Ehrlichia chaffeensis, an obligatory intracellular rickettsial pathogen, enters and replicates in monocytes/macrophages and several non-phagocytic cells. E. chaffeensis entry into mammalian cells is essential not only for causing the emerging zoonosis, human monocytic ehrlichiosis, but also for its survival. It remains unclear if E. chaffeensis has evolved a specific surface protein that functions as an ‘invasin’ to mediate its entry. We report a novel entry triggering protein of Ehrlichia, EtpE that functions as an invasin. EtpE is an outer membrane protein and an antibody against EtpE  greatly inhibited E. chaffeensis binding, entry and infection of both phagocytes and non-phagocytes. EtpE-C-immunization of mice significantly inhibited E. chaffeensis infection. EtpE-C-coated latex beads, used to investigate whether EtpE-C can mediate cell invasion, entered both phagocytes and non-phagocytes and the entry was blocked by compounds that block E. chaffeensis entry. None of these compounds blocked uptake of non-coated beads by phagocytes. Yeast two-hybrid screening revealed that DNase X, a glycosylphosphatidyl inositol-anchored mammalian cell-surface protein binds EtpE-C. This was confirmed by far-Western blotting, affinity pull-down, co-immunoprecipitation, immunofluorescence labeling, and live-cell image analysis. EtpE-C-coated beads entered bone marrow-derived macrophages (BMDMs) from wild-type mice, whereas they neither bound nor entered BMDMs from DNase X-/- mice. Antibody against DNase X or DNase X knock-down by small interfering RNA impaired E. chaffeensis binding, entry, and infection. E. chaffeensis entry and infection rates of BMDMs from DNase X-/- mice and bacterial load in the peripheral blood in experimentally infected DNase X-/- mice, were significantly lower than those from wild-type mice. Thus this obligatory intracellular pathogen evolved a unique protein EtpE that binds DNase X to enter and infect eukaryotic cells. This study is the first to demonstrate the invasin and its mammalian receptor, and their in vivo relevance in any ehrlichial species. Ehrlichia chaffeensis and is transmitted by the bite of infected ticks. This bacterium has special ability to enter and replicate inside human white blood cells and this feature is very essential for the bacterial survival. How E. chaffeensis enters host cells has been a mystery. The present study revealed that E. chaffeensis outer-surface protein named EtpE binds a specific host cell-surface protein, DNase X, and this ligand-receptor interaction is required to induce bacterial entry into its host cells. In order to test whether E. chaffeensis infection can be prevented by EtpE immunization, mice were immunized with the recombinant EtpE protein, and challenged with live E. chaffeensis. Infection was significantly reduced in the EtpE protein-immunized mice compared to controls. Mice lacking DNase X were also resistant to infection. This study shows EtpE-mediated entry pathway of E. chaffeensis is important in infecting mammals and EtpE can be incorporated into a future HME vaccine design.Human monocytic ehrlichiosis (HME), discovered in 1986, was designated as a nationally notifiable disease by Centers for Disease Control and Prevention in 1998. HME is one of the most prevalent, life-threatening emerging infectious diseases in the United States. HME is caused by a bacterium,  Ehrlichia chaffeensis causes human monocytic ehrlichiosis (HME), an emerging tick-borne zoonosis. From the site of infected tick bite on human skin, E. chaffeensis infects monocytes and spreads via the bloodstream to various tissues, causing a systemic febrile disease. HME is characterized by fever, headache, myalgia, thrombocytopenia, leucopenia, and elevated liver-enzyme levels, but complications such as pulmonary insufficiency, renal failure, encephalopathy, and disseminated intravascular coagulation can cause death E. chaffeensis is a small obligatory intracellular bacterium. It belongs to the family Anaplasmataceae in the order Rickettsiales that includes many understudied pathogens of veterinary and public health importance E. chaffeensis is a polymorphic bacterium (0.2–2.0 µm in diameter), and can be morphologically categorized as small dense-cored cells (DCs) or large reticulate cells (RCs) Chlamydia and larger viruses such as Vaccinia virus. By light microscopy, it is not possible to distinguish individual RCs and DCs, since E. chaffeensis aggregates inside eukaryotic host cells. The characteristic clump of intracellular E. chaffeensis organisms is termed as “morula” (mulberry in Latin) E. chaffeensis, and hence, the disease was named as “monocytic ehrlichiosis” to distinguish it from “granulocytic ehrlichiosis” caused by infection with granulocyte-tropic Ehrlichia sp. E. chaffeensis can replicate well in several mammalian cell lines including canine histiocytic leukemia (DH82), human acute leukemia (THP-1), human promyelocytic leukemia (HL-60), human embryonic kidney (HEK293), and monkey endothelial (RF/6A) cells E. chaffeensis to sustain its life, since its small genome of 1.18 Mb lacks a large portion of metabolic genes that are required for free living E. chaffeensis also lacks LPS, peptidoglycan, lipoteichoic acid, and flagella that engage Toll-like or NOD-like receptors, or scavenger receptors E. chaffeensis entry and subsequent infection of THP-1 cells, but not binding are almost completely inhibited by monodansylcadaverine (MDC), a transglutaminase inhibitor Neorickettsia risticii (formerly Ehrlichia risticii) entry and infection of P388D1 cells, vesicular stomatitis virus uptake and receptor-mediated endocytosis of α2-macroglobulin by Swiss 3T3 mouse cells, but not the uptake of latex beads by P388D1 mouse macrophages E. chaffeensis entry into THP-1 cells, leading to productive infection, is dependent on the host-cell surface lipid rafts and glycosylphosphatidyl inositol (GPI)-anchored proteins E. chaffeensis entry site E. chaffeensis replicates in the membrane-bound compartment resembling an early endosome as it contains early endosome antigen 1 (EEA1), Rab5, and transferrin receptor E. chaffeensis entry are unknown; particularly regarding the involvement of any specific bacterial surface protein that can function as an invasin and its cognitive host-cell receptor Entry into the permissive eukaryotic host cells is essential for E. chaffeensis strains revealed that a hypothetical protein, ECH1038, consists of highly conserved N- and C-terminal segments flanking its strain-variable central region E. chaffeensisEhrlichia, EtpE), particularly its C-terminal conserved region (EtpE-C), is critical for E. chaffeensis binding, entry, and infection of several different host cell types. In order to study whether EtpE-C can mediate the invasion into permissive host cells in the absence of other E. chaffeensis factors, we used latex beads coated with EtpE-C. The coated beads entered both phagocytes and non-phagocytes in a manner similar to E. chaffeensis entry. Subsequent investigation led to the discovery that DNase X, a host cell surface GPI-anchored protein, is the receptor of EtpE-C mediating the entry of E. chaffeensis into several mammalian cell types permissive to its replication.The comparative genome hybridization study of r 222,638, pI: 7.0) and is predicted to be an outer membrane protein with an N-terminal secretion signal by PSORT analysis E. chaffeensis strains of distinct virulence, suggesting that these two regions are indispensable for E. chaffeensis. The amino acid sequence alignment of EtpE orthologs in the three strains of E. chaffeensis, Arkansas, Wakulla, and Liberty are shown  consists of 1963 amino acid residues  and C-terminal (residues 1658–1965) of EtpE as C-terminal histidine-tagged fusion proteins (rEtpE-N and rEtpE-C) and antisera were prepared against rEtpE-N and rEtpE-C. Western blot analysis using these antibodies showed that full-length EtpE was expressed by 82 cells . DH82 ceE. chaffeensis in human monocytes, the pathogen's primary in vivo target cells, EtpE expression was determined in E. chaffeensis cultured in human primary macrophages derived from peripheral blood monocytes by double immunofluorescence labeling after paraformaldehyde (PFA) fixation and saponin permeabilization. E. chaffeensis major outer membrane protein P28 E. chaffeensis in human macrophages, and localized at bacterial membrane like P28 To determine whether EtpE is expressed by E. chaffeensis bound to the surface of DH82 cells. Unlike methanol or acetone fixation, PFA fixation does not allow antibody penetration across biological membranes unless with subsequent permeabilization, thereby limiting antibody staining to molecules exposed to the cell surface E. chaffeensis had a beaded (rosary-like) pattern encircling individual bacterium, in contrast to P28 that had a uniform ring pattern  are referred to as phagocytes. Phagocytes are very efficient in bacteria and particle uptake as they have an array of dedicated phagocytic receptors, including pathogen pattern recognition receptors, mannose receptors, scavenger receptors, receptors for immunoglobulin (FcR) and complement (CR3) that utilize opsonins for ingestion, to name a few in vitro antibody neutralization of EtpE as they lack the response to opsonization and will not readily take-up opsonized particles. E. chaffeensis was pre-incubated with mouse anti-EtpE-C or preimmune mouse sera, and then incubated with RF/6A cells. Binding and entry were determined by immunofluorescence labeling of E. chaffeensis with anti-P28 at 30 min and 2 h post-incubation/infection (pi), respectively. Infection was determined at 48 h pi by quantitative real-time PCR (qPCR). Anti-EtpE-C blocked E. chaffeensis binding, entry, and subsequently infection by approximately 80% compared to preimmune serum  against P28 did not block binding or entry of E. chaffeensis  response is mounted against this protein during infection and disease.Because EtpE is highly expressed by E. chaffeensis in immunocompetent mice E. chaffeensis binding, consequently entry and infection in vitro, EtpE was expressed by E. chaffeensis in vivo and that a humoral immune response was mounted in infected mammals, we decided to examine whether rEtpE-C immunization could confer protection in mice from E. chaffeensis challenge. C3H/HeJ strain of mice was used, since this strain was reported to serve as a useful model for studying E. chaffeensis infection E. chaffeensis. The E. chaffeensis load in the blood from rEtpE-C-immunized mice at 5 days post challenge was significantly lower than that of non-immunized mice  coated with rEtpE-C protein. The presence of rEtpE-C on beads was confirmed by dot-blot analysis (data not shown) and immunofluorescence labeling with antiserum against EtpE-C  E. chaffeensis bound to DH82 cells 2+ channel blocker) that block E. chaffeensis entry into THP-1 cells E. chaffeensis entry into RF/6A cells . One of the clones contained an additional plasmid encoding S-adenosyl methionine-dependent methyltransferase but the coding sequence was out-of-frame; this prey construct alone did not support yeast growth when co-transformed with bait vector to test their interaction. All sequence hits corresponded to the C-terminal fragment of DNase X (residues 105–302). DNase X, one of the human DNase I–family endonucleases, is expressed on the cell surface as a GPI-anchored protein and also localized at early endocytic vesicles, endoplasmic reticulum, and Golgi E. chaffeensis for 30 min . When DH82 cells were pre-incubated with monoclonal anti-DNase X IgG to block the surface-exposed DNase X, E. chaffeensis binding, entry, and overall infection were significantly reduced compared with the control mouse IgG-treated DH82 cells  ability of E. chaffeensis to bind and enter non-phagocytes vs. inability of non-coated beads to enter non-phagocytes, 2) inhibition of E. chaffeensis entry into phagocytes and non-phagocytes by MDC, genistein, verapamil, and PI-PLC vs. lack of inhibition of entry of non-coated beads into phagocytes by these compounds, 3) DNase X-dependent entry of E. chaffeensis into phagocytes and non-phagocytes, vs. DNase X-independent entry of non-coated beads into phagocytes, and 4) colocalization of DNase X and E. chaffeensis during entry vs. lack of colocalization of non-coated beads with DNase X. Remarkably, coating with a fragment of a single protein, EtpE-C made the beads to bind and enter non-phagocytes and phagocytes like E. chaffeensis does. Similarities between EtpE-C-coated beads and E. chaffeensis include 1) signaling pathways for invasion sensitive to MDC, genistein, verapamil, and PI-PLC, 2) DNase X-dependent adhesion and invasion, 3) colocalization with DNase X at entry foci during invasion, and 4) ultrastructure of entry process  that is closely related to E. chaffeensisN. risticii infects P388D1 macrophages, but FcR-mediated entry kills this bacterium and Fab fragment of the immune IgG blocks binding and entry of N. risticiiE. chaffeensis infection by rEtpE-C immunization awaits further study. Nonetheless, our results from in vitro neutralization studies suggest a direct inhibition of E. chaffeensis binding by anti-EtpE-C antibody and support the importance of antibodies in ehrlichial immunity. A recent study showed that EtpE mRNA is highly expressed by E. chaffeensis in cell lines derived from the ticks, Amblyomma americanum and Ixodes scapularis using E. chaffeensis genome microarrays E. chaffeensis is transmitted between mammals by tick bite, expression of EtpE in tick cells suggests an indispensable role for this protein throughout the E. chaffeensis life cycle.The present study showed that not only antibody against rEtpE-C neutralizes Ehrlichia spp. Compared to DNase I, DNase X has an extra hydrophobic stretch at its C-terminus −/− mice and their reproduction is normal DNase X-mediated uptake is the first entry pathway to be uncovered for −/− BMDMs. Un-opsonized non-coated latex beads are taken up by macrophages predominantly via scavenger receptors rEtpE-C-coated beads, unlike non-coated beads or rECH0825-coated beads, were unable to bind or enter DNase XE. chaffeensis seems to be by a ‘zipper mechanism’ E. chaffeensis binding and invasion of its target cells is depicted in E. chaffeensis attachment. This binding elicits signals that culminate in host cytoskeletal remodeling, filopodial formation embracing the bacteria and engulfment of the bound bacteria into an early endosome in the host cell. This receptor-mediated endocytosis can be specifically disrupted by genistein, verapamil or MDC.The EtpE-DNase X-mediated entry of host cells by Rickettsia conorii using in vitro cultured cells. For invasion, R. conorii uses an autotransporter protein OmpB as the ligand and Ku70, one of the DNA-binding components of the DNA-dependent protein kinase of mammalian cells as the receptor in vivo infection by Rickettsia spp. is unknown. OmpB was originally identified as an invasive rickettsial protein capable of mediating binding and entry of host cells in R. japonica, another spotted fever group Rickettsia closely related to R. conoriiAnaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, binds to fucosylated P-selectin glycoprotein ligand-1 (PSGL-1) as a key initial receptor component A. phagocytophilum to infect neutrophils in mice and for the bacterium to colonize ticks A. phagocytophilum adhesin, the peptidoglycan-associated lipoprotein, OmpA Ehrlichia invasin-receptor pair and is a critical advancement as this provides the first evidence for the importance of a given invasin-receptor pair in vivo for any pathogens belonging to the order Rickettsiales. In contrast to the almost complete abrogation of entry of rEtpE-C-coated beads into DNase X−/− BMDMs, E. chaffeensis entry into DNase X−/− BMDMs was reduced by 60% compared to wild-type BMDMs. This suggests existence of additional mammalian receptors for E. chaffeensis infection. Similarly R. conorii invasion of embryonic fibroblasts derived from Ku70−/− mice is reduced by 50% compared to the cells derived from the wild-type litter mates E. coli expressing another autotransporter protein of R. conorii, OmpA, can invade endothelial cells by interacting with α2β1 integrin R. rickettsiiRickettsia and Ehrlichia spp. are phylogenetically related , and share similar wild animal reservoirs and vector ticks in nature, E. chaffeensis EtpE and R. conorii invasin OmpB/OmpA are uniquely evolved in them, respectively Among members of the order Rickettsiales, the bacterial ligand and the cognitive host cell receptor pair for invasion have been reported only for In light of severe and potentially fatal outcomes of HME, the limited choice of antibiotics and lack of prophylactic measures, further understanding of invasion mechanisms is of great importance. Such information will assist in the development of new preventive and therapeutic measures against HME and similar diseases by specific pharmacological or immunologic disruption of invasin and the host cell receptor interaction.All animal experiments were performed in accordance with The Ohio State University Institutional Animal Care and Use Committee guidelines and approved e-protocol numbers 2009A0186 and 2008A0066. The University program has Full Continued Accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC-I) under #000028 dated June 9, 2000 and has a Public Health Services assurance renewal #A3261-01 dated February 5, 2007 through February 28, 2015. The program is licensed by the USDA, #1-R-014, and is in full compliance with Animal Welfare Regulations.E. chaffeensis Arkansas type strain was propagated in DH82 cells, and host cell–free E. chaffeensis was obtained by controlled sonication as described −/− mice as described E. chaffeensis challenge was performed 10 days following the last immunization as described −/−E. chaffeensis-infected THP-1 cells . DNA was extracted from blood samples using a QIAamp blood kit (Qiagen), and subjected to qPCR using E. chaffeensis 16S rDNA and mouse glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene primers.Two groups of C3H/HeJ mice  (Jackson Laboratories) received either minced SDS-polyacrylamide gel containing 50 µg of rEtpE-C or minced gel alone, with Quil A  as adjuvant for a total of three times at 14-day intervals. E. chaffeensis chromosomal DNA as template. The fragments were cloned into pET33b(+) vector (Novagen); recombinant proteins were expressed in E. coli BL21 (DE3) and purified by Ni-affinity chromatography. The antibody against rEtpE-C was produced in ICR mice (Harlan), and the antibody against rEtpE-N was produced in rabbits.DNA fragments encoding EtpE-C and EtpE-N were amplified by PCR with Phusion high-fidelity DNA polymerase (NEB) using E. chaffeensis-infected dogs  Affinity-purified rEtpE-C and rEtpE-N (5 µg each) were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with sera from 5′-AATCCATGGAATTGTTGTCATTAGTTGGTGGGCATCG-3′ (5′ NcoI site underlined) and reverse primer 5′-TCGACGGATCCAATCCCCTTCCAGCATTAATTTTATCAAAGG-3′ (5′ BamHI site underlined), and the product was ligated into pGBKT7. pGBKT7-EtpE-C was transformed into Saccharomyces cerevisiae strain AH109 and selected by the ability to grow on SD agar plates lacking tryptophan. The expression of bait protein EtpE-C in yeast was examined by Western blotting. The human bone marrow MATCHMAKER cDNA library (Clontech) that was fused with GAL4-activating domain in pGADT7 was transformed in S. cerevisiae strain Y187 (Clontech). Library clones expressing interacting prey proteins were screened with yeast mating. Positive clones were selected by their ability to grow on SD quadruple drop-out (SD/QDO) plates lacking adenine, histidine, leucine, and tryptophan, and verified on SD/QDO plates containing X-gal. Positive clones were then isolated, and the prey plasmids were purified and sequenced after they were transformed into E. coli TOP10F′ competent cells (Invitrogen). The interaction was confirmed by re-shuttling the purified prey plasmid into S. cerevisiae AH109 transformed with bait plasmid and by nutritional selection in SD/QDO plates.Yeast two-hybrid screening was performed using Matchmaker Two-Hybrid System (Clontech) according to manufacturer's instructions. The bait plasmid pGBKT7-EtpE-C was constructed by the fusion of EtpE-C with the GAL4 DNA-binding domain in pGBKT7 (Clontech) by PCR. EtpE-C coding sequence was amplified using the forward primer −/− or congenic wild-type mice and suspension culture of THP-1 cells were incubated with E. chaffeensis freshly isolated from infected cells at approximate multiplicity of infection (MOI) of 200, unless otherwise noted, for 30 to 45 min for binding assays or 2 to 4 h for internalization assays at 37°C in 5% CO2/95% air. Cells were washed with phosphate-buffered saline  to remove unbound bacteria and labeled with antibodies as described E. chaffeensis P28 E. chaffeensisz-axis of the cells, or an LSM 510 laser-scanning confocal microscope (Carl Zeiss). For immunostaining of live bacteria, host cell-free E. chaffeensis was incubated with anti-EtpE-C, anti-EtpE-N or E. chaffeensis P28 for 1 h at room temperature followed by fixing with 3% PFA and labeling with AF555-conjugated goat anti-mouse or anti-rabbit antibodies. To further demonstrate the surface exposure of EtpE, host cell-free E. chaffeensis was incubated with either pronase E (Sigma) at a concentration of 2 mg/ml in PBS or vehicle control for 15 min at 37°C Coverslip cultures of DH82, HEK293, RF/6A cells, or macrophages differentiated from human peripheral blood monocytes or established from bone-marrow of DNase X6 cells of E. chaffeensis-infected THP-1 cells/ml in 2 ml of methionine cysteine-deficient RPMI 1640 medium  supplemented with 10% FBS and 2 mM l-Gln were incubated with cycloheximide (Sigma) at 10 µg/ml at 37°C for 1 h. A metabolic labeling reagent  was added and the mixture was incubated further at 37°C for 24 h. The radiolabeled E. chaffeensis was released by sonication and washed by centrifugation at 10,000×g for 10 min. To study the effect of rabbit anti-P28 on E. chaffeensis binding and entry, radiolabeled E. chaffeensis cells  preincubated with Fab fragment of anti-P28 IgG  or Fab fragment of normal rabbit IgG (0.5 mg/ml) were added to 1×106 THP-1 cells in 0.4 ml of RPMI 1640 medium containing 10% FBS and 2 mM l-Gln and incubated at 4°C for 2 h. The uptake of E. chaffeensis was evaluated following removal of bound E. chaffeensis cells by incubation with pronase E at 2 mg/ml in PBS at 37°C for 10 min after incubation of E. chaffeensis with THP-1 cells at 37°C for 3 h. THP-1 cells were washed by centrifugation at 375×g for 5 min, the cells then were dissolved in 0.6 N NaOH and 0.5% SDS, and the radioactivity was measured in a scintillation counter.Approximately 10E. chaffeensis preincubated with 25 µg/ml of mouse anti-rEtpE-C, rabbit anti-rEtpE-N, or preimmune mouse or rabbit sera for 1 h at 4°C were used to infect THP-1, RF/6A, or DH82 cells. Alternatively, E. chaffeensis was added to DH82 cells preincubated with 10 µg/ml of monoclonal anti-DNase X or control mouse monoclonal antibody for 30 min at 25°C in serum-free DMEM. Binding, internalization, and infection were determined at 30 min, 2 h and 48 h pi, respectively. HEK293 cells in 24-well plates were transfected with 50 nM DNase X siRNA (Santa Cruz Biotechnology) or scrambled control siRNA using Lipofectamine 2000 (Invitrogen). A second transfection with 50 nM of DNase X and scrambled siRNAs was performed 30 h after the first transfection. An aliquot of cells were harvested at 24 h after the second transfection to determine the protein amount of DNase X by Western blotting and densitometry analysis with anti-DNase X and rabbit anti-actin (Sigma). The other aliquot of cells were incubated with E. chaffeensis and incubated for an additional 48 h to evaluate infection. Infection was determined by qPCR of E. chaffeensis 16S rRNA gene relative to host cell G3PDH gene E. chaffeensis for 30 min and lysed in NP-40 lysis buffer. The lysate was immunoprecipitated with anti-EtpE-C (2 µg)-bound protein A agarose or control mouse IgG (2 µg)-bound agarose beads. The precipitate was resuspended in 2× SDS-sample buffer and subjected to Western blotting with anti-DNase X antibody.Far-Western blotting was performed using 5 µg of rEtpE-C and rECH0825 that were separated by SDS-PAGE, transferred to a nitrocellulose membrane and renatured with serial guanidinium-HCl treatment followed by incubation with THP-1 cell lysate in NP-40 lysis buffer  as described 6 beads in 200 µl of 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 8.0 were incubated with 1 µg of rEtpE-C or rEtpE-N proteins in 5–7 µl 7 M urea in 50 mM sodium phosphate buffer, pH 7.2 at 4°C overnight with mixing at 20 rpm. MES buffer (150 µl) was sequentially added to the mixture every 15 min and incubated at room temperature, rotating at 20 rpm eventually diluting to around 200 times the original volume of urea buffer. rECH0825 and rGroEL were treated similarly, but without urea. The coated beads were collected by low speed centrifugation, washed twice in MES buffer and resuspended in complete DMEM or advanced MEM media, then gently sonicated to disperse the beads. Protein coating of the beads were confirmed by dot blot assay and/or immunofluorescence labeling. Freshly prepared protein-coated or non-coated beads were added at a multiplicity of approximately 50 beads per cell for co-localization studies and 500 beads per cell for quantitation of binding and internalization studies. The beads were incubated with HEK293 or RF/6A cells for 1 h at 37°C. Unbound beads were removed by washing and cells were fixed for immunofluorescence labeling to detect the localization of DNase X or EEA1 . To study the effect of MDC, genistein, or verapamil on bead internalization, RF/6A cells were incubated with these chemicals at a final concentration of 100 µM or 0.1% DMSO solvent control for 30 min, then with rEtpE-C-coated beads for 8 h at 37°C. BMDMs from wild-type mice were pre-incubated with MDC or genistein (100 µM), PI-PLC (5 U/ml) or 0.1% DMSO for 30 min and then incubated with rEtpE-C-coated or non-coated beads for 45 min at 37°C. PI-PLC-treated cells were washed prior to addition of rEtpE-C-coated beads. The cells were washed and treated with 0.25% trypsin at 37°C for 10 min to remove surface-bound beads. The detached cells were further washed by low-speed centrifugation and later cytocentrifuged onto a glass slide and fixed with 3% PFA to observe internalized beads. To estimate the number of bound beads, a similar procedure for observing internalized beads was followed except that the beads were incubated with BMDM for 30 min at 4°C and following incubation the cells were washed to remove loosely bound beads and directly fixed with 3% PFA without trypsin treatment. For scanning electron microscopy, rEtpE-C-coated beads were incubated with RF/6A cells for 2 h at 37°C and processed as described previously Sulfate-modified fluorescent red polystyrene beads  at 3–4×102/95% air. Time-lapse images were acquired at an interval of 10 s for 5 to 20 min through a 60×1.42 NA oil immersion lens with an inverted Olympus IX-70 microscope, in 0.4-µm steps in the z-axis using the attached Applied Precision motorized stage (DeltaVision deconvolution microscope). All stacks of images were deconvoluted using SoftWoRx software and the time-lapse images of a single focal plane of 0.4-µm focal depth at the cell surface were exported as a video.RF/6A cells were cultured in 35-mm glass bottom dishes (Wilco), transfected with DNase X-GFP, and incubated with rEtpE-C-coated beads at 4°C for 1 h to facilitate bead binding, but prevent internalization. Unbound beads were washed off, cells were replenished with medium lacking phenol red, and the samples moved to a controlled environmental chamber at 37°C with under 5% COt-test. P<0.05 was considered to be significant.Statistical analysis was performed by unpaired two-tailed Student's Figure S1Alignment of Amino acid sequence of EtpE orthologs among three sequenced Ehrlichia chaffeensis strains, related to.E. chaffeensis EtpEs of Arkansas, Wakulla, and Liberty , were aligned by Clustal W using MegAlign. The red shade represents residues that match the consensus.(TIF)Click here for additional data file.Figure S2Alignment of Amino acid sequence of EtpE orthologs among three sequenced Ehrlichia species, related to.E. chaffeensis Arkansas EtpE, E. canis Jake Ecaj_0838 and E. ruminantium Welgevonden Erum7970 , were aligned by Clustal W using MegAlign. The red shade represents residues that match the consensus.(TIF)Click here for additional data file.Figure S3EtpE is bacterial surface exposed, related to. Immunofluorescence image showing host cell-free E. chaffeensis that was either treated with pronase E or PBS control. Cells were processed for double immunostaining with anti-EtpE-C and anti-CtrA with or without saponin permeabilization as described to distinguish extracellular and internalized bacteria. When bacteria were treated with pronase E, the surface immunofluorescence staining of EtpE was abolished completely, but not that of the internal control CtrA. Scale bar, 1 µm.(TIF)Click here for additional data file.Figure S4Anti-EtpE-C neutralizes E. chaffeensis binding and entry into THP-1 cells, related to. (A) Numbers of E. chaffeensis (Ech) bound to THP-1 cells at 30 min pi. E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and incubated with THP-1 cells for 30 min. Unbound E. chaffeensis was washed away, cells were fixed with PFA and E. chaffeensis was labeled with anti-P28 without permeabilization. E. chaffeensis in 100 cells was scored. (B) Numbers of E. chaffeensis internalized into THP-1 cells at 2 h pi. Purified host cell-free E. chaffeensis was pretreated with anti-rEtpE-C or preimmune mouse serum and incubated with THP-1 cells for 2 h. To distinguish intracellular from bound E. chaffeensis, unbound E. chaffeensis was washed away, and cells were processed for two rounds of immunostaining with anti-P28: first without permeabilization to detect bound but not internalized E. chaffeensis (AF555–conjugated secondary antibody), and another round with saponin permeabilization to detect total E. chaffeensis, i.e., bound plus internalized (AF488–conjugated secondary antibody). The black bar represents total E. chaffeensis, and the white bar represents internalized E. chaffeensis . E. chaffeensis in 100 cells was scored. qPCR for E. chaffeensis 16S rDNA was normalized with human G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (P<0.05).(TIF)Click here for additional data file.Figure S5Anti-P28 does not inhibit binding or uptake of E. chaffeensis by THP-1 cells, related to. (A) Relative radioactivity representing numbers of E. chaffeensis bound to THP-1 cells. Host cell-free radiolabeled E. chaffeensis preincubated with Fab fragment of rabbit anti-P28 IgG or pre-immune rabbit IgG were incubated with THP-1 cells for 2 h at 4°C. Unbound E. chaffeensis was washed away, and radioactivity of bound E. chaffeensis was measured. (B) Relative radioactivity representing numbers of E. chaffeensis internalized into THP-1 cells. Host cell-free radiolabeled E. chaffeensis preincubated with Fab fragment of rabbit anti-P28 IgG or pre-immune rabbit IgG was incubated with THP-1 cells for 3 h at 37°C. Bound un-internalized E. chaffeensis was removed by pronase E treatment, radioactivity of internalized E. chaffeensis measured. Data represent the mean and standard deviation of triplicate samples and are representative of two independent experiments.(TIF)Click here for additional data file.Figure S6Anti-EtpE-N is not effective in neutralizing E. chaffeensis infection in vitro and N-terminus of EtpE is less surface-accessible in live E. chaffeensis than its C-terminus, related to. (A) Infection of RF/6A cells with E. chaffeensis. E. chaffeensis was pretreated with anti-EtpE-N or preimmune rabbit serum and used to infect RF/6A cells; cells were harvested at 48 h pi. qPCR for E. chaffeensis 16S rDNA was normalized with monkey G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (P<0.05). (B) Immunofluorescence labeling of live host cell-free E. chaffeensis. Unfixed E. chaffeensis was first incubated with anti-EtpE-C, EtpE-N, or P28 (ECHP28); then fixed and labeled with AF555–conjugated secondary antibodies. Scale bar, 10 µm.(TIF)Click here for additional data file.Figure S7MDC blocks entry of E. chaffeensis into non-phagocytic RF/6A cells, related to. Immunofluorescence labeling of E. chaffeensis incubated with RF/6A cells pre-treated with MDC or DMSO control. At 3 h pi, cells were treated with trypsin to remove un-internalized E. chaffeensis and then labeled with anti-P28. Scale bar, 10 µm. Bar graph shows quantitation by scoring E. chaffeensis (Ech) in 100 cells (right panel). Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. * Significantly different (P<0.05).(TIF)Click here for additional data file.Figure S8rEtpE-C-coated beads recruit DNase X to the areas of binding, related to. rEtpE-C-coated or non-coated latex beads were incubated with canine primary macrophages derived from peripheral blood monocytes (A) or DH82 cells (B) at 37°C for 30 min, and labeled with anti-DNase X without permeabilization. rEtpE-C-coated beads recruited surface exposed DNase X to their sites of binding and clustered, whereas non-coated beads did not colocalize with DNase X on the cell surface. A single z-plane, of an optical section thickness of 0.4-µm, at cell surface by deconvolution microscopy was shown. Scale bar, 5 µm.(TIF)Click here for additional data file.Figure S9Binding of rEtpE-C-coated beads is dependent on DNase X, related to. (A) Fluorescence and DIC merged images of rEtpE-C-coated, rECH0825-coated and non-coated beads incubated with BMDMs from wild-type and DNase X−/− mice. Beads were incubated with cells for 30 min at 4°C followed by rigorous washing with PBS to remove unbound or loosely-adherent beads. Scale bar, 5 µm. (B) Numbers of internalized rEtpE-C-coated beads/cell of similar experiment as (A), relative to the number of rECH0825-coated beads bound to wild-type BMDM set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (P<0.05).(TIF)Click here for additional data file.Movie S1DNase X is recruited to the sites of binding of rEtpE-C-coated beads on cell surface, related to. Time-lapse video of rEtpE-C-coated latex beads binding to RF/6A cells ectopically expressing DNaseX-GFP. Coated beads were incubated with DNaseX-GFP-expressing RF/6A cells at 4°C, and the time 0 min was set upon raising the temperature to 37°C. The DNase X-GFP patchy signal is initially ∼3 µm away from the beads and redistributes towards the beads, eventually merging with the beads at around 5:47 min. A single z-plane, of an optical section thickness of 0.4-µm, at cell surface by deconvolution microscopy was shown. Scale bar, 30 µm.(MOV)Click here for additional data file.Movie S2rEtpE-C-coated beads cluster with surface DNase X in phagocytic cells, related for. A 3-D view reconstructed from serial z-stack images (for a combined z-section width of 7.2 µm) of human primary macrophages derived from peripheral blood monocytes incubated with rEtpE-C-coated beads at 30 min pi, labeled with α-DNase X without permeabilization. rEtpE-C-coated beads cluster and colocalize with DNase X on the cell surface.(MOV)Click here for additional data file.Movie S3Non-coated beads do not cluster with surface DNase X in phagocytic cells, related for. A 3-D view reconstructed from serial z-stack images (for a combined z-section width of 7.2 µm) of human primary macrophages derived from peripheral blood monocytes incubated with non-coated beads at 30 min pi, labeled with α-DNase X without permeabilization. Non-coated beads are randomly dispersed and not associated with DNase X, unlike the rEtpE-C-coated beads.(MOV)Click here for additional data file.


Diterpenoids are widely distributed throughout the plant kingdom and posses interesting biological activities.Electrospray tandem mass spectrometry approach is widely used for the rapid characterization of natural products. This paper describes the gas-phased ESI-MS/MS fragmentation of abietane-type diterpenoids and their novel dimeric conjugate, salvialeriafone  showed the characteristic losses of water, carbonmonoxide and propene molecules, while analysis in negative ion mode showed the characteristic losses of water, carbon monoxide, methane molecules and methyl radical. Results demonstrated the differences in the product ions and base peaks due to the differences in the skeleton. A novel dimeric conjugate, salvialeriafone (1) showed characteristic fragmentation pattern and was found to be more prone to form radical ions, as compared to monomeric diterpenoids. The fragmentation pathways of characteristic fragments were proposed with the aid of HRESIMS.ESI-QqTOF-MS (positive ion mode) of diterpenoids 1) and related diterpenoids 2–6 were conducted and their characteristic fragments were identified. The knowledge of the fragmentation pattern of these diterpenoids will be useful for the characterization of new dimers of this class of compounds.Extensive tandem mass spectrometric studies of salvialeriafone ( Diterpenoids constitute a large class of chemically diverse metabolites, widely distributed throughout the plant kingdom with more than 12,000 known examples, antiplaSalvia constitutes the largest of the plant family Labiatea with 900 species wide-spread throughout the world. The genus has yielded various classes of natural products, including the major class of terpenoids, particularly the diterpenoids. Diterpenoids of genus Salvia are abietane and neo-clerodane types+, 611.3297  [M+H-H2O]+, 553.2412  [M+H-C3H6-CH4]+. Fragments due to the loss of CO and other consequent lossess did not appear in the MS/MS spectra of compound 1 . All diterpenoids showed the removal of water molecule, i.e. [M+H-H2O]+ except compound 4 which showed the removal of methanol due to the presence of methoxy group followed by the loss of water molecule. A loss of water molecule followed by the loss of CO was observed in compounds 2, 3, 5, and 6, while compounds having OH group in ring B (compounds 3 and 5) showed the loss of two water molecules, i.e. [M+H-2H2O]+. All analyzed compounds showed the removal of propene moiety directly from the [M+H]+ and/or after the removal of H2O and CO groups. These compounds also showed the neutral lossess of ethene and butane molecules. Compounds 3 and 5 having same molecular formula but difference of skeleton in ring B produced differentiated base peak and some other abundant peaks .The fragmentation pattern of momeric diterpenoids eriafone . All diteriafone . All dit1–6 produced abundant [M-H]- ions, which were selected as the precursor ions to produce MS/MS spectra. The QTOF-MS/MS the low-energy collision induced dissociation tandem mass spectrometry experiments (CID-MS/MS) were optimized and developed through ramping collision voltage to induce more product ions and the optimum collision energy for recording product ion spectra of diterpenoids in negative ion mode is ranging from 30 to 45 eV HRESIMS data of all compounds in negative ion mode is presented in Table 2O, CO, CH4, and CH3 radical, similar to positive ion mode, except the loss of CH4, and CH3 radical Table All diterpenoids 1, which is a dimer of two diterpenoids, showed the loss of propene radical as a base peak i.e. [M-H-C3H7].- at m/z 584.2777 corresponding to the molecular formula C36H40O7 . The other ions were found to be at m/z 566 [M-H-C3H7-H2O]-, 556 [M-H-C3H7-CO]-, 538 [M-H-C3H7-H2O-CO]- and 528 [M-H-C3H7-2CO]- . The other abundant ions were m/z 297 [M-H-CH4]-, 269 [M-H-CH4-CO]- and 227 [M-H-CH4-CO-C3H6]-, while the minor ions at m/z 298 [M-H-CH3]-, 270 [M-H-CH3-CO]- and 148 [M-H-CH3-CO]- were produced due to the loss of CH3 radical. Compounds 3 and 5, having same molecular formula, showed similar losses of H2O, CO, CO2 from the precursor ions. The ions are produced at m/z 327 [M-H-H2O]-, 301 [M-H-CO2]-, 317 [M-H-CO]- , 299 [M-H-CO-H2O]- and 271 [M-H-2CO-H2O]-. As the compound 5 have different skeleton in which the ring B has been modified it produce a base peak at m/z 179.0734 due to the fragment ion C10H11O3- , while in compound 3 the base peak was appeared at m/z 317 [M-H-CO]-. Compound 6 yielded the base peak at m/z 269 [M-H-CO]- and the other ions were found to be at m/z 281 [M-H-CH4]-, 282 [M-H-CH3]-., 253 [M-H-CO-CH4]- and 254 [M-H-CH3-CO]-. The product ion spectra of compound 1 in negative ionization was very simple with only a few peaks in mass range of ≥ m/z 499. Unlike compound 1, compounds 2–6 did not show the lossess of propene and propyl radical in negative ionization. Fragmentation pathway for the fragments formed due to the removal of CO, CH4, and CH3 through negative ionization has also been proposed and shown in Scheme 6 is taken as a representive of monomeric diterpenoids).In monomeric compound 1) have been studied by using ESI-QqTOF-MS/MS in both positive and negative ionization mode. It has been observed that many characteristic neutral losses and formation of key fragment ions can provide important structural information about the basic skeleton of abietane-type diterpenoids having dimeric linkages. The dimeric conjugate showed somewhat different pattern and less fragmentation as compared to monomeric diterpenoid analogue. The knowledge of fragmentation pattern is immensely helpful for the rapid characterization of abietane-type diterpenoids through liquid chromatography coupled with mass spectrometry in complex mixtures such as plant extracts or herbal formulations by utilizing their analytical amount.In conclusion, fragmentation pattern of six abietane-type diterpenoids and one novel dimeric conjugate diterpenoid, salvialeriafone (Authors declare that they have no competing interests.1–6, useful discussions and participated in manuscript preparation. All authors read and approved the final manuscript.SGM: Supervised the whole study and participated in manuscript preparation. MG: Involved in performing experimental and manuscript preparation. AH: Participated in purification of standard compounds. MIC: Involved in supervision of isolation of compounds


Disassembly of the viral capsid following penetration into the cytoplasm, or uncoating, is a poorly understood stage of retrovirus infection. Based on previous studies of HIV-1 CA mutants exhibiting altered capsid stability, we concluded that formation of a capsid of optimal intrinsic stability is crucial for HIV-1 infection.To further examine the connection between HIV-1 capsid stability and infectivity, we isolated second-site suppressors of HIV-1 mutants exhibiting unstable (P38A) or hyperstable (E45A) capsids. We identified the respective suppressor mutations, T216I and R132T, which restored virus replication in a human T cell line and markedly enhanced the fitness of the original mutants as revealed in single-cycle infection assays. Analysis of the corresponding purified N-terminal domain CA proteins by NMR spectroscopy demonstrated that the E45A and R132T mutations induced structural changes that are localized to the regions of the mutations, while the P38A mutation resulted in changes extending to neighboring regions in space. Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation. Nonetheless, the R132T mutation rescued the selective infectivity impairment exhibited by the E45A mutant in aphidicolin-arrested cells, and the double mutant regained sensitivity to the small molecule inhibitor PF74. The T216I mutation rescued the impaired ability of the P38A mutant virus to abrogate restriction by TRIMCyp and TRIM5α.The second-site suppressor mutations in CA that we have identified rescue virus infection without correcting the intrinsic capsid stability defects associated with the P38A and E45A mutations. The suppressors also restored wild type virus function in several cell-based assays. We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability-altering mutations can be mitigated by additional mutations that affect interactions with host factors in target cells or the consequences of these interactions. The ability of mutations at other CA surfaces to compensate for effects at the NTD-NTD interface further indicates that uncoating in target cells is controlled by multiple intersubunit interfaces in the viral capsid. During retrovirus maturation, the viral capsid protein (CA) assembles into a shell, referred to as the capsid, surrounding the viral genomic ribonucleoprotein (RNP) complex. HIV-1 CA consists of 231 residues that fold into two distinct domains connected by a flexible linker. Various surfaces of CA are involved in HIV-1 capsid formation. Interactions between the N-terminal domains (NTDs) form hexamers through an intersubunit NTD-NTD interface, while the C-terminal domains (CTDs) form dimers that connect adjacent hexamers through a CTD-CTD interface . The exiFollowing cell entry, the HIV-1 particle releases its core into the host cytoplasm. Subsequently, the core undergoes an uncoating process, which we define as disassembly or dissociation of the viral capsid from the internal RNP complex . The detin vitro, was competent for reverse transcription but impaired for nuclear import - or U-[15 N]-labeled CA-NTD samples in NMR buffer  using Bruker Avance 700 and 600 MHz spectrometers, equipped with 5 mm, triple resonance and z-axis gradient cryoprobes. Complete backbone assignments of the wild-type CA-NTD were obtained using 3D HNCACB and HN(CO)CACB experiments on U--labeled proteins. Complete assignments of the 2D 1H-15 N HSQC spectra for the E45A, R132T and E45A/R132T mutant proteins were obtained unambiguously by direct comparison with the spectrum of wild-type protein since the spectra of the mutants are essentially identical to that of wild-type, except for few resonances arising from amino acids around the mutation sites. The assignment of the P38A mutant spectra were less straightforward since more (~ 20) resonances exhibited changes > 0.1 ppm from the wild type. Nevertheless, all but two resonances (Trp23 and Met55) were unambiguously assigned. Structural perturbations created by the mutations were assessed by evaluating the combined amide chemical shift differences (Δδ) between wild-type and mutant using the equationExperiments were conducted at 25°C on U-[HN δand ΔN δrepresenting the 1HN and 15 N chemical shift differences, respectively.with Δ4 cells/well) were plated in 48-well plates. Aphidicolin (Sigma-Aldrich) was added to a final concentration of 2 μg/ml upon seeding [To generate cell cycle-arrested target cells for infectivity assay, HeLa-P4 cells  in triplicate, in the presence or absence of the reverse transcriptase inhibitor Efavirenz (1 μM). Total DNA was harvested at 8 hours post-inoculation and was purified using the DNeasy kit (Qiagen). DNA was eluted in 100 μl of water. Five microliter aliquots were assayed for 2escribed .3H47.pdb)[Molecular graphics images in Figure 3H47.pdb).HIV-1: Human immunodeficiency virus type 1; NTD: Amino-terminal domain; RNP: Ribonucleoprotein; PIC: Preintegration complex; CTD: Carboxy-terminal domain; NMR: Nuclear magnetic resonance; RSV: Rous sarcoma virus; VLPs: Virus-like particles; DTT: Dithiothreitol; VSV-G: Vesicular stomatitis virus glycoprotein.The authors declare that they have no competing interests.RY, AMG, and CA conceived and designed the experiments; RY, JS, I-JLB, and JA performed the experiments; JHS and JM produced the structural figures; RY, I-JB, JA, AMG, and CA wrote the manuscript. All authors read and approved the final manuscript.Figure showing monitoring of structural perturbations of CA-NTD caused by mutations with 1H-15 N HSQC NMR. Superimposed 1H-15 N HSQC spectra of 15 N-labeled wild-type (black) and mutants (red), E45A (A), P38A (B), E45A/R132T (C) and R132T (D). The peaks showing substantial perturbation due to the mutation are labeled with assignments.Click here for file


K-RAS oncogene is widely mutated in human cancers. Activating mutations in K-RAS give rise to constitutive signalling through the MAPK/ERK and PI3K/AKT pathways promoting increased cell division, reduced apoptosis and transformation. The majority of activating mutations in K-RAS are located in codons 12 and 13. In a human colorectal cancer we identified a novel K-RAS co-mutation that altered codons 19 and 20 resulting in transitions at both codons (L19F/T20A) in the same allele. Using focus forming transformation assays in vitro , we showed that co-mutation of L19F/T20A in K-RAS demonstrated intermediate transforming ability that was greater than that of individual L19F and T20A mutants, but less than that of G12D and G12V K-RAS mutants. This demonstrated the synergistic effects of co-mutation of codons 19 and 20 and illustrated that co-mutation of these codons is functionally significant.The  RAS family genes occur in approximately 20-30% of all human cancers, with mutations in the K-RAS gene comprising ~80% of these mutations [K-RAS mutations have been documented in the majority of human cancer types with pancreatic (~90% of these cancers) and colorectal (~40%) cancers demonstrating the highest incidence of mutations in this gene [K-RAS codons 12 and 13 are the most common sites of oncogenic activation with over 90% of documented mutations being found in these codons [K-RAS both encode glycine residues. The incorporation of other amino acids, most commonly aspartate and valine at codon 12 and aspartate at codon 13 [K-RAS, although mutations at these positions occur at a much lower prevalences  than codon 12 and 13 mutations [RAS has several effects on rodent cells in vitro, including the establishment of a transformed phenotype with anchorage independent growth in soft agar, transformed focus formation, as well as tumour formation following injection into animals [K-RAS accelerates intestinal adenoma formation in vivo on both a mutant Apc background and a Msh2-null background, increases proliferation, modulates apoptosis and alters gene expression patterns [K-RAS codon 19 from leucine to phenylalanine (L19F) and codon 20 from threonine to alanine (T20A), in a colorectal cancer and we demonstrate that it causes transformation in vitro and is therefore a rare, but functionally significant co-mutation of K-RAS.Mutations in utations . K-RAS morectal ~0% cancere codons . Codons codon 13 , brings codon 13 . Mutatioorectal ~0% cancer animals . Our prepatterns -15. In tK-RAS in a panel 186 colorectal adenocarcinoma samples, obtained as part of the European Prospective Investigation into Cancer and Nutrition (EPIC) Norfolk study cohort, identified 41 cancers harbouring mutations in K-RAS [K-RAS exon 1 harbouring the L19F/T20A mutations. Analysis of sequencing traces from individual bacterial clones identified solely double mutant (L19F/T20A) alleles . These data demonstrated that this L19F/T20A co-mutation of K-RAS confers oncogenic activation capable of inducing transformed focus formation to a greater degree than single mutations at either codons 19 or 20 or wildtype K-RAS cDNA, but to a lesser degree than codon 12 mutants of K-RAS.A series of full length human C. elegans RAS homologue let-60. C. elegans carrying this mutation showed a temperature-sensitive multivulval phenotype. In a mammalian system, at body temperature, H-RAS (L19F) protein had a reduced rate of GTP hydrolysis relative to wildtype H-RAS, suggesting that H-RAS L19F conferred an increased level of activation [H-RAS with the incorporated L19F mutation failed to demonstrate increased focus formation above that of controls, a similar observation to that made here using L19F K-RAS. A second report, however, describes L19F as causing increased cell proliferation, anchorage-independent growth, increased tumourigenicity in nude mice and elevated levels of RAS-GTP [K-RAS mutations outside of codons 12 and 13 also described mildly increased focus formation by L19F mutants [K-RAS mutation was described to induce formation of ~5 foci whereas positive controls (G12V and G12D) were observed to develop ~80 - 90 foci. These data are not mutually exclusive with our observations, as the mean focus count observed here with the G12D mutant was 35 colonies per plate, thus a mild focus forming ability of the L19F mutation may not have been detected. The observed differences between the number of colonies formed in the study by Smith and colleagues [It is not surprising that the T20A substitution alone was not sufficiently oncogenic to cause focus formation as this amino acid alteration has never been found previously in human cancers. However, the L19F substitution has been documented, albeit in a limited number of studies, and may be expected to have some transforming potential. One study described analysis of L19F in the tivation . However RAS-GTP . Additio mutants . In thislleagues  and thosK-RAS is the first description of this genetic change, which confers a greater transforming ability than individual mutations in these codons. The rare observed frequency of this double amino acid change giving rise to oncogenic K-RAS in human cancers, compared with that of the individual L19F amino acid change, despite its increased oncogenic potential, may be due to the lower likelihood of 2 base changes occurring at these positions in the same allele during cancer development.Our identification and analysis of the L19F/T20A double mutation in To assess the potential structural effects of the L19F/T20A amino acid changes we modelled their side chains into a shortened wildtype K-RAS protein ribbon structure bound to a GTP analogue Figure . Codons K-RAS mutations affecting codons 15 and 22 have also been reported in human colorectal cancers [ cancers ,24. AlthThe authors declare that they have no competing interests.All authors contributed to the study design, analyses and manuscript preparation. All authors read and approved the final manuscript.Experimental methods and statistical testing. Additional File Click here for file


Escherichia coli. The fermentation system described here paves the way for the development of an economical process for microbial production of flavonoids.Due to increasing concerns about food safety and environmental issues, bio-based production of flavonoids from safe, inexpensive, and renewable substrates is increasingly attracting attention. Here, the complete biosynthetic pathway, consisting of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydrogenase (CM/PDH), tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was constructed using pre-made modules to overproduce (2S)-naringenin from D-glucose. Modular pathway engineering strategies were applied to the production of the flavonoid precursor (2S)-naringenin from L-tyrosine to investigate the metabolic space for efficient conversion. Modular expression was combinatorially tuned by modifying plasmid gene copy numbers and promoter strengths to identify an optimally balanced pathway. Furthermore, a new modular pathway from D-glucose to L-tyrosine was assembled and re-optimized with the identified optimal modules to enable de novo synthesis of (2S)-naringenin. Once this metabolic balance was achieved, the optimum strain was capable of producing 100.64 mg/L (2S)-naringenin directly from D-glucose, which is the highest production titer from D-glucose in  This coAlthough significant progress has been made recently in improving strain titers and yields Escherichia coli.To achieve direct (2S)-naringenin production from D-glucose, it has become clear from previous studies that efficient conversion of L-tyrosine to (2S)-naringenin is the limiting factor 4Cl were used. Various combinations of ampicillin (100 µg/mL), kanamycin (40 µg/mL), chloramphenicol (20 µg/mL), and streptomycin (40 µg/mL) were added to cultures of plasmid-bearing E. coli strains. The primers and plasmids used in this study are given in Rhodotorula glutinis, 4CL (GenBank ID: KF765780) from Petroselinum crispum, CHS (GenBank ID: KF765781) from Petunia X hybrida, CHI (GenBank ID: KF765782) from Medicago sativa, and matB (GenBank ID: KF765783) and matC (GenBank ID: KF765784) from Rhizobium trifolii for E. coli expression were synthesized by GeneScript . The accession numbers for unmodified genes of TAL, 4CL, CHS, CHI, matB, and matC are DQ013364, X13325, AF233638, M91079, AAC83455, and AAC83457, respectively. Gene sequences of these modified genes are provided in the E. coli JM109 was used for plasmid propagation and BL21 (DE3) was used for flavonoid production. Polymerase chain reaction (PCR) was performed using pfu turbo polymerase .Luria broth (LB) and MOPS minimal medium phenicol 0 µg/mL, 600 of 1.65 had been reached, an additional 25 mL of fresh MOPS medium, a final aliquot of isopropyl-β-D-thiogalactopyranoside (IPTG) (taking the concentration to 1 mM), and a final aliquot of L-tyrosine (taking the concentration to 3 mM) were added. Cultures were subsequently conducted at 30°C for (2S)-naringenin production. (2S)-Naringenin concentrations were measured after a total fermentation time of 48 h. For malonyl-CoA availability experiments, 1 g/L of sodium malonate dibasic (Sigma) was added twice, resulting in a total concentration of 2 g/L.For flavonoid production from L-tyrosine, strains were first cultured in 25 mL of MOPS medium at 37°C with 220 rpm orbital shaking. After an OD600 of 1.65 was reached, after which an additional 25 mL of fresh MOPS medium and a final aliquot of IPTG (taking the concentration to 1 mM) were added. Cultures were subsequently conducted at 30°C for (2S)-naringenin production. For malonyl-CoA availability experiments, sodium malonate dibasic (Sigma) was added at a concentrations of 2 g/L (1 g/L added twice).For flavonoid production from D-glucose, strains were first cultured in 25 mL of MOPS medium until an ODp-coumaric acid production, E. coli cells were separated through centrifugation . To quantify levels of (2S)-naringenin, 1 mL of supernatant was extracted with an equal volume of ethyl acetate . After vortexing and centrifugation , the top organic layer was separated and evaporated to dryness, and the remaining residue was resolubilized with 1 mL of methanol . Samples were analyzed by high-performance liquid chromatography (HPLC), using an Agilent 1100 series instrument and a reverse-phase Gemini NX-C18 column (5×110 mm) maintained at 25°C. (2S)-Naringenin was separated by elution with an acetonitrile/water gradient at a flow rate of 1.0 mL/min under the following conditions: 10% to 40% acetonitrile (vol/vol) for 10 min, 40% acetonitrile (vol/vol) for 5 min, 40% to 10% acetonitrile (vol/vol) for 2 min. The retention times under these conditions for standard authentic samples of (2S)-naringenin  and p-coumaric acid  were 14.085 and 10.481 min, respectively. The recombinant product was detected by monitoring absorbance at 280 nm.To analyze (2S)-naringenin and −) accumulated on a liquid chromatography-mass spectrophotometer (LC-MS)  equipped with an electrospray ionization (ESI) source. HPLC separations were performed using an Agilent Zorbax Extend-C18 column  under the gradient elution mode at a flow rate of 0.7 mL/min. Mobile phases A and B were water and acetonitrile, respectively, both containing 0.1% formic acid. Gradient elution was conducted as follows: 0–20 min for 5–60% B with a linear gradient, followed by 20–30 min of 100% B 2) flow, 1.5 L/min; drying gas (N2) flow, 200 kPa; ion accumulation time, 30 ms; scan range m/z, 100–1000 for MS1, 100–500 for MS2. The instruments used for quantitative measurements were operated in the selected ion monitoring (SIM) mode. Data-dependent tandem mass spectrometry experiments were controlled using Shimadzu Composition Formula Predictor software. The negative ion value by LC-MS of the authentic (2S)-naringenin compound was 271.0591 [M-H]−.The compound was identified by the area of major mass spectra signals . Primers Pf_aroG(NcoI) and Pr_aroG(HindIII), Pf_tyrA(NdeI) and Pr_tyrA(BlnI) were used to clone the wild-type (WT) aroG and tyrA with pfu turbo polymerase. These PCR products were then ligated into T-vector pMDTM19 (Simple) , which resulted in plasmids T-aroG(WT) and T-tyrA(WT). Two amino acid substitutions were performed to obtain feedback-inhibition-resistant (fbr) tyrA gene (tyrAfbr): Met-53-Ile in the chorismate mutase domain and Ala-354-Val in the prephenate dehydrogenase domain aroG gene (aroGfbr) fbr(53) and Pr_tyrAfbr(53), Pf_tyrAfbr(354) and Pr_tyrAfbr(354), Pf_aroGfbr(146) and Pr_aroGfbr(146) were used to perform these substitutions. The resulting plasmids were named T-tyrAfbr and T-aroGfbr, respectively.Chromosomal DNA from fbr was constructed by cloning tyrAfbr from T-tyrAfbr into NdeI/BlnI sites of pCDFDuet-1. pCDF-tyrAfbr-aroGfbr was constructed by cloning aroGfbr from T-aroGfbr into NcoI/HindIII sites of pCDF-tyrAfbr. To construct pCDF-KanFRT-tyrAfbr-aroGfbr, primers Pf_KanFRT(pfoI) and Pr_KanFRT(EcoNI) were used to amplify an FRT-flanked kanamycin resistance gene (Kan) (KanFRT) on the plasmid pKD13 pfoI and EcoNI, this product was ligated to pfoI/EcoNI sites of pCDF-tyrAfbr-aroGfbr.pCDF-tyrAKanFRT-tyrAfbr-aroGfbr cassette was integrated into the lacZ locus of E. coli BL21 using a lambda-red recombination-based method KanFRT-tyrAfbr-aroGfbr was amplified from pCDF-KanFRT-tyrAfbraroGfbr with primers Pf_KanFRT-tyrAfbr-aroGfbr (CCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGAAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTGTGTAGGCTGGAGCTGCTTCG) and Pr_KanFRT-tyrAfbr-aroGfbr (CAAAAGTTTGTGTTTTTTAAATAGTACATAATGGATTTCCTTACGCGAAATACGGGCAGACATGGCCTGCCCGGTTATTACCTAGGTTACTGGCGATTGTCATT). Both primers incorporated 80 bp of homology with the ends of the lacZ gene to facilitate integration into the proper locus. Following transformation of the cassette into E. coli BL21, colonies were verified by colony PCR and sequencing. Excision of FRT-flanked kan from the resulting strains E. coli lacZ::KanFRT-tyrAfbr-aroGfbr was mediated by transformation with FLP recombinase-expressing pCP20 as described in the literature The All constructed plasmids were verified by both colony PCR and Sanger sequencing. Primers and plasmids used in this study are listed in E. coli. Hence, selecting appropriate genetic sources for the enzymes in a pathway remains a challenging task.Previous studies have demonstrated that efficient conversion of L-tyrosine to (2S)-naringenin or resveratrol is the limiting factor for de novo synthesis of (2S)-naringenin or resveratrol Rhodotorula glutinis, since this enzyme had been previously shown to have the highest in vitro enzyme activity toward L-tyrosine p-coumaric acid to (2S)-naringenin, including 4CL from Petroselinum crispum (Pc4CL), CHS from Petunia X hybrida, and CHI from Medicago sativa, were chosen because these enzymes had been successfully utilized in previous studies E. coli, a recombinant malonate assimilation pathway from Rhizobium trifolii (matB and matC) was utilized E. coli expression and synthesized in order to improve the expression of these enzymes.As the first step of this pathway, TAL was chosen from the red yeast matB and matC were placed in an individual module to modulate the amount of malonyl-CoA, which is the bottleneck when overproducing flavonoids in E. coli. Therefore, this synthetic pathway was partitioned into three modules: (1) module one consisted of genes coding for TAL and 4CL; (2) module two consisted of genes coding for CHS and CHI; (3) module three consisted of genes matB and matC to modulate the amount of malonyl-CoA , pETDuet-1 (pBR322 origin), pCDFDuet-1 (CDF origin), and pACYCDuet-1 (p15A origin) were assigned from the published copy numbers for the origin of replication, which are 10, 20, 40, and 100, respectively Total modular expression was calculated using promoter strengths and gene copy numbers. The strengths of On the basis of previous studies Trc), while the metabolic space between modules one and two was varied. It was found that an appropriate metabolic space between modules one and two resulted in better yields. Similarly, in the second (S4–S5), third (S6–S7), fourth (S8–S9), fifth (S10–S11), and sixth (S12–S13) rounds, module three was expressed at constant values with different gene copy numbers and promoters, while the metabolic space between modules one and two was exhaustively explored. Finally, it was found that an appropriate metabolic space between modules one and two with special modular expression of module three (p15A × T7) would result in the best (2S)-naringenin production  and erythrose 4-phosphate (E4P) catalyzed by DAHP synthase, which possesses three isoforms . The second rate-limiting step in the L-tyrosine biosynthesis is found at the chorismate branch point with chorismate mutase/prephenate dehydrogenase  aroGfbr) tyrAfbr) tyrAfbr and aroGfbr was assembled with the identified optimal modules to enable de novo synthesis of (2S)-naringenin.When the major pathway bottlenecks from L-tyrosine to (2S)-naringenin were alleviated, the next step was assembling a modular pathway from D-glucose to L-tyrosine to precede the identified optimal modules to enable de novo synthesis of (2S)-naringenin. To obtain (2S)-naringenin from D-glucose, strains exhibiting an enhanced capacity for L-tyrosine synthesis need to be constructed. In lacZ locus of E. coli BL21 under the T7 promoter. This resulted in the highest production titer of 100.64 mg/L (S16) directly from D-glucose  and the other with a low gene copy number (10), were modulated to rebalance the entire pathway. By expressing this module on the plasmid pRSFDuet-1 (highest gene copy number) or pCOLADuet-1 (lowest gene copy number), it was observed that the titer of the product (2S)-naringenin increased when the expression of this module decreased from the highest level to the lowest level (S14–S15). Our engineering methods require the use of antibiotic cassettes and plasmid-based expression, while marker and origin of replication incompatibilities can oftentimes arise between hosts and tools. Besides, the antibiotic cassettes and plasmid-based expression would result in an increase in the metabolic burden of host cells. Therefore, this new module was integrated into the -glucose . It was i. e., pCDF-Trc-TAL-Trc-4CL, pET-CHS, and pACYC-matC-matB, was transformed into BL21 (DE3) strain integrated with tyrAfbr and aroGfbr. No (2S)-naringenin could be detected under the same culture conditions and analytical conditions as mentioned above.Previous studies demonstrated that naringein chalcone could be converted to (2S)-naringenin or (2R)-naringenin spontaneously without CHI by raising the pH of the culture broth p-coumaric acid The biosynthesis of flavonoids from safe, inexpensive, and renewable substrates is increasingly attracting attention due to concerns about food safety and environmetal issues E. coli. This proves the necessity of varying the expressions of modules simultaneously.Previous studies have demonstrated the feasibility of de novo production of (2S)-naringenin In our previous work, for de novo production of (2S)-pinocembrin, the overall pathway from D-glucose to (2S)-pinocembrin was divided into four modules and expressions of the modules were varied simultaneously by modifying plasmid gene copy numbers p-coumaric acid compared to the initial strain. However, p-coumaric acid accumulation was still observed in these strains. Further studies are required to achieve more efficient conversion of p-coumaric acid to (2S)-naringenin. In some particularly relevant studies, previous researchers have found that simultaneous deletion of genes sdhA, adhE, brnQ, and citE and overexpression of acetyl-CoA synthase, acetyl-CoA carboxylase, biotin ligase, and pantothenate kinase fumC and sucC and overexpression of ACC, PGK, GAPD, and PDH The engineered strains exhibited a significantly increased titer of (2S)-naringenin and a decreased titer of Supporting Information S1DNA sequences of optimized genes.(DOC)Click here for additional data file.Supporting Information S2Description of plasmid constructs and further information.(DOC)Click here for additional data file.


Rhipicephalus haemaphysaloides.Ticks and tick-borne diseases affect animal and human health worldwide and cause significant economic losses in the animal industry. Functional molecular research is important to understand the biological characteristics of ticks at the molecular level. Enzymes and enzyme inhibitory molecules play very important roles in tick physiology, and the cystatins are tight-binding inhibitors of papain-like cysteine proteases. To this end, a novel cystatin, designated RHcyst-1, was isolated from the tick Escherichia coli, and its inhibitory activity against cathepsin L, B, C, H, and S, as well as papain, was identified by fluorogenic substrate analysis. Expression analysis of RHcyst-1 at different tick stages was performed by quantitative reverse transcription - PCR (qRT-PCR). An RNAi experiment for RHcyst-1 was performed to determine its function for tick physiology.The full-length gene of RHcyst-1 was cloning by RACE. The recombinant protein of RHcyst-1 was expressed in a glutathione S-transferase (GST)-fused soluble form in The full-length cDNA of RHcyst-1 is 471 bp, including an intact open reading frame encoding an expected protein of 98 amino acids, without a signal peptide, having a predicted molecular weight of ~11 kDa and an isoelectric point of 5.66. A sequence analysis showed that it has significant homology with the known type 1 cystatins. The results of proteinase inhibition assays showed that rRHcyst-1 can effectively inhibit the six cysteine proteases’ enzyme activities. An investigation of the RHcyst-1 genes’ expression profile showed that it was more richly transcribed in the embryo (egg) stage. A disruption of the RHcyst-1 gene showed a significant decrease in the rate of tick hatching.Our results suggested that RHcyst-1 may be involved in the early embryonic development of ticks. Ticks rank first as arthropod vectors of fungi, protozoa, rickettsiae, bacteria, and viruses, which cause diseases in non-human vertebrates, and rank second only to mosquitoes as vectors of human pathogens . CystatiRhipicephalus haemaphysaloides is a widespread tick species in China and other south Asian countries, where it transmits animal babesiosis and human Kyasanur Forest disease [R. haemaphysaloides. This molecule exhibited significant inhibitory activities against cysteine proteinases and may be involved in tick embryonic development. disease . In thisR. haemaphysaloides has been maintained by feeding on rabbits for several generations in our laboratory at the Chinese Academy of Agricultural Sciences  [The Hubei strain of , China) . The expRapid amplification of cDNA ends (RACE) was conducted using a SMARTer RACE cDNA amplification kit  following the manual’s instructions. Cloning was performed using primers from highly conserved regions of cystatin . The genE. coli expression vector . The accuracy of the insertion in the resulting plasmid was confirmed by sequencing. The cystatin gene was expressed as a glutathione S-transferase (GST)-fusion protein in the E. coli BL21 (DE3) strain according to the manufacturer’s instructions (Amersham Pharmacia Biotech). The resulting E. coli cells were washed three times with phosphate-buffered saline (PBS), lysed in PBS containing 1% Triton X-100, sonicated, and then centrifuged at 12,000 × g for 10 min at 4°C. Supernatants containing the soluble GST fusion protein were purified with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. The purified proteins were dialyzed against PBS for further use. The empty pGEX-4T-1 vector was used to produce the control GST protein, which was expressed and purified using the same procedure as that for the cystatin-GST fusion protein. Recombinant protein expression and purification analyses were carried out by standard SDS–PAGE [The open reading frame (ORF) of the cystatin gene in the pGEM-T vector was subcloned into the pGEX-4T-1 50) was measured. Recombinant protein was preincubated with each enzyme (0.15 μM) in an assay buffer for 30 min. Then, 0.25 mM of the protease-specific substrates was added to each well and residual enzyme activity monitored [To calculate the inhibitory activity of the recombinant protein, the concentration of rRHcyst-1 at which a 50% inhibition of the proteolytic enzymes’ activities was achieved .cDNA of RHcyst-1 in the pGEM-T vector was amplified by PCR using oligonucleotides, including the T7 promoter sequence at the 5′ end of both primers, as follows: 5′-GGATCCTAATACGACTCACTATAGGGTAGCAGCGCGCGCGATTAG-3′ and 5′-GGATCCTAATACGACTCACTATAGGCGCGTAAAATTTGTTCCTT-3′ for the gene encoding RHcyst-1, and 5′-GGATCCTAATACGACTCACTATAGGGCTTCCATCTTCCAGGGATACG-3′ and 5′-GGATCCTAATACGACTCACTATAGGCGTCCACAAACACAACTCCTCC-3′ for the gene encoding luciferase. The PCR products were gel-purified and used to synthesize RNA by in vitro transcription with T7 RNA polymerase  according to the manufacturer’s protocol. The double-stranded (ds) RNA injection was administered as described previously . In totaR. haemaphysaloides using the RACE method. The full-length cDNA of RHcyst-1 is 471 bp, as shown in Figure http://smart.embl-heidelberg.de/) detected the cystatin-like domain in the putative amino acid sequence (position 3–97). A BLASTP analysis of the predicted polypeptide sequence against all non-redundant databases accessed through GenBank revealed significant similarity scores with members of the cystatin type 1 family of other species. The identities of the putative RHcyst-1 amino acid sequences with the type 1 cystatin of Boophilus microplus (GenBank accession number: ABG36931), Dermacentor variabilis (GenBank accession number: ACF35512), and Haemaphysalis longicornis (GenBank accession number: ABZ89553) were 90%, 86%, and 81%, respectively, as shown in Figure We found a novel cystatin gene, RHcyst-1, from the tick -PAGE analysis.The gene encoding RHcyst-1 was ligated into the bacterial expression vector pGEX-4T-1, and the recombinant was successfully expressed as GST-fusion protein with an expected size of 37 kDa Figure . rRHcystTo investigate the efficiency of RHcyst-1 in inhibiting its overlapping target enzymes, purified recombinant cystatin was used to test the inhibitory activity against cysteine proteases. The results show that: rRHcyst-1 can effectively inhibit cathepsin L, B, C, H, and S, as well as papain, enzyme activities, as shown in Figure To determine the expression profile of RHcyst-1, total RNA samples from four different tick developmental stages were subjected to real-time PCR. RHcyst-1 mRNA transcripts were detected in eggs (embryos), larvae, nymphs, and adults. RHcyst-1 was most highly expressed in the embryo stage, as shown in Figure Unfed adult ticks were injected with RHcyst-1 or luciferase dsRNA and then allowed to feed on a rabbit. Gene-specific silencing was confirmed by real-time PCR. Within 5 days of dsRNA treatment, 95.6% of the RHcyst-1 transcripts in ticks were silenced as shown in Figure R. haemaphysaloides. The characteristics of the putative RHcyst-1 amino acid sequence indicate that it is a member of the type 1 cystatins.The present study describes the sequence of a novel cystatin, RHcyst-1, from the tick The capacities of various cystatins to inhibit the activity of cysteine proteases have been characterized. In this study, the GST-fused recombinant cystatin efficiently inhibited the activity of cathepsin L, B, C, H, and S, and papain. rRHcyst-1 effectively inhibited cathepsin S, and it also had quite a high inhibitory efficiency against cathepsin L. However, rRHcyst-1 showed relatively less inhibitory activity against cathepsin B, perhaps due to an occluding loop in the cathepsin B active site that limits the access of both substrates and inhibitors to the active site . The lacIxodes scapularis (GenBank accession number: AAY66864), which was the first type 1 cystatin identified in the I. scapularis’ sialome [R. microplus because this cystatin was found to inhibit, in addition to human cathepsin L, a vitellin-degrading cysteine endopeptidase of R. microplus. Hlcyst-1 (GenBank accession number: ABZ89553) was localized in epithelial cells of the tick midgut alongside with HlCPL-A, a cathepsin L-like cysteine protease of H. longicornis [H. longicornis, with their strongest expression levels occurring at 48 h in the midgut cells [All known type 1 tick cystatins, except RHcyst-1, were discovered from five different hard tick species of four genera. Only two type 1 tick cystatins have been biochemically analyzed thoroughly enough to identify their roles in tick physiology . Bmcysta sialome ,23. Lima sialome  suggestegicornis . HlCPL-Agicornis . Hlcyst-ut cells . Thus, HIn this study, RHcyst-1 was found to be highly expressed in the embryo (egg) stage, and we hypothesize that it plays a role in early embryonic development. To further confirm the function of RHcyst-1 in tick physiology, an RNAi experiment was performed. The disruption of the RHcyst-1 gene led to a significant decrease in the rate of egg hatching. However, there are several cystatin homologs which may compensate the role of the silenced cystatin, thus affect the result of RHcyst-1 RNAi. And this hypothesis will be tested in our future work. In conclusion, our results suggested that RHcyst-1 may be involved in tick early embryonic development.


Specific somatic mutations in c-kit have been associated with acute myeloid leukemia (AML) and are markers of a poor prognosis in AML. Various methods have been used to detect the c-kit gene mutation; however, the suitability of these methods in the clinical management of AML remains unclear. The current study developed a novel method, using modified hybridization probes and melting curve analysis, for detecting c-kit mutations in exon 17. Dual-labeled self-quenched oligonucleotide probes containing two segments, labeled with carboxyrhodamine or hexachlorofluorescein, were designed to detect sequences around the D816 or N820/N822 mutation hot spots in exon 17 of c-kit. The exon 17 region of c-kit was amplified by polymerase chain reaction using control plasmids carrying wild-type or mutant sequences, or genomic DNA derived from AML patients. Melting curve analysis of the amplification products was performed using a self-quenched probe. The results showed that the detection sensitivity, assayed using mutation-positive control plasmids, was 10% for the N820G mutation and 5% for the six other mutations; N822K(A), N822K(G), D816V, D816Y, D816H and D816F. In addition, c-kit mutations were identified in six of the 12 samples from the core-binding factor (CBF)-AML patients. This demonstrates that the novel method developed in the present study, is simple, rapid, specific and highly sensitive, and may facilitate the diagnosis and treatment of CBF-AML.The  AML1-ETO and CBFB-MYH11 fusion genes, respectively. As AML1 encodes the α subunit of the core-binding factor (CBF) and CBFB encodes its β subunit, these two gene fusions interfere with normal CBF function. Therefore, AML with AML1-ETO or CBFB-MYH11 is termed CBF-AML and accounts for 15% of AML cases worldwide . The experimental procedures were performed following the guidelines of the Xiamen University Medical Research Council and were approved by the ethics committee of the Zhongshan Hospital of Xiamen University.Bone marrow (2 ml) or peripheral blood (5 ml) samples were collected from 107 patients with leukemia at the Zhongshan Hospital of Xiamen University , between July 2008 and January 2010. All patients were diagnosed in accordance with the leukemia diagnostic standards , which wGenomic DNA was extracted using a Qiagen genomic DNA extraction kit  within 24 h of the collection of the blood samples. The DNA concentration was measured using spectrophotometry ; the absorbance was measured at 260 nm and the DNA samples were diluted to a concentration of 10 ng/ml.2 group at its 3′ end to prevent probe extension by the DNA polymerase  and Tm Utility v1.3 (Sangon Biotech (Shanghai) Co., Ltd.  software packages were used to design the primers and probes. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The probes contained the following two segments: i) A self-quenched probe segment labeled with a carboxyrhodamine (ROX) fluorophore at its 5′ end (the first three basic groups were thiophosphorylated to prevent shearing of the fluorophore-carrying basic group by the DNA polymerase) and a black hole quencher (BHQ) at its 3′ end; and ii) a probe segment labeled with a hexachlorofluorescein (HEX) fluorophore at its 5′ end and a NHlymerase , Table Ic-kit WT or mutant sequences: D816WT, D816V, D816Y, D816H, D816F, N822K(A), N822K(G) and N820G. D816WT contained the WT sequence, while in D816V, the GAC codon for amino acid 816 was mutated to GTC, resulting in a D (aspartic acid) to V  change. The relevant plasmid sequences are listed in Using genomic DNA from 293T human embryonic kidney cells as the template, mutation-positive control plasmids were constructed using the overlap extension PCR method ,16. The 4)2SO4, 6.7 μM EDTA and 0.085 mg/ml bovine serum albumin], 4 mM Mg2+, 0.2 mM dNTPs, 1 pmol upstream primer, 10 pmol downstream primer, 2 pmol D816-ROX probe, 2 pmol N822-HEX probe, 1 unit of Taq HS, 5 μl of template DNA and ddH2O in a final volume of 25 μl.The PCR reactions contained 1× sequence-specific primer buffer , and a N820 mutation (N820G). The results of the sensitivity analysis are shown in To test the sensitivity of the novel system, the plasmid mixtures containing the plasmid with the WT The results of the melting curve and sequencing analyses for the 12 CBF-AML patient-derived samples are shown in c-kit mutation-positive samples, three different mutations were identified: A D816H mutation (one sample), a D816V mutation (three samples) and a N822K(A) mutation  and an additional peak at 46°C, indicating the presence of a mutation at N820 or N822. Sequencing analysis of the 12 samples supported the melting curve data. Among the five c-kit mutation rate in samples from patients with other types of leukemia, the novel method was used to analyze 95 non-CBF-AML samples, including 58 AML (negative for AML1-ETO and CBFB-MYH11), 25 acute lymphoblastic leukemia, 10 chronic myelocytic leukemia and two chronic eosinophilic leukemia samples. Representative data for the 31 samples are shown in c-kit mutations.To assess the c-kit in t AML has been reported in the extracellular domain (encoded in exon 8), the juxtamembrane domain (encoded in exons 10 and 11) and the A-loop domain with tyrosine kinase activity (encoded in exon 17). Certain previous studies reported that the D816V mutation (in exon 17) confers increased tumor growth and antiapoptotic potential compared with mutations in the extracellular or juxtamembrane domains  comparable with those previously reported; 12.8–46.8% (c-kit mutations in non-CBF-AML cases, c-kit mutations were also analyzed in 95 samples obtained from non-CBF-AML patients. As predicted, no c-kit mutations were identified, which indicates that the c-kit mutation is rare in non-CBF-AML cases.In the present study, .8–46.8% –21. FurtIn conclusion, the method described in the present study is simple and rapid, and exhibits high sensitivity and specificity. This modified probe method may facilitate the classification and individual treatment of patients with CBF-AML.


Escherichia coli cultures. We have found that methionine supplementation reduced the frequency of persisters at mild (37°C) and elevated (42°C) temperatures, as well as in the presence of acetate. Homoserine-o-succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway, is prone to aggregation under many stress conditions, resulting in a methionine limitation in E. coli growth. Overexpression of MetA induced the greatest number of persisters at 42°C, which is correlated to an increased level of aggregated MetA. Substitution of the native metA gene on the E. coli K-12 WE chromosome by a mutant gene encoding the stabilized MetA led to reduction in persisters at the elevated temperature and in the presence of acetate, as well as lower aggregation of the mutated MetA. Decreased persister formation at 42°C was confirmed also in E. coli K-12 W3110 and a fast-growing WErph+ mutant harboring the stabilized MetA. Thus, this is the first study to demonstrate manipulation of persister frequency under stressful conditions by stabilization of a single aggregation-prone protein, MetA.Bacterial persisters are a small subpopulation of cells that exhibit multi-drug tolerance without genetic changes. Generally, persistence is associated with a dormant state in which the microbial cells are metabolically inactive. The bacterial response to unfavorable environmental conditions  induces the accumulation of aggregated proteins and enhances formation of persister cells in  Escherichia coli, Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, Gardnerella vaginalis, Salmonella enterica, Acinetobacter baumannii, Bordetella petrii and Mycobacterium tuberculosisA small subpopulation of bacterial cells, designated persisters, which are able to survive lethal antibiotic treatment and produce a new population of antibiotic-sensitive cells genetically identical to the originals was first described by Joseph W. Bigger Acinetobacter baumannii and P. aeruginosa increased in the presence of aminoglycosides or β-lactams E. coli cells exposed to thermal stress accumulated a large number of aggregated proteins et al. showed that an increased level of protein aggregates in E. coli stationary-phase cells was strongly correlated with a higher frequency of persister formation E. coli persisters under normal or stressful conditions. Homoserine o-succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway in vitro and completely aggregates at temperatures of 44°C and higher, resulting in methionine limitation of E. coli growth Persistence arises from the dormant state when the bacterial cells are metabolically inactive E. coli K-12 WE at mild (37°C) or elevated (42°C) temperatures, as well as in the presence of sodium acetate. Overexpression of MetA resulted in increased persister formation at 42°C and an enhanced level of aggregated MetA. Stabilized MetA mutant accelerated growth in the WE strain at the higher temperature (44°C) and in the presence of sodium acetate, decreased the frequency of persisters under heat and weak-acidic conditions and was less aggregation-prone. Strain W3110 and fast-growing mutants of strain WE expressing the wild-type and stabilized MetAs yielded similar results.In this study, we have shown that exogenous methionine reduced the frequency of persister cells in the strain E. coli K-12 cells. Generally, our experiments confirmed that the stress response and dormancy appeared to be alternative strategies for cell survival We showed the influence of a single aggregation-prone protein on persister formation in E. coli strains were grown in minimal M9 medium E. coli strains in M9 glucose medium at different temperatures was studied in a TVS126MB automatic growth-measuring incubator . The specific growth rate  was calculated through linear regression analysis of ln(X/X0) data using Sigma Plot software, where the initial OD600 (X0) was 0.15 at the zero time point and X represents the OD600 values measured every 10 min in an exponentially growing culture for 1 h.The strains and plasmids employed in this study are listed in rph-1 gene in the WE strain was replaced with the chloramphenicol-resistance gene using the λ Red recombination system GGAAGTCCGTATAATGCGCAGCCACATTTGTTTCAAGCCGGAGATTTCAATATGGTTGGCAGCATCACCCGAC), reverse primer RG2 (GCGACTCATCAGTCGCCTTAAAAATCAGTTTGCCAGCGCCGCCTTCTGCGTCGCGTAGCACCAGGCGTTTAAGG), Vent polymerase  and the plasmid pACYC184  as a template . The Δrph::cat mutant of strain WE-LYD was obtained through transduction with P1vir using the WEΔrph donor strain. For the rph-kan cassette construction, the kanamycin-resistance gene from the plasmid pKD13 rph gene was amplified from E. coli ATCC 9637 genomic DNA using the primers RG3 (CGCCTCGGATCCGGAAGAAAAATGCCGCTCTG) and RG4 (GTTAAAGCAGTACGGCAGGTC) and cloned in the BamH1 site of pUC18-Kan resulted in the pUC18Rph-Kan plasmid which was used for the rph-kan cassette amplification with the primers RG5 (CGTTCATTGCCCACTCCATGTG) and RG6 (GAATCCACCAACGCTTCAGC). The 3.5-kb PCR product was transferred into WEΔrph(pKD46) using the λ Red recombination system + was generated by P1vir phage transduction with the WErph+ donor strain. The kanamycin-resistance gene was eliminated from strains WErph+ and WE-LYDrph+ upon exposure to plasmid pCP20-encoded FLP recombinase rph gene from the genomic DNA of strains WErph+ and WE-LYDrph+ was synthesized using the primers RG7 (GTCATACTGCGGATCATAGACG) and RG8 (GTTAAAGCAGTACGGCAGGTC), followed by sequencing with the primers RG9 (GGAGAGGTGGAAGGATTATAGC) and RG10 (GAATCCACCAACGCTTCAGC).The native PBAD-metA cassette. The PBAD promoter was amplified from the template plasmid pBAD/HisA  using Vent polymerase  with a first pair of primers, bad1 (CATACTCCCGCCATTCAGAGAAG) and bad2 (GTCCGGCACACGAATCGGCATGGTTAATTCCTCCTGTTAGC). The metA gene was synthesized using a second pair of primers, bad3 (GCTAACAGGAGGAATTAACCATGCCGATTCGTGTGCCGGAC) and bad4 (CGCCTCAGATCTCGTATGGCGTGATCTGGTAGAC), with E. coli WE genomic DNA as a template. The PCR products from the two first reactions were then used as templates in a second PCR with the primers bad1 and bad4. The resulting PCR product was digested with BglII and cloned into HincII-BamHI sites of the plasmid pUC18-Kan. The kan-PBAD-metA cassette was synthesized through PCR with the template plasmid pUC18-Kan-PBAD-metA, Vent polymerase  and a pair of primers, bad5 (GAATACTAATAACCATTTTCTCTCCTTTTAGTCATTCTTATATTCTAACG CTTGAGCGATTGTGTAGGCTG) and bad6 (CGTATGGCGTGATCTGGTAGACGTAATAGTTGAGCCAG), then gel-purified and transferred into freshly prepared E. coli WE (pKD46) cells via electroporation, as described previously kan-PBAD-metA cassette was synthesized from the genomic DNA of kanamycin-resistant clones and sequenced.A two-step PCR procedure was used to construct the The MetAs were purified as described previously Cultures were grown in 50 ml of M9 glucose medium for 16 h or in LB medium for 24 h at 37°C or 42°C. Soluble and insoluble protein fractions were purified as previously described 600 of 0.1 in fresh medium (M9 glucose or LB) supplemented with ampicillin (200 µg/ml) or ofloxacin (5 µg/ml) and incubated at 37°C for 10 hours. Samples were taken every hour and plated on LB agar for colony counting. The values represent the means of three independent experiments. The frequency of persister formation was determined as the relationship between the CFU of surviving bacteria and the total CFU before the addition of antibiotics. The error bars indicate the standard errors.Bacteria grown overnight in M9 glucose medium for 16 h or in LB medium for 24 h were diluted to an ODP value was <0.05.The significance of differences between mean values of two measured parameters was assessed using two-tailed t test with unequal variances. Differences were considered significant when the E. coli growth in the defined medium was impaired at elevated temperatures due to methionine limitation resulting from the extreme inherent instability of the first enzyme in the methionine biosynthetic pathway, MetA E. coli K-12 WE cells. Strain WE of E. coli K-12 grown in M9 glucose medium at 37 and 42°C for 16 h was treated with ampicillin, and the frequency of persisters was determined by plating samples on LA plates , which catalyzes the first unique step in the de novo methionine biosynthetic pathway, is inherently unstable and prone to aggregation As seen in metN) lacking the L-methionine ABC transporter MetN metN) mutant did not affect the number of persister cells tolerant to ampicillin at 37°C. At 42°C, however, the number of persisters was 6–15 times lower in the presence of L-methionine than in methionine-free medium (p<0.05)  . We assuThus, these results showed that the formation of persisters was dependent on the availability of methionine and might be linked to the solubility of MetA.metA) and WE harboring the wild-type metA gene metA gene, like deletion of rmf, relE, or mazF, did not affect persister production To determine whether the MetA participates in persistence, we compared the level of persistence to ampicillin in a pair of isogenic strains, JW9673.Previous investigations have shown that et al. found that the number of persisters corresponded to the level of protein aggregation Leszczynska Tm) of the wild-type and mutant proteins using differential scanning calorimetry (DSC). Both of these proteins contain a C-terminal six-histidine tag and were purified as described in the Tm than wild-type MetA , evidence of the increased thermal stability of the MetA-LYD mutant.As MetA aggregation increased persister production, we studied the effect of MetA stabilization on the persister frequency at mild (37°C) and elevated temperatures (42°C). Strain WE-LYD, which harbors three stabilizing mutations in MetA , had been constructed previously A pair of isogenic strains, WE and WE-LYD, was used for the study of persister formation at 37°C and 42°C in M9 glucose medium. Both strains displayed similar numbers of persisters at 37°C . At 42°CmetA transcription at elevated temperatures We have detected 12.5-fold more aggregated wild-type MetA at 42°C compared to 37°C, but the level of stabilized MetA increased only 9.5 times . The relE. coli strains, we substituted the native metA gene on the W3110 chromosome with the metA-LYD mutant and constructed fast-growing mutants of the WE and WE-LYD strains. Previous studies have shown that the genomes of E. coli K-12 strains MG1655 and W3110 harbor a GC deletion within the 3′-terminal part of the rph gene that causes partial auxotrophy for pyrimidines, resulting in a growth defect for K-12 strains rph gene from E. coli strain ATCC 9637 (W) does not contain this mutation, we substituted the defective rph gene in the K-12 WE and WE-LYD strains with the rph gene from the ATCC 9637 strain. The resulting WErph+ and WE-LYDrph+ mutant strains grew 13–15% faster at 37° and at 42°C than the parental strains  affects persister formation under weak-acidic conditions.A previous study reported that acetate induced protein aggregation and increased the frequency of persisters in cspH, htrA, ibpAB, htpX, and clpB) were upregulated in the cell samples with the higher frequencies of persisters E. coli cells Interest in microbial persistence is rising; however, the mechanisms underlying persister formation are not fully understood. Populations containing higher numbers of dormant or slow-growing cells were highly persistent under antibiotic treatment, a phenomenon associated with decreased biosynthetic activity in vitro at temperatures of approximately 25°C, with the maximum level of unfolding at 44°C, resulting in complete aggregation with any subsequent rise in temperature MetA is a heat-shock protein E. coli growth in defined medium et al. Methionine added to the culture medium to repress transcription of the methionine-biosynthetic genes E. coli survival under antibiotic challenge?’ If unstable MetA offers a selective advantage, more stable MetA may not evolve.Increased persister formation with inherently unstable MetA raises an intriguing question: ‘Does inherently instable MetA favor E. coli under stressful conditions. A higher frequency of persisters was correlated with an increased amount of aggregated MetA. Stabilization of the unstable MetA enzyme resulted in decreased aggregation and thus in reduced persister formation at elevated temperature and in the presence of acetate.In summary, we found that the first enzyme in the methionine biosynthetic pathway, MetA, affects the level of persisters in Thus, we have shown a possibility to correct persister formation by manipulating the thermostability of the single enzyme, MetA.Table S1Effect of stabilized MetA protein on E.coli growth at different temperatures or in the presence of sodium acetate. Strains were grown in M9 glucose medium (pH 7.0 or 6.0) with or without sodium acetate (20 mM) in an automatic growth-measuring incubator at indicated temperatures for 24 h. The specific growth rate μ (h−1) was calculated through linear regression analysis of ln(X/X0) data with Sigma Plot software, where the initial OD600 (X0) was 0.1–0.15 at the zero time point, and X represents the OD600 values measured every 10 min in an exponentially growing culture over 1 h.(DOC)Click here for additional data file.