KCNE1 D85N potassium channel variant prolongs QT interval by inhibiting IKs (KCNQ1) and IKr (KCNH2) currents and is therefore a suitable candidate for a modifier gene in LQTS.Long QT syndrome (LQTS) is an inherited ion channel disorder manifesting with prolongation of the cardiac repolarization phase and severe ventricular arrhythmias. The common KCNQ1 G589D (n = 492), KCNQ1 IVS7-2A>G (n = 66), KCNH2 L552S (n = 73), and KCNH2 R176W (n = 88). We also investigated the association between D85N and clinical variables reflecting the severity of the disease.We studied the effect of D85N on age-, sex-, and heart rate-adjusted QT-interval duration by linear regression in LQTS patients carrying the Finnish founder mutations p = 0.003) in males with KCNQ1 G589D (n = 213), but not in females with G589D (n = 279). In linear regression, the interaction between D85N genotype and sex was significant (p = 0.028). Within the KCNQ1 G589D mutation group, KCNE1 D85N carriers were more often probands of the family (p = 0.042) and were more likely to use beta blocker medication (p = 0.010) than non-carriers. The number of D85N carriers in other founder mutation groups was too small to assess its effects.D85N was associated with a QT prolongation by 26 ms (SE 8.6, KCNE1 D85N is a sex-specific QT-interval modifier in type 1 LQTS and may also associate with increased severity of disease. Our data warrant additional studies on the role of KCNE1 D85N in other genetically homogeneous groups of LQTS patients.We propose that  LQTS is caused by mutations of the ion channel genes controlling the repolarization phase of the cardiac action potential cycle [Ks potassium channel [KCNQ1 gene, and the rapidly activating IKr potassium channel [KCNH2 gene. Mutations in KCNE1 underlie the LQT5 subtype of LQTS [KCNE1 mutations may cause sensorineural hearing loss in association with LQTS, or Jervell-Lange-Nielsen syndrome [Long QT syndrome (LQTS) is an inherited arrhythmia disorder associated with risk of al cycle ,2. The p channel ,4, encod channel , encoded of LQTS  and acco of LQTS . In homosyndrome .KCNE1 was originally detected by Tesson et al. [Ks potassium channel, when studied in Xenopus oocytes [KCNE1 D85N has a minor allele frequency of 0.8-1.4% [A common variant D85N of n et al. . This va oocytes , and to  oocytes . In gene0.8-1.4% -13 and i0.8-1.4% -14. More0.8-1.4% ,11, as w0.8-1.4% .KCNE1 D85N would also modify the QT interval and/or the clinical picture in patients with genetically homogeneous forms of LQTS in which the variability caused by the disease-causing mutation itself can be controlled for. To this end, we took advantage of the unique situation in Finland where four different mutations account for approximately 70% of the known spectrum of LQTS genes [KCNQ1 mutation carriers.These findings prompted us to study whether TS genes  and wherTS genes . Our resKCNQ1 G589D , 66 carriers of KCNQ1 IVS7-2A>G , 73 carriers of KCNH2 L552S (2 of them homozygous), and 88 carriers of KCNH2 R176W. These four founder mutations account for 70%, and KCNQ1 G589D alone 50%, of all Finnish LQTS patients with an established molecular diagnosis of LQTS [The study sample consisted of all available (n = 712) carriers of the Finnish LQTS founder mutations, including 492 carriers of  of LQTS . The colSubjects taking any known QT-prolonging medication at the moment of electrocardiogram (ECG) recording were excluded from the study. QT intervals were measured manually by a single cardiologist (H.S.) using the mean of two consecutive QT intervals in lead II in standard 12-lead ECG. Heart rate-corrected QT (QTc) was calculated using the Bazett's formula (QT/√RR). The study was performed in accordance with the Declaration of Helsinki and written informed consent was obtained from all participants. The Ethics Review Committee of the Department of Medicine, University of Helsinki, approved the study.KCNE1 253G>A  was genotyped in DNA from venous blood samples using polymerase chain reaction with primers 5'-GAGATTGGAGTGGTGGATGGA-3' and 5'-CACCCCTTACAACAGCCAAAA-3' followed by Lwe I digestion  and agarose gel electrophoresis. Previously identified heterozygous and major and minor homozygous samples served as controls in the assay.KCNE1 D85N on age-, sex-, and heart rate  -adjusted QT interval was studied by linear regression analysis using 1-df additive model . Because gender differences were detected, an additional model included also a multiplicative interaction term between KCNE1 D85N genotype and sex. In KCNQ1 G589D carriers, association of D85N with proband/family member status , appearance of syncope , use of beta blocker medication , and occurrence of pacemaker or implantable cardioverter-defibrillator  was studied by Fisher exact test. Statistical analyses were performed with SPSS 17.0 . A p value < 0.05 was considered statistically significant.Normality of continuous distributions was reviewed visually. Non-normally distributed age was normalized by Blom's method. The effect of KCNE1 genotype D85D, 21 had the heterozygous genotype D85N, and 2 were minor homozygotes (N85N)  were KCNQ1 G589D carriers and were siblings from the same family. As a whole, the founder mutation carriers were derived from 126 LQTS families. The number of KCNE1 D85N carriers in type 2 LQTS was too small for statistical comparisons and therefore the analyses were confined to KCNQ1 G589D carriers and the combined group of founder mutation carriers.Of the 712 LQTS founder mutation carriers, 689 had the KCNQ1 G589D (n = 492) was the largest group of founder mutation carriers. A QT-prolonging effect of 26 ms  was detected in males with G589D (n = 213), whereas D85N did not prolong QT interval in females with G589D (n = 279) (Table KCNQ1 G589D carriers with KCNE1 N85 (n = 8) were of age ≤16. To account for the possible confounding effect of age, male G589D carriers ≤16 years were analyzed also as a separate group. Even in this analysis, the effect of N85 allele on QT interval remained essentially the same . The majority (9 out of 10) of the female KCNQ1 G589D carriers with KCNE1 D85N were adults ≥18 years . In order to further study the gender specificity of D85N, a multiplicative interaction term for D85N genotype and sex was introduced into the linear model. This interaction term received a significance of p = 0.028, indicating that D85N indeed has a sex-specific effect for QT interval in KCNQ1 G589D carriers.Patients with 9D n = 49 was the KCNE1 D85N was associated with a 13 ms (SE 6.0) prolongation of QT interval per each N85 minor allele . The proportion of subjects using beta blocker medication was also higher in D85N heterozygotes (81%) than in D85D homozygotes . The median age at which the medication was started was 15 years (range 0-58 years) for the D85N heterozygotes and 8 years (range 5-11 years) for the N85N minor homozygotes. A similar but non-significant trend for difference between non-carriers and heterozygotes was observed in the occurrence of syncope and use of pacemaker or implantable-cardioverter defibrillator , and a sex-specific analysis revealed that the N85 allele prolongs QT interval by 26 ms in males but not in females  than in the general population (10 ms). This result suggests that KCNQ1 and KCNE1 mutations and variants may interact with each other, at least in males, to produce an even more pronounced QT-prolonging effect than when occurring separately. This interaction could be caused by the coassembly of the two proteins to form a functional IKs channel. However, due to small number of subjects with the KCNE1 N85 allele and LQT1 mutations other than KCNQ1 G589D, we cannot draw conclusions on the general applicability of this effect in LQT1, nor provide sufficient data on a possible similar relation in LQT2. It should be emphasized that the association between D85N and QT interval in type 1 LQTS should be replicated in another material. However, we realize that this is not an easy task considering the population prevalence of D85N. Thus, any replication material should contain hundreds of genetically uniform patients with type 1 LQTS.In the present study, we found that the QT-prolonging effect of KCNE1 D85N variant may be associated with a more severe phenotype of type 1 LQTS, as the proportions of probands and users of beta blocker medication were significantly higher in D85N heterozygotes than in non-carriers (Figure et al. [torsades de pointes studied by Mank-Seymour et al. [et al. [KCNE1 D85N variation as determinant of life-threatening and fatal arrhythmias will be explored. Polymorphisms in another gene, NOS1AP, have also been associated with QT prolongation and cardiac events in LQTS patients [Our study also provides evidence that the s Figure . Indirece et al.  identifir et al. . Sotoode [et al.  suggestepatients .KCNE1 D85N variation has a gender-dependent QT-prolonging effect in KCNQ1 G589D mutation carriers and could thus complicate the symptoms of LQTS. Previously, this variant has not been studied in vivo in a large cohort of LQTS mutation carriers. As KCNE1 D85N is relatively frequent occurring in 2-3% of the general population, this interaction between KCNQ1 and KCNE1 could ultimately have direct implications in counseling of LQTS patients. However, future studies are required to assess the direct risks of arrhythmias and/or sudden death in LQTS patients carrying KCNE1 N85.Our study suggests that The authors declare that they have no competing interests.AML participated in the design of the study, performed the molecular genetic and statistical analyses, and drafted the manuscript. AM participated in the design of the study, data analysis, and writing of the manuscript. HS collected the patient material and participated in the design of the study, data analysis, and writing of the manuscript. KK participated in the design of the study, data analysis, and writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/11/prepub


Ciona intestinalis.Cross inhibition between NK4 and TBX1 transcription factors specifies heart versus pharyngeal muscle fates by promoting the activation of tissue-specific regulators in distinct precursors within the cardiopharyngeal lineage of the ascidian,  NKX2-5, ISL1, and TBX1. This ontogenetic kinship is dramatically reflected in the DiGeorge/Cardio-Velo-Facial syndrome (DGS/CVFS), where mutations of TBX1 cause malformations in the pharyngeal apparatus and cardiac outflow tract. Cardiac progenitors of the first heart field (FHF) do not require TBX1 and segregate precociously from common progenitors of the second heart field (SHF) and pharyngeal muscles. However, the cellular and molecular mechanisms that govern heart versus pharyngeal muscle specification within this lineage remain elusive. Here, we harness the simplicity of the ascidian larva to show that, following asymmetric cell division of common progenitors, NK4/NKX2-5 promotes GATAa/GATA4/5/6 expression and cardiac specification in the second heart precursors by antagonizing Tbx1/10-mediated inhibition of GATAa and activation of Collier/Olf/EBF (COE), the determinant of atrial siphon muscle (ASM) specification. Our results uncover essential regulatory connections between the conserved cardio-pharyngeal factor Tbx1/10 and muscle determinant COE, as well as a mutual antagonism between NK4 and Tbx1/10 activities upstream of GATAa and COE. The latter cross-antagonism underlies a fundamental heart versus pharyngeal muscle fate choice that occurs in a conserved lineage of cardio-pharyngeal progenitors. We propose that this basic ontogenetic motif underlies cardiac and pharyngeal muscle development and evolution in chordates.The heart and head muscles share common developmental origins and genetic underpinnings in vertebrates, including humans. Parts of the heart and cranio-facial musculature derive from common mesodermal progenitors that express  NKX2-5 and TBX1, which govern heart and head muscle development, cause prevalent congenital defects. Recent studies using vertebrate models have shown that the heart and pharyngeal head muscle cells derive from common progenitors in the early embryo. To better understand the genetic mechanisms by which these progenitors select one of the two developmental trajectories, we studied the activity of these transcription factors in a simple invertebrate chordate model, the sea squirt Ciona intestinalis. We show that the sea squirt homolog of NKX2-5 promotes early heart specification by inhibiting the formation of pharyngeal muscles. Conversely, the TBX1 homolog determines pharyngeal muscle fate by inhibiting GATAa and thereby the heart program it instructs, as well as promoting the pharyngeal muscle program through activation of COE (Collier/Olf-1/EBF), a recently identified regulator of skeletal muscle differentiation. Finally, we show that the NKX2-5 homolog protein directly binds to the COE gene to repress its activity. Notably, these antagonistic interactions occur in heart and pharyngeal precursors immediately following the division of their pluripotent mother cells, thus contributing to their respective fate choice. These mechanistic insights into the process of early heart versus head muscle specification in this simple chordate provide the grounds for establishing the etiology of human congenital cardio-craniofacial defects.Mutations in the regulatory genes encoding the transcription factors  TBX1/Tbx1, ISL1/Islet-1, and NKX2-5/Nkx2.5The combined cardiac and craniofacial malformations observed in Cardio-Velo-Facial/Di George Syndrome patients arise from complex sets of defects in the development of the pharyngeal apparatus Ciona intestinalis recently emerged as a simple and relevant model for chordate heart development Mesp and gives birth to four cells: two heart progenitors, the trunk ventral cells (TVCs), activate FoxF and GATAa and migrate into the trunk in response to an FGF signal, while their two sister cells form anterior tail muscles (ATMs) Mesp or FoxF enhancers allows the visualization and molecular manipulation of B7.5 lineage cells or TVCs, respectively Islet, and contributes to inhibiting the heart program specifically in the ASMs (myosin heavy chain 3 (MHC3) in the atrial siphon primordium. During metamorphosis, ASM precursors continue to divide and give birth to definitive ASM and longitudinal muscle (LoM) precursors, which migrate into the body wall MHC2+ cardiomyocytes  signaling, FoxF, GATAa, and Bone Morphogenetic Protein (BMP) signaling  assays on Mesp>nls:lacZ-expressing larvae indicated that NK4 expression persists at low levels until 20 hpf in both the ASMs, FHPs, and SHPs   ectively  [21]. Ins (OSMs)  [21],[30s 28/35; . In thess 25/35; , while iCOE is expressed immediately after the asymmetric division of the secondary TVCs  of control 20 hpf larvae contained four βgal+/COE+ and six βgal+/COE− cells per electroporated half  of electroporated larvae. Mosaic incorporation of the transgenes reduced the number of COE+ cells to 1–3 per half in 38% (16/29) of the larvae. The average number of COE+ cells was reduced to 2.2±0.4 (n = 29), and the number of βgal+, COE− cells increased to 6.3±0.4 , which is consistent with the above observation. Targeted dnNK4 expression increased the number of ASM cells to 8.5±2.8, including 6.6±0.4 MHC3+ cells  is up-regulated by COE in the ASM Islet is expressed in all TVC daughter cells, but more highly in the ASMs compared to the heart precursors  of the larvae, thus reducing the average number of Tbx1/10+ cells to 0.3±0.1 per side . Electroporation of U6>shTbx1/10 constructs down-regulated the endogenous Tbx1/10 mRNA as evaluated by FISH . In 19 and 20 hpf larvae, TVC divisions had resumed and double FISH-IHC assays using COE and GATAa probes showed that Tbx1/10 knock-down caused ectopic activation of GATAa in the lateral-most TVC derivative, while COE expression was markedly down-regulated (GATAa and promote COE expression in the ASMs.We first used short hairpin RNA (shRNA)-mediated RNA interference (RNAi) to knock down Tbx1/10 activity and test whether it is required to promote  by FISH . Notablyegulated . These rCOE activation observed upon dnNK4 expression depends upon Tbx1/10 activity by co-electroporating FoxF>dnNK4 and U6>shTbx1/10 constructs and assaying COE and GATAa expression in 19 and 20 hpf larvae by double FISH-IHC  to 5.1±0.3 in FoxF>Tbx1/10-electroporated larvae . These cells likely represent the FHPs, suggesting that Tbx1/10 or dnNK4 alone are not sufficient to promote COE expression in the FHPs.We reasoned that the absence of t 20 hpf . Tbx1/10per half , resultiGATAa is reactivated in the FHPs, where its expression appeared independent of NK4 activity, while the delayed reactivation in the SHPs may require NK4 function. We reasoned that persisting, NK4-sensitive, Tbx1/10 activity could be antagonizing GATAa expression specifically in the SHPs, but not in the FHPs that do not express Tbx1/10. We tested this possibility by mis-expressing Tbx1/10 in all TVC progenitors and assayed GATAa expression at 18 hpf. Tbx1/10 mis-expression eliminated all TVC-specific expression of GATAa in 57% (28/49) of the larvae, causing the average number of GATAa+ cells to significantly decrease from 2.5±0.2 to 0.75±0.15 cells per side  of the larvae to express COE in all TVC derivatives  and FHPs  to evaluate NK4 binding to the endogenous COE enhancer. We used the Brachyury enhancer, which is only active in the notochord COE enhancer enrichment over input was 2.3±0.1-, 3.1±0.1-, and 5.9±0.1-fold greater in the NK4-FLAG ChIP samples compared to mock, GFP-FLAG, and dnNK4-FLAG control samples, respectively  will uncover additional regulators of ASM versus heart fate specification. Second, since NK4 transcripts were detected throughout the TVC lineage, and high levels of NK4 activity were sufficient to repress Tbx1/10 and COE expression, what prevents NK4-mediated inhibition of Tbx1/10 and COE expression in the secondary TVCs and ASM? Since differential regulatory inputs between the heart and ASM precursors occur following stereotyped oriented asymmetric divisions, we reason that differences between the SHPs and ASM arise as consequences of the asymmetric secondary TVC division. This may contribute to the observed delay in ectopic COE expression in the FoxF>dnNK4-expressing larvae, even though these cells maintained Tbx1/10 transcription. Future studies ought to identify the molecular mechanisms that link oriented asymmetrical TVC division to the secondary TVC and ASM-specific inhibition of NK4-mediated repression of Tbx1/10 and COE.Our model also raises several questions that should be addressed in future studies. First, the above argument does not formally rule out the possibility that early TVC inputs  atrioventricular canal (AVC)-specific enhancer to directly repress the latter in the mouse myocardium The NK4-Tbx1/10 antagonism possibly occurring at the Drosophila ortholog of Tbx1/10, org-1, contributes to myogenesis in the visceral mesoderm COE orthologs, Ebf2 and −3, as essential determinants of MyoD expression and head muscle development in XenopusEbf2 expression in mouse embryonic stem cells COE orthologs during pharyngeal muscle development in vertebrates and tunicates.Studies in vertebrates and flies have documented a requirement for Tbx1 activity in noncardiac muscle development. The Nkx2.5 mutant mice display profound defects in the formation of SHF derivatives, which fail to proliferate, but form FHF-derived left ventricle tissue, and loss of Nkx2.5 function also up-regulates Islet1 expression in the mouse cardiogenic mesoderm gata4+/nkx2.5+ part of the anterior lateral plate mesoderm and fail to proliferate normally in nkx2.5 morphant embryos, while initial SHF specification appeared to occur normally as judged by latent TGF β binding protein 3 (ltbp3) expression nkx2.5 and nkx2.7 caused an increase in atrial tissue formation at the apparent expense of the ventricle Isl1+ SHF progenitors Drosophila, the NK4 ortholog tinman inhibits Collier expression during muscle-type diversification COE-dependent branchiomeric muscle precursors downstream of Tbx1/10 in Nkx2.5 mutants. We propose that a conserved cross-antagonism between NK4 and Tbx1/10 orthologs acts upstream of GATAa and COE orthologs for heart versus pharyngeal muscle fate specification within an ancestral lineage of cardio-pharyngeal progenitors in tunicates and vertebrates, where this simple ontogenetic motif underlies heart and head muscles development and evolution. More specifically, it is tantalizing to speculate that prolonged Tbx1 expression in the amniote second heart field has fostered the emergence of novel heart compartments by antagonizing heart differentiation, thus maintaining a proliferative progenitor state in the SHF and expanding on the ancestral heterochrony between first and second heart fields' specification that is observed in extant chordates.Established lineage topologies within the mouse cardio-pharyngeal mesoderm show that precursors of the FHF diverge before the separation of the SHF from pharyngeal/branchiomeric muscle progenitors Ciona intestinalis adults were purchased from M-Rep . Isolation of gametes, fertilization, dechorionation, electroporation, and development were conducted as described Gravid NK4 and Tbx1/10 (Genbank Accession: KC196542) were amplified by RT-PCR from total RNA isolated from 16 hpf Ciona intestinalis larvae using the following primers: NK4_CDS_F, GGGCCCAAACCATGATTCCTAGTCCGGTTGGATCGACT-3′5′-AAA; NK4_CDS_R, GCTCAGCTCACGTGCACAGCCCAAGCTTAT-3′5′-TT. The CDS were cloned downstream of the FoxF minimal TVC enhancer (FoxF-TVC) fused to the basal promoter of the Friend of GATA gene (bpFOG) The coding sequences (CDS) of NK4 was introduced by overlapping PCR strategy using the primers NK4_N448K _F, 5′-AAGATCTGGTTCCAAAAGCGTCGATACAAATGTAAACGAATGCGACAAGA-3′ and NK4_N448K_R, ACATTTGTATCGACGCTTTTGGAACCAGATCTTGACCTGGGTGGAAGT.The N448K point mutation of Mesp enhancer (Mesp>lifeact:mCherry) to visualize the cell cortices of B7.5 lineage cells. Mesp>nls:lacZ was used as described previously mCherry was fused to N-terminal LifeAct peptide Double FISH-IHC was performed following a protocol modified from Christiaen et al. Samples were mounted in 50% glycerol/PBS with 2% DABCO. Images were acquired with a Leica TCS SP5 or a Leica TCS SP8 X confocal microscope, using 20× or 63× objectives. Z-stacks were acquired 1.5 µm (20×) or 1 µm (63×) z steps. Maximum projections were processed with maximum projection tools from the LEICA software LAS-AF.3. Quantifications were exported into Excel for further analysis.The measurements of juvenile heart volume were conducted using Volocity 5 . Volume of Mesp>nls:lacZ labeled heart cells was detected on confocal stacks. Three filters were set for objective detection as following: (1) regions of interest were segmented by percentage intensity , (2) noise was removed from objects with a Fine Filter setting, And (3) touching objects separated with object size guide as 100 µmhttp://www.broadinstitute.org/rnai/public/seq/search) algorithms. Candidate 21-mer target sites were trimmed to 20 mer, and those that begin with A or G and end with A were further considered. Candidate 20 mer were BLASTed against the Ciona intestinalis genome on the Ghost database and selected to avoid polymorphic region as well as 20 mer with potential off-targets . Target sense sequences were reverse-complemented, and sense and antisense sequences were combined to mimick the hairpin structure corresponding to the microRNA miR-2213 Ciona U6 promoter GCACAAGTccACttAGATTcTGTAATTaGtAACATAATCTGGGTAAACTTGTGttttt; shTbx1-A-R, aattaaaaaCACAAGTTTACCCAGATTATGTTaCtAATTACAgAATCTaaGTggACTTGTGC; shTbx1-B-F, gCTGGTtAaaCAttGATATcTGTAATTaGtAACATATATCGGTGGGTGACCAGTtttt; shTbx1-B-R, aattaaaaACTGGTCACCCACCGATATATGTTaCtAATTACAgATATCaaTGttTaACCAGc; shTbx1-C-F, gGTtGGTCcaCATAGTTATcTGTAATTaGtAACATATAACTATGGTGACCGACTtttt; shTbx1-C-R, aattaaaaAGTCGGTCACCATAGTTATATGTTaCtAATTACAgATAACTATGtgGACCaACc; shTbx1-D-F:, gTTCACTgaaAGtAtGATAcTGTAATTaGtAACATTATCGTGCTGGTAGTGAATtttt; and shTbx1-D-R, aattaaaaATTCACTACCAGCACGATAATGTTaCtAATTACAgTATCaTaCTttcAGTGAAc. Sensor assays using a GFP:Tbx1/10 fusion indicated that shRNA constructs A, B and A, D were the most efficient pairwise combinations (unpublished data). The data showed used 30 µg each of U6>shTbx1/10-A and -B per electroporation.The coding sequence of Ci-Tbx1/10 was used to identify siRNA target sites with the DSIR A 2xFLAG tag was cloned to the C terminus of coding sequences to make the following constructs: FoxF>GFP:2xFLAG, FoxF>dnNK4:2xFLAG, FoxF>NK4:2xFLAG. We used 50 µg of plasmid for each electroporation. The larvae from three electroporations were combined as one ChIP sample. All samples were harvested at 16 hpf and cross-linked at 22°C for 15 min by adding formaldehyde to the final concentration of 1%. Chromatin extracted from larvae was sheared into 400 bp fragments by S220 Focused-ultrasonicator . The cross-linked enhancer fragments were incubated with DYKDDDDK tag antibody  overnight and then captured by Dynabeads M-280 sheep anti-Rabbit IgG . Then, enriched DNA were treated by RNaseA and proteinase K and purified using NucleoSpin Gel and PCR Clean-up kit .COE enhancer. Two pairs of primers were also used to amplify the enhancer region of Ciona Brachyury5′ GTCGCTGAGGAAGTCATCATT 3′; En_CiCOE_R1 5′ GAACCAATAAGGACGGGTGAA 3′; En_CiCOE_F2 5′ TGGCGGCTGGCTAATTAAA 3′; En_CiCOE_R2 5′ TTCCCTGCAGACAAATAATGGA 3′; En_CiBra_F1 5′ GGCGCACTTTCAACAAACA 3′; En_CiBra_R1 5′ TCTGCCTCCAAATCACACTC 3′; En_CiBra_F2 5′ CACGCAAGACAATGGGAAAG 3′; En_CiBra_R2 5′ GGTGGCGCTCTATGTTTACT 3′. Quantitative PCR was performed using the SYBR Green method on a LightCycler 480 system . Each qPCR was performed in technical triplicates, and the average values were used for subsequent calculations. Fold enrichments were calculated using the ΔΔCt method: For each ChIP sample and corresponding input, the ΔCt for En_COE_1 and En_COE_2 versus the average Brachyury was calculated first. Then ChIP versus input ΔΔCt was calculated for each En_CiCOE primer pair and each sample. Finally, fold enrichment over mock control was calculated for each replicate. Average fold enrichments relative to mock controls are showed in Each ChIP sample was obtained in three independent biological replicates. Two pairs of primers were designed within the minimal Figure S1Conserved features of NKX2-5 homedomain proteins across species. (A) Alignment of NK4 orthologues across species using ClustalX 2.1 . CiNK4, Ciona intestinalis, NP_001071957, 623 aa CsNKX2-5, Ciona savignyi, BAA25399, 595aa; GgNKX2-5, Gallus gallus, NP_990495, 294 aa XlNKX2-5, Xenopus laevis, NP_001080190, 299 aa MmNKX2-5, Mus musculus, NP_032726, 318 aa HsNKX2-5, Homo sapiens, NP_004378, 324 aa DrNKX2-5, Danio rerio, NP_571496, 314 aa SkNKX2-3/5, Saccoglossus kowalevskii, NP_001158401, 268 aaTinman, Drosophila melanogaster, NP_524433, 416 aa CiNK4 lacks the Nkx2-5 box and GIRAW motif. (B) Domain structure of CiNK4. Asparagine (N) at amino acid residue position 448, which is critical for DNA binding capability of the homeobox (TIF)Click here for additional data file.Figure S2Endogenous expressions of NK4. (A–D) NK4 endogenous expression pattern during TVC lineage specification. Mesp>LifeAct:mCherry (blue) and Mesp>nls:lacZ (red) were detected by immunostaining, indicating cell boundary and nucleus, respectively; NK4 mRNAs (green) were detected by FISH. NK4 transcripts are present throughout the TVC lineage and endoderm from 12 to 20 hpf at 18°C. Scale bar, 25 µm. (A–D) FISH detection of NK4 transcripts (green) at 48 hpf (E), 72 hpf (F). Red, Mesp>nls:lacZ positive B7.5 lineage cells; the heart areas (dotted line square) were rescanned at higher magnification . NK4 transcripts were not detected in the juvenile heart. Scale bar, 10 µm.(TIF)Click here for additional data file.Figure S3COE expression initiates prior to that of Islet. (A) Proportions of embryos and larva showing COE (blue) or Islet (red) expression at indicated stages of TVC development  Initiation of COE expression visualized in early ASMs by two nuclear dots, indicating that this cDNA probe labels nascent transcripts. Ventral view of larva with TVC lineage progenitors in early class 2 pattern. Larva is electroporated with Mesp>nls:lacZ to mark the TVC lineage (red). DAPI channel to visualize the nucleus (blue). COE transcripts are detected by FISH (green). TVC lineage cells (squared) are magnified. (C–E) Islet nascent transcripts detected by Islet intron-specific probe (green). TVC lineage is marked with Mesp>nls:lacZ (red). Islet transcription is activated in all the TVC derivatives around 18 hpf, but exclusively in the ASM precursors (open arrowheads) at 20 hpf. Scale bar, 25 µm.(TIF)Click here for additional data file.Figure S4NK4 represses COE in TVC derivatives.  FISH of COE (green) in 28 hpf (A–C) and 24 hpf (E–G) larvae; FoxF>dnNK4 induces ectopic COE expression and results in extra-numerary TVC lineage cells to contribute to the formation of the ASM ring (B). Conversely, FoxF>NK4 represses COE and inhibits ASM ring formation (C). Mesp>nls:lacZ (red) labels B7.5 lineage cells; COE transcripts (green) detected by FISH. White dotted lines indicate the midline. Scale bar, 25 µm. Counts of COE+/−  and GATAa+/− (J) cells following manipulations of NK4 activity at 28 hpf (D), 24 hpf (H), and 18 hpf . Student's t tests compare each experimental condition to the control. **p<0.05 and *0.05<p<0.1. The significant increase in GATAa− cell numbers observed in (J) is due to a slight increase in the total number of cells per half in these larvae, which could be caused by premature divisions in the dnNK4 condition (see text for details). (K) FISH-IHC with COE intronic probes at indicated stages of larvae electroporated with indicated constructs. Note that no ectopic COE expression was detected before 17–17.5 hpf.(TIF)Click here for additional data file.Figure S5NK4 regulates heart and ASM specification via COE. (A–F) Lateral views of 28 hpf larvae, showing the ventral heart precursors and dorso-lateral ASM rings. Mesp>nls:lacZ (red immunostaining) marks B7.5 lineage cells. Larvae hybridized with digoxigenin-conjugated MHC3 probe (green). (A) In control larvae, four to six of the ASM ring cells express MHC3. (B) FoxF>COE:WRPW abolishes ASM migration, ring formation, and MHC3 expression in TVC derivatives. (C–C′) FoxF>COEΔ321 has mild or no significant effects on second migration, causes ASMs to cluster instead of forming a ring in 31% (23/74) (the cluster phenotype is scored independently) of the larvae (C′), and inhibits MHC3 expression. (D) FoxF>NK4 increases the number of migrating and MHC3+ cells. (E) FoxF>COE:WRPW inhibits the effects of FoxF>dnNK4; neither second migration nor MHC3 expression are observed. (F) FoxF>COEΔ321 also inhibits the effects of FoxF>dnNK4; fewer cells migrates to the siphon placode and fewer MHC3+ cells are observed in the ASM ring. (G) Numbers of MHC3+/− cells per half in indicates conditions. (H) Numbers of cells that migrates to the Atrial Siphon Primordium per half. Student's t tests compare each experimental condition to the control. **p<0.05.(TIF)Click here for additional data file.Figure S6NK4 represses Islet in TVC lineage cells. FISH of Islet in 20 (A–C), 24 (D–F), and 28 hpf (G–I) larvae. In control larvae, Islet is expressed at higher levels in ASM precursors than in heart precursors. Targeted expression of dnNK4 causes up-regulation of Islet in heart precursors, while overexpression of NK4 inhibits Islet in all TVC derivatives. Asterisk, Islet expression in endodermal cells; dotted line, ventral midline. Scale bar, 25 µm.(TIF)Click here for additional data file.Figure S7Endogenous expression pattern of GATAa during TVC lineage specification.GATAa mature (green) (A–D) and nascent (E–L) (green) transcripts were detected by FISH. Mesp>nls:LacZ (red) were detected by immunostaining to indicate cell nucleus. ASM precursors, open arrowheads; SHPs, white arrowheads; FHPs, arrows. Dotted lines indicate the midline. Scale bar, 10 µm. GATAa transcription is active in the TVCs at stage 23 (corresponding to 14 hpf at 16°C)  and then stopped after the first asymmetric division . The transcription reactivates at 16 hpf in FHP (H–L).(TIF)Click here for additional data file.Figure S8Mapping a minimal ASM enhancer for COE. (A) Snapshots of the ANISEED and VISTA browsers showing the KH2008 transcript models for COE and sequence conservation between Ciona intestinalis and C. savignyi. (B) Map and coordinates (relative to the +1 ATG) of the genomic fragments that were cloned upstream of bpFOG>mCherry and tested in reporter gene expression assays. Percentages indicate the percentage of 21 hpf larvae showing ASM-specific mCherry expression among Mesp>GFP+ larvae. These constructs mapped a minimal ASM enhancer encompassing a conserved noncoding sequence ∼2.5 kb upstream of COE. (C) Example of a 21 hpf larva showing mCherry expression in the ASM (arrow) but not in the heart (plain arrowhead), where only GFP is detected because Mesp>GFP is active early and marks all the descendants of the B7.5 blastomeres.(TIF)Click here for additional data file.Table S1Heart volumes in control and experimental juveniles. Fertilized eggs were electroporated with Mesp>nls:lacZ and indicated constructs driven by FoxF(TVC)bpFOG. Electroporated larvae were raised until 72 hpf juvenile stage, fixed, and stained for MHC2, MHC3, and β-galactosidase expression using FISH-IHC. The total volume of βgal+ nuclei per half was measured using Volocity (see the 3.(DOCX)Click here for additional data file.Table S2Relative qPCR values for ChIP samples. Values were obtained as described in the p values are for indicated pair-wise comparisons and were performed in Excel using the TTEST function with two-tail distributions for two-sample of unequal variance.(DOCX)Click here for additional data file.


Evidence also suggests a potential role of LTB4 in COPD disease mechanisms including recruitment of neutrophils to the lung. The aim of the current study was to see if these SNPs and those spanning the receptor genes for LTB4 (LTB4R1 and LTB4R2) influence baseline lung function and COPD susceptibility/severity in smokers.We have previously shown evidence that polymorphisms within genes controlling leukotriene BALOX5AP, six LTA4H and six LTB4R single nucleotide polymorphisms (SNPs) were genotyped in a UK Smoking Cohort (n = 992). Association with baseline lung function (FEV1 and FEV1/FVC ratio) was determined by linear regression. Logistic regression was used to compare smoking controls (n = 176) with spirometry-defined COPD cases (n = 599) and to more severe COPD cases .Eight ALOX5AP, LTA4H or LTB4R survived correction for multiple testing. However, we showed modest association with LTA4H rs1978331C (intron 11) with increased FEV1 (p = 0.029) and with increased FEV1/FVC ratio (p = 0.020).No association with ALOX5AP, LTA4H and the LTB4R locus are not major determinants of baseline lung function in smokers, but provide tentative evidence for LTA4H rs1978331C (intron 11) in determining baseline FEV1 and FEV1/FVC ratio in Caucasian Smokers in addition to our previously identified role in asthma susceptibility.These data suggest that polymorphisms spanning  ALOX5, although existing data did not support their inclusion [LTA4H and ALOX5AP in 20,288 individuals from the general population (the SpiroMeta consortium) and did not identify these genes as containing major determinants of lung function in this large general population cohort [It is important to acknowledge the limitations of our study. Other SNPs spanning these large genes could be important. There may also be another functional variant in linkage disequilibrium with rs1978331. We cannot exclude the contribution of polymorphisms spanning other 5-LO pathway genes nclusion ,30,31. Tn cohort .4 production and activity are major determinants of baseline lung function in smokers and do not determine susceptibility to develop COPD. However, rs1978331  may have a modest effect on lung function parameters in smokers. Heterozygosity of this polymorphism has previously been correlated with LTB4 production, asthma and TB. These findings may be important when considering potential approaches to target LTA4H in COPD.In conclusion, these data did not confirm the hypothesis that polymorphisms in genes specific to LTB1: Forced expiratory volume in one second: FEV1/FVC: Ratio of FEV1 to FVC; FLAP: 5-lipoxygenase activating protein; FVC: Forced vital capacity; GOLD: Global Initiative for Obstructive Lung Diseases; LD: Linkage disequilibrium; LTA4, B4, C4, D4, E4: Leukotriene A4, B4, C4, D4, E4; LTA4H: Leukotriene A4 hydrolase; LTB4R1/2: Leukotriene B4 receptor 1/2; LTC4S: Leukotriene C4 synthase; MAF: Minor allele frequency; MI: Myocardial infarction; OR: Odds ratio; PGP Proline-glycine-proline; PostBD: Post bronchodilator; SNP: Single nucleotide polymorphism; TB: Tuberculosis; UK: United Kingdom; UTR: Untranslated region.5-LO: (pathway) 5-lipoxygenase (pathway); 95% CI: 95% confidence interval; ALOX5: 5-lipoxygenase; ALOX5AP: 5-lipoxygenase activating protein; COPD:Chronic obstructive pulmonary disease; CysLT: Cysteinyl leukotriene; CYSLTR1: Cysteinyl leukotriene receptor 1; FEVThe authors declare that they have no competing interests.IS and AST designed the study and drafted the manuscript. AST completed the statistical analyses. SGP, MFM, AJW and MJC recruited and clinically characterised subjects. All authors contributed to the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/173/prepub1 and FEV1/FVC ratio) and ALOX5AP, LTA4H and LTB4R SNPs in the smokers (n = 992)Baseline lung function (FEV. This table shows the results of the association analysis between leukotriene pathway SNPs and baseline FEV1 and FEV1/FVC using the additive model. Covariates included in the model were age, gender, height and pack years. Associations with p < 0.05 are shown in bold black.Click here for file


PB2-H357N variant demonstrated growth ability comparable to the WT in A549 cells, but replicated well in PK15, LA-4 cells and in mice with an enhanced pathogenic phenotype. Despite such mutations are rare in nature, they could be observed in avian H5 and H7 subtype viruses which were currently recognized to pose potential threat to human. Our findings indicated that pH1N1 may adapt well in mammals when acquiring these mutations. Therefore, future molecular epidemiological surveillance should include scrutiny of both markers because of their potential impact on pathogenesis.Influenza A virus can infect a wide variety of animal species with illness ranging from mild to severe, and is a continual cause for concern. Genetic mutations that occur either naturally or during viral adaptation in a poorly susceptible host are key mechanisms underlying the evolution and virulence of influenza A virus. Here, the variants containing PA-A36T or PB2-H357N observed in the mouse-adapted descendants of 2009 pandemic H1N1 virus (pH1N1), A/Sichuan/1/2009 (SC), were characterized. Both mutations enhanced polymerase activity in mammalian cells. These effects were confirmed using recombinant SC virus containing polymerase genes with wild type (WT) or mutant PA or PB2. The PA-A36T mutant showed enhanced growth property compared to the WT in both human A549 cells and porcine PK15 cells  Influenza A viruses are highly infectious to a variety of mammalian and avian species. Individual viruses are host-specific; however, interspecies transmission of influenza A viruses is not uncommon. Many evidences suggested that genetic mutations in viral proteins, including envelope hemagglutinin (HA) and neuraminidase (NA) glycoproteins The 2009 pandemic influenza A (H1N1) virus is a swine-origin reassortant with human, avian and swine influenza virus genes. In particular, the polymerase contains PB2 and PA of avian origin, PB1 derived from human viruses and NP from the classical swine lineage. This novel virus was transmitted efficiently from human to human and was more pathogenic than seasonal influenza viruses in both the naive pediatric population and animal models th 2012, less than 1% (including H5 and H7 subtypes) carried these mutations in nature. However, little is known about the impacts of these two rare mutations on virus properties. In this study we fully characterized the variants containing substitution PA-A36T or PB2-H357N in vitro and in vivo.One or multiple mutations in HA , NA (at position 63), and polymerase (at position 357 of PB2 or 36 of PA ) were found in our recent work on the adaptation of pH1N1 virus, A/Sichuan/1/2009(SC) after 15 passage times in mice Human embryonic kidney (293T) cells, human type II alveolar epithelial (A549) cell, Madin-Darby canine kidney (MDCK), porcine kidney (PK15) and mouse lung adenoma (LA-4) cells were obtained from the American Type Culture Collection. 293T and A549 cells were maintained in Dulbecco's modified Eagle's medium  and MDCK and PK15 were in minimum essential medium , respectively, supplemented with 10% fetal bovine serum , glutamine , HEPES , penicillin (100 units/ml), and streptomycin . LA-4 cells were propagated in Ham's F12K medium  with 2 mM L-glutamine and 15% FBS.All eight gene segments of influenza virus A/Sichuan/1/2009  were amplified by RT-PCR and cloned into a modified version of the bidirectional expression plasmid pCQI, derived from pHW2K. Mutations(PA-A36T and PB2-H357N) were introduced into the plasmids to generate mutant segments using a QuikChange™ Site-Directed Mutagenesis Kit , following the manufacturer's instructions. The presence of the introduced mutation and the absence of additional unwanted mutations were verified by sequencing of the whole cDNA.50) was calculated using the Reed–Muench formula Recombinant viruses SC_WT and SC_PA-A36T/ SC_PB2-H357N were generated by reverse-genetics method as described as previously 4 /well in 96-well plates) using the PolyFect  reagent, according to the manufacturer's instructions. Mock transfections were performed with polI-Gluc alone. Gluc activity in supernatants was analyzed using a Gluc assay Kit . Each sample was determined in triplicates.polI-Gluc (0.1 µg) A549, PK15 and LA-4 cells were infected with rescued viruses at a multiplicity of infection (MOI) of 0.0001, and incubated in the appropriate medium containing 2 mg/L N-p-tosyl-L-phenylalaninechloromethyl ketone-treated (TPCK) trypsin  at 35 or 39°C. At 12, 24, 48, 60, 72, and 96 hour post-inoculation (hpi), supernatants were harvested and virus titer was determined using MDCK cells as described 50), groups of five 6-week-old female BALB/c mice obtained from Vital Reviver, PRC, were anesthetized with isoflurane and intranasally inoculated with 50 µl of 10-fold serial dilutions of viruses in phosphate-buffered saline (PBS). The MLD50 values were calculated by the method of Reed-Muench 50 causing half fatality. To determine morbidity and mortality, we inoculated groups of 6-week-old female BALB/c mice intranasally with the wildtype and recombinant viruses at the indicated dose in 50 µl volumes, or mock inoculated with PBS. Body weight was monitored continuously for 14 days. Infected mice with more than 30% body loss were recorded as death cases. To determine virus replication in the lungs of infected mice, mice were inoculated intranasally with 104 TCID50 of the indicated viruses and three per group were euthanized at 12, 24, 48, and 72 hpi. The right lobe of the lung was homogenized using an ultra-turrax disperser . Homogenates were collected in 1 ml virus collection medium supplemented with 0.2% BSA fraction V and 1% antibiotic-antimycotic. Virus titers were determined by TCID50 assay.Our mice experiment protocol was approved by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC (IORG0000724). To determine the 50% mouse lethal dose . A th 2012, 5,875 and 5,633 sequences were filtered from initial 14,594 and 14,740 PA and PB2 sequences, respectively, and used for polymorphic analysis. Comprehensive analysis showed that residues at these two positions were highly conserved and 99.5% and 98.8% of PA and PB2 gene contained the same residue as SC_WT, respectively. Amino acid at position 357 in PB2 or 36 in PA displayed polymorphic, and the substitutions similar to SC_PB2-H357N and SC_PA-A36T were rare . Notably, the strongest RNP activity of PA-A36T than PB2-357N and WT was detected at both 37 and 39°C, and was nearly 10-, and 20-fold increase as compared with WT, respectively .In order to investigate whether the PA-A36T or PB2-H357N mutations affect the enzymatic activity of RdRp, we analyzed the reconstituted ribonucleoprotein complexes polymerase activities  in 293T cells at different temperatures using a mini-genome replication assay as described previously 5–107 TCID50/ml at 48 hpi. in the human lung epithelia cell A549 and porcine kidney cell PK15. The mutation of PA-A36T exhibited significantly elevated growth ability in both A549 and PK15 cells with virus titers of more than 10-fold higher than those of SC_WT at 24 hpi and the trend maintained throughout the time course . Significantly higher virus titer of SC_PB2-H357N mutant than SC_WT was found in PK15 cells at 24 hpi. and the titer maintained at a higher level at following time points although the differences did not reach statistic significance . In consistent with the polymerase activity profile at different temperatures , PB2-H357N has an MLD50 of 103.5 TCID50. Mice were inoculated intranasally with 104 TCID50 of SC_WT, SC_PB2-H357N, and SC_PA-A36T. Approximately 30% weight loss was detected in SC_PB2-H357N-infected mice together with an 80% mortality rate  at 48 hpi , while did not differ from that of SC_WT at any following time points. Our study showed that the SC_PB2-H357N mutant displayed marked replication activity in mice and caused severe infection and high mortality.To determine if SC_PB2-H357N/SC_PA-A36T replicated more efficiently than SC_WT virus culation . In linet 48 hpi . SimilarThe polymerase complex of influenza A virus, known to function in many aspects of viral replication and to interact with host factors, therefore, play a critical role during viral adaptation process. Unlike 1957 H2N2 and 1968 H3N2 pandemic viruses caused by reassortants between avian and human influenza viruses, 1918 H1N1 influenza virus derived its polymerase genes directly from avian source before the pandemic emergence. The finding that ten consensus residues observed in 1918 H1N1 polymerases and those from other mammalian viruses is indicative of its human adaptation The variants with the detected mutations were characterized in this study. Both mutations enhanced the polymerase activity remarkably. Furthermore, the activity of WT and mutations displayed a temperature-dependent pattern and highly active at higher temperature. The mutations, particularly PA A36T, exhibited the strongest up-regulation effect at 39°C than that of either 33°C or 37°C. Interestingly, the viruses containing the mutations demonstrated similar temperature-dependent effects on viral replication in mammalian cells from different species. Although the mutants showed varied replication properties in different cells, better growth of the mutants with PA-36T or PB2-357N than WT in mammalian cells as well as increased RNP activity are indicative of their mammalian adaptation. A synergistic effect on polymerase activity rendered by dual PA-36T and PB2-357N substitutions was observed (data not shown) and the mutant containing PA-A36T and PB2-H357N accompanied with HA-D222G was detected in our mice-adaptive experiments. However, the variant with dual mutations, PA-36T and PB2-357N, showed nearly identical virulence to that of WT in mice model (data not shown). Of note, it was recently identified that synergistic actions of HA D222G, K163E and PA F35L in pH1N1 result in an enhanced pathogenicity in mice model As a part of hetrotrimeric RdRp, PA functions in diverse aspects of the complex including endonuclease, protease and viral RNA binding 7GTP Residues 242–252(N-site), 318–483(C-site) and 533–577 are thought to be the cap-binding domain on PB2. Position 357 is located at the C-terminal of strand β4 which was mapped to positions 318 to 483 and was involved in viral transcription via snatching the cap of host mRNA. H357A mutation could decrease PB2's cap binding activity and thus reduce mRNA transcription while H357W showed an enhanced binding to mSystematic analyzing PA and PB2 sequences from all available data of GenBank showed high conservation of the both residues in field isolates and less than 1% viruses possessed mice adaptive mutations. Such mutations exclusively presented in avian viruses including H5 and H7 subtypes. Although whether the mutation pattern for pH1N1, observed here, represents a preferred viral strategy to gain virulence is still unclear, careful monitoring these virulence enhancing mutations is critical important for pandemic preparedness and response due to increasing interspecies transmission events of pH1N1 virus into other hosts have been documented Table S1Natural isolates with PB2-357N or PA-36T.(DOCX)Click here for additional data file.


Campylobacter jejuni is the principal cause of bacterial food-borne infections. The mechanism(s) that contribute to bacterial survival and disease are still poorly understood. In other bacterial species, type VI secretion systems (T6SS) are increasingly recognized to contribute to bacterial pathogenesis by toxic effects on host cells or competing bacterial species. Here we report the presence of a functional Type VI secretion system in C. jejuni. Proteome and genetic analyses revealed that C. jejuni strain 108 contains a 17-kb T6SS gene cluster consisting of 13 T6SS-conserved genes, including the T6SS hallmark genes hcp and vgrG. The cluster lacks an ortholog of the ClpV ATPase considered important for T6SS function. The sequence and organization of the C. jejuni T6SS genes resemble those of the T6SS located on the HHGI1 pathogenicity island of Helicobacter hepaticus. The C. jejuni T6SS is integrated into the earlier acquired Campylobacter integrated element CJIE3 and is present in about 10% of C. jejuni isolates including several isolates derived from patients with the rare clinical feature of C. jejuni bacteremia. Targeted mutagenesis of C. jejuni T6SS genes revealed T6SS-dependent secretion of the Hcp needle protein into the culture supernatant. Infection assays provided evidence that the C. jejuni T6SS confers contact-dependent cytotoxicity towards red blood cells but not macrophages. This trait was observed only in a capsule-deficient bacterial phenotype. The unique C. jejuni T6SS phenotype of capsule-sensitive contact-mediated hemolysis represents a novel evolutionary pathway of T6SS in bacteria and expands the repertoire of virulence properties associated with T6SS.The pathogen  Campylobacter jejuni contains a functional T6SS gene cluster. The cluster comprises 13 conserved T6SS genes including genes encoding the typical T6SS Hcp and VgrG proteins. The gene cluster is part of a larger DNA element and is present in about 10% of C. jejuni strains including several blood isolates. The identified C. jejuni T6SS has unique properties compared to similar systems in other bacterial species. C. jejuni T6SS lacks the ClpV ATPase that supposedly energizes part of T6SS function in other species, causes contact-dependent lysis of red blood cells, and requires downregulation of the C. jejuni capsule polysaccharide to be effective. The unique cytotoxic properties of C. jejuni T6SS, the effect of the capsule on T6SS function, and the possible association with systemic C. jejuni infection broaden the scope of the existing bacterial T6SS phenotypes and point to a different evolution of C. jejuni T6SS compared to other bacterial species.Bacteria contain a number of secretion systems to export macromolecules to the environment. The bacterial type VI secretion system (T6SS) forms a needle-like structure that delivers toxic effector molecules to neighboring eukaryotic and/or prokaryotic cells. Here we report that the important human pathogen  Vibrio cholerae and Pseudomonas aeruginosaGram-negative bacteria have evolved at least six types of protein secretion systems (type I–VI) to export proteins to the periplasmic space or the environment V. cholerae have been visualized in vivo, supporting the model that the T6SS sheath is a dynamic contractile structure that projects the T6SS spike into the target cell analogous to bacteriophage entry Structurally the T6SS consists of a membrane-associated assembly platform and a needle structure that transports effector molecules into neighboring bacteria or eukaryotic cells. A number of the T6SS core proteins show similarity to elements of tailed bacteriophages. Examples are the baseplate gp-25-like protein VCA109, the tail sheath-like proteins TssB and TssC (VipA/VipB), the tail subunit-like hemolysin co-regulated protein (Hcp) that polymerizes into the T6SS needle structure, and the valine-glycine repeat protein (VgrG) that forms the spike of the nanotube V. cholerae that binds membrane lipids Pseudomonas aeruginosaThe hallmark of a functional T6SS is the presence of Hcp and VgrG in the culture supernatant Campylobacter jejuni is one of the principal bacterial food-borne pathogens causing millions of cases of gastroenteritis worldwide. Yet, the pathogenesis of C. jejuni infections is still poorly understood and a limited number of potential virulence determinants have been identified C. jejuni. The T6SS gene cluster is part of an integration element present in the genomes of a subset of C. jejuni strains. The system shows several unique traits compared to other bacterial T6SS including contact-dependent lysis of red blood cells and capsule expression-sensitive T6SS function.C. jejuni strain 108 using two-dimensional gel electrophoresis and liquid chromatography mass-spectrometry (LC-MS) revealed a ∼20 kDa protein that contained 4 peptide sequences most similar to a Campyobacter coli protein annotated in the NCBI database either as hypothetical protein or as putative hemolysin co-regulated protein (Hcp)  . We amplC. jejuni strain 108, we determined the flanking regions of the hcp gene by primer walking. This strategy yielded a putative C. jejuni T6SS gene cluster of ∼17 kilobases consisting of 13 open reading frames with tight intergenic spacing (less than 30 bp)  . The genepaticus .C. jejuni hcp gene (tssD) of strain 108 is located in reverse orientation between the tssJ-M genes (C. jejuni TssJ (17 kDa) has a putative lipoprotein signal peptidase cleavage site (LFFCA/CSSVV) and a serine residue at position +2 which may sort the protein to the outer membrane C. jejuni TssK (53 kDa) lacks an apparent signal sequence and resembles a conserved T6SS protein with unknown function. The putative C. jejuni TssL (30 kDa) and TssM (137 kDa) proteins are predicted to have transmembrane domains and coiled-coil structures. Both proteins share characteristics with the IcmH/DotU and IcmF proteins originally identified as non-essential components of a type IV secretion system (T4SS) that facilitate the translocation of bacterial effector proteins into eukaryotic target cells C. jejuni TssM contains a Walker A motif (GXXGXGKT/S) in its cytoplasmic N-terminal domain implicated in the ATP hydrolysis energizing the recruitment of Hcp to the TssL-TssM membrane complex Closer inspection revealed that in the first group of genes the -M genes . The proC. jejuni strain 108 comprises the proteins TssB, TssC, TssE, TssF and TssG (C. jejuni TssE (15 kDa) has remote homology to the bacteriophage T4 baseplate protein gp25 and to the V. cholerae ortholog VCA0109 that is essential for Hcp secretion C. jejuni proteins TssF (66 kDa) and TssG (38 kDa), both predicted to be inner membrane proteins, are homologous to conserved T6SS components of unknown function, although the TssF ortholog in Rhizobium leguminosarum (ImpG) is involved in plant root infection C. jejuni TssA (50 kDa) and TagH (35 kDa) proteins resemble T6SS hypothetical proteins. C. jejuni TagH appears to have a forkhead-associated domain (FHA) that may confer phosphoprotein-specific protein-protein interactions and thus may have a regulatory function. The C. jejuni T6SS gene cluster lacks a ClpV-ATPase ortholog implicated in the depolymerization and recycling of the T6SS tubular sheath proteins that may wrap the Hcp inner tube structure A second group of predicted T6SS components in and TssG . TssB  element of the VgrG protein family and with the bacteriophage T4 tail spike protein C. jejuni strain 108 lacks the extended biological effector domain that is often associated with modulation of eukaryotic cell function C. jejuni strain 108 and its most related orthologs in several other species are summarized in The protein encoded by the C. jejuni T6SS is 26.5%, compared to about 30% for the C. jejuni genome. In C. jejuni strain 108 the T6SS cluster is flanked at the amino- and carboxyterminal ends by orthologs (98% similarity at the amino acid level) of respectively CJE1139 and CJE1141/CJ1142 of strain RM1221. These genes are located on CJIE3, an integrated element present in the genome of several C. jejuni strains including RM1221 tssL (VgrG) gene at the carboxyterminal end of T6SS of C. jejuni strain 108. This gene organization strongly suggests that the T6SS of C. jejuni 108 is localized within CJIE3 and has inserted between the genes CJE1139 and CJE1141/CJE1142 of strain RM1221. Analysis of C. jejuni strain 108 for the presence of CJIE3 by PCR using a specific primerset C. jejuni strain 108 between genes CJE1139 and CJE1141/CJE1142 in the CJIE3 element of C. jejuni strain RM1221.T6SS gene clusters are usually located on pathogenicity islands or chromosomal regions that show a bias towards bacterial survival or virulence C. jejuni T6SS on genetic element CJIE3 in strain 108 and the variable presence of T6SS genes in other C. jejuni genomes led us to determine the T6SS prevalence in C. jejuni. To this end, we analyzed 80 Campylobacter strains for the presence of the hcp gene using PCR. Both human and animal isolates from different regions of the world were analyzed  culture supernatants using the Hcp-specific antiserum demonstrated Hcp in both fractions  is required for T6SS-dependent Hcp secretion C. jejuni 108ΔTssM. The disrupted gene had inserted in the same orientation as the parent gene as verified by PCR. Western blot analysis of the TssM mutant demonstrated the presence of Hcp in whole bacterial lysates but not in the culture supernatant  vasH appears absent from the T6SS locus of C. jejuni 108, we tested the effect of genetic inactivation of C. jejuni rpoN on Hcp expression. This mutant has a defect in flagella assembly and displays a motility deficient phenotype C. jejuni.The presence of large quantities of C. jejuni T6SS we first tested the potential competitive advantage of C. jejuni strain 108 towards other microorganisms. In several bacterial species the presence of T6SS facilitates survival in mixed populations, often through the production of antibacterial toxins that are injected into neighboring bacteria C. jejuni strain 108 with E. coli DH5α either in broth or on agar plates for up to 5 days did not reveal a selective growth advantage for the T6SS expressing strain. Similar results were obtained when C. jejuni strain 108 was incubated with the TS6SS-negative C. jejuni strain 81116ΔCPS which has similar growth requirements and growth rate as strain 108.In search for a biological function of the C. jejuni T6SS appeared to lack the extended VgrG protein often associated with cytotoxicity toward host cells, we next tested the effects of C. jejuni strains 108 and 108ΔHcp on eukaryotic cells including Caco-2 intestinal epithelial cells and red blood cells. Confocal laser microscopy on Caco-2 cells infected with strain 108 demonstrated that internalized C. jejuni remained in a CD63-positive endolysosomal compartment for up to 24 h C. jejuni. This showed that C. jejuni 108 caused strong hemolysis compared to mutant strain 108ΔHcp (C. jejuni grown on agar plates for 3 days followed by growth in HI broth for 8 h (3 p/8 h) failed to consistently induce substantial hemolysis . C. jejuemolysis .C. jejuni surface capsule polysaccharide (CPS) interfered with the contact-dependent hemolysis. To test this hypothesis we first analyzed the CPS of C. jejuni 108 at different age of culture using Alcian blue staining. This revealed variable intensity of capsule staining with highest levels of capsule expression for C. jejuni grown on agar plates for 3 days and then in HI broth for 16 h (3 p/16 h)  . At earl p/16 h) . This se p/16 h) .C. jejuni 108ΔCPS for hemolytic activity. The mutant was constructed by allelic exchange of the kpsM gene with a disrupted copy of this gene. Unexpectedly, C. jejuni 108ΔCPS displayed strong cytotoxicity at all tested time points except during early exponential growth phase (3 d/8 h) , irrespective the presence of a functional T6SS  . The cyt or tssM , indicatemolysis . Togethenal T6SS .C. jejuni culture fails to show the T6SS phenotype even in the capsule-negative background. To address the point, we compared relative transcript levels of hcp in 3 p/8 h and 3 p/16 h cultures for C. jejuni strains 108 and 108ΔCPS. Real-time PCR analysis showed 5–10 fold more hcp transcript for the 3 p/16 cultures compared to 3 p/8 h bacteria both for the parent and cps mutant strain (C. jejuni hcp mRNA levels vary between growth conditions and suggest that in the early logarithmic phase C. jejuni Hcp levels may be insufficiently expressed to induce the hemolytic phenotype.The issue that remained to be resolved was why the 3 d/8 h t strain . These rC. jejuni. The T6SS gene cluster is present in about 10% of the C. jejuni isolates and has inserted into C. jejuni integrated element 3 (CJIE3). The function is evidenced by the secretion of the hallmark hemolysin co-regulated protein Hcp. C. jejuni T6SS is special among the T6SS family because it confers contact-dependent cytotoxicity toward red blood cells and because its function requires down-regulation of the polysaccharide capsule. The capsule controlled cytotoxicity of C. jejuni T6SS adds a new element to the growing repertoire of T6SS regulation mechanisms and phenotypes.Type VI secretion systems are bacterial nano-injection machines that transport macromolecules into neighboring prokaryotic or eukaryotic cells. The toxic effector molecules serve to outcompete other bacterial species C. jejuni consists of 13 genes that most resemble the T6SS genes of C. coli and H. hepaticus both with regard to gene organization and content (C. jejuni T6SS proteins include the TssJ-M proteins encoding base plate components of the secretion apparatus C. jejuni T6SS locus lacks the frequently found TssH gene (COG0542) encoding a ClpV ATPase implicated in the recycling of the TssB/TssC tubular sheath V. choleraeC. jejuni genome partakes in T6SS function, although analysis of the (incomplete) genome of strain 108 has thus far failed to detect a ClpV homologue in strain 108 (unpublished results).The T6SS cluster of  content . In addiC. jejuni T6SS genes are only present in isolates that carry the integrative element CJIE3 (integrated into the 3′ end of an arginyl-tRNA). This element is likely plasmid derived and appears to consist of modular regions of unknown function that differ between CJIE3-positive strains C. jejuni isolates and those used in the Parker study, is approximately 18%. Yet, only approximately 10% of the isolates contained the T6SS locus. Based on the difference in T6SS flanking regions in our isolates, we assume that the T6SS genes have been acquired en bloc and inserted at different positions into the previously acquired CJIE3.The C. jejuni T6SS as a secretion apparatus is evident from the accumulation of Hcp in the culture medium and the defective Hcp secretion (but intact production) in the constructed tssM-defective strain  C. jejuni T6SS protein secretion  complementation of the hcp mutant with an intact copy of the gene restored the phenotype, (iii) hemolysis was minimal for strain 81116 that lacks the T6SS gene cluster. The low level of hemolysis measured after prolonged incubation with the mutant strains (C. jejuni by increasing the availability of nutrient sources such as iron and nicotinamide adenine dinucleotide (NAD) that are abundantly present in erythrocytes.Our results indicate that toxicity  and the  strains  may be aC. jejuni T6SS phenotype was the explanation of the initially highly variable results by changes in the level of capsule expression. The use of constructed capsule- and Hcp-negative mutant strains unequivocally demonstrate C. jejuni polysaccharide capsule as a key determinant controlling T6SS-induced hemolysis. To our knowledge capsule expression has not previously been implicated as a factor influencing the activity of T6SS. We propose that the polysaccharide capsule inhibits T6SS function by acting as a steric barrier that prevents the T6SS needle structure to puncture the host cells. In addition, needle length may be of importance as suggested by the poor hemolytic activity of capsule-negative strains at early growth phase when C. jejuni hcp transcript levels were much lower  and absence in many disease isolates indicate that the T6SS is not an essential virulence factor in intestinal infections but rather may be advantageous for the bacterium in a distinct niche. We did note that four out of the eight TSS6-positive C. jejuni isolates identified in this study were derived from human patients with Campylobacter bacteremia. This seems disproportionally high considering that C. jejuni bacteremia occurs in <0.2% of intestinal infections, often in immunocompromised patients C. jejuni growth in human blood from healthy donors. This resulted resulted in rapid bacterial killing irrespective the presence of T6SS, indicating that in healthy donors bacterial killing mechanisms dominate over the possible growth advantage of T6SS-positive strains due to e.g. the release of nutrients from red blood cells. Similar assays in horse blood revealed no differences in bacterial growth/survival between strain 108 and the T6SS mutant  the T6SS system is functional, (iii) C. jejuni T6SS confers cytotoxicity toward red blood cells, and (iv) the T6SS phenotype requires down regulation of the polysaccharide capsule. To our knowledge, T6SS-mediated hemolysis and an effect of capsule on T6SS function has never been reported for other bacterial species. C. jejuni T6SS represents a novel direction in the evolution of T6SS, expands the existing repertoire of T6SS-mediated effects on eukaryotic cells, and may contribute to systemic C. jejuni infection.Overall, our study provides the first evidence that (i) ∼10% of Campylobacter was grown at 37°C under micro-aerobic conditions  either on Saponin agar plates containing agar base II medium  with 5% saponin lysed horse blood, or in 5 ml of Heart Infusion broth (HI) (Oxoid) in 25 cm2 tissue culture flasks at 160 rpm. Escherichia coli were grown in Luria-Bertani medium at 37°C. When appropriate, growth media were supplemented with ampicillin (100 µg/ml), kanamycin (20 µg/ml), or chloramphenicol (20 µg/ml).The bacterial strains used in this study are listed in Supporting Information . CampyloC. jejuni 108 genomic DNA as template, 2.5 U of Super Taq+ polymerase (HT Biotechnology Ltd. UK), 50 pmol of each primer, and 0.1 mM of dNTPs in a final volume of 50 µl of 10 mM of Tris-HCl, 1.5 mM of MgCl2 and 50 mM KCl. Standard PCR conditions were heating for 2 min at 95°C, followed by 35 cycles of 15 s at 95°C, 15 s at 52°C, and 5 min at 72°C in a BioRad iCycler. Amplified products were subjected to agarose gel electrophoresis, purified with the Qiaex II gel extraction kit (Qiagen), and cloned using pGEM-Teasy (Promega) or PJet (Fermentas) in E. coli DH5α. Primers T7 and Sp6 were used to determine the nucleotide sequences of the pGEM-Teasy inserts. The PJet1.2 forward and reverse primers were used to sequence the pJet1.2 inserts . Sequences were analyzed and aligned using the DNASTAR software package (Lasergene). The complete sequence of the T6SS gene cluster of C. jejuni strain 108 is deposited at GenBank (Accession number: JX436460).The nucleotide sequence of the primers used for cloning and sequencing the T6SS locus are listed in Supporting Information . DNA frahcp and tssM gene with their flanking sequences of strain 108 were amplified by PCR as described above. PCR products were cloned into pGEM-T easy. Inverse PCR on pGEMhcp was used to delete 166 bp of hcp and to introduce a BamHI restriction site. Plasmid pGEMtssM was cut with bglII. The chloramphenicol resistance gene (Cm) from pAV35 was ligated into the created BamHI site in hcp and into the BglII site in tssM, yielding pGEMhcp::cm and pGEMtssM::cm, respectively. Knockout plasmids carrying the Cm gene in the same orientation as the hcp and tssM genes were used to transform C. jejuni 108 by electroporation. Mutants were selected on saponin agar plates containing 20 µg/ml of chloramphenicol. Disruption of the genes was verified by PCR.Primers used for construction of mutants are listed in Supporting Information C. jejuni 81116ΔCPS the kpsM gene with flanking sequences was amplified with primers listed in BglII restriction site. The 2,000 bp tetracycline resistance gene from pTetO was inserted into this BglII site, yielding pGEM-T easy kpsM::tet. Natural transformation was used to introduce the knockout plasmid into C. jejuni 81116. Mutants were selected on saponin agar plates containing 15 µg/ml of tetracycline. BglII digestion was used to replace the tetO gene in the pGEM-T easy kpsM::tet construct with the Cm gene from pAV35. The resulting plasmid pGEMkpsM::cm was introduced in C. jejuni 108 via electroporation, yielding strain 108ΔCPS. Chloramphenicol resistant transformants were selected and gene disruption was confirmed by PCR.For construction of hcp gene including its ribosomal binding site was PCR amplified from strain C. jejuni 108 using primers 276 and 277. After digestion with XhoI and XbaI, the resulting 531 bp fragment was cloned into the pMA1 vector behind the C. jejuni metK promoter hcp. The plasmid was introduced by electroporation into C. jejuni 108 wild type and the hcp mutant, and kanamycin resistant transformants were selected.The hcp gene was PCR amplified from C. jejuni 108 genomic DNA with primers 210 and 211 and fused to a C-terminal 6× histidine tag using the XbaI–XhoI sites of pSCodon1 (Delphi genetics SA). Selection of pSCodon1hcp was performed in E. coli CYS21, while E. coli SE1 was used to express the Hcp protein . An overnight culture of E. coli pSCodon1hcp in Staby Switch auto-inducible medium  was used to isolate His-tagged Hcp from the soluble cytosolic fraction. Recombinant protein was purified under native conditions using Ni-NTA agarose and 250 mM of imidazole in the elution buffer as described in the manual (Qiagen). Eluted fractions were analyzed by SDS-PAGE. Hcp-positive fractions were pooled and dialyzed for 20 h against 3 liter of 50 mM of Tris-HCl buffer (pH 7.6) containing 200 mM of KCl, 10 mM of MgCl2, 0.1 mM of EDTA, 10% glycerol, and then against the same buffer containing 50% glycerol.The Two New Zealand White rabbits (SPF) were immunized four times  with 100 µg of purified Hcp protein using the classical anti-protein protocol . Serum aliquots were collected  and stored at −20°C. Dilutions of sera were tested by Western blot analysis for Hcp reactivity. All immunizations and handling of animals were performed by Eurogentec, Belgium.For protein detection, aliquots of bacterial cultures  were pelleted  and dissolved in the same volume of Laemmli electrophoresis solution containing 30 mM of Tris-HCl (pH 6.8), 4% SDS, 0.025% bromophenol blue and 20% glycerol. The bacterial supernatant was subjected to high speed centrifugation  to remove supramolecular structures and mixed at a 3∶1 (v/v) ratio with 3×-concentrated Laemmli solution. After boiling (10 min), the equivalent of 10 µl of bacterial culture was loaded onto a 12% SDS-polyacrylamide gel. Proteins were transferred to PVDF membranes . After blocking , 0.1% Tween 20), Hcp was detected with rabbit anti-Hcp antiserum  in combination with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) and Super signal west pico chemiluminescent substrate (Pierce).Two-dimensional gel electrophoresis was generously carried out by Dr. Bas van Balkom as previously described 9) were suspended in 100 µl of Laemmli buffer. Next, 30 µl of 20 mg/ml proteinase K in water was added and the mixture was incubated at 55°C for 2 h. Capsular polysaccharides were separated on a 12% SDS-PAGE gel. Gels were washed twice in water and stained (1 h) with filtered 0.5% Acian Blue 8GX (Sigma) in 2% acetic acid/40% methanol. Gels were destained in 2% acetic acid/40% methanol until bands became visible For capsule detection, bacteria (2×10C. jejuni grown on saponin agar plates for 3 or 7 days were transferred to HI broth (OD550: 0.05) in 25 cm2 flask and shaken (160 rpm) for 8 h or 16 h at 37°C under microaerophillic conditions. Bacterial pellets from solid and broth media were suspended in PBS to OD550 of 1. One ml of this bacterial suspension was then mixed with 0.25 ml of a 5% (v/v) horse erythrocyte suspension (Biotrading) kept in PBS with 0.4 mM of CaCl2 in a 1.5 ml plastic tube with a perforated cap. After incubation , the tubes were mixed and centrifuged . The OD420 of the supernatants was then measured as indicator of the degree of hemolysis. Negative (with PBS without bacteria) and positive  controls were included in all assays. Cytotoxicity was scored as percentage of cell lysis of the positive control. Data are expressed as the mean ± SEM of at least three independent experiments.2 atmosphere. Prior the use the medium was replaced by 1 ml of fresh DMEM (without FCS). C. jejuni strains 108, 108ΔHcp, 108ΔCPS, 108ΔCPS/ΔHcp and (as control) Salmonella Typhimurium strain SL1344 were grown in HI broth, collected by centrifugation , and added to the macrophages at a bacteria to host cell ratio of 20. After 2 h of incubation , the extracellular Salmonella were removed and 1 ml of DMEM+50 µg/ml gentamicin was added. The cells were incubated for an additional 10 h before total cellular LDH and LDH release from the cells was determined with the Cytotoxicity Detection Kit according to the protocol of the manufacturer (Roche). The supernatant or the lysed cells stained with the kit was analyzed on the FLUOstar omega (BMG Labtech) at 492 nM and 690 nM for wavelength correction. Cytotoxicity was determined by calculating the percentage of LDH released after background subtraction. C. jejuni had no effect on the LDH itself or the assay. Presented results are from three individual assays performed in triplicate. Data were analyzed using Graphpad Prism software.Cytotoxicity toward macrophages was determined using J774A.1 macrophages (ATCC) grown for 24 h in a 24-well plate in DMEM+10% FCS at 37°C in a 10% COC. jejuni strain 108, 108ΔCPS and 108ΔCPS/ΔHcp grown in HI broth  were collected by centrifugation  and suspended in 50 µl of HI broth. Bacteria (109 CFU) were, added to 3 ml of heparinized human or defibrinated horse blood in 35 mm petri dishes and incubated at 37°C under micro-aerobic conditions. After 0, 6, 24 and 48 h, 10 µl aliquots were taken, serially diluted in PBS, and plated onto saponin agar plates. Bacterial survival was determined by counting the number of colony forming units after 48 h of incubation at 37°C under micro-aerobic conditions.C. jejuni strain 108 and 108ΔCPS grown in 25 ml of HI Broth for 8 or 16 h at 37°C under micro-aerobic conditions, using the RNA-Bee kit  according to the manufacturer's specifications. Isolated RNA was treated with 1 µg of DNase (Fermentas) per µg of RNA for 30 min at 37°C, after which the DNase was inactivated by heating at 65°C for 10 min in the presence of EDTA . Real time RT-PCR analysis was performed using the Brilliant III Ultrafast SYBR Green QRT-PCR master mix . The PCR mixture (20 µl) contained 40 ng of DNase treated RNA, 20 pmol of the Hcp gene specific primers hcp-RT forw (5′-ACCCGATTTATATCTATTGCCAAT-3′) and hcp-RT rev (5′-GAAGGTTCCACACAAGGTTTGAT-3′), 10 µl of 2× SYBR green mix, 0.2 µl of 100 mM of DTT and 1 µl of RT/RNAse block. The reverse transcriptase cycle was 50°C for 10 min, followed by a PCR initial activation step of 95°C for 3 min. The mixtures were then amplified in 45 cycles of 95°C for 5 sec and 60°C for 10 sec in a Light Cycler 480 (Roche). Total hcp mRNA in each sample was normalized against the internal controls gyrA and rpoA. Three independent experiments with two independent preparations of RNA were analyzed by real-time RT-PCR.Total bacterial RNA was extracted from Figure S1Growth of C. jejuni strain 108, 108ΔCPS and 108ΔCPS/ΔHcp in blood.(TIF)Click here for additional data file.Table S1Strains and plasmids used in this study.(DOC)Click here for additional data file.Table S2Primers used for cloning and sequencing of the T6SS locus of C. jejuni strain 108.(DOC)Click here for additional data file.Table S3Primers used for construction of mutants and expression vectors.(DOC)Click here for additional data file.


Cellular restriction factors responding to herpesvirus infection include the ND10 components PML, Sp100 and hDaxx. During the initial stages of HSV-1 infection, novel sub-nuclear structures containing these ND10 proteins form in association with incoming viral genomes. We report that several cellular DNA damage response proteins also relocate to sites associated with incoming viral genomes where they contribute to the cellular front line defense. We show that recruitment of DNA repair proteins to these sites is independent of ND10 components, and instead is coordinated by the cellular ubiquitin ligases RNF8 and RNF168. The viral protein ICP0 targets RNF8 and RNF168 for degradation, thereby preventing the deposition of repressive ubiquitin marks and counteracting this repair protein recruitment. This study highlights important parallels between recognition of cellular DNA damage and recognition of viral genomes, and adds RNF8 and RNF168 to the list of factors contributing to the intrinsic antiviral defense against herpesvirus infection. The cellular DNA damage response pathway monitors damage to genomic DNA. We investigated whether cellular DNA damage response proteins can also respond to incoming viral genetic material and how they impact virus growth. Using Herpes Simplex Virus type 1 (HSV-1), we present evidence that DNA repair proteins are activated at the earliest times post-infection, and that they physically accumulate at sites associated with incoming viral genomes. A subset of these DNA repair proteins deposit repressive ubiquitin marks, recruit other DNA repair proteins, and limit transcription from the viral genomes. We demonstrate that the virus overcomes this anti-viral defense by targeting key DNA repair proteins for degradation. Our study adds these DNA repair protein mediators to the list of intrinsic antiviral defense factors active against HSV-1, and demonstrates that many aspects of the cellular recognition of foreign DNA parallel the recognition and response to cellular damage. Mammalian cells have evolved complex defenses to protect themselves from viral infections. Innate and adaptive immune responses are well-characterized, but resistance mediated by pre-existing cellular factors has recently emerged as another important arm of antiviral defense. In contrast to the canonical immune responses, which are slower acting and initiated by virus-induced signaling cascades, the pre-existing cellular factors are poised to protect the cell before the virus has even entered Herpes simplex virus type 1 (HSV-1) is a common human pathogen that causes life-long recurrent disease. Lytic HSV-1 infection is characterized by transcription in a temporal cascade of immediate-early (IE), early (E), and late (L) gene products. The immediate early (IE) genes create a favorable intracellular environment for the virus, and regulate the expression of the E and L genes. The IE protein ICP0 is one of the first viral proteins expressed during HSV-1 infection , Abcam (SUMO1 and SUMO2/3), Rockland (ATM S1981-P), Santa Cruz , Millipore , Calbiochem (BRCA1), Research Diagnostics Inc. (GAPDH), Covance (HA), Transduction Laboratories (DNA-PKcs), and Sigma (FLAG). Rabbit antisera to Mdc1 was from J. Chen. The 58S monoclonal antibody to ICP4 was generated from an ATCC hybridoma cell line For immunoblotting, lysates prepared by standard methods. For immunofluorescence, cells were fixed with 4% paraformaldehyde for 15 min and extracted with 0.5% Triton X-100 in PBS for 10 min. For certain antibodies, cells were pre-treated with 0.5% Triton X-100 in PBS for 10 min prior to fixation. Nuclei were visualized by staining with DAPI. Images were acquired using a Leica TCS SP2 confocal microscope.6 cells were infected with WT or ICP0-null virus at an MOI of 0.01 and harvested at 2 and 5 hpi. 75% of the cell pellet was used for RNA extraction and 25% for DNA purification. 1 µg RNA was reverse transcribed using SuperScriptIII RT (Invitrogen) and oligo dT in a 20 µl reaction. qPCR was run in triplicate with 3 µl cDNA or 100 ng genomic DNA using SYBR Green PCR master mix (ABI) on an ABI 7900HT system. ICP27 transcript was detected using primers GCATCCTTCGTGTTTGTCATT (F) and GCATCTTCTCTCCGACCCCG (R) CTGGAAGTCCAACTACTTCC (F) and TGCTGCATCTGCTTGGAGCC (R).2X10Figure S1(A) Simplified overview of the cellular response to DNA damage, indicating the positions of key proteins used in our studies (B) HFF cells were treated with 50 µg/ml α-amanitin for 1 hr prior to infection with ICP0-null HSV-1 at an MOI of 10 for 4 hrs in the presence of 50 µg/ml α-amanitin. Cells were fixed and stained for ICP4 (to confirm that transcription was blocked), and localization of γH2AX and 53BP1 was assessed. Nuclei were stained with DAPI as shown in the merged image. (C) HFF cells were infected with wild-type or ICP0-null HSV-1 at an MOI of 0.001 or 0.1, respectively for 1 hr, and virus was replaced with media containing 1% human serum to limit viral spread. Cells were fixed at 24 hpi, stained for ICP4, and localization of γH2AX (left graph) or 53BP1 (right graph) was assessed in 100 asymmetrically infected cells at edges of plaques.Overview of the cellular response to DNA damage, requirements for recruitment of 53BP1 to sites associated with ICP0-null HSV-1 genomes and quantification of the recruitment phenotype. (TIF)Click here for additional data file.Figure S2(A) HFF cells were infected with ICP0-null HSV-1 at an MOI of 0.1 for 1 hr, and virus was replaced with media containing 1% human serum. Cells were fixed at 24 hpi, and the correlation between pixels positive for 53BP1 and pixels positive for γH2AX (top panel), 53BP1 and ICP4 (middle panel) or γH2AX and ICP4 (bottom panel) was assessed in one representative asymmetrically infected triple-labeled cell. (B) Cells were infected as in A, and stained to assess ICP4, 53BP1, and PML localization. The Manders' overlap co-efficient was determined for ICP4 and 53BP1 overlap compared to ICP4 and PML overlap in one representative asymmetrically infected triple-labeled cell.Sites of DNA repair protein accumulation are distinct from viral genomes and de novo ND10 structures. (TIF)Click here for additional data file.Figure S3(A) HepaRG cells were infected with lentivirus expressing shRNA specific to RNF8 as described in the (B) HepaRG shRNF8 cells were irradiated with 10 Gy γIR and the localization of 53BP1 was assessed by immunofluorescence.Characterization of RNF8-depleted cells. (TIF)Click here for additional data file.Figure S4(A) Control HepaRG cells or cells in which RNF8 had been depleted using shRNA were infected with wild-type virus at an MOI of 0.001 or ICP0-null HSV-1 at an MOI of 0.1 for 1 hr, and virus was replaced with media containing 1% human serum. Cells were fixed at 24 hpi, stained for ICP4, and γH2AX localization was assessed in asymmetrically infected cells at edges of plaques. (B) RIDDLE cells or RIDDLE cells with HA-tagged RNF168 reconstituted were infected and analyzed as in A.γH2AX accumulation at sites associated with incoming HSV-1 genomes is not dependent on RNF8 or RNF168. (TIF)Click here for additional data file.Figure S5(A) HFF cells were infected with wild-type or ICP0-null HSV-1 at an MOI of 0.001 or 0.1 respectively for 1 hr, and virus was replaced with media containing 1% human serum. Cells were pre-extracted before fixation at 24 hpi, and localization of uH2A was assessed in asymmetrically infected cells at edges of plaques. (B) HFF cells were infected with ICP0-null HSV-1 at an MOI 0.1 for 1 hr, and virus was replaced with media containing 1% human serum. Cells were fixed at 24 hpi, and stained for ICP4, PML, and FK2 (conjugated ubiquitin) or ICP4, 53BP1, and FK2. The correlation between pixels positive for 53BP1 and FK2 (top panel), or PML and ICP4 (bottom panel) was assessed in the images shown.uH2A and conjugated ubiquitin accumulation at sites of incoming ICP0-null HSV-1 genomes. (TIF)Click here for additional data file.Figure S6(A) RNF8-/- MEFs transduced with control retrovirus or retrovirus expressing RNF8 were infected with WT or ICP0 null virus at an MOI of 0.01. ICP27 transcripts were detected at 2 or 5 hrs post infection and normalized to a cellular control. In both cell lines, ICP0 null virus was less transcriptionally competent than wild-type, in keeping with the known phenotype of this mutant virus. Experiments were performed in duplicate and averaged. Results are representative of three independent experiments and error is one standard deviation of the duplicate samples. (B) RIDDLE cells complemented either with empty vector or RNF168 were treated with lentivirus expressing either control shRNA or shRNA targeting RNF8. Levels of RNF8 and uH2A were assessed by western blot. (C) Cells depleted for RNF168 and/or RNF8 were infected with wild-type or ICP0-null virus. Relative probabilities of plaque formation were calculated by comparing the numbers of plaques on the different cell lines at each separate dilution of virus.RNF8 represses viral genomes and is part of the intrinsic anti-viral defense. (TIF)Click here for additional data file.