Parasporins from a Caribbean Island: Evidence for a Globally Dispersed <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">thuringiensis</Emphasis> Strain

Parasporins represent a new functional class of Cry (crystal protein) toxins produced by the bacterium Bacillus thuringiensis (Bt). Unlike Cry toxins that demonstrate activity mainly against some insect cells, parasporins are characterized as being non-hemolytic, yet capable of preferentially killing some human cancer cells. Globally, six different parasporin types, PS1–PS6, based on protein sequence homology, have been identified in only four countries (Japan, Vietnam, India, and Canada). Herein we report the results of a screening study of 160 Bt isolates collected from the Caribbean island of Trinidad. One isolate (strain 64-1-94) was shown to kill human cancer cells and to contain one ps6 and two ps1 parasporin genes. The two ps1 genes were located approximately 6 kb apart from each other, sharing a similar spatial arrangement, and high sequence homology, with two plasmid-located ps1 genes, ps1Aa6 and ps1Ad1, recently isolated from a Japanese strain. Evidence is also presented that a parasporin gene reported previously for a Canadian strain, ps1Aa2, is most likely derived from a recombination event between these same two genes found in the Trinidadian and Japanese strains. Notably, all three strains share a ps6 parasporin gene, presumably located on a separate plasmid. These data suggest that the global population of ps1 genes may be have originated from a single pair of parasporin genes. Given the large geographical distance between the collection sites, which are located on both continental land masses and islands at sea, ps1 genes are able to retain a remarkable level of homology not easily explained.     

Characterization of a Novel <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Phenotype Possessing Multiple Appendages Attached to a Parasporal Body

Bacillus thuringiensis is a bacterium best known for its production of crystal-like bodies comprised of one or more Cry-proteins, which can be toxic to insects, nematodes or cancer cells. Although strains of B. thuringiensis have occasionally been observed with filamentous appendages attached to their spores, appendages in association with their parasporal bodies are extremely rare. Herein we report the characterization of Bt1-88, a bacterial strain isolated from the Caribbean that produces a spore–crystal complex containing six long appendages, each comprised of numerous thinner filaments approximately 10 nm in diameter and 2.5 μm in length. Each of the multi-filament appendages was attached to a single, small parasporal body located at one end of the bacterial spore. Biochemical tests, 16S rDNA gene sequencing, and the identification of two Cry proteins by partial protein sequencing (putatively Cry1A and Cry2A), unambiguously identified Bt1-88 as a strain of B. thuringiensis. Bt1-88 represents the second reported strain of B. thuringiensis possessing a parasporal body/appendage phenotype characterized by one or more long appendages, comprised of numerous filaments in association with a parasporal body. This finding suggests that Bt1-88 is a member of a new phenotypic class of B. thuringiensis, in which the parasporal body may perform a novel structural role through its association with multi-filament appendages.     

Identification of native Bacillus thuringiensis strain from South India having specific cytocidal activity against cancer cells

Aim: To identify the parasporin‐producing, indigenous Bacillus thuringiensis strains that specifically targets human cancer cells in Madurai, Tamil Nadu, South India. Methods and Results: Alkali‐solubilized inclusion proteins from the 82 nonclonal indigenous isolates of B. thuringiensis were analysed for their cytotoxicity against two human cancer cell lines, U‐937 (human histiocytic lymphoma) and HCT‐250 (adherent human colon cancer cells). Activated inclusion protein from one of the isolates, B. thuringiensis LDC‐391, was found to be highly cytotoxic to HCT‐250 and moderately toxic to U‐937, but nontoxic to normal lymphocytes. This strain did not show any insecticidal activity against the lepidopteran and dipteran larvae tested, as well as it was nonhaemolytic on human erythrocytes. The Western‐blotting analysis showed that the putative 180 kDa cytotoxic protein from the isolate B. thuringiensis LDC‐391 cross‐reacted with the reference antisera of 81‐kDa parasporin‐1. Conclusions: Our observations imply that B. thuringiensis LDC‐391 is different from the already reported parasporin producers, as it is showing variation in the target specificity. Significance and Impact of the Study: Characterizing these proteins can pave the way to alleviate problems associated with neoplastic transformation and cancer progression.     

Characterization of the anti-cancer-cell parasporal proteins of a Bacillus thuringiensis isolate

An unusual activity, associated with non-insecticidal and non-haemolytic parasporal inclusion proteins of a Bacillus thuringiensis soil isolate, designated 89-T-26-17, was characterized. The parasporal inclusion of this isolate was bipyramidal, rounded at both ends, containing proteins of 180, 150, 120, 100, and 88 kDa. No homologies with the Cry and Cyt proteins of B. thuringiensis were detected based on N-terminal sequences. Proteolytic processing of the inclusion proteins by proteinase K, trypsin, and chymotrypsin produced a major protein of 64 kDa exhibiting cytocidal activity against human leukaemic T cells and uterus cervix cancer (HeLa) cells. The protease-activated proteins showed no cytotoxicity to normal T cells.     

Binding immunoglobulin protein resolves rheumatoid synovitis: a xenogeneic study using rheumatoid arthritis synovial membrane transplants in SCID mice

Introduction  

Binding immunoglobulin protein (BiP) has previously shown powerful anti-inflammatory properties in the collagen-induced arthritis (CIA) model, where a single dose of BiP has proved to be both a long-term prophylactic and therapeutic. In both CIA and human in vitro studies, BiP induced regulatory T cells. The present investigation looked at the anti-inflammatory effect of BiP on inflamed human synovial tissue transplanted into severe combined immunodeficient mice (SCID), a chimaeric in vivo model previously used to test the efficacy of biologic therapies.     

Investigation of a novel Bacillus thuringiensis gene encoding a parasporal protein, parasporin-4, that preferentially kills human leukemic T cells

A novel gene encoding a leukemic cell-killing parasporal protein, designated parasporin-4, was cloned from an isolate of Bacillus thuringiensis serovar shandongiensis. The amino acid sequence of the parasporin-4, as deduced from the gene sequence, had low-level homologies of <30% with the established B. thuringiensis Cry proteins including the three known parasporins. When the gene was expressed in a recombinant of Escherichia coli BL21(DE3), the parasporin-4 formed intracellular inclusion bodies. Alkali-solubilized and proteinase K-activated inclusion protein exhibited strong cytotoxic activity against human leukemic T cells (MOLT-4) and weak for normal T cells, but no adverse effect on human uterus cervix cancer cells (HeLa).     

Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1

Background  

The promoter of the keratin 18 (K18) gene is 5- to 10-fold more active in tumorigenic (T-type) cell clones derived from the SW613-S human colon carcinoma cell line than in non-tumorigenic (NT-type) clones. We have reported previously that the mechanism responsible for this differential activity is acting on the minimal K18 promoter (TATA box and initiation site). This mechanism does not require the binding of a factor to a specific site on the DNA but involves the acetylation of a non-histone substrate. To get further insight into this mechanism, we investigated the effect of the adenovirus E1A protein on the activity of the K18 promoter, both in T and NT cells.     

Heterologous expression in <Emphasis Type="Italic">Tritrichomonas foetus</Emphasis> of functional <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> AP65 adhesin

Background  

Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor.     

Equol an isoflavonoid: potential for improved prostate health, <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>evidence

Background  

To determine: in vitro binding affinity of equol for 5alpha-dihydrotestosterone (5alpha-DHT), in vitro effects of equol treatment in human prostate cancer (LNCap) cells, and in vivo effects of equol on rat prostate weight and circulating levels of sex steroid hormones.     

Sub-chronic lung inflammation after airway exposures to <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> biopesticides in mice

Background  

The aim of the present study was to assess possible health effects of airway exposures to Bacillus thuringiensis (Bt) based biopesticides in mice. Endpoints were lung inflammation evaluated by presence of inflammatory cells in bronchoalveolar lavage fluid (BALF), clearance of bacteria from the lung lumen and histological alterations of the lungs. Hazard identifications of the biopesticides were carried out using intratracheal (i.t.) instillation, followed by an inhalation study. The two commercial biopesticides used were based on the Bt. subspecies kurstaki and israelensis, respectively. Groups of BALB/c mice were i.t instilled with one bolus (3.5 × 10⁵ or 3.4 × 10⁶ colony forming units (CFU) per mouse) of either biopesticide. Control mice were instilled with sterile water. BALFs were collected and the inflammatory cells were counted and differentiated. The BALFs were also subjected to CFU counts.     

Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

Background  

Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099) of the S-layer protein and EGFP (enhanced green fluorescent protein), in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells.     

Evidence for glycosylation on a DNA-binding protein of <Emphasis Type="Italic">Salmonella enterica</Emphasis>

Background  

All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column.     

Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

Background  

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.     

An Investigation of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in Rectal-Collected Fecal Samples of Cows

In order to better understand the range and role of Bacillus thuringiensis (Bt) and its toxins in nature, we have undertaken a study of Bt taken directly from the rectum of 117 cows from 37 farms on the Caribbean island of Trinidad. Thirty-seven fecal samples (32%) were found to contain at least one Bt. Generally only one or two isolates with a particular crystal morphology were isolated from any one sample, however, a few samples contained more, up to 11 isolates, suggesting post-ingestion amplification. Bioassays using larvae of Musca domestica, Caenorhabditis elegans and Tetrahymena pyriformis showed no observable toxicity in gross bioassays. Very small dot-like parasporal bodies, not generally characteristic of Bt, were isolated from 44% of the samples, which in many instances appeared unstable and whose relation to Bt Cry protein-containing parasporal bodies is unknown. In conclusion, we find little evidence for a host adapted strain of Bt in the cows examined, nor toxicity to organisms that might logically be associated with either the cow or its feces. The presence of a large number of isolates containing small dot-like parasporal bodies, possibly either poly-β-hydroxybutyrate storage bodies or Cry proteins, was unexpected and merits further investigation.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

Targeted protein depletion in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by activation of a bidirectional degron

Background  

Tools for in vivo manipulation of protein abundance or activity are highly beneficial for life science research. Protein stability can be efficiently controlled by conditional degrons, which induce target protein degradation at restrictive conditions.     

In vitro cytotoxicity of Gymnema montanum in human leukaemia HL‐60 cells; induction of apoptosis by mitochondrial membrane potential collapse

Objectives

Gymnema montanum Hook, an Indian Ayurvedic medicinal plant, is used traditionally to treat a variety of ailments. Here, we report anti‐cancer effects and molecular mechanisms of ethanolic extract of G. montanum (GLEt) on human leukaemia HL‐60 cells, compared to peripheral blood mononuclear cells.

Materials and methods

HL‐60 cells were treated with different concentrations of GLEt (10–50 μg/ml) and cytotoxicity was assessed by MTT assay. Levels of lipid peroxidation, antioxidants, mitochondrial membrane potential and caspase‐3 were measured. Further, apoptosis was studied using annexin‐V staining and the cell cycle was analyzed by flow cytometry.

Results

GLEt had a potent cytotoxic effect on HL‐60 cells (IC₅₀‐20 μg/ml), yet was not toxic to normal peripheral blood mononuclear cells. Exposure of HL‐60 cells to GLEt led to elevated levels of malonaldehyde formation, but to reduced glutathione, superoxide dismutase, catalase and glutathione peroxidase activities (P < 0.05). Induction of apoptosis was confirmed by observing annexin‐V positive cells, associated with loss of mitochondrial membrane potential. Cell cycle arrest at G0/G1 was observed in GLEt‐treated HL‐60 cells, indicating its potential at inducing their apoptosis.

Conclusions

Findings of the present study suggest that G. montanum induced apoptosis in the human leukaemic cancer cells, mediated by collapse of mitochondrial membrane potential, generation of reactive oxygen species and depletion of intracellular antioxidant potential.

     

Tooth loss and its relationship with protein intake by elderly Brazilians—A structural equation modelling approach

Objective

This study aimed at assessing the relationship between self‐perceived tooth loss and wearing dentures, on the one hand, and the consumption of protein, on the other hand, among the elderly population of Botucatu, SP. Food consumption tends to decrease with ageing, especially protein intake, and one of the causes could be the precariousness of oral health. Several risk factors associated with deficient dietary protein intake have been identified, namely greater physical dependence, reduced caloric intake and food insecurity, but no studies have analysed whether tooth loss and prostheses interfere with protein intake.

Methods

An interview was conducted among 365 elderly individuals, in which we examined oral health‐related quality of life (OHRQoL) as the only latent variable, in a 24‐hour nutritional assessment dietary recall repeated 3 times, conducted in person by a trained nutritionist and also performed an analysis of nutritional needs using the Nutrition Data System Research (NDSR) Program.

Results

The structural equation model, performed using Stata v.14, showed that lack of teeth (standardised coefficient [SC] = 0.21, P < .001), and prosthesis use (SC = ?0.21, P < .001) was associated with OHRQoL. Lack of teeth had a direct effect on the consumption of animal protein (SC = 0.08, P = .02), a strong total effect on animal protein intake (SC = 0.51, P = .04) and a medium effect on total protein intake (SC = 0.20, P = .03), adjusted for confounders (depression and medical problems).

Conclusion

Tooth loss had a strong and significant total effect on animal protein intake and a medium effect on total protein intake among elderly Brazilians.