mutants are similar those displayed by B. mallei and B. pseudomallei T6SS-1 mutants, i.e. these strains do not stimulate MNGC formation and are attenuated in a mouse model of acute pneumonic B. thailandensis infection B. thailandensis activates T6SS-1 expression and results in Hcp1 production. Curiously, however, in contrast to B. mallei and B. pseudomallei, growth of B. thailandensis in M9G and M9CG media did not induce Hcp1 production. These findings suggest that the regulatory mechanisms controlling T6SS-1 expression in B. thailandensis may differ from those used by the pathogens. Although VirAG plays a role in regulating T6SS-1, it appears that under the conditions tested, divalent metals do not. Whether B. thailandensis is an appropriate surrogate for studying the regulation of this virulence-associated T6SS requires additional investigation. It is tempting to speculate that differences in the regulatory mechanisms governing virulence factor expression in these three Burkholderia species may account, in part, for the differences in their pathogenicity.in vitro growth conditions that induce Hcp1 production in B. mallei and B. pseudomallei. We demonstrated that virG is expressed under these conditions and is required for T6SS-1 gene expression. In addition, we showed that iron and zinc down-regulate tssA, hcp1 and bimA expression. Other genes  under the control of VirAG did not appear to be as tightly regulated by these metals. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc. The regulation of T6SS-1 appears to be complex and may involve VirG-independent mechanisms that have not yet been identified. Nevertheless, the media conditions described in this study will facilitate additional investigations aimed at characterizing the functions of these important intracellular virulence factors at a molecular level and will be useful for identifying in vivo expressed antigens for use in the development of glanders and melioidosis diagnostics and vaccines.In summary, we have identified


While there is much information about the burden of influenza A(H1N1)pdm09 in North America, little data exist on its burden in South America.During April to December 2009, we actively searched for persons with severe acute respiratory infection and influenza-like illness (ILI) in three sentinel cities. A proportion of case-patients provided swabs for influenza testing. We estimated the number of case-patients that would have tested positive for influenza by multiplying the number of untested case-patients by the proportion who tested positive. We estimated rates by dividing the estimated number of case-patients by the census population after adjusting for the proportion of case-patients with missing illness onset information and ILI case-patients who visited physicians multiple times for one illness event.We estimated that the influenza A(H1N1)pdm09 mortality rate per 100,000 person-years (py) ranged from 1.5 among persons aged 5–44 years to 5.6 among persons aged ≥65 years. A(H1N1)pdm09 hospitalization rates per 100,000 py ranged between 26.9 among children aged <5 years to 41.8 among persons aged ≥65 years. Influenza A(H1N1)pdm09 ILI rates per 100 py ranged between 1.6 among children aged <5 to 17.1 among persons aged 45–64 years. While 9 (53%) of 17 influenza A(H1N1)pdm09 decedents with available data had obesity and 7 (17%) of 40 had diabetes, less than 4% of surviving influenza A(H1N1)pdm09 case-patients had these pre-existing conditions (p≤0.001).Influenza A(H1N1)pdm09 caused a similar burden of disease in Argentina as in other countries. Such disease burden suggests the potential value of timely influenza vaccinations. Early during the 2009 pandemic, the number of deaths attributed to influenza A(H1N1)pdm09 in Argentina pdm09 who died as a result of their illness may have been similar to that elsewhere. In order to explore whether Argentina's influenza A(H1N1)pdm09 burden was higher or similar to the burden documented elsewhere, we use active facility-based influenza surveillance and health utilization surveys from three cities in Argentina to estimate rates of influenza A(H1N1)pdm09-associated mortality, hospitalization, and influenza-like illnesses.This study was conducted in three cities in Argentina, Mar de Plata , Tucumán  , and Santa Fe  that comprised 6% of the total population of 40 million persons in Argentina. During April–December 2009 , surveillance staff actively searched city and hospital ledgers for all decedents with a history, in the previous two-weeks, of influenza-like illness (ILI), defined as persons with sudden onset fever [≥38°C], with cough or sore throat For each SARI and ILI case patient, staff recorded the person's age; sex; date of illness onset; history of asthma or chronic obstructive pulmonary disease, diabetes, obesity, pregnancy; and survival status. As part of clinical care, a proportion of these case-patients provided both nasal and throat swabs for influenza testing during the course of their acute illness We conducted door to door cross-sectional surveys in each of the sentinel city populations during May–November 2010, the seasonal influenza epidemic months in Argentina, to determine if household members had developed ILI, if they had sought care, and the proportion who visited a physician multiple times for a single illness event . We assumed that the proportion of case-patients visiting physicians multiple times were similar during 2009 and 2010 and used bootstrapping to determine the 95% confidence interval for this proportion. We also estimated the proportion of persons in the population who were pregnant, obese, diabetic, asthmatic, or had chronic obstructive pulmonary disease and developed ILI.For each case-patient  of a particular age group, we estimated the number of case-patients associated with influenza A(H1N1)pdm09 illness each week by adding case-patients which tested positive for influenza A(H1N1)pdm09 to the number of untested case-patients who may have tested positive for influenza A(H1N1)pdm09 if a respiratory sample had been obtained . We obtaWe adjusted each numerator by the proportion of persons without information about their date of illness onset (i.e. epidemiologic week of illness) and, in the case of ILI case-patients, for the proportion of persons who sought care multiple times per illness event as estimated by the health utilization surveys . Next, wThe research protocol was reviewed and approved by the Argentina Ministry of Health and the Argentina office of the Pan American Health Organization. Health authorities strived to maintain case-patient confidentiality by protecting data elements that help third parties identify them. Health utilization survey participants provided verbal informed consent prior to participation.Investigators identified 108 decedents with a history of ILI during the study period Table 1 Investigators identified 1,622 SARI case-patients Table 2 Clinicians at the three sentinel cities reported 101,179 ILI cases to the national obligatory surveillance system when physicians submitted samples for respiratory virus testing Table 3.Staff interviewed 14,535 households with 22,066 household members (mean 1.5 persons per household). Seven hundred fifty-two (3.4%) of the 22,066 household members reported a history of ILI during the month before the interview. The ILI case patients had a median age of 20 years (IQR 7–40 years) and 339 (45%) of 752 were male Table 4.We estimated that the influenza A(H1N1)pdm09-associated ILI rates per 100 py were 1.6 (95% CI 3.8–0.32) among children aged <5 years, 15.0 (95% CI 26.6–5.7) among persons aged 5–44 years, 17.1 (95% CI 42.8–1.7) among persons aged 45–64, and 7.1 (95% CI 9.5–5.2) among persons aged ≥65 years Table 3.Decedents were more likely than other case-patients to be pregnant , obese , or diabetic . While 9 (53%) of 17 influenza A(H1N1)pdm09 decedents with available data had obesity and 7 (17%) of 40 had diabetes, less than 4% of surviving influenza A(H1N1)pdm09 case-patients had these pre-existing conditions (p≤0.001).Our findings suggest that influenza A(H1N1)pdm09 caused a significant burden of disease in Argentina during 2009. If we assume that influenza activity was similar throughout country, we could multiply the age specific influenza-associated rates from Santa Fe, Tucumán, and Mar del Plata and their 95% confidence intervals by the census population of Argentina  to estimate that approximately 1,300,000  persons visited clinicians for ILI, 10,000  were hospitalized, and 990 died  throughout the country as a result of the 2009 pandemic.Our mortality pandemic rates were more conservative than those estimated using linear models of 2009 Argentina pneumonia and influenza mortality data  a method that is not well suited to differentiate the impact of influenza from that or RSV Influenza A(H1N1)pdm09 mortality and hospitalization rates were also similar to those of seasonal influenza Influenza A(H1N1)pdm09-associated hospitalizations were similar to those reported in Australia during 2009  A higher proportion of influenza decedents were pregnant, obese, or diabetic when compared with surviving ILI case-patients Our study found that the proportion of decedents with positive 2009 H1N1 samples (66%) was significantly higher than that of hospitalized SARI case-patients (42%) and ILI case-patients (25%). Such a finding suggests that while influenza was identified in a fraction of ILI case-patients This study had several important limitations. We assumed that after accounting for case-definition, age-group, and epidemiologic week, the proportion of tested and untested case-patients with influenza A(H1N1)pdm09 was likely similar. This may be incorrect if physicians were more likely to test severely ill younger case-patients without pre-existing medical conditions, if the laboratory used one assay preferentially to test severely ill case-patients, and if the probability of testing positive for influenza A(H1N1)pdm09 was greater among severely ill case-patients. Although improbable, it is mathematically feasible that all un-tested case-patients had influenza A(H1N1)pdm09 infection, pdm09-associated ILI rates would have been 50/100py, the hospitalization rates 48/100,000py, and the mortality rates 3.3/100,000py). Conversely, all un-tested case-patients may have been infected with other pathogens and not influenza pdm09-associated ILI rates would have been 1/100py, the hospitalization rates 11/100,000py, and the mortality rates 2.0/100,000py). Last, we assumed that proportion of persons seeking care multiple times for ILI was similar during the 2010 and 2009 epidemic periods.Our study suggests that influenza A(H1N1)pdm09 burden in Argentina was similar to that elsewhere and caused a large number of deaths, hospitalizations, and cases of ILI. Indeed, the majority of decedents with a history of ILI identified during the pandemic tested positive for influenza A(H1N1)pdm09. Influenza-associated mortality and hospitalization rates were similar to those elsewhere and to those of Argentina during seasonal influenza epidemics. Such findings suggest that it may be prudent to examine interventions used during the pandemic to determine their potential value to prevent and mitigate Argentina's annual seasonal influenza burden.Appendix S1Equation for calculating the rates of influenza A(H1N1)pdm09-associated mortality among decedents with a history of influenza-like illness (ILI) at three sentinel cities(DOC)Click here for additional data file.


A novel 2009 swine-origin influenza A H1N1 virus (S-OIV H1N1) has been transmitted among humans worldwide. However, the pathogenesis of this virus in human airway epithelial cells and mammals is not well understood.in vitro and in vivo may have been mediated by different mechanisms.In this study, we showed that a 2009 A (H1N1) influenza virus strain, A/Beijing/501/2009, isolated from a human patient, caused typical influenza-like symptoms including weight loss, fluctuations in body temperature, and pulmonary pathological changes in ferrets. We demonstrated that the human lung adenocarcinoma epithelial cell line A549 was susceptible to infection and that the infected cells underwent apoptosis at 24 h post-infection. In contrast to the seasonal H1N1 influenza virus, the 2009 A (H1N1) influenza virus strain A/Beijing/501/2009 induced more cell death involving caspase-3-dependent apoptosis in A549 cells. Additionally, ferrets infected with the A/Beijing/501/2009 H1N1 virus strain exhibited increased body temperature, greater weight loss, and higher viral titers in the lungs. Therefore, the A/Beijing/501/2009 H1N1 isolate successfully infected the lungs of ferrets and caused more pathological lesions than the seasonal influenza virus. Our findings demonstrate that the difference in virulence of the 2009 pandemic H1N1 influenza virus and the seasonal H1N1 influenza virus in vitro and that the pathological alterations in the lungs of S-OIV H1N1-infected ferrets are much more severe.Our understanding of the pathogenesis of the 2009 A (H1N1) influenza virus infection in both humans and animals is broadened by our findings that apoptotic cell death is involved in the cytopathic effect observed  In April 2009, an outbreak of influenza in North American was found to be caused by a new swine-origin influenza A (H1N1) virus that has since become prevalent in human populations and has spread worldwide This 2009 pandemic H1N1 virus can cause human respiratory disease, but its pathogenesis remains poorly understood. In our previous studies, we showed that the S-OIV H1N1 A/Beijing/501/2009 virus replicated in a C57BL/6 mouse model with acute lung injury, and the mice exhibited immune responses mimicking human clinical disease Mustela putorius furo) are a suitable animal model for studying human influenza virus infections because they are susceptible to natural infection and can develop respiratory disease and lung pathology similar to humans with influenza virus infections Previous animal studies have indicated that the 2009 pandemic H1N1 is slightly more pathogenic than the contemporary seasonal H1N1 viruses All procedures were conducted under protocols approved by the Institute of Animal Care and Use Committee (ID: SYXK 2007-005) at AMMS, all facilities were accredited by the AMMS Animal Care and Ethics Committee, and guidelines for ferret housing, environment and comfort described in the Guide For The Care and Use of Laboratory Animals, National Research Council, were strictly adhered to. All infections and sample collections were performed under 5% isoflurane anaesthesia and all efforts were made to minimize suffering.Subject provided written informed consent for participation in the study.Patient: A 69-year-old male entered the 302 Military Hospital on 22 May 2009 with a high fever (37.9°C) that started 15 hours prior. He complained of diffuse pain predominantly in the lower abdomen, nausea, vomiting, runny nose, sore throat, coughing, weakness and fever blisters on his lips. A 5-ml sample of a saline wash of his throat was sent to the laboratory.3.5 TCID50 (50% tissue culture infective doses) in the presence of trypsin (1 µg/ml) and 101.5 TCID50 without trypsin. The virus strain was verified by the inhibition assay using anti-sera to A/California/07/2009 (1∶320) and A/Sichuan/01/2009 (1∶320).A 0.2-ml volume of the throat wash was inoculated into the allantoic cavities of 10-day-old SPF embryonated eggs and were incubated at 34°C for 72 h. The first two passages (three and five eggs) were negative, but on the third passage, three of five eggs showed hemagglutination titers of 1∶16, 1∶4, and 1∶32, respectively. Blind passages were performed using samples from a pool of infected allantoic fluids. MDCK cells (Madin-Darby canine kidney cells) were simultaneously infected with virus from the eggs, and positive results were confirmed by the hemagglutination of supernatants and a hemadsorption assay at 4°C. The virus titers were 1050) for each virus was determined by serial dilution of the virus in Madin-Darby canine kidney (MDCK) cells  and calculated by the method developed by Reed and Muench The influenza A H1N1 virus A/Beijing/501/2009 (BJ501) was isolated from a patient from Beijing in 2009 2. The primary antibodies anti-PARP and anti-caspase 3 were purchased from Cell Signaling Technology Company. The anti-β-actin antibody was purchased from Sigma-Aldrich.The human lung adenocarcinoma epithelial cell line A549 was purchased from ATCC and cultured in DMEM (Hyclone) supplemented with 10% FBS (Hyclone) and 100 U/ml penicillin/streptomycin at 37°C with 5% CO5 cells/ml. Influenza virus or allantoic fluid was added to the wells the next day. Each group was represented by triplicate wells. After incubating the samples for the indicated times, 20 µl of CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) was added to each well, and the samples were incubated at 37°C for another 2 h. The absorbance was then measured at 490 nm.Cells were seeded in 96-well plates at a density of 1×105 cells/ml in 12-well plates. H1N1 virus or an equal volume of allantoic fluid was added to the wells the next day, and the cells were incubated for another 24 h. Cells were lysed in lysis buffer (RIPA lysis buffer containing protease inhibitors), and proteins were denatured at 97°C for 10 min and analyzed by western blot.Cells were seeded at 1×105 cells/ml on cover slips in 24-well plates. One day later, virus infection was performed at an MOI of 3.0, and cells were incubated for another 24 h. Apoptotic cells were characterized by positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining following the manufacturer’s instructions . Briefly, cells were fixed with paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100. The nonspecific endogenous peroxidases were inactivated with 3% hydrogen peroxide in methanol. After the TdT reaction, dUTPs were visualized using HRP-conjugated antibody and 3,3′-diaminobenzidine (DAB).Cells were seeded at a density of 1×10TUNEL staining was also performed on the paraffin-embedded sections of ferret lung tissue. In short, after deparaffinization and rehydration, sections were digested with proteinase K at a concentration of 20 µg/ml for 15 minutes. The nonspecific endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol. After the TdT reaction, dUTPs were visualized using HRP-conjugated antibody and DAB. The slides were counterstained with haematoxylin. 100 random lung fields per group were captured at a 400× magnification and the percentage of TUNEL positive area was calculated by the Image Plus software.SiRNA against caspase-3 were purchased from Sangon Biotech (Shanghai) Co., Ltd. siRNA-1, UGGAUUAUCCUGAGAUGGGTT (nucleotides [nt’s] 351 to 369), and siRNA-2, AGUGAAGCAAAUCAGAAACTT (nt’s 2148 to 2166) 7 TCID50 (200 µl) of the BJ501, CA07 or seasonal H1N1 viruses, respectively. At days 3 and 7 post-infection, 3 ferrets per group were euthanized and the nasal turbinates, lungs, spleen, kidney, liver and brain were harvested for virological and pathological examination. In the virological examination, tissues were homogenized in MEM medium containing antibiotics to make a 10% w/v tissue homogenates for both the day 3 and day 7 post-infection groups. The supernatant of tissue homogenates obtained after low-speed centrifugation were titrated into 24- and 96-well culture plates containing MDCK cells, and the titers are expressed as log10TCID50/g tissue We used 10–12 week old female ferrets  that were tested negative for circulating influenza virus by the serological hemagglutination inhibition (HI) assay. The baseline body temperatures and body weights were measured prior to infection. Nine ferrets per group were anaesthetized by intramuscular injection of ketamine and xylazine  and intranasally inoculated with 10The lung tissues of infected ferrets were harvested and immediately fixed in 4% neutral formalin for 48 h and then embedded in paraffin. Ultrathin sections were obtained and stained with hematoxylin-eosin, and the histopathological alterations were determined using light microscopy. The number of infiltrating cells was counted in 100 microscopic fields for each group at a magnification of 1000 X.All data are shown as the means ± S.E.M. Measurements at single time points were analyzed using an ANOVA, and if they demonstrated significance, they were further analyzed by a two-tailed t-test. p<0.05 indicates statistical significance.A549 cells were infected with A/Beijing/501 H1N1, a virus strain that emerged in Beijing, and the resulting cytotoxicity was compared with the toxicity induced by the A/California/07/2009 H1N1 virus strain and a seasonal H1N1 strain. We used the human lung adenocarcinoma epithelial cell line A549 because these cells originate from the pulmonary alveoli and are sensitive to virus infection. The cells were infected with the three H1N1 strains at multiplicities of infection (MOIs) of 1, 3, and 10, and the cells were incubated at 37°C. The cell viability was measured using the MTT assay at 12 and 24 h post-infection. The cell viability did not decrease among any of the influenza H1N1-infected A549 cells at 12 h post-infection ; howeverTo elucidate the type of cell death induced during A/Beijing/501 H1N1 virus infection, we investigated whether A/Beijing/501 H1N1 virus infection induces apoptotic cell death. First, we analyzed hallmarks of apoptosis, PARP and caspase-3 activation, by immunoblot analysis. Twenty-four hours after infection, activated caspase-3 was only detected in A549 cells infected with A/Beijing/501 H1N1 virus at MOI 10 but not in the mock-infected control, seasonal H1N1-infected cells or A/California07 H1N1-infected cells. PARP, a death substrate that is required for caspase-3 activation, was detected as an 89-kDa, proteolytically cleaved fragment in all three H1N1 influenza virus-infected cells. With increasing MOIs, the PARP cleaved protein bands gradually increased in the A/Beijing/501 H1N1-infected groups. These results suggest that apoptosis contributed to the A/Beijing/501 H1N1-induced cell death, and the cell death was caspase-3 dependent .To further confirm our observation, we performed the TUNEL assay, which uses terminal deoxynucleotidyltransferase (TdT) to catalyze the addition of FITC-labeled dUTPs onto nicks in DNA 7 TCID50 of virus. The animals were monitored for clinical signs and were weighed daily as an indicator of the disease. Both the A/Beijing/501 H1N1 and the A/California/07 H1N1 viruses caused sneezing, ruffled fur, decreased appetite, and nasal discharge in the ferrets. The mean maximum weight loss was 10% for animals inoculated with the A/Beijing/501 H1N1 influenza virus at two days post-infection. The body weight of the ferrets infected with any of the 2009 A (H1N1) influenza viruses gradually returned to the original weight by 7 days post-infection. On the contrary, there was no significant weight loss observed in the seasonal H1N1 infected-ferrets  (3.85 versus 104.0 TCID50 per gram of tissue) and lungs (104.6 versus 104.3 TCID50 per gram of tissue) of inoculated ferrets, whereas the seasonal influenza virus was not detectable in the lungs. Seven days post-inoculation, all virus strains were detected at relatively low titers in the nasal turbinates, trachea and lungs. Interestingly, the A/Beijing/501 H1N1 strain was detected in brains of the animals (101.25 TCID50 per gram of tissue) at 3 days post-infection, and the virus was below the detection limit 7 days post-inoculation. No virus was detected in the liver, spleen, or kidneys of animals inoculated with any of the viruses at 3 or 7 days after inoculation. These results show that the 2009 A (H1N1) influenza virus can be cleared from the respiratory tract within 7 days post-infection.To evaluate the replication of the viruses in different tissues, three animals of each group were sacrificed at days 3 and 7 post-infection. Sections of the nasal turbinates, trachea, lungs, liver, spleen, kidney, and brain tissue were homogenized and used for the determination of virus titers by means of endpoint titration in MDCK cells. Among the tissue types, the virus replicated efficiently in the upper and lower respiratory tract of virus infected-ferrets and reached detectable titers at 3 days post-infection. The seasonal and 2009 A (H1N1) influenza viruses were both detectable in the nasal turbinates of inoculated ferrets at 3 days after inoculation, whereas the A/Beijing/501 H1N1 influenza virus- and A/California/07 H1N1 influenza virus-infected group yielded slightly higher virus titers (10ctively) . The A/BWe further analyzed caspase 3 and PARP activation in the lung tissues of ferrets by immunoblot analysis. At 3 dpi, activated caspase-3 was detected in A/Beijing501 H1N1-infected lung tissues of ferrets, but not in the CA07 H1N1-infected lung tissues . And TUNAs reported previously, the swine-origin 2009 A (H1N1) viruses cause more severe pathological lung damage than the seasonal strains Humans infected with the 2009 pandemic H1N1 suffer from fever, cough, sore throat, diarrhea, and vomiting Our study using a ferret model has demonstrated that the 2009 pandemic H1N1 strain causes more severe clinical signs and symptoms  and produces higher viral titers than does the seasonal H1N1 influenza virus.Programmed cell death, or apoptosis plays an important role in the pathogenesis of many infectious disease, including those caused by virus In the 2009 pandemic H1N1-infected groups, weight loss was more prominent, and the animals required a longer recovery period, which may have resulted from the tissue damage caused by profound cytokine production and broader tissue tropism of pandemic strains as reported by others in this field In conclusion, the more severe pathological signs in some humans or animals infected with the 2009 pandemic H1N1influenza virus compared to the seasonal H1N1 influenza virus appear to be attributed to the severe apoptotic cell death in human airway epithelial cells and the ability of the virus to replicate in the lungs of infected ferrets.Figure S150 in MDCK cells.The growth kinetics of seasonal H1N1, A/Beijing/501 H1N1 and A/CA/07 H1N1 virus in A549 cells. A549 cells were inoculated with 10 MOI of virus. At the indicated times, cells were collected and virus titers were determined by TCID(TIF)Click here for additional data file.Figure S2A/Beijing/501 induces apoptosis in the lung tissue of ferrets. (A)Western blot analysis of CA07 and BJ501 H1N1-infected lung tissue of ferrets with anti-caspase 3, anti-PARP and anti-β-actin antibodies at 3 dpi. (B–C) Ferrets were infected with seasonal H1N1, A/Beijing/501 H1N1 and A/CA/07 H1N1 influenza viruses. At 3 and 7 days post-infection, the lung tissues of ferrets were examined by TUNEL assay and statistical analysis of relative proportion of TUNEL positive cells. For quantification, 100 random lung fields per group were captured at a 400× magnification and the percentage of TUNEL positive area was calculated by the Image Plus software. **p<0.001(TIF)Click here for additional data file.


All seven hot spot mutp53 proteins  were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition  TP53 gene by point mutations is a common event in human cancers  Inactivation of the in vivoThe p53 protein displays classical features of a sequence-specific transcriptional factor, including a transactivation domain, a sequence-specific DNA binding core domain  that plays a crucial role in recognition of the p53 target sites (p53CON), and an oligomerization domain (aa ∼325–∼356). In addition, p53 is unique due to a second autonomous DNA binding site at the extreme C-terminus  in vitro recognition of certain non-B DNA structures. For example, four-way junctions, hairpins, G-quadruplexes and structures formed by CTG.CAG trinucleotide repeats are strongly bound by G245S More than 80% of the known cancer-associated p53 mutations are located within the core domain, where six hot spots  represent about 40% of all p53 mutations MSP/MST1CD95/Fas/Apo1ID2EGR1ID4Insensitivity to drugs, resistance to apoptosis, enhanced cell proliferation and/or migration, increased chromosomal instability and nonhomologous recombination are attributed to all hot spot mutp53 proteins, such as mutp53GOFs. Proposed multiple mechanisms include transcriptional and nontranscriptional activities: a) physical interaction with p53 family members p63 and p73; b) interaction with and recruitment by other transcription factors to their consensus binding sites ; c) physical interaction with other cellular proteins  and d) direct interaction with structure-specific (non-B DNA secondary structures) and sequence-specific DNA elements or chromatin landscape Formation of non-B DNA secondary structures is facilitated under favorable conditions (e.g. negative supercoiling and stabilization by bound protein) in vitro binding to supercoiled plasmid DNA (scDNA) as a DNA substrate mimicking some conformational and topological features of DNA in cells. It was shown Our previous studies had focused on the investigation of wtp53 in vitro in detail using purified mutp53 proteins , extracts from cancer cell lines and in cells by ChIP. Similarly to wtp53, we observed mutp53 preference for scDNA with more negative superhelix density. In order to confirm these phenomena in cells, we analyzed mutp53 dependent repression of selected target genes in H1299 and Saos2. We demonstrated that DNA supercoiling strongly enhanced the level of mutp53-mediated repression of the BAX and MSP/MST1 promoters.In this study, we have analyzed for the first time the interaction of seven hot spot mutp53 proteins  with supercoiled, linear and relaxed circular DNA of plasmids lacking or containing p53CON or mutp53 binding sites (detected by chromatin immunoprecipitation (ChIP)). SCS-binding of mutp53 proteins has been tested AGACATGCCTAGACATGCCTCTCACTGATGTGTAGCGGTGCT, MSPR: TGTCCAACAGAGTAACCATTAGCC) and cloned into the EcoRV site in pBSK. Plasmids pAA3, pAB10, pAA12 are derivates of the pCRII plasmid containing repetitive sequences - mutp53BSs (SmaI restriction enzyme (Takara) was used for linearization of pBSK (linBSK), pPGM1 (linPGM1) and pMSP (linMSP); similarly ScaI (Takara) was used for linearization of pCRII based plasmid DNAs. Relaxed DNA (relDNA) was prepared with topoisomerase I (Takara) according to a protocol described previously Plasmids (pT7-7) encoding full length human wtp53  and CΔ30-p53 (aa 1–363) were kindly provided by C. Midgley utp53BSs  describeThe p53 proteins were purified according to a protocol described previously y) on the amount of p53 proteins . Mean values of three independent experiments were plotted in the graph.ScDNA  or linDNA  were mixed with p53 proteins at p53 tetramer/DNA molar ratios between 0.25–20 and incubated in binding buffer  for 30 min either on ice or 25°C to reach equilibrium as described previously A mix of scDNA (native superhelix density) and linDNA in equimolar ratio were incubated with p53 proteins at p53 tetramer/DNA molar ratios between 1–20 in binding buffer for 30 min either on ice or 25°C to reach equilibrium as described previously P53 proteins were preincubated with affinity purified mouse mAbs (50–300 ng) at 25°C for 20 min and incubation continued with addition of mixture of sc/linDNA  or scDNA (200 ng) for 15 min on ice or at 25°C Proteins from agarose gels were blotted onto a nitrocellulose transfer membrane  and p53 was detected by primary mouse monoclonal antibody DO1. Details of the procedures are described in The Ab-p53-DNA complexes were prepared by mixing the DO1 or anti-GST antibody (400 ng) with the purified protein (50 ng) or whole cell lysate (15–30 µg) in binding buffer , followed by 20 min incubation on ice. Then, 300 ng of scDNA (pMSP) and/or the same amount of the linDNAs were mixed with the given immune complexes and incubated in the binding buffer for 30 min on ice. Magnetic beads (15 µl of suspension per sample) coated with protein G , were added to Ab-p53-DNA complexes after washing in binding buffer and incubated with the beads for 30 min at 10°C while shaking mildly. Beads were separated from the assay using a magnetic particle concentrator. Finally, after triplicate washing in binding buffer, the DNA was released from the beads by heating at 65°C in 15 µl of 1.0% SDS for 5 min and analyzed by agarose gel electrophoresis. The electrophoresis conditions  were used for separation of bound DNA. In these conditions scDNA migrates faster than linDNA.2. The luciferase reporter construct containing the mutp53 recognition site MSP/MST1 Human Saos2  osteosarcoma cells and the human non-small lung carcinoma cell line H1299 , both p53 null cell lines, were grown in DMEM medium supplemented with 5% FBS and penicillin/streptomycin (Gibco). All cultures were incubated at 37°C with 5% COT-Rex is a Tet-regulated mammalian expression system based on the binding of tetracycline or doxocycline to a Tet repressor (TR) resulting in promoter activation of the appropriate Tet-inducible gene CTTTATGTTTTTGGCGTCTTCC; RV3: CTA GCAAAATAGGCTGTCCC) and primers targeting MSP/MST1 site in pMSP plasmid (BT7: GCGCGTAATACGACTCACTA and MSP: CTCACTGATGTGTAGCGGTGCT). For quantitative analysis, PCR was carried out for 25 cycles.H1299 cells (10 cm dish) transfected with plasmids pGL3-MSP and pMSP (2 µg of each) in sc and lin forms and p53 expression vector  were crosslinked with formaldehyde and subjected to chromatin immunoprecipitation (ChiP) assays as previously described with the following modifications 5) were transfected using Effectine (Qiagen) or Lipofectamine (Invitrogene) according to the manufacturer’s instructions at 80% confluence. When appropriate, 500 ng of the p53 expression vector or empty vector pCDNA3.1 were used. In the stress condition cells were exposed to 0.1 µM doxorubicin for 16 h −ΔΔCT algorithm. The following primer sets were used:H1299 and Saos2 cells (2×10BAX-F (GCCCTTTTGCTTCAGGGTTT)+BAX-R (TCCAATGTCCAGCCCATGAT)GAPDH-R (AAGGTCATCCCTGAGCTGAA)+GAPDH-R (CCCCTCTTCAAGGGGTCTAC)Q-MSP-F (ACAAGCCGCAGTTCACGTTT)+Q-MSP-R (TCTCCTCCAGTTGTGCATGC)HPRT1-Q1 (TGACACTGGCAAAACAATGCA)+HPRT1-Q2 (GGTCCTTTTCACCAGCAAGCT).In order to analyze the DNA binding properties of mutant p53 proteins, we investigated their interaction with scDNA at native superhelix density  by agarose gel electrophoresis and immunoblotting techniques. Full length hot spot mutp53 proteins  , S3. UndThis result indicates that in the absence of the CTDBS hot spot mutp53 proteins R175H, R273H and R248W have lost the ability to selectively recognize scDNA.Several mutp53 binding sites were identified by p53 specific chromatin imunoprecipitation in different cell systems for some of mutp53 target genes as shown for some representative examples in To examine the role of DNA topology in mutp53 recognition of different mutp53BSs we analyzed the interaction of mutp53 proteins with sc and lin forms of mutp53BSs. We observed differences in the recognition of the sc form of mutp53BS-MSP  into H1299 cells together with mutp53 expression vectors (pCDNA3.1-G245S or R273H). Binding of G245S to scpGL3-MSP/linMSP and R273H to scMSP/relpGL3-MSP was studied by ChIP assay performed after 24 h with DO1 and control antibody (whole mouse IgG). Significant preference for the sc form of MSP/MST1 site was observed for both G245S , lane 2 To analyze, whether the DNA topology of a p53 binding site (p53BS) has an effect on (mut and wt) p53-driven transcription, luciferase reporter assays were performed using supercoiled, relaxed or linearized reporter vectors, pGL3-BAX, pGL3-MDM2 and pGL3-MSP. Equal amounts of the described reporter vectors were transfected, either in their sc, lin (not shown) or rel forms, together with plasmid pRLSV40  and p53 expression plasmids. At first, we investigated the effect of DNA topology on mutp53 repression of the BAX promoter region, as described for hot spot mutp53 proteins by Expression of mutp53  resulted in the repression of the sc form of the pGL3-BAX reporter, compared to vector and in accordance with findings obtained earlier Finally, sc and rel forms of the MSP/MST1 binding site in the pGL3 promoter vector were transfected into Saos2 cells and luciferase assays were performed under the same condition as described above for pGL3-BAX. The strongest repression of the pGL3-MSP reporter was observed for its sc form upon R175H and R273H expression .BAXMSP/MST1 gene expression, induced by wtp53 and mutp53, were compared against the level of mock transfection (pCDNA empty vector); both unstressed . Cells were transfected with vectors bearing coding sequences for wtp53, particular mutp53s or a pCDNA empty vector in conditions similar to the luciferase reporter assays. Alterations in stressed  and doxostressed  were mea99 cells  and S7B.99 cells  and with99 cells , S7B. Doed cells  and S7A.ed cells , S7 at led cells . MSP/MSTWe have shown that DNA topology influences the mutp53-driven repression on the system of BAX and MSP/MST1 promoters, regulated by mutp53s in H1299 and Saos2 cells.In our previous studies we described the selective binding of the tumor suppressor p53 to topologically constrained DNA in vitro and in cells.Considering these facts, we investigated the ability of hot spot mutp53 proteins to bind selectively scDNA. Furthermore, we studied the influence of DNA topology on mutp53BS recognition both Here we show for the first time that hot spot mutp53 proteins  are inherently capable of structure-selective binding to supercoiled plasmid DNA independently of the sequence context by EMSA and MBIP methods. This binding varies somewhat depending on the p53 mutation, but changes dramatically with the topological state (superhelix density) of the DNA. We observed slight differences among the p53 mutants in scDNA binding, without any apparent correlation with the type of mutation  were identified in cells . Our luciferase assay data show that the strongest mutp53 protein R273H repression was for the sc form of the reporter containing the mutp53-MSP binding site. These data correlated with in vitro and in cells binding studies. R273H, the best binder to MSP/MST1 binding site in scDNA, is also a good repressor of the MSP/MST1 reporter. Furthermore, in ex vivo binding assays, R273H from several cancer cell lines displayed significant SCS-binding also in an in cells setting. In case of the BAX promotor, transrepression by hot spot mutp53s has been observed by different laboratories using the luciferase assay BAX is more complicated in vivo, where many other transcription factors are involved . Interestingly, in vitro mutp53 R273H interaction with ChIP-sequences, intronic regions of regulated genes, is positively influenced by the presentation of the genomic elements in supercoiled form to a significant extent EGFR, MDR-1 and c-myc (reviewed in MSP/MST1 and BAX genes. Based on these recent findings we hypothesize that abundant (overexpressed) mutp53 in cells can interact stably in vivo with already existing non-B DNA structures formed within chromatin filaments due to DNA supercoiling, specific DNA sequences  and combinations of other transcription factors. The bound proteins can physically cover the DNA by forming higher-order oligomeric forms  retain the ability of wtp53 to bind with high selectivity to scDNA. Moreover, preferential binding to more tightly packed DNA molecules containing mutp53 MSP/MST1 binding site was detected also in cells by chromatin immunoprecipitation techniques. We hypothesize that DNA topology is important for mutp53 recognition of cognate sites and may significantly contribute to repression of mutant p53 target genes.Figure S1Purity of p53 and CΔ30-p53 proteins. Purity of recombinant full length p53 proteins (lanes 1–7) and p53CΔ30 forms (lanes 8–11) in quantities ranging between approximately 750 and 1000 ng, is demonstrated on Coomassie blue stained 12.5% SDS-polyacrylamide gels. Standard polypeptide markers (lanes M) and 1 µg of BSA were used as controls.(TIF)Click here for additional data file.Figure S2Comparison of mutp53 binding to scDNA and linDNA by EMSA and binding of baculoviral p53 protein G245Si to DNA .A–C) Mutp53 proteins G245S, R273H and wtp53 were bound to scDNA , linDNA  and to linDNA with p53CON sequence  in p53/DNA molar ratios as indicated in the figure for 20 min at 4°C before separation on 1% 0.33x TBE agarose gel. Graphs on the right were plotted on the basis of Et-Br stained agarose gels; free DNA substrates labeled with arrows were measured by densitometry. Graphs show the evaluation of p53-DNA binding as the dependence of % of bound DNA (axis y) on the amount of input of p53 proteins in the reaction . Bound DNA (%) were calculated as % of decrease of free DNA after binding of p53 in comparison to input DNAs . D) Binding of mutp53i protein isolated from insect cells infected recombinant baculovirus. G245Si was incubated with 200 ng of scDNA , 200 ng linDNA , both  at molar ratios of p53/DNA 0.5–2, as indicated in the figure, for 20 min at 4°C before separation on 1.3% 0.33x TBE agarose gel. Binding of G245Si to DNA was detected by ethidium bromide staining (linDNA migrated faster than scDNA).(TIF)Click here for additional data file.Figure S3Binding of CΔ30-R273H to scDNA, linDNA, and their mix in sc/lin competition assays. A, B) Effect of CΔ30 deletion on p53 protein binding to scBSK and linBSK (similarly to C) Loss of SCS-binding of CΔ30-R273H. Binding of CΔ30-R273H to scDNA in the presence of linDNA  at molar ratios of p53/DNA, as indicated bellow, was detected in the 1.3% 0.33x TBE agarose gel by ethidium bromide staining (linDNA migrated faster than scDNA).(TIF)Click here for additional data file.Figure S4Influence of C-terminal antibodies on binding of R273H to scDNA and sc/lin competition. A) R273H (100 ng) was pre-incubated with 50–200 ng of Bp5310.1 (lanes 3–6), PAb421 (lanes 8–11) and ICA9 (lanes 13–16) at 25°C; scDNA binding expreriment (200 ng pBSK) was performed at RT for 5 h in 1% 0.33x TBE agarose gel. Controls: DNA (lane 1); complex without Ab . B) Similarly R273H (100 ng) was pre-incubated with 50–300 ng of Bp5310.1 (lanes 4–8), PAb421 (lanes 9–12) and ICA9 (lanes 13–16) at 25°C; sc/lin DNA binding expreriment (200 ng pBSK and 200 ng pBSK/SmaI) was performed at 4°C for 8 h in 1.5% 0.33x TBE agarose gel. Complexes of Ab-p53-linDNA were observed with Bp5310.1 (lanes 5–8) and PAb421 (lanes 10–12). Controls: DNAs ; p53+sc/lin mixture (lane 4). C) Evaluation of binding of R23H to MSP/MST1 mutp53BS in competition assay from (TIF)Click here for additional data file.Figure S5Mutant p53 recognition of mutp53BS (repetitive ChIP sequences) in scDNA. Repetitive ChIP sequences scAA3, scAB10, scAA12 were isolated as R273H binding sites (Tab. S1) from U251 cells A) R273H binding to scpCRII (lanes 2–3), scAA3 (lanes 5–7), scAB10 (lanes 9–11) was compared at p53/DNA molar ratios of 5–10. B) Mutp53 proteins (R273H and R175H) and wtp53 bound scDNA form of scAA3 and scAA12 differently; binding to linear form was already shown A) and B) were the same as in C) Binding of R273H from H1299 lysate to scAB10 (lane 5), linAB10 (lane 6) and to scAB10/linAB10 mixture (lane 7) by MBIP assays. Lanes 2–4  were a control for input DNA . After DO1 immunoprecipitation DNAs were analyzed on 1% TAE gel (scDNA migrates faster than lin or oc forms). The same conditions as in (PDF)Click here for additional data file.Figure S6Influence of DNA topology on mutp53-driven trans-activation/repression measured by luciferase reporter assay. A) PCR control of DNA transfection for experiments on B, C) Dual Luciferase Assay showing influence of p53 proteins on gene promoters . H1299 cells were transiently transfected with p53 expression plasmids based on pCDNA3.1 or pCDNA3.1 alone (CMV) together with the reporter plasmids expressing the firefly luciferase gene under the transcriptional control of the indicated gene promoters and a reference plasmid with the renilla gene under control of the SV40 promoter. Experiments were analyzed 16–20 h post transfection and carried out in triplicates and at least three independent times; standards deviations are indicated. Representative western blot analysis of p53 and actin was performed using 50 µg of samples. B) Effect of MDM2 promoter topology on p53 transcriptional regulation. Both wtp53 and G245S activated scMDM2 and relMDM2. But R248W and R273H effects on sc and rel form of pGL3-MDM2 reporter were moderate. C) Effects of wtp53 and mutp53 on vectors pGL3-basic, pGL3-promoter and repetitive ChIP sequences pGL3-AA12.(TIF)Click here for additional data file.Figure S7Levels of down-regulation of BAX and MSP/MST1 mRNA in H1299 and Saos-2 cells overexpressing Mutant p53s without stress and after doxorubicin treatment. H1299 and Saos2 cells were transfected by p53 constructs or empty vector in the same conditions as in A) and MSP/MST1 (B) were determined by quantitative real-time reverse transcription-PCR. BAX and MSP/MST1 values were normalized by GAPDH, HPRT1 or Actin. The values are the average of three biological independent experiments.(TIF)Click here for additional data file.Figure S8Non-B DNA structures in scDNA  predicted by computational methods and detected by S1 treatment. A)In silico analysis of MSP/MST1  B) The sequence of pBSK was analyzed using the UNAFold software package C) ScDNAs  were treated with S1 nuclease followed ScaI restrictase digestion (PDF)Click here for additional data file.Table S1Examples of mutant p53 binding sites identified by ChIP and confirmed by in vitro mup53 binding analysis, by luciferase assay or on the level of mutp53 target gene transcription.(DOC)Click here for additional data file.


If and how these two signal 3 cytokines compensate each other in T cell activation during different infections is so far unknown. Using CD8 T cells lacking receptors for IL-12, type I IFN or both, we show that the expansion of CD8 T cells depends on type I IFN (LCMV infection), type I IFN and IL-12 (Listeria and vesicular stomatitis virus infection) or is largely independent of the two cytokines (vaccinia virus infection). Furthermore, we show that CD8 T cells lacking IL-12 and type I IFN signals are impaired in cytokine production and cytolytic activity in the context of VSV and Listeria infection. These effector CD8 T cells fail to express KLRG1, thereby exhibiting a memory-like phenotype which correlated with lower expression of the transcription factor T-bet and higher expression of Eomes. This indicates that the variable interplay of both signal 3 cytokines is mandatory for cell fate decision of CD8 T cells in the context of different infections. Furthermore our results demonstrate that the pathogen-induced overall inflammatory milieu and not the antigen load and/or the quality of antigen presentation critically determine the signal 3 dependence of CD8 T cells.Signal 3 cytokines, such as IL-12 or type I IFN, support expansion and differentiation of CD8 T cells  Furthermore, both cytokines facilitated continued gene expression relevant for CD8 T cell differentiation by chromatin remodeling via histone acetylation In vivo, expansion and survival of CD8 T cells is strictly dependent on a direct type I IFN signal during lymphocytic choriomeningitis virus (LCMV) infection but less critical after vaccinia virus (VV) or Listeria monocytogenes infections Listeria infections, replaces type I IFN as third signal, however a mechanistic proof is still lacking Listeria infection but not after viral infections with LCMV, VSV or VV Activation of CD8 T cells depends on three signals: TCR engagement , costimulation  and an inflammatory stimulus  via cytokines such as interleukin 12 (IL-12) or type I interferons (type I IFN). Both signal 3 cytokines have been shown to support expansion and effector functions of CD8 T cells Listeria infection Listeria) using CD8 T cells with one defined antigen-specificity lacking receptors for IL-12, type I IFN, or both in an adoptive transfer system. Results reveal a complex pattern of signal 3 dependence of T cells for activation, expansion and cell fate decision in the different infections, with CD8 T cells being either largely independent or differentially dependent on one or both signal 3 cytokines. Although type I IFN can substitute IL-12 signals for expansion and effector functions in some infections, IL-12 predominantly determines high expression levels of the transcription factor T-bet, and therefore regulates the cell fate decision between SLEC and MPEC. Furthermore by using experimental approaches to separate antigen-specific signal 1 from the cytokine milieu, our results demonstrate that the pathogen-induced overall inflammatory milieu and not the antigen load and/or the quality of antigen presentation critically determines the signal 3 dependence of CD8 T cells.During the early phase of infection CD8 T cells differentiate into short-lived effector cells (SLEC) and memory-precursor effector cells (MPEC), also called effector and memory cytolytic T-lymphocytes (CTL). These effector T cell subpopulations can be distinguished according to their surface marker expression. SLEC (or effector CTL) express high levels of KLRG1 and low levels of CD127 Animal care and use was approved by the Regierungspraesidium Freiburg (G-08/93).tm1Jm) mice and IFN-α receptor deficient IFNARKO (B6.129S7-Ifnar1tm1Agt) from Jackson Laboratory or Dr. U. Kalinke (Hannover) C57BL/6 (B6) mice were obtained from Harlan Winkelmann, IL-12Rβ2-deficient (IL-12RKO) (Il12rb2GP) and recombinant vesicular stomatitis virus (rVSVGP) Listeria monocytogenes (rListeriaGP33) 6 PFU of rVVGP or rVSVGP, and 2×104 CFU of rListeriaGP33 i.v.LCMV WE and LCMV8.7, recombinant vaccinia virus (rVVb. P14 T cells that lack either the type I IFN receptor (P14.IFNARKO), the IL-12 receptor (P14.IL-12RKO) or both (P14.DOKO) were generated by crossing. Spleen cells from donor mice were purified using a negative CD8 purification kit (Miltenyi Biotec), according to the manufactureŕs instructions. Equal numbers ) of donor T cells were transferred i.v. into sex-matched B6 mice. After adoptive transfer of P14 T cells, recipient mice were infected with LCMV, rVVGP, rVSVGP or recombinant rListeriaGP33, all expressing the epitope gp33-41. P14 T cells were traced in recipients using anti-Thy1.1 monoclonal antibody.P14 T cells express a transgenic TCR specific for the LCMV glycoprotein epitope gp33-41 in the context of H-2 DFor BrdU incorporation experiments, recipient mice were treated with 2 mg BrdU i.p. at day 4 after infection.To expand CD8 T cells in H8 mice independently of an infection, P14.WT and P14.IFNARKO T cells were adoptively transferred into H8 mice. One day after transfer, recipient H8 mice were treated with 6 µg anti-CD40 antibody . Intracellular staining for T-bet and Eomes was done directly ex vivo. Staining for incorporated BrdU was done using the BrdU staining kit (BD) according to the manufactureŕs instructions.All antibodies were purchased from eBioscience. For analysis of intracellular cytokines, 10All flow cytometry was analyzed on a FACSCalibur or FACSCanto (BD).GP, rVSVGP or recombinant rListeriaGP33 as described. Infected mice were sacrificed 12, 24, 48, 72 and 96 hours after infection. Blood sera were obtained by centrifugation.For detection of the IL-12 and type I IFN levels, mice were infected with either LCMV WE, rVVIL-12 levels in sera and spleen homogenates were analysed using ELISA . Type I IFN levels were detected by VSV protection assay based on a protocol by Vogel et al. 51Cr release assay using gp33-41 peptide loaded EL-4 cells as target cells or adeno peptide loaded EL-4 cells as a control. Five days after infection with rVSVGP or rListeriaGP33, P14.WT, P14.IFNARKO, P14.IL-12RKO and P14.DOKO effectors were isolated from whole spleen cell suspensions using Thy1.1-PE antibody staining and anti-PE magnetic bead separation as described above. Effector and target cells were incubated together for 5 hours at 37°C. Duplicate wells were assayed for each E:T ratio starting with a ratio of 10∶1 (105 effector cells to 104 target cells) and % specific lysis was calculated.Cytolytic activity of transferred P14 T cells was determined in a standard Statistical analysis was determined by one-way ANOVA followed by Bonferroni’s multiple comparison post-test or Student’s unpaired t-test. P values p<0.01 was considered as significant.GP), recombinant vesicular stomatitis virus (rVSVGP) or recombinant Listeria monocytogenes (rListeriaGP33) all expressing the GP33 epitope.To examine the impact and possible compensatory effects of IL-12 and type I IFN as signal 3 to CD8 T cell responses in the context of infections, P14 T cells that lack either the type I IFN receptor (P14.IFNARKO), the IL-12 receptor (P14.IL-12RKO) or both (P14.DOKO) were adoptively transferred in B6 recipient mice. Thereafter recipient mice were infected with LCMV, recombinant vaccinia virus  to 3 (P14.IL-12RKO) compared to P14.WT T cells  in order to obtain enough cells for analysis. Since high input numbers of TCR transgenic T cells may influence T cell differentiation and potentially the dependence of T cells on signal 3, we performed transfers with more physiological P14 T cell numbers (104). Under low precursor frequency conditions, P14.WT and P14.DOKO T cells exhibited the same signal 3 dependence in the context of the infections used compared to our findings after high cell number transfer  .After VSV infection, CD8 T cells deficient in IL-12 signaling expanded comparable to wild-type T cells, whereas T cells that lack type I IFN or both cytokine signals were limited in their expansion . For a d numbers . In cont numbers  A. The r numbers , indicat P14.WT) . In addi P14.WT)  in the cin vitro. On a per-cell basis, P14.DOKO T cells tend to produce less IFN-γ compared to P14.WT T cells, as seen in a reduced MFI  and the induced local inflammatory milieu. To address this question in more detail we established two experimental approaches, in which the delivery of the antigen-specific signal 1 and the co-stimulatory signal 2 are separated from the pathogen-induced inflammatory milieu. To this end we chose the LCMV infection model, since CD8 T cell expansion is strictly dependent on type I IFN as signal 3. First we established a co-infection model using rVVIn a second experimental approach we separated antigen load and antigen presentation from the pathogen-induced inflammatory milieu in an even more radical way. We used H8 mice as recipient mice for P14 T cells , or neither type I IFN nor IL-12 (VV). Interestingly, during VSV infection, type I IFN substitutes IL-12 as third signal, whereas during Listeria infection both cytokines are needed for expansion of CD8 T cells. Similar results were reported by Obar et al. in the OT-1 T cell adoptive transfer system, with the difference that IL-12 could not fully compensate a lack in type I IFN signaling for T cell expansion during a VSV infection In this study we tested the hypothesis that type I IFN and IL-12, two signal 3 cytokines, compensate each other in supporting CD8 T cell activation in the context of different infections. Using CD8 T cells that lack either type I IFN receptors, IL-12 receptors or both in four different infections, we could show a complex pattern of the requirement for those two third signals. Our results indicate that the infection typical inflammatory milieu dictates whether the expansion of CD8 T cells depends on type I IFN (LCMV), type I IFN and IL-12 , infection with different recombinant VV and use of different application routes may have an impact on signal 3 dependence of T cell responses. However, in a recent study Obar et al demonstrated that OT-1 T cells also critically depend on both type I IFN and IL-12 for CD8 T cells expansion in the context of a Listeria infection It was postulated that the formation of memory T cells is regulated directly by IL-12 and type I IFN Listeria infection in vitroin vivo model indicates that mainly direct IL-12 signals determine the high expression of T-bet whereas type I IFN have a modulating effect, as CD8 T cells lacking IL-12 signals but not type I IFN signals exhibited diminished expression levels compared to wild-type cells. The impact of type I IFN on regulation of T-bet is only evident if T-bet expression levels between P14.IL-12RKO and P14.DOKO T cells are compared, showing that T-bet expression in P14.DOKO T cells was further reduced. Of note, Eomes expression levels are exclusively enhanced if both cytokines, IL-12 and type I IFN, are missing, although Eomes expression preferentially increased relative to T-bet already if only IL-12 signals were missing. It is tempting to speculate that T-bet expression regulates Eomes, with high levels of T-bet repressing Eomes expression. In addition, the overall expression levels of Eomes were lower after Listeria infection compared to VSV infection, a finding that might be explained by higher IL-12 levels induced after Listeria infection which is in accordance with previous studies demonstrating that IL-12 mediates repression of Eomes in vivo. During both, VSV and Listeria infection, T cells lacking type I IFN and IL-12 signals down-regulated T-bet and up-regulated Eomes, thereby showing a memory CTL phenotype, with high CD127 and low KLRG1 expression. In addition, these T cells were impaired in IFN-γ expression and exhibited reduced cytolytic activity. A recent study demonstrated that the early cytokine milieu induced by Listeria or VSV infection, differentially determines effector T cell differentiation into SLECs and MPECs. Additionally, these authors showed that strong inflammation and thus IL-12 production induced by CpG application promoted CD8 T cell differentiation into a SLEC phenotype during VSV infection. Interestingly, for the IL-12 independent SLEC differentiation of CD8 T cells during VSV infection the transcription factor Ebi3 was critically involved. It is tempting to speculate that IL-27 may be involved in the differentiation of CD8 T cell into a KLRG-1high/CD127low phenotype and regarded as an alternative signal 3 cytokine To determine the contribution of type I IFN and IL-12 to the transcriptional regulation in CD8 T cell differentiation, we analyzed the expression of two T-box transcription factors T-bet and Eomes. Both transcription factors are induced in effector CD8 T cells and regulate IFN-γ expression and cytolytic activity GP/LCMV8.7 and rVSVGP/LCMV8.7 we could demonstrate that the LCMV8.7-induced inflammatory milieu determines the signal 3 dependence of CD8 T cells. CD8 T cells deficient in type I IFN receptor were not able to expand in the context of a rVVGP or rVSVGP infection, when a LCMV typical inflammatory milieu is induced simultaneously. Of note, the LCMV8.7 isolate by itself is not recognized by P14 T cells, due to a mutation in the GP33 epitope and therefore will only determine the inflammatory milieu. Thus, in presence of a LCMV inflammatory milieu CD8 T cells are strictly dependent on a direct type I IFN signal for survival. This is supported by the fact that type I IFN receptor deficient CD8 T cells enter cell cycle and undergo cell divisions in vivo but are failing at clonal expansion Besides the differential impact of signal 3 cytokines on the cell fate decision of effector CD8 T cells, we would like to emphasize the direct role of a signal 3 for survival of effector T cell in the context of a given infection. To analyze signal 3 requirements for T cell survival in an acute infection in more detail, we established two independent experimental settings, in which the delivery of the antigen-specific signal 1 and the co-stimulatory signal 2 are separated from the pathogen-induced inflammatory milieu. First, using co-infection experiments with rVVTransfer of P14.WT and P14.IFNARKO T cells into H8 mice further demonstrated the role of signal 3 as a survival factor for effector T cell. H8 mice ubiquitously express the GP33 epitope and therefore antigen load and presentation is independent of an infection. After activation of APC by application of an agonistic anti-CD40 antibody, both P14 T cell populations expanded to the same extent. However, if anti-CD40 treatment was combined with a LCMV8.7 infection P14.IFNARKO T cells initially expanded but then dramatically crashed. Thus, in the context of a LCMV-induced inflammatory milieu even already expanded CD8 T cells are strictly dependent on a direct type I IFN signal for survival. We thus think that signal 3 cytokines may have an important role as a kind of fail-safe mechanism to ensure effector T cell survival in a given inflammatory milieu.in vivo. However, IL-12 signals seem to regulate transcription factor expression, thereby influencing the cell fate decision of CD8 T cells. Most significant, the relevance of IL-12 and type I IFN as signal 3 for T cell differentiation and survival is determined by the pathogen-induced inflammatory milieu. This knowledge of the modulatory capacity of signal 3 cytokines on CD8 T cells may have important implications for the improvement of classical vaccination strategies as well as for the development of new approaches in therapeutic vaccination, in persistent infection and cancer.In summary, our results help to clarify the requirements for signal 3 cytokines for CD8 T cell expansion, differentiation and survival in the context of different infections. We showed that type I IFN signals can partially compensate IL-12 signals in CD8 T cell expansion and cytolytic activity Figure S1Adoptive Transfer with low numbers of P14 T cells. 104 Thy1.1+ P14.WT, P14.IL-12RKO, P14.IFNARKO or P14.DOKO T cells were transferred into B6 mice (Thy1.2+) followed by infection with rVSVGP, rVVGP, LCMV or rListeriaGP33. Kinetics of P14.WT (•) and P14.IFNARKO, P14.IL12RKO and P14. DOKO (○). T cells gated on CD8+ T cells in the blood at indicated time points. Values are expressed as mean ± SEM (n = 3). Results are representative of three independent experiments.(TIF)Click here for additional data file.Figure S2Analysis of P14 T cell after VSV and Listeria infection. P14.WT, P14.IL-12RKO, P14.IFNARKO or P14.DOKO T cells were transferred into B6 mice followed by infection with A, rVSVGP or B, rListeriaGP33. Analysis of CD44 and CD62L expression on spleen cells at day 5 after infection is shown. Plots are gated on CD8, Thy1.1 (P14) cells. Expression of IL-2 and CD107a of P14 T cells after short time restimulation in vitro is indicated. Percentages of cytokine positive P14 T cells of total P14 T cells, as well as MFI of cytokine expression are depicted. Values are expressed as mean ± SEM (n = 3). * p<0,01 (Student́s unpaired t-test). Results are representative of three independent experiments.(TIF)Click here for additional data file.Figure S3Expansion of P14 T cells after i.v. or i.p. infection with rVVGP. 105 Thy1.1, P14.WT, P14.IL-12RKO, P14.IFNARKO or P14.DOKO T cells were transferred into B6 mice (Thy1.2) followed by either A, intravenously or B, intraperitoneally infection with rVVGP. Kinetics of P14.WT (•) and P14.IFNARKO, P14.IL12RKO and P14. DOKO (○). T cells gated on CD8+ T cells in the blood at indicated time points. Values are expressed as mean ± SEM (n = 3).(TIF)Click here for additional data file.


H3f3b.The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b knockout (KO) mice exhibit a semilethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in mouse embryonic fibroblasts (MEFs) with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile.Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development. The nucleosome is a dynamic structure whose constituent histones undergo a vast number of posttranslational modifications (PTMs), which influence nucleosomal function. In addition, the histones can be entirely replaced by deposition of variant histones carrying their own unique complements of PTMs. For example, canonical histone H3 proteins, H3.1 and H3.2, can be replaced by the histone variant, H3.3. Deposition of H3.3 is thought to produce a nucleosomal state that is less structured and associated with a more transcriptionally active state . One notAnother H3 variant, CENP-A is essential for centromere function where a role for histone H3.3 is also implicated . H3.3 maThe unique functions of the histone H3.3 protein are conferred by the few amino acids by which it varies from canonical histone H3 proteins, and by the distinct expression of H3.3 throughout the cell cycle. H3.3 protein differs by only four and five unique residues from histones H3.2 and H3.1, respectively. Residues S31, A87, I89, and G90 are completely unique to H3.3. Histone H3.3 phosphorylated at Serine31 (H3.3S31P) is enriched at domains bordering centromeres during metaphase and may function in mitosis , while aH3f3a and H3f3b (H3.3A and H3.3B in Drosophila), encode the H3.3 protein, while more than a dozen histone H3.1 and H3.2 genes encode the canonical histone H3.1 and H3.2 proteins. The H3f3a and H3f3b genes also are uniquely located outside canonical histone gene clusters, and their gene structure is distinct from that of canonical histone genes in that they have introns and they also produce mRNAs with polyadenylated tails. When compared to each other, H3f3a and H3f3b also have distinct gene expression patterns, untranslated regions and promoters  and [NCBI-GEO:GSE35321], respectively.in-situ hybridization; FL: Floxed; H3K4me3: Histone H3 lysine 4 tri-methylation; H3K9ac: Histone H3 lysine 9 acetylation; H3.3S31P: Histone H3.3 phosphorylated at serine 31; ICC-IC: Immunocytochemistry-Immunofluorescence; KO: Knockout; MCM: Minichromosome maintenance complex; MEF: Mouse embryonic fibroblasts; MFI: Mean fluorescence intensity; MSC: Mesenchymal stem cell; PML: Promyelocytic leukemia; PTM: Posttranslational modification; RD: Replication dependent; RI: Replication independent; RT: Room temperature; TSS: Transcription start site; WT: Wildtype.ChIP-Seq: Chromatin immunoprecipitation-sequencing; ES: Embryonic stem cells; FISH: Fluorescent The authors declare that they have no competing interests.KB conducted all mouse breeding and genotyping to produce the H3f3b knockout strain. KB also did the majority of experiments conducted that are reported in this manuscript. BY conducted most of the immunostaining studies of MEFs, did the kinetochore staining and quantitative analysis, and aided in data interpretation. BB conducted data analysis from the ChIP-Seq studies and conducted other experiments including cell cycle and cell shape analysis, JR aided in expression microarray analysis, HO conducted the ChIP-Seq sequencing and aided in data analysis, RC prepared ChIPs and aided in the ChIP-Seq studies, and PK conceived of the project, supervised the studies, did experimental design and data interpretation, and together with KB did the majority of the writing of the manuscript. All authors read, helped write and edit, and have approved the manuscript.A) Genomic location, (B) genotyping genomic PCR, and (C) southern blotting assays/probes for H3f3b.(Click here for fileA-B) H3f3a mRNA levels in (A) other knockout (KO) mouse embryonic fibroblasts (MEF) lines produced and (B) KO embryonic tissue. (C) H3.3 protein levels of total H3.3 protein levels in other MEF lines produced. (D) Western quantification of H3.3 protein levels in KO 1, 2, 49 and 52. (E) Western quantification of H3.3S31P protein levels in KO1, 2, 49 and 52 relative to H3.3 protein levels.(Click here for fileA) (top) Example of normal wildtype (WT) karyotype. (Bottom) Example of knockout 1 (KO1) karyotype exhibiting breaks. (B) Abnormal shape factor and evidence of elevated levels of endoreduplication in the KO, DAPI staining and quantitation. Arrows indicate nuclei with possible endoreduplication. (C) Metaphase spread of mouse embryonic fibroblasts (MEF) line KO2 show endoreduplication. (D) DAPI/CREST staining for mouse acrocentric chromosomes and centromeric regions in WT 63 and KO 56. Scale bar = 10 um. (E) Immunostaining for total H3 in WT 46 and KO 49 (MEFs). Scale bar = 20 um.(Click here for filein-situ hybridization (FISH) (green) on wildtype (WT) and H3f3b knockout (KO) cells either arrested in (i) metaphase or (ii) unsynchronized in interphase.Telomere fluorescent Click here for fileA) Total RNA levels in wildtype (WT) and knockout (KO) mouse embryonic fibroblasts (MEFs). (B) qPCR validation of genes downregulated on microarray. (C) Histone genes with upregulated expression in constitutive KO – array data. (D) Immunoblots of histone H2b and H4. (E) Western blot and associated quantification of CENP-A protein in KO 49 and 52 versus mean WT.(Click here for fileP <0.05).Gene expression analysis on constitutive and conditional mouse embryonic fibroblasts (MEFs) for genes that were up- or downregulated by at least 2 fold.  for genes that were up- or downregulated by at least 1.5 fold.Click here for fileOntological expression analysis on constitutive mouse embryonic fibroblasts (MEFs) for genes upregulated.Click here for fileOntological expression analysis on constitutive mouse embryonic fibroblasts (MEFs) for genes downregulated.Click here for fileBasic statistics from the alignment and peak calling portion of the chromatin immunoprecipitation-sequencing (ChIP-Seq) analysis. Sequencing reads were aligned using Bowtie, followed by peak calling using both PeakRanger and Sole Search.Click here for fileA) Chromatin immunoprecipitation-sequencing (ChIP-Seq) global peak overlap between two biological replicates for each condition. (B) Average profile of ChIP-Seq peaks in the region from -1000 to +1000 bp around the transcription start site (TSS) of genes.(Click here for fileChromatin immunoprecipitation-sequencing (ChIP-Seq) differential peak analysis. Differential H3K4me3 and H3K9ac peaks were identified using R package DiffBind with False Discovery Rate < 0.1. Peak annotations within 30 kb of each peak were generated using Galaxy Cistrome tool peak2gene.Click here for fileOntology of genes within 30 kb of H3K4me3 peaks that are significantly different in knockout (KO) mouse embryonic fibroblasts (MEFs) 1 and 2 versus wildtype (WT).Click here for fileHistogram of H3K4me3 peaks that are significantly reduced (top) or increased (bottom) in the knockout (KO) compared to wildtype (WT), based on two biological replicates for each genotype. Histograms are divided into one plot for each chromosome. Chromosomal location is shown on the x-axis, scaled from 0 (centromere) to 1 (telomere); the y-axis shows fold change of significantly changed peaks (FDR <0.1). Graphs were produced using the R package ggplot2.Click here for fileSupplemental Methods.Click here for fileAntibody list.Click here for file