Bt Cells Research Articles

Super-Resolution Imaging of Live Anaerobic Bacteria: Assembly and Dynamics of the B. Theta Starch Utilization System

The human gut hosts trillions of bacteria that directly influence human health. The majority of these gut microbiota play an important role in nutrition by metabolizing host-indigestible complex glycans into short-chain fatty acids. Bacteroides thetaiotaomicron (Bt), a prominent bacterial symbiont in the distal gut, metabolizes over a dozen complex glycans using membrane-associated protein complexes. The Starch Utilization System (Sus) is the first recognized multi-protein complex in Bt cells that is essential for growth on starch. Bt-Sus uses eight proteins (SusRABCDEFG) to process starch; though, only SusD and SusG are vital for cellular proliferation in the presence of starch. SusCDEFG localize in the outer membrane and likely form a complex to facilitate starch binding, degradation and import. However, conventional biochemical methods have been unable to completely reveal the assembly and dynamics of these proteins in response to starch. We have applied single-molecule super-resolution imaging to characterize the Sus complex response to different sugars in live Bt cells under anaerobic conditions. HaloTag-labeled SusG was tracked concurrently with other fluorescent antibody-labeled Sus proteins. Our initial protein correlation studies demonstrated that simple sugars such as glucose or maltose do not induce Sus complex assembly. On the other hand, incubation of Bt cells with amylopectin, a constituent of starch, enhanced the colocalization of Sus proteins suggesting starch-mediated assembly of the Sus complex. Furthermore, we have used single-molecule tracking experiments to quantify Sus protein movement in the presence and absence of starch in live cells. We have discovered two distinct modes of SusG: a diffusing population and a stationary population. This latter group is mainly observed for cells on amylose-coated beads and is consistent with starch-bound SusG.

Comparison of quantitative PCR and culture-based methods for evaluating dispersal of Bacillus thuringiensis endospores at a bioterrorism hoax crime scene

Since the anthrax mail attacks of 2001, law enforcement agencies have processed thousands of suspicious mail incidents globally, many of which are hoax bioterrorism threats. Bio-insecticide preparations containing Bacillus thuringiensis (Bt) spores have been involved in several such threats in Australia, leading to the requirement for rapid and sensitive detection techniques for this organism, a close relative of Bacillus anthracis. Here we describe the development of a quantitative PCR (qPCR) method for the detection of Bt crystal toxin gene cry1, and evaluation of the method's effectiveness during a hoax bioterrorism event in 2009. When combined with moist wipe sampling, the cry1 qPCR was a rapid, reliable, and sensitive diagnostic tool for detecting and quantifying Bt contamination, and mapping endospore dispersal within a mail sorting facility. Results from the cry1 qPCR were validated by viable counts of the same samples on Bacillus-selective agar (PEMBA), which revealed a similar pattern of contamination. Extensive and persistent contamination of the facility was detected, both within the affected mailroom, and extending into office areas up to 30m distant from the source event, emphasising the need for improved containment procedures for suspicious mail items, both during and post-event. The cry1 qPCR enables detection of both viable and non-viable Bt spores and cells, which is important for historical crime scenes or scenes subjected to decontamination. This work provides a new rapid method to add to the forensics toolbox for crime scenes suspected to be contaminated with biological agents.

Inactivation of Pathogens in Red Blood Cells by a Modified Treatment Process Utilizing S-303 and Glutathione.

Background: Treatment of red blood cells (RBC) with 0.2 mM S-303 (an acridine compound) and 2.0 mM glutathione (GSH) has previously been shown to inactivate a variety of pathogens, including viruses, bacteria, protozoan parasites and contaminating leukocytes. During Phase III clinical trials antibodies to S-303-treated RBC were detected in a few trial participants. These antibodies were later determined to be specific to residual acridine bound to the RBC surface. As a result of these antibodies, a modification of the S-303 treatment process has been developed to reduce the potential immunogenicity of S-303-treated RBC. This modified process utilizes 0.2 mM S-303 and 20 mM GSH. In this abstract we report data from an on-going series of studies designed to evaluate the efficacy of bacterial and viral inactivation in RBC using the modified S-303 treatment process.Methods: Leukoreduced RBC units of approximately 280 mL with a hematocrit of approximately 60% were prepared in AS-3 storage medium. RBC units were inoculated with approximately 6 logs/mL of organism when possible, and an aliquot was removed to serve as the untreated, input control. GSH in a solution of 2 equivalents NaOH was added to the inoculated units to a final concentration of 20 mM and mixed well. Following a 5 to 10-minute room temperature incubation, S-303 was added to a final concentration of 0.2 mM and the units were again mixed well and incubated at 20 to 25 °C for three hours. After incubation, samples were removed and assayed to detect residual viable organisms. Control preparations were titered immediately after preparation and again after the 3-hour incubation. Bacterial titers were determined by colony formation on agar plates and viral titers were determined by plaque formation in MT-2 cells (HIV), BT cells (BVDV), vero E6 cells (VSV) and A549 cells (Adenovirus). Bacteria were chosen to represent Gram positive and Gram negative organisms of relevance to transfusion. Vesicular stomatis virus (VSV) was used as a representative negative sense RNA virus, and Adenovirus 5 as a representative non-enveloped DNA virus. HIV and bovine viral diarrhea virus (BVDV, model for HCV) inactivation was evaluated because of their relevance to blood transfusion. Replicate experiments were performed for each organism and the results are expressed as the mean and standard deviation (SD) of the observed inactivation levels. Inactivation is expressed as log reduction.Results: See table below. Both Gram positive and Gram negative bacteria were effectively inactivated by treatment with the modified S-303 process. This process also resulted in effective inactivation of both the enveloped and non-enveloped viruses tested.Conclusion: The modified S-303 process effectively inactivates bacterial and viral pathogens in RBC.OrganismLog Reductiona (Mean ±SD)Staphylococcus aureus≥6.9 ±0.4Staphylococcus epidermidis≥7.0 ±0.1Yersinia enterocolitica4.1 ±0.8Escherichia coli6.1 ±0.7Serratia marcescens4.0 ±0.4HIV>5.8 ±0.2BVDV>4.4 ±0.2Vesicular stomatitis virus (VSV)4.2 ±0.1Adenovirus 5≥8.0 ±0.3

Cost-effective production of Bacillus thuringiensis by solid-state fermentation

Production of Bacillus thuringiensis (Bt) was standardized on wheat bran based media in 250 ml Erlenmeyer flasks. Scale-up of Bt production on the best medium in plastic tubs with aeration at 8 h intervals starting 16 h after incubation yielded a significant increase in spore count and toxin content of the product. Maximum lysis of Bt cells was obtained by 60 h of incubation at 30 °C. This protocol was suitable for production of Bt strains and local isolates. The Bt produced proved highly effective at 0.1% concentration against larvae of castor semilooper, Achaea janata L, resulting in complete mortality by three days in laboratory bioassays. In field trials, the population of castor semilooper larvae on the castor bean crop was reduced significantly by three days after application. The cost for material production of 1 kg of Bt was approximately US $ 0.70.

Infection with the noncytopathic BVDV-2 strain 890 prevents replication of superinfecting cytopathic BVDV-1 RNA in BT and MDOK cells

Infection with the noncytopathic BVDV-2 strain 890 prevents replication of superinfecting cytopathic BVDV-1 RNA in BT and MDOK cells

Improvement of bioinsecticides production through mutagenesis of Bacillus thuringiensis by u.v. and nitrous acid affecting metabolic pathways and/or delta-endotoxin synthesis.

The present work aimed to obtain bioinsecticide over-producing mutants through classical mutagenesis of vegetative cells of Bacillus thuringiensis (Bt) by using u.v. and nitrous acid, and to evidence the involvement of cell metabolism in delta-endotoxin synthesis. Vegetative cells of Bt were treated by nitrous acid (0.17 mg ml(-1)) or exposed to u.v. rays (emitted at a wave length of 240 nm). The isolated survivors were screened on the basis of the production of delta-endotoxins and biomass in glucose and/or in gruel-based media at two aeration conditions. Bioinsecticide over-producing mutants were obtained with high frequencies because random mutations were shown to affect cell metabolism at different pathways related to the regulation of delta-endotoxin synthesis. Classical mutagenesis of Bt cells lead to the isolation of a large variety of delta-endotoxin over-producing mutants that could be classified into six groups based on the location of the mutations, particularly in metabolism pathways and delta-endotoxin synthesis. High frequencies of delta-endotoxin over-producing mutants of Bt could be obtained through classical mutagenesis of vegetative cells. This should contribute to a significant reduction of production and utilization costs of Bt bioinsecticides.

S-acetyl-glutathione selectively induces apoptosis in human lymphoma cells through a GSH-independent mechanism

Reduced apoptosis is associated to cancer development. Agents able to restore the programmed cell death responsiveness of cancer cells are foreseen as potential effective cancer therapies. In this study, we report that a glutathione-S-derivative, S-acetyl-glutathione (Sag), induces significant apoptosis in three human lymphoma cell lines, including Daudi, Raji and Jurkat cells while it had no or little effect on either Hut-78 lymphoma cells or the normal BT lymphocytes. We used Annexin-V FACS analysis and DNA laddering to demonstrate that Sag activated apoptosis in the three sensitive cell lines in a dose- and time-dependent-fashion. Using mercury orange staining and FACS analysis, we showed that Sag generated an intracellular GSH depletion in Daudi, Raji and Jurkat cells but not in Hut-78 or the normal BT cells. These data provide direct evidence that Sag specifically activates programmed cell death in lymphoma cells through, at least in part, a depletion of intracellular GSH rather than an increase, as previously suggested. Because of its selective effect on cancer cells, Sag appears as a promising new lymphoma cell apoptosis inducer with potential clinical value for lymphoma patients.

A Mathematical Model for Vegetative Growth ofBacillus Thuringiensis

A structured but unsegregated model was developed for the vegetative growth of Bacillus thuringiensis (Bt) cells. This model included cell growth, limiting-substrate consumption, production and consumption of acetic acid, and production of poly-β-hydroxy butyric acid. The parameter estimation and model validation were conducted using data from carbon-limited batch experiments. The model successfully predicted batch behavior of biomass and glucose concentrations.

High throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods

The development and application of a novel, sensitive TaqMan fluorescent probe-based product enhanced RT test (F-PERT) for the detection of retrovirus are described. The assay allows discrimination between the amplification signals generated by genuine positive signals that result from retroviral RT activity and the RT-like activity from DNA polymerases. The RT-like activity from DNA polymerases was suppressed by the addition of activated calf-thymus DNA with no reduction in the RT activity. A linear relationship between threshold cycle ( C T) and the number of virus particles was demonstrated, allowing quantification of retroviruses in unknown samples. The F-PERT assay was able to detect a wide range of retroviral RT activities, including that from porcine endogenous retrovirus (PoERV), murine leukaemia virus (MLV), simian foamy virus (SFV), simian immunodeficiency virus (SIV mac) and squirrel monkey retrovirus (SMRV). The detection limit of SMRV, MLV and PoERV was approximately 100 virion particles and the test was able to detect at least 10 2 molecules of purified RT enzyme. RT activity was not detected in cellular lysates and supernatants from MRC-5, BT, VERO, or Raji cells, whereas RT activity was detected in C127I, Mus dunni, K-Balb, BHK-21, CHO-K1, SP2/0-Ag14 and NSO cell supernatants. RT activity was also detected in the Spodoptera cell line Sf9.

Expression of HGF/SF in mesothelioma cell lines and its effects on cell motility, proliferation and morphology.

The expression of hepatocyte growth factor/scatter factor (HGF/SF) was studied in 12 mesothelioma cell lines characterized by either an epithelioid or a fibroblast-like phenotype. Conditioned media from these lines were analysed by bioassay and ELISA, and HGF/SF was detected in three cell lines, all with a fibroblast-like or mixed morphology. None of eight epithelioid cell lines expressed the factor. Thus, for these cell lines, the ability to secrete HGF/SF correlated with the cell phenotype. Following on from these observations, two cell lines, BR and BT, with a fibroblast-like and an epithelioid phenotype, respectively, were further investigated. Both cell lines expressed the Met receptor but only BR secreted HGF/SF. Both cell lines responded to exogenous HGF/SF treatment by a change of morphology but in different ways: BR became more elongated and bipolar, while BT formed more spread-out cell colonies. HGF/SF acted as a paracrine effector on the epithelioid BT cells and stimulated both cell-spreading and proliferation. Interestingly, BT cells spread but did not scatter in response to exogenous HGF/SF. In contrast BR cells showed only some stimulation of cell motility with HGF/SF and no increase in cell proliferation was observed. Because HGF/SF was previously found in the pleural effusion fluids of patients with malignant mesothelioma and in paraffin-embedded tumour tissues, it is concluded that HGF/SF may well stimulate the growth and spread of malignant mesothelioma in vivo by paracrine and/or autocrine mechanisms.

The nucleotide sequence of the 5′-untranslated region of bovine viral diarrhoea virus: its use as a probe in rapid detection of bovine viral diarrhoea viruses and border disease viruses

A 289 bp cDNA fragment from the 5′-untranslated region (UTR) of 16 bovine viral diarrhoea virus (BVDV) isolates was amplified by reverse transcription and polymerase chain reaction, and sequenced by dideoxy DNA sequencing. The sequence showed greater than 90% homology between the isolates and BVDV NADL in this region, and greater than 97% homology within a 72 base sub-region (nt 314-386). The 289 bp fragment was then used as a probe for rapid detection of BVDV and border disease virus (BDV) from cell culture samples by dot-blot hybridization. This probe hybridized to 100% of BVDV isolates ( n = 78) and 100% of BDV isolates ( n = 9), but not to the uninfected BT cells or other bovine infectious agents. A shorter probe from the more conserved sub-region also was tested for hybridization with some of the isolates, and the results were similar to those using the longer probe. These results suggest that the 5′-UTR is highly conserved among BVDV and BDV isolates, and may be used as a potential probe for rapid detection of BVDV and BDV in clinical and cell culture samples from cattle and sheep.

Comparative activity of macrolides against Toxoplasma gondii demonstrating utility of an in vitro microassay

The utility of spiramycin for preventing transplacental transmission of toxoplasmosis and the efficacy of conventional macrolides against Toxoplasma gondii are subjects of active debate. An in vitro microassay was developed to determine the relative inhibitory activity against T. gondii of 24 conventional macrolides derived from erythromycin and tylosin (14- and 16-membered macrolides, respectively). Macrolides and T. gondii RH tachyzoites were added to monolayers of BT cells grown in 96-well plates. Plates were incubated for 20 h at 37 degrees C, and the growth of T. gondii was then measured by the selective incorporation of [3H]uracil in trichloroacetic acid-precipitable material during an additional incubation of 20 h. Dose-response curves and 50 and 90% inhibitory concentrations (IC50 and IC90, respectively) were determined for each drug. Microscopic examination was performed on stained replicates of the infected monolayers, and the relative toxicities of the drugs for host cells were determined. Spiramycin and tylosin showed only limited activity against T. gondii (IC50 of 20.16 and 20.00 micrograms/ml, respectively). Erythromycin and azithromycin had a better anti-Toxoplasma activity with IC50 of 14.38 and 8.61 micrograms/ml, respectively, whereas drugs like desmycosin, dirithromycin, and roxithromycin had no detectable activity. Although many macrolides inhibited intracellular proliferation of T. gondii, azithromycin was the only macrolide demonstrating prolonged inhibitory activity on the replication of intracellular tachyzoites. We conclude that conventional 14- and 16-membered macrolides often interfere with the growth of, but may not kill, T. gondii RH tachyzoites in vitro.

Host range mutant of human immunodeficiency virus type 1: modification of cell tropism by a single point mutation at the neutralization epitope in the env gene

We have isolated a variant of human immunodeficiency virus type 1 (HIV-1) which is highly infectious to fibroblastlike cells (BT cells) derived from human brain as well as CD4-positive T cells. This variant HIV-1, named HIV[GUN-1V], was obtained by infecting BT cells with a prototype HIV-1 isolate, named HIV[GUN-1WT], which is highly infectious to T cells but barely infectious to BT cells. HIV[GUN-1V] infects BT cells productively and this infection appeared to be mediated by CD4. To elucidate the viral gene responsible for the host range difference between the variant and prototype HIV-1s, we cloned and analyzed the provirus genomes of the two viruses. Examination of the infectivities of BT cells by various recombinant viruses and analyses of the nucleotide sequences of HIV[GUN-1V] and HIV[GUN-1WT] showed that a single nucleotide exchange was responsible for their difference in infectivity of BT cells: HIV[GUN-1V] contains a thymine residue instead of the cytosine residue in HIV[GUN-1WT] at position 931 of the env coding sequence. Replacement of cytosine by thymine at this position of the env coding sequence of the HIV[GUN-1WT] genome induced the ability to infect BT cells. The base exchange at this position was expected to change amino acid 311 of the envelope glycoprotein, gp120, from proline to serine, which is located in a variable region containing type-specific immunodominant epitopes. Thus, HIV[GUN-1V] acquired a wider host range than HIV[GUN-1WT] by a single point mutation in the env gene.

Cell and Virus Sensitivity Studies with Recombinant Human Alpha Interferons

The cell sensitivity of recombinant human alpha interferons (rIFN-alpha) of greater than 95% purity (A,D and the hybrid A/D) and crude nature (B and F) was studied in human (WISH, HeLa, AG1732), bovine (MDBK, BT), monkey (Vero), mouse (L), rabbit (RK-13), and hamster (BHK-21) cells. Based on an activity of 100% in WISH cells, the other cells responded to rIFN-alpha A as follows: AG1732 (90%), HeLa (94%), and MDBK and BT cells (170-190%). Rabbit, mouse, and hamster cells had a relative sensitivity of less than 1%. rIFN-alpha B and F were essentially equivalent to rIFN-alpha A in terms of cell sensitivity, but MDBK and BT cells were about 20 times more sensitive to rIFN-alpha D than were WISH cells and rIFN-alpha D was 1/5 to 1/10 as active on L cells as on WISH cells. The activity of the hybrid IFN A/D on bovine, mouse, and human cells was similar. The inhibitory dose50 (U/ml) of rIFN-alpha A, B, D, and F against virus infections in WISH cells were: vesicular stomatitis virus (1-4), rhinovirus types 1 and 42 (2-18), and herpes simplex virus (HSV) type 2 (45-70). Type 1 HSV, Semliki Forest (SFV) and encephalomyocarditis (EMC) viruses were tested against only rIFN-alpha A and D where SFV and EMC were the most sensitive to both IFNs (ID50-SFV, 0.2 U/ml, EMC, 1.2 U/ml) while 13 U/ml of rIFN-alpha A and D inhibited Type 1 HSV. The various rIFN-alpha s did not exhibit different antiviral spectra in vitro. When tested in mice rIFN-alpha A did not protect against infections with SFV, EMC, HSV, or influenza viruses. rIFN-alpha D and A/D protected mice infected with EMC, SFV, or HSV.

Cell-mediated cytotoxicity in carcinoma of the human urinary bladder

Lymphocytes from patients with tumors of the bladder or other unrelated tissues or with non-malignant genitourinary (GU) conditions and from normal subjects were tested in a microcytotoxicity assay against long- and short-term cultures (T24 and BT) derived from bladder carcinoma and several other target cell types, to determine the validity of the hypothesis that cell-mediated cytotoxicity (CMC) in bladder cancer patients is a specific disease-related phenomenon. At the effector cell level, lymphocytes from bladder cancer patients displayed uniformly greater cytotoxicity for T24 and BT cells than those from normal donors. This proclivity was shared by the lymphocytes of patients with non-GU cancers but not of those with non-malignant GU disorders. At the target cell level, CMC was observed less frequently against non-bladder tumor targets than against T24 and BT cells and the CMC of bladder cancer patients did not differ significantly from that of the other groups. The pattern of CMC observed against the bladder tumor-derived targets was thus one of target cell sensitivity rather than tumor-specificity and disease-related only to the extent that the CMC of patients with cancer was greater overall than of healthy subjects. Abrogation of CMC by passage of lymphocytes through immunoglobulin-coated columns indicated that the effector cells were principally of non-T type, bearing a superficial resemblance to those in normal individuals which induce non-disease related CMC.