[go: up one dir, main page]
More Web Proxy on the site http://driver.im/
You seem to have javascript disabled. Please note that many of the page functionalities won't work as expected without javascript enabled.
 
 
Sign in to use this feature.

Years

Between: -

Subjects

remove_circle_outline
remove_circle_outline
remove_circle_outline
remove_circle_outline
remove_circle_outline

Journals

Article Types

Countries / Regions

Search Results (25)

Search Parameters:
Keywords = cytotoxin K

Order results
Result details
Results per page
Select all
Export citation of selected articles as:
19 pages, 579 KiB  
Article
Characterization of the Bacillus cereus Group Isolated from Ready-to-Eat Foods in Poland by Whole-Genome Sequencing
by Joanna Kowalska, Elżbieta Maćkiw, Dorota Korsak and Jacek Postupolski
Foods 2024, 13(20), 3266; https://doi.org/10.3390/foods13203266 - 14 Oct 2024
Viewed by 1222
Abstract
Bacillus cereus sensu lato can contaminate food and cause food poisoning by producing toxins such as cereulide, toxin BL, and cytotoxin K. In this study, we retrospectively analyzed B. cereus sensu lato from retail food products and food poisoning cases using PCR methods to [...] Read more.
Bacillus cereus sensu lato can contaminate food and cause food poisoning by producing toxins such as cereulide, toxin BL, and cytotoxin K. In this study, we retrospectively analyzed B. cereus sensu lato from retail food products and food poisoning cases using PCR methods to determine their virulence profiles. A new toxin profile, encoding all four toxins (hbl, nhe, cytK, ces), was found in 0.4% of isolates. The toxin profiles, classified into A-J, revealed that 91.8% harbored nhe genes, while hbl, cytK, and ces were detected in 43.8%, 46.9%, and 4.2% of isolates, respectively. Whole-genome sequencing (WGS) identified four distinct species within the B. cereus group, with 21 isolates closely related to B. cereus sensu stricte, 25 to B. mosaicus, 2 to B. toyonensis, and 1 to B. mycoides. Three novel sequence types (STs 3297, 3298, 3299) were discovered. Antibiotic resistance genes were common, with 100% of isolates carrying beta-lactam resistance genes. Fosfomycin (80%), vancomycin (8%), streptothricin (6%), tetracycline (4%), and macrolide resistance (2%) genes were also detected. These results highlight the genetic diversity and antibiotic resistance potential of B. cereus sensu lato strains in Polish food products. Full article
(This article belongs to the Section Food Microbiology)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>SNP phylogeny of <span class="html-italic">B. cereus</span> group. The CSI Phylogeny 1.4 tool provided by the Center for Genomic Epidemiology, accessed at <a href="http://www.genomicepidemiology.org" target="_blank">www.genomicepidemiology.org</a> (accessed on 6 May 2024). We generated phylogenetic trees based on our datasets and used our first genome as a reference. To visualize the Newick files that were generated, we utilized iTol (<a href="https://itol.embl.de/" target="_blank">https://itol.embl.de/</a> (accessed on 6 May 2024)).</p>
Full article ">
14 pages, 4311 KiB  
Article
A High-Homology Region Provides the Possibility of Detecting β-Barrel Pore-Forming Toxins from Various Bacterial Species
by Alexey S. Nagel, Olesya S. Vetrova, Natalia V. Rudenko, Anna P. Karatovskaya, Anna V. Zamyatina, Zhanna I. Andreeva-Kovalevskaya, Vadim I. Salyamov, Nadezhda A. Egorova, Alexander V. Siunov, Tatiana D. Ivanova, Khanafi M. Boziev, Fedor A. Brovko and Alexander S. Solonin
Int. J. Mol. Sci. 2024, 25(10), 5327; https://doi.org/10.3390/ijms25105327 - 14 May 2024
Viewed by 1022
Abstract
The pathogenicity of many bacteria, including Bacillus cereus and Staphylococcus aureus, depends on pore-forming toxins (PFTs), which cause the lysis of host cells by forming pores in the membranes of eukaryotic cells. Bioinformatic analysis revealed a region homologous to the Lys171-Gly250 sequence [...] Read more.
The pathogenicity of many bacteria, including Bacillus cereus and Staphylococcus aureus, depends on pore-forming toxins (PFTs), which cause the lysis of host cells by forming pores in the membranes of eukaryotic cells. Bioinformatic analysis revealed a region homologous to the Lys171-Gly250 sequence in hemolysin II (HlyII) from B. cereus in over 600 PFTs, which we designated as a “homologous peptide”. Three β-barrel PFTs were used for a detailed comparative analysis. Two of them—HlyII and cytotoxin K2 (CytK2)—are synthesized in Bacillus cereus sensu lato; the third, S. aureus α-toxin (Hla), is the most investigated representative of the family. Protein modeling showed certain amino acids of the homologous peptide to be located on the surface of the monomeric forms of these β-barrel PFTs. We obtained monoclonal antibodies against both a cloned homologous peptide and a 14-membered synthetic peptide, DSFNTFYGNQLFMK, as part of the homologous peptide. The HlyII, CytK2, and Hla regions recognized by the obtained antibodies, as well as an antibody capable of suppressing the hemolytic activity of CytK2, were identified in the course of this work. Antibodies capable of recognizing PFTs of various origins can be useful tools for both identification and suppression of the cytolytic activity of PFTs. Full article
(This article belongs to the Collection Feature Papers in Molecular Immunology)
Show Figures

Figure 1

Figure 1
<p>The taxonomic distribution of significant hits with an E-value less than 0.003 from the search for the HlyII Lys171-Gly250 sequence (HP) using the HMMER [<a href="#B12-ijms-25-05327" class="html-bibr">12</a>] tool in the UniProtKB [<a href="#B12-ijms-25-05327" class="html-bibr">12</a>,<a href="#B13-ijms-25-05327" class="html-bibr">13</a>] database.</p>
Full article ">Figure 2
<p>Comparison of the HlyII Lys171-Gly250 sequence with the β-barrel PFT sequences. 1—Hemolysin II of <span class="html-italic">B. cereus</span>; 2—cytotoxin K2 of <span class="html-italic">B. cereus</span>; 3—α-hemolysin of <span class="html-italic">S. aureus</span>; 4—necrotizing enteritis toxin NetG of <span class="html-italic">C. perfringens;</span> 5—leukotoxin domain protein A of <span class="html-italic">C. perfringens</span>; 6—β-channel forming cytolysin of <span class="html-italic">C. septicum</span>; 7—leukocidin family pore-forming toxin of <span class="html-italic">C. botulinum</span>. Positions with identical amino acid residues are highlighted in green. Positions where similar properties among groups of amino acids are conserved are marked in yellow. Orange refers to an identical section.</p>
Full article ">Figure 3
<p>Amino acid sequence of the cloned HP against which HP-series mAbs were obtained. The amino acids are numbered in relation to the sequence of the full-length hemolysin II. Positions with identical amino acid residues for Hla, CytK2, and HlyII are shown in green. Yellow indicates positions where similar properties between groups of amino acids are preserved. Orange refers to an identical section. The sequence including the HlyIILCTD [<a href="#B15-ijms-25-05327" class="html-bibr">15</a>] region is underlined. The thrombin recognition site, linker, and six histidine residues are highlighted in bold. For the synthetic peptide against which the SHP-series mAbs were obtained, part of the stem domain and triangle region [<a href="#B14-ijms-25-05327" class="html-bibr">14</a>] are noted with signatures.</p>
Full article ">Figure 4
<p>(<b>a</b>) Location of the homologous regions on models of the water-soluble monomer proteins Hla (Uniprot: Q2G1X0), CytK2 (Uniprot: Q81GS6), and HlyII (Uniprot: Q81AN8), with structures predicted by AlphaFold [<a href="#B18-ijms-25-05327" class="html-bibr">18</a>,<a href="#B19-ijms-25-05327" class="html-bibr">19</a>]. Positions with identical amino acid residues for Hla, CytK2, and HlyII are shown in green. Yellow indicates the homologous amino acids of the three selected PFTs. The identical section is highlighted in orange. (<b>b</b>) Homologous peptide alignment in Hla, CytK2, and HlyII. Orange refers to an identical section.</p>
Full article ">Figure 5
<p>(<b>a</b>) Comparison of the interaction of the HP-series mAbs (concentration, 5 μg/mL) with <span class="html-italic">B. cereus</span> CytK2, <span class="html-italic">S. aureus</span> Hla, and HlyIILCTD, HlyII, and HlyII∆CTD of <span class="html-italic">B. cereus</span> during the sorption from a concentration of 1 μg/mL of toxins in ELISA. Data are represented as the means ± SDs of 5 independent repeats (<span class="html-italic">n</span> = 5). * A statistically significant difference (<span class="html-italic">p</span> &lt; 0.05, Mann–Whitney). (<b>b</b>) Immunoblotting of HlyII, HlyIILCTD, CytK2 of <span class="html-italic">B. cereus</span>, Hla of <span class="html-italic">S. aureus</span>, and HlyII∆CTD of <span class="html-italic">B. cereus</span> with the mAbs against the HP. Before electrophoretic separation, the samples were boiled in 1% SDS for 10 min. The names of the mAbs are given above the lanes. The image was obtained by combining four immunoblots.</p>
Full article ">Figure 6
<p>(<b>a</b>) Comparison of the interaction of the SHP-series mAbs (concentration, 5 μg/mL) with <span class="html-italic">B. cereus</span> CytK2, <span class="html-italic">S. aureus</span> Hla, and HlyIILCTD, HlyII, and HlyII∆CTD of <span class="html-italic">B. cereus</span> during the sorption from a concentration of 1 μg/mL of toxins in ELISA. Data are represented as the means ± SDs of 5 independent repeats (<span class="html-italic">n</span> = 5). * A statistically significant difference when comparing the interaction of antibodies with different antigens. (<span class="html-italic">p</span> &lt; 0.05, Mann–Whitney); ** a statistically significant difference when comparing the antibody binding to HlyIILCTD (<span class="html-italic">p</span> &lt; 0.05, Mann–Whitney). (<b>b</b>) Immunoblotting of CytK2 of <span class="html-italic">B. cereus,</span> Hla of <span class="html-italic">S. aureus</span>, and HlyII, HlyIILCTD, and HlyII∆CTD of <span class="html-italic">B. cereus</span> with the mAbs against the SHP. Before electrophoretic separation, the samples were boiled in 1% SDS for 10 min. The names of the mAbs are given above the lanes. The image was obtained by combining two immunoblots.</p>
Full article ">Figure 7
<p>Suppression of the hemolysis of the HP-5 mAb during an attack of rabbit erythrocytes by various PFTs. (<b>a</b>) Hemolytic activity of PFTs without the addition of the mAb (blue line) and after the addition of the mAb to a final concentration of 0.4 μM (orange line). (<b>b</b>) Hemolytic activity of the PFTs after the addition of the mAb at different concentrations. The hemolytic activity curves without the addition of the mAb are shown on the left. Colored arrows indicate points on the curves corresponding to the concentrations of the pore-forming toxin incubated with the mAb. On the right, suppression of hemolysis depending on the concentration of the mAb. The color of the arrows corresponds to the color of the lines. Data are represented as the means ± SDs of 5 independent repeats (<span class="html-italic">n</span> = 5).</p>
Full article ">Figure 7 Cont.
<p>Suppression of the hemolysis of the HP-5 mAb during an attack of rabbit erythrocytes by various PFTs. (<b>a</b>) Hemolytic activity of PFTs without the addition of the mAb (blue line) and after the addition of the mAb to a final concentration of 0.4 μM (orange line). (<b>b</b>) Hemolytic activity of the PFTs after the addition of the mAb at different concentrations. The hemolytic activity curves without the addition of the mAb are shown on the left. Colored arrows indicate points on the curves corresponding to the concentrations of the pore-forming toxin incubated with the mAb. On the right, suppression of hemolysis depending on the concentration of the mAb. The color of the arrows corresponds to the color of the lines. Data are represented as the means ± SDs of 5 independent repeats (<span class="html-italic">n</span> = 5).</p>
Full article ">Figure 8
<p>Location of putative regions containing epitopes to the HP-1, HP-3, HP-4, HP-5, and HP-7 mAbs on the HlyII (UniProt: Q81AN8) water-soluble monomer model. Blue: HP-1, HP-3, and HP-7; red: HP-5 and SHP-series mAbs (including an identical (Tyr219-Met225) section (orange)); cyan: HP-4 (includes a section of HlyIILCTD).</p>
Full article ">
16 pages, 2726 KiB  
Article
Transcriptional Activation of a Pro-Inflammatory Response (NF-κB, AP-1, IL-1β) by the Vibrio cholerae Cytotoxin (VCC) Monomer through the MAPK Signaling Pathway in the THP-1 Human Macrophage Cell Line
by Julio Rodrigo Escartín-Gutiérrez, Mariana Ponce-Figueroa, Miguel Ángel Torres-Vega, Leopoldo Aguilar-Faisal and Paula Figueroa-Arredondo
Int. J. Mol. Sci. 2023, 24(8), 7272; https://doi.org/10.3390/ijms24087272 - 14 Apr 2023
Cited by 5 | Viewed by 2150
Abstract
This study describes, to some extent, the VCC contribution as an early stimulation of the macrophage lineage. Regarding the onset of the innate immune response caused by infection, the β form of IL-1 is the most important interleukin involved in the onset of [...] Read more.
This study describes, to some extent, the VCC contribution as an early stimulation of the macrophage lineage. Regarding the onset of the innate immune response caused by infection, the β form of IL-1 is the most important interleukin involved in the onset of the inflammatory innate response. Activated macrophages treated in vitro with VCC induced the activation of the MAPK signaling pathway in a one-hour period, with the activation of transcriptional regulators for a surviving and pro-inflammatory response, suggesting an explanation inspired and supported by the inflammasome physiology. The mechanism of IL-1β production induced by VCC has been gracefully outlined in murine models, using bacterial knockdown mutants and purified molecules; nevertheless, the knowledge of this mechanism in the human immune system is still under study. This work shows the soluble form of 65 kDa of the Vibrio cholerae cytotoxin (also known as hemolysin), as it is secreted by the bacteria, inducing the production of IL-1β in the human macrophage cell line THP-1. The mechanism involves triggering the early activation of the signaling pathway MAPKs pERK and p38, with the subsequent activation of (p50) NF-κB and AP-1 (cJun and cFos), determined by real-time quantitation. The evidence shown here supports that the monomeric soluble form of the VCC in the macrophage acts as a modulator of the innate immune response, which is consistent with the assembly of the NLRP3 inflammasome actively releasing IL-1β. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Infection for Pathogenic Bacteria)
Show Figures

Figure 1

Figure 1
<p>Standardization of responsive concentrations of VCC. A restricted concentration of VCC (40 pg/mL) induces pro-IL-1β after 6 h of treatment without cytoplasmic vacuolization in differentiated THP-1 macrophages. (<b>A</b>) Chronological sequence by inverted optical microscopy. The cells were treated with 40 pg/mL toxin and later monitored (0, 60, 90 and 180 min) during incubation and photographed from an inverted optical microscope (40× Olympus IX71). Black arrows indicate a few characteristic vacuoles at 90 min; yet, this is not considered a vacuolating effect (where at least 50% of the cells are vacuolated). The results shown were arbitrarily chosen, but they are representative of three independent experiments. (<b>B</b>) Western blot showing Pro IL-1β by treatments with 40 pg/mL of VCC after 6 h of incubation with no vacuolating effect. The expression of pro IL-1β protein with different treatments (LPS + PMA, LPS, VCC, VCC + ATP) at 6 h was measured by a western blot assay. WB of the β-actin protein level is an internal control.</p>
Full article ">Figure 2
<p>IL-1β release under VCC treatment. THP-1 macrophages were treated with 40 pg/mL of VCC. A positive control of LPS (1 µg/mL) was used. The release of IL-1β was determined by ELISA. ELISA shows IL-1β release under treatments with 40 pg/mL of VCC after 30, 60 and 120 min. Results in the figure are representative of three performed experiments. W/T, without treatment. Mean ± SD. <span class="html-italic">p</span> &lt; 0.001, significantly different from the group treated with LPS (ANOVA plus the Student–Newman–Keuls method).</p>
Full article ">Figure 3
<p>VCC activates the phosphorylation of p38 in THP-1 macrophages. THP-1 macrophages were treated with 40 pg/mL of VCC at 10, 15, 30 and 60 min. A positive control of LPS (1 µg/mL) was used. Cell lysates were analyzed by the Western blot assay. (<b>A</b>) Phosphorylation of p38 after treatments with VCC. The results illustrated here are from a single experiment representing three separate experiments. In the upper panel of the Western blot, changes in the phosphorylation of P-p38 were identified, while in the middle panel, it is observed that the content of non-phosphorylated p38 appears constantly in all the analyzed samples. The lower panel shows the β-actin protein considered an internal control. (<b>B</b>) Kinetic of treatment with VCC. Results in the graphic are representative of three performed experiments. W/T, without treatment. Mean ± SD. * <span class="html-italic">p</span> &lt; 0.001, significantly different from the group treated with LPS (ANOVA plus the Student–Newman–Keuls method).</p>
Full article ">Figure 4
<p>VCC activates the phosphorylation of ERK in THP-1. Differentiated THP-1 macrophages were treated with the VCC at 10, 15, 30 and 60 min. The positive control was LPS (1 µg/mL). Cell lysates were analyzed by Western blot assays. (<b>A</b>) Western blot showing ERK phosphorylation under treatments with 40 pg/mL of VCC at 10, 15, 30 and 60 min. The results illustrated are a single experiment representative of three; changes in the phosphorylation of p-ERK are identified in the heading of the Western blot; below this, it is shown that the ERK content is constant in all the samples analyzed. The lower panel shows the β-actin protein at a level considered as an internal control. (<b>B</b>) Kinetics of Erk phosphorylation induced by treatments with VCC. The graphic represents results from three experiments. W/T, without treatment. Mean ± SD. * <span class="html-italic">p</span> &lt; 0.001, significantly different from the group treated with LPS (ANOVA plus the Student–Newman–Keuls method).</p>
Full article ">Figure 5
<p>The VCC initiates messenger RNA expression of the transcription factor AP-1. Kinetics of transcriptional activation in THP-1 cells were performed by treatments with 40 pg/mL VCC during 10, 15, 30 and 60 min. LPS (1 µg/mL) was the positive control. (<b>A</b>) Transcriptional activation of Fos. VCC starts the transcriptional regulation of the subunits of AP-1 Fos and Jun; then, its mRNA expression was studied by qp-PCR. (<b>B</b>) Transcriptional activation of Jun. VCC starts the transcriptional regulation of subunits of Jun, and its mRNA expression was evaluated using qp-PCR. Changes in transcriptional expression were established in comparison with the untreated control and then normalized with the housekeeping gene NAPDH. The graphic includes results from three performed experiments. W/T, without treatment. Mean ± SD. * <span class="html-italic">p</span> &lt; 0.001, significantly different from the group without treatment (ANOVA plus the Student–Newman–Keuls method).</p>
Full article ">Figure 6
<p>The VCC initiates the expression of the transcription factor NF-κB. THP-1 cells were treated with the VCC toxin at 10, 15, 30 and 60 min. LPS (1 µg/mL) was used as a positive control. The VCC starts the transcriptional regulation of subunits of NF-κB, p50. The mRNA expressions were evaluated using qp-PCR. Changes in expression were compared with the control without treatment and normalized with the housekeeping gene NAPDH. The graphic includes results from three performed experiments. W/T, without treatment. Mean ± SD. ** <span class="html-italic">p</span> &lt; 0.05, significantly different from that without treatment (ANOVA plus the Student–Newman–Keuls method).</p>
Full article ">Figure 7
<p>The VCC initiates the expression of the transcription factor NF-κB. (<b>A</b>) THP-1 cells were exposed to VCC (0, 10, 15, 30, 60 min and 24 h); the LDH activity was detected and determined from the collected conditioned media. Data are the mean ± SD from three independent experiments. W/T, without treatment. * <span class="html-italic">p</span> &lt; 0.001 (ANOVA plus the Student–Newman–Keuls method). (<b>B</b>) Expression of Caspase-1 protein with different treatments (LPS and PMA, LPS, VCC, VCC + ATP) at 6 h was measured by a western blot assay. The β-actin protein level is shown as the internal control.</p>
Full article ">Figure 8
<p>Model mechanism proposed for the monomeric VCC starting the innate immune response. The monomeric form of the VCC behaves as a PAMP; it binds its membrane receptor (TLR 4), turning on a signaling cascade activating MAPKs (ERK, p38 and perhaps JNK). The MAPKs then start the activation of the transcription factors AP-1 and NF-κB, starting the pro-inflammatory and innate response involved with the synthesis of the classic IL-1β.</p>
Full article ">Figure 9
<p>Proposed time lapse of activated MAPKs and their transcriptional activators. Phosphorylated MAPK proteins and activated transcripts of AP-1 (Fos, Jun) and NF-κB elicited by treatments with the monomeric VCC on THP-1-treated cells.</p>
Full article ">
18 pages, 3961 KiB  
Article
The Potassium Channel Blocker β-Bungarotoxin from the Krait Bungarus multicinctus Venom Manifests Antiprotozoal Activity
by Alexey V. Osipov, Elena G. Cheremnykh, Rustam H. Ziganshin, Vladislav G. Starkov, Trang Thuy Thi Nguyen, Khoa Cuu Nguyen, Dung Tien Le, Anh Ngoc Hoang, Victor I. Tsetlin and Yuri N. Utkin
Biomedicines 2023, 11(4), 1115; https://doi.org/10.3390/biomedicines11041115 - 7 Apr 2023
Cited by 2 | Viewed by 1964
Abstract
Protozoal infections are a world-wide problem. The toxicity and somewhat low effectiveness of the existing drugs require the search for new ways of protozoa suppression. Snake venom contains structurally diverse components manifesting antiprotozoal activity; for example, those in cobra venom are cytotoxins. In [...] Read more.
Protozoal infections are a world-wide problem. The toxicity and somewhat low effectiveness of the existing drugs require the search for new ways of protozoa suppression. Snake venom contains structurally diverse components manifesting antiprotozoal activity; for example, those in cobra venom are cytotoxins. In this work, we aimed to characterize a novel antiprotozoal component(s) in the Bungarus multicinctus krait venom using the ciliate Tetrahymena pyriformis as a model organism. To determine the toxicity of the substances under study, surviving ciliates were registered automatically by an original BioLaT-3.2 instrument. The krait venom was separated by three-step liquid chromatography and the toxicity of the obtained fractions against T. pyriformis was analyzed. As a result, 21 kDa protein toxic to Tetrahymena was isolated and its amino acid sequence was determined by MALDI TOF MS and high-resolution mass spectrometry. It was found that antiprotozoal activity was manifested by β-bungarotoxin (β-Bgt) differing from the known toxins by two amino acid residues. Inactivation of β-Bgt phospholipolytic activity with p-bromophenacyl bromide did not change its antiprotozoal activity. Thus, this is the first demonstration of the antiprotozoal activity of β-Bgt, which is shown to be independent of its phospholipolytic activity. Full article
(This article belongs to the Special Issue Editorial Board Members’ Collection Series in Drug Discovery)
Show Figures

Figure 1

Figure 1
<p>Separation of <span class="html-italic">B. multicinctus</span> venom. (<b>a</b>) Separation of the crude venom by gel-filtration chromatography on Superdex 75 column. (<b>b</b>) Separation of active fraction IV using ion-exchange chromatography on HEMA 1000 CM column. The asterisk indicates the active fraction. (<b>c</b>) Fraction 18 was separated into individual components by RP-HPLC using Jupiter C18 column.</p>
Full article ">Figure 2
<p>Images of <span class="html-italic">T. pyriformis</span> cells. The alive (<b>a</b>) and dead (<b>b</b>) cells are shown. The images were obtained using MBS-10 microscope at a magnification of 56.</p>
Full article ">Figure 3
<p>Toxicity to <span class="html-italic">Tetrahymena</span> of the fractions obtained by gel-filtration to <span class="html-italic">Tetrahymena</span>. (<b>a</b>) Activity of fractions I-VI at concentration of 0.1 mg/mL. (<b>b</b>) Activity of fractions VII-XII at concentrations of 2 and 20 mg/mL.</p>
Full article ">Figure 4
<p>Toxicity to <span class="html-italic">Tetrahymena</span> of the fractions obtained by ion-exchange chromatography. (<b>a</b>) Activity determination in short-time experiment. (<b>b</b>) Activity of fractions at 24 h experiment. Survival rate is the ratio of the number of live ciliates after 24 h of incubation to the number of ciliates at the beginning of the experiment. Three independent measurements were performed for each fraction. For all fractions, the coefficient of variation (CV) did not exceed 0.07, or 7% of the mean.</p>
Full article ">Figure 5
<p>The toxicity of fraction IV-18-2 to <span class="html-italic">Tetrahymena</span>. (<b>a</b>) Short-time experiment. (<b>b</b>) 24 h experiment. Survival rate is the ratio of the number of live ciliates after 24 h of incubation to the number of ciliates at the beginning of the experiment. Three independent measurements were performed for each concentration. For all concentrations, the coefficient of variation (CV) did not exceed 0.07, or 7% of the mean.</p>
Full article ">Figure 6
<p>High resolution mass spectrum of compound IV-18-2. z = 11.</p>
Full article ">Figure 7
<p>Peptide mass fingerprinting for the protein of fraction IV-18-2. The peptides found in tryptic (<b>a</b>) and Glu-C (<b>b</b>) digests and matching the shown amino acid sequence are indicated by blue lines. C on the red background shows carbamidomethylated cysteine residues, d on the yellow background shows aspartic acid produced by deamidation of asparagine and o on the blue background—methionine sulfoxide. The amino acid sequence is for the A-chain (gi|6523113) precursors of β-Bgt.</p>
Full article ">Figure 8
<p>Peptide mass fingerprinting for the protein of fraction IV-18-2. The peptides found in tryptic (<b>a</b>) and Glu-C (<b>b</b>) digests and matching the shown amino acid sequence are indicated by blue lines. C on the red background shows carbamidomethylated cysteine residues and d on the yellow background shows aspartic acid produced by deamidation of asparagine. The amino acid sequence is for the B-chain (gi|82207097) precursors of β-Bgt, in which Arg45 was replaced by Glu and Asn67 by Asp.</p>
Full article ">Figure 9
<p>Toxicity to the <span class="html-italic">Tetrahymena</span> of fraction IV-18-2 (β-Bgt) and modified toxin (β-Bgt-M) in 24 h experiment. Survival rate is the ratio of the number of live ciliates after 24 h of incubation to the number of ciliates at the beginning of the experiment. Three independent measurements were performed for each concentration.</p>
Full article ">
17 pages, 1734 KiB  
Article
Characterization and Genetic Diversity of Bacillus cereus Strains Isolated from Baby Wipes
by Laurenda Carter, Mei-Chiung J. Huang, Kyuyoung Han, Jayanthi Gangiredla, Jenny Yee, Hannah R. Chase, Flavia Negrete and Ben D. Tall
Microorganisms 2022, 10(9), 1779; https://doi.org/10.3390/microorganisms10091779 - 3 Sep 2022
Cited by 3 | Viewed by 2246
Abstract
Bacillus cereus, a ubiquitous environmental microorganism known to cause foodborne illness, was isolated from samples taken from imported baby wipes from two different countries. These strains were characterized using a comprehensive molecular approach involving endpoint PCR, whole genome sequencing (WGS), comparative genomics, [...] Read more.
Bacillus cereus, a ubiquitous environmental microorganism known to cause foodborne illness, was isolated from samples taken from imported baby wipes from two different countries. These strains were characterized using a comprehensive molecular approach involving endpoint PCR, whole genome sequencing (WGS), comparative genomics, and biochemical analyses. A multiplex endpoint PCR assay was used to identify the enterotoxins: hemolysin BL, nonhemolytic enterotoxin, cytotoxin K, and enterotoxin FM toxin genes. Phylogenetically, the strains clustered into two major groups according to sequence type (ST) and singleton. We used the Center for Food Safety and Applied Nutrition (CFSAN) GalaxyTrakr BTyper computational tool to characterize the strains further. As an additional means of characterization, we investigated the possible role of carbohydrate transport systems and their role in nutrient uptake by performing a BLAST analysis of the 40 B. cereus genomes recovered from baby wipes. This study outlines a multifaceted workflow that uses the analysis of enterotoxigenic potential, bioinformatics, genomic diversity, genotype, phenotype, and carbohydrate utilization as a comprehensive strategy to characterize these B. cereus strains isolated from baby wipes and further our understanding of the phylogenetic relatedness of strains associated with baby wipe production facilities that could potentially pose an infection risk to a vulnerable infant population. Full article
(This article belongs to the Special Issue An Update on Bacillus)
Show Figures

Figure 1

Figure 1
<p>Phylogenetic and evolutionary history among 55 <span class="html-italic">B. cereus</span> strains isolated from baby wipes and other cosmetics products in comparison with NCBI reference genomes was inferred using the Neighbor-Joining method [<a href="#B15-microorganisms-10-01779" class="html-bibr">15</a>]. The optimal tree with the sum of branch length = 1.41269531 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method [<a href="#B16-microorganisms-10-01779" class="html-bibr">16</a>] and are in the units of the number of base differences per sequence. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 431,768 positions in the final dataset. Evolutionary analyses were conducted in MEGA X 10.0 [<a href="#B17-microorganisms-10-01779" class="html-bibr">17</a>]. The scale bar represents a 0.050 base substitution per site. The baby wipe <span class="html-italic">B. cereus</span> strains clustered into two distinct phylogenetically related groups according to ST. For example, the ST266 strains clustered with ST266 reference strain BcQ1 and the ST1295 strains clustered separately in a related but distinct clade with the ST90 strains, which grouped with the ST90 reference strain Bc FRI-35. The baby wipe and cosmetic product isolates are identified by red circles. The green circles represent the closest Bc reference strains.</p>
Full article ">Figure 2
<p>The heatmap represents the presence/absence of <span class="html-italic">B. cereus</span> virulence, antimicrobial resistance genes (AMR), <span class="html-italic">panC</span> phylogenic typing genes, and ST designations identified in baby wipe strains using BTyper. All the baby wipes strains possessed <span class="html-italic">nheBAC</span>, <span class="html-italic">entAFM</span>, <span class="html-italic">bpsEFH</span>, <span class="html-italic">cerAB</span>, <span class="html-italic">clo</span>, <span class="html-italic">plcABR</span>, and <span class="html-italic">inhA1A2</span>. ST1295 strains possessed the additional virulence factor, <span class="html-italic">cytK2</span>. The lone ST2103 strain carried <span class="html-italic">cytK2</span>, <span class="html-italic">hblDAC</span>, <span class="html-italic">bceT,</span> and <span class="html-italic">hblB</span> as additional virulence factors. There were seven AMR genes which included (GLY) <span class="html-italic">VanY-Pt2</span>, (GLY) <span class="html-italic">VanYF-Pp</span>, (MLS)<span class="html-italic">LsaB</span>, (GLY) <span class="html-italic">VanS-Pt2,</span> and (GLY) <span class="html-italic">VanR-Pt</span>, along with (GLY) <span class="html-italic">VanR-M</span> and (Bla) <span class="html-italic">BLA-1</span> found in all the strains. All strains except for Bc57 ST2103 were identified as <span class="html-italic">panC</span>-positive. Bc57 ST2013 was identified as a <span class="html-italic">panD</span>-positive strain. The presence of the gene is indicated in blue, and the absence is indicated in gray. The measured parameters were consistent with the ST of the strains.</p>
Full article ">Figure 3
<p>Comparison of carbohydrate utilization capabilities of the 40 <span class="html-italic">B. cereus</span> isolates from baby wipes.</p>
Full article ">Figure 4
<p>The presence/absence of genes related to carbohydrate utilization shows the closest agreement to ST found in all the strains showing differences in gene composition. P = presence of a gene; A = absence of a gene; Bc14579 is <span class="html-italic">B. cereus</span> ATCC 14579; ST57 is highlighted in light blue, ST1295 is highlighted in gray, ST266 is highlighted in yellow.</p>
Full article ">
12 pages, 313 KiB  
Article
Toxigenic Genes, Pathogenic Potential and Antimicrobial Resistance of Bacillus cereus Group Isolated from Ice Cream and Characterized by Whole Genome Sequencing
by Rosa Fraccalvieri, Angelica Bianco, Laura Maria Difato, Loredana Capozzi, Laura Del Sambro, Domenico Simone, Roberta Catanzariti, Marta Caruso, Domenico Galante, Giovanni Normanno, Lucia Palazzo, Maria Tempesta and Antonio Parisi
Foods 2022, 11(16), 2480; https://doi.org/10.3390/foods11162480 - 17 Aug 2022
Cited by 13 | Viewed by 2312
Abstract
Bacillus cereus is isolated from a variety of foods where it may cause food spoilage and/or food poisoning due to its toxigenic and pathogenic nature. In this study, we identified members of B. cereus groups in 65% of the ice cream samples analyzed, [...] Read more.
Bacillus cereus is isolated from a variety of foods where it may cause food spoilage and/or food poisoning due to its toxigenic and pathogenic nature. In this study, we identified members of B. cereus groups in 65% of the ice cream samples analyzed, which were characterized based on multi locus variable number tandem repeats analysis (MLVA) and whole genome sequencing (WGS). The MLVA revealed that 36 strains showed different allelic profiles. Analyses of WGS data enabled the identification of three members of the B. cereus group: B. cereus sensu stricto, B. mosaicus and B. thuringiensis. Based on the multi locus sequence typing (MLST) scheme, the strains were classified in 27 sequence types (STs), including ST26 that causes food poisoning. Toxin genes’ detection revealed the presence of the genes encoding nonhemolytic enterotoxin (NHE), hemolysin BL (HBL), cytotoxin K (cytK) and cereulide (ces) in 100%, 44%, 42% and 8% of the strains, respectively. The identification of eleven antimicrobial resistance (AMR) genes predicted the resistance to five different antimicrobials, and the resistance to beta-lactam antibiotics was confirmed with a phenotypic antimicrobial test. Taken together, the results showed that the B. cereus strains isolated from ice cream were a potential hazard for consumer safety. Full article
(This article belongs to the Section Food Microbiology)
24 pages, 3269 KiB  
Article
Strains Associated with Two 2020 Welder Anthrax Cases in the United States Belong to Separate Lineages within Bacillus cereus sensu lato
by Laura M. Carroll, Chung K. Marston, Cari B. Kolton, Christopher A. Gulvik, Jay E. Gee, Zachary P. Weiner and Jasna Kovac
Pathogens 2022, 11(8), 856; https://doi.org/10.3390/pathogens11080856 - 29 Jul 2022
Cited by 3 | Viewed by 4973
Abstract
Anthrax-causing members of Bacillus cereus sensu lato (s.l.) pose a serious threat to public health. While most anthrax-causing strains resemble B. anthracis phenotypically, rare cases of anthrax-like illness caused by strains resembling “B. cereus” have been reported. Here, whole-genome [...] Read more.
Anthrax-causing members of Bacillus cereus sensu lato (s.l.) pose a serious threat to public health. While most anthrax-causing strains resemble B. anthracis phenotypically, rare cases of anthrax-like illness caused by strains resembling “B. cereus” have been reported. Here, whole-genome sequencing was used to characterize three B. cereus s.l. isolates associated with two 2020 welder anthrax cases in the United States, which resembled “B. cereus” phenotypically. Comparison of the three genomes sequenced here to all publicly available, high-quality B. cereus s.l. genomes (n = 2890 total genomes) demonstrated that genomes associated with each case effectively belonged to separate species at the conventional 95% average nucleotide identity prokaryotic species threshold. Two PubMLST sequence type 78 (ST78) genomes affiliated with a case in Louisiana were most closely related to B. tropicus and possessed genes encoding the Bps exopolysaccharide capsule, as well as hemolysin BL (Hbl) and cytotoxin K (CytK). Comparatively, a ST108 genome associated with a case in Texas was most closely related to B. anthracis; however, like other anthrax-causing strains most closely related to B. anthracis, this genome did not possess Bps-, Hbl-, or CytK-encoding genes. Overall, results presented here provide insights into the evolution of anthrax-causing B. cereus s.l. Full article
(This article belongs to the Special Issue Anthrax—a Threat beyond Bacillus anthracis)
Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Maximum likelihood phylogeny constructed using core genes detected among 607 genomes assigned to the Genome Taxonomy Database (GTDB) <span class="html-italic">Bacillus</span> (<span class="html-italic">B.</span>) <span class="html-italic">anthracis</span>, <span class="html-italic">B. paranthracis</span>, and <span class="html-italic">B. tropicus</span> species, plus outgroup genome <span class="html-italic">B. cereus sensu lato</span> (<span class="html-italic">s.l.</span>) strain FSL W8-0169 (National Center for Biotechnology Information [NCBI] RefSeq Assembly Accession GCF_001583695.1; omitted for readability). Branch colors and clade labels denote GTDB species assignments or, for <span class="html-italic">B. anthracis</span> and Group III “<span class="html-italic">B. cereus</span>”, historical species assignments (per the United States Food and Drug Administration’s Bacteriological Analytical Manual [FDA BAM]). The heatmap to the right of the phylogeny denotes the following (from left to right): (i) whether a genome was sequenced in this study or publicly available (“Origin”); (ii) selected PubMLST lineages assigned using seven-gene multi-locus sequence typing, to which the three genomes sequenced in this study were assigned (“MLST”); (iii) whether a genome possessed two or more anthrax toxin-encoding genes (<span class="html-italic">cya, lef, pagA</span>) or not (“Anthrax”); (iv) whether a genome possessed three or more cereulide synthetase (emetic toxin)-encoding genes (<span class="html-italic">cesABCD</span>) or not (“Emetic”); (v) whether a genome possessed four or more polyglutamate capsule-encoding genes (<span class="html-italic">capBCADE</span>) or not (“Cap”); (vi) whether a genome possessed six or more Bps exopolysaccharide-encoding genes (<span class="html-italic">bpsXABCDEFGH</span>) or not (“Bps”); (vii) whether a genome possessed two or more hyaluronic acid capsule-encoding genes <span class="html-italic">(hasABC)</span> or not (“Has”); (viii) whether a genome possessed three or more hemolysin BL diarrheal enterotoxin-encoding genes (<span class="html-italic">hblABCD</span>) or not (“Hbl”); (ix) whether a genome possessed cytotoxin K-encoding <span class="html-italic">cytK-2</span> or not (“CytK-2”); (x) the <span class="html-italic">panC</span> Group to which each genome was assigned (using BTyper3 and an eight-group scheme; “<span class="html-italic">panC</span>”). The phylogeny was rooted along the outgroup genome (omitted for readability), with branch lengths reported in substitutions per site.</p>
Full article ">Figure 2
<p>Maximum likelihood phylogeny constructed using core genes detected among 55 genomes assigned to the Genome Taxonomy Database (GTDB) <span class="html-italic">B. tropicus</span> species, plus GTDB <span class="html-italic">B. paranthracis</span> outgroup genome <span class="html-italic">B. cereus s.l.</span> strain AH187 (NCBI RefSeq Assembly Accession GCF_000021225.1; omitted for readability). A predicted anthrax toxin gene gain event among PubMLST Sequence Type 78 (ST78) genomes is denoted by a black arrow. The heatmap to the right of the phylogeny denotes the following (from left to right): (i) whether a genome was sequenced in this study or publicly available (“Origin”); (ii) selected PubMLST lineages assigned using seven-gene multi-locus sequence typing, to which genomes sequenced in this study were assigned (“MLST”); (iii) whether a genome possessed two or more anthrax toxin-encoding genes (<span class="html-italic">cya, lef, pagA</span>) or not (“Anthrax”); (iv) whether a genome possessed four or more polyglutamate capsule-encoding genes (<span class="html-italic">capBCADE</span>) or not (“Cap”); (v) whether a genome possessed six or more Bps exopolysaccharide-encoding genes (<span class="html-italic">bpsXABCDEFGH</span>) or not (“Bps”); (vi) whether a genome possessed two or more hyaluronic acid capsule-encoding genes <span class="html-italic">(hasABC)</span> or not (“Has”). The phylogeny was rooted along the outgroup genome (omitted for readability), with branch lengths reported in substitutions per site. For complete ancestral state reconstruction results, see <a href="#app1-pathogens-11-00856" class="html-app">Supplementary Figure S3</a>.</p>
Full article ">Figure 3
<p>Geographic origins of anthrax toxin gene-harboring <span class="html-italic">B. cereus s.l.</span> genomes, which do not belong to the clonal, historical <span class="html-italic">B. anthracis</span> lineage. Regions are colored by the associated number of anthrax toxin gene-harboring genomes assigned to (<b>A</b>) sequence type 78 (ST78) or (<b>B</b>) the ST365 clonal complex (CC) within PubMLST. For each of (<b>A</b>) ST78 and (<b>B</b>) the ST365 CC, one anthrax toxin gene-harboring genome with an unknown origin was excluded from the map.</p>
Full article ">Figure 4
<p>Maximum likelihood phylogeny constructed using core genes detected among 326 genomes assigned to the Genome Taxonomy Database (GTDB) <span class="html-italic">B. anthracis</span> species, plus GTDB <span class="html-italic">B. paranthracis</span> outgroup genome <span class="html-italic">B. cereus s.l.</span> strain AH187 (NCBI RefSeq Assembly Accession GCF_000021225.1; omitted for readability). Predicted anthrax toxin gene gain events among PubMLST Sequence Type 365 Clonal Complex (ST365 CC) genomes are denoted by black arrows. Branch colors and clade labels differentiate genomes that are members of the clonal, historical <span class="html-italic">B. anthracis</span> lineage (darker pink) from genomes that are not (lighter pink). The heatmap to the right of the phylogeny denotes the following (from left to right): (i) whether a genome was sequenced in this study or publicly available (“Origin”); (ii) selected PubMLST lineages assigned using seven-gene multi-locus sequence typing, to which the genomes sequenced in this study were assigned (“MLST”); (iii) whether a genome possessed two or more anthrax toxin-encoding genes (<span class="html-italic">cya, lef, pagA</span>) or not (“Anthrax”); (iv) whether a genome possessed four or more polyglutamate capsule-encoding genes (<span class="html-italic">capBCADE</span>) or not (“Cap”); (v) whether a genome possessed two or more hyaluronic acid capsule-encoding genes <span class="html-italic">(hasABC)</span> or not (“Has”); (vi) the <span class="html-italic">panC</span> Group to which each genome was assigned (using BTyper3 and an eight-group scheme; “<span class="html-italic">panC</span>”). The phylogeny was rooted along the outgroup genome (omitted for readability), with branch lengths reported in substitutions per site. For complete ancestral state reconstruction results, see <a href="#app1-pathogens-11-00856" class="html-app">Supplementary Figure S4</a>.</p>
Full article ">Figure 5
<p>Inferred parameters for the Finitely Many Genes (FMG) model among genomes assigned to the following taxonomic units: (i) the <span class="html-italic">B. mosaicus</span> genomospecies within the 2020 <span class="html-italic">B. cereus s.l.</span> Genomospecies-Subspecies-Biovar (GSB) framework (<span class="html-italic">n</span> = 664 genomes)<span class="html-italic">;</span> (ii) the clonal, historical <span class="html-italic">B. anthracis</span> lineage most commonly associated with anthrax toxin production (also known as <span class="html-italic">B. mosaicus</span> subsp. <span class="html-italic">anthracis</span> within the 2020 GSB framework; <span class="html-italic">n</span> = 119 genomes); (iii) the Genome Taxonomy Database (GTDB) <span class="html-italic">B. anthracis</span> species (<span class="html-italic">n</span> = 180 genomes)<span class="html-italic">;</span> (iv) GTDB’s <span class="html-italic">B. anthracis</span>, <span class="html-italic">B. paranthracis</span>, and <span class="html-italic">B. tropicus</span> species (<span class="html-italic">n</span> = 415 genomes); (v) GTDB’s <span class="html-italic">B. tropicus</span> species (<span class="html-italic">n</span> = 46 genomes). FMG parameters were estimated using Panaroo, with gray bars denoting the 2.5 and 97.5% confidence interval bounds for each parameter (obtained using 100 bootstrap replicates).</p>
Full article ">Figure 6
<p>Results of non-metric multidimensional scaling (NMDS) performed using the presence and absence of pan-genome orthologous gene clusters detected among anthrax toxin gene-harboring members of (i) the clonal, historical <span class="html-italic">B. anthracis</span> lineage typically associated with anthrax toxin production (also known as <span class="html-italic">B. mosaicus</span> subsp. <span class="html-italic">anthracis</span> within the 2020 Genomospecies-Subspecies-Biovar [GSB] framework); (ii) the PubMLST ST365 Clonal Complex (CC;, i.e., anthrax-causing “<span class="html-italic">B. cereus</span>”-like genomes, which are most closely related to the <span class="html-italic">B. anthracis</span> species type strain genome but are not members of the 2020 GSB <span class="html-italic">B. anthracis</span> subspecies); (iii) PubMLST ST78 (i.e., anthrax-causing “<span class="html-italic">B. cereus</span>”-like genomes, which are most closely related to the <span class="html-italic">B. tropicus</span> species type strain genome). Points represent genomes, while shaded regions and convex hulls correspond to the anthrax toxin gene-harboring lineage to which each genome belongs. Lineages differed significantly based on pan-genome orthologous gene cluster presence/absence (Bonferroni-corrected ANOSIM and PERMANOVA <span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">
11 pages, 2913 KiB  
Article
Dichloroacetyl Amides of 3,5-Bis(benzylidene)-4-piperidones Displaying Greater Toxicity to Neoplasms than to Non-Malignant Cells
by Mohammad Hossain, Praveen K. Roayapalley, Hiroshi Sakagami, Keitaro Satoh, Kenjiro Bandow, Umashankar Das and Jonathan R. Dimmock
Medicines 2022, 9(6), 35; https://doi.org/10.3390/medicines9060035 - 8 Jun 2022
Viewed by 2398
Abstract
A series of 3,5-bis(benzylidene)-1-dichloroacetyl-4-piperidones 1al was evaluated against Ca9-22, HSC-2, HSC-3, and HSC-4 squamous cell carcinomas. Virtually all of the compounds displayed potent cytotoxicity, with 83% of the CC50 values being submicromolar and several CC50 values being in the [...] Read more.
A series of 3,5-bis(benzylidene)-1-dichloroacetyl-4-piperidones 1al was evaluated against Ca9-22, HSC-2, HSC-3, and HSC-4 squamous cell carcinomas. Virtually all of the compounds displayed potent cytotoxicity, with 83% of the CC50 values being submicromolar and several CC50 values being in the double digit nanomolar range. The compounds were appreciably less toxic to human HGF, HPLF, and HPC non-malignant cells, which led to some noteworthy selectivity index (SI) figures. From these studies, 1d,g,k emerged as the lead molecules in terms of their potencies and SI values. A Quantitative Structure-Activity Relationship (QSAR) study revealed that cytotoxic potencies and potency–selectivity expression figures increased when the magnitude of the sigma values in the aryl rings was elevated. The modes of action of the representative cytotoxins in Ca9-22 cells were found to include G2/M arrest and stimulation of the cells to undergo mitosis and cause poly(ADP-ribose) polymerase (PARP) and procaspase 3 cleavage. Full article
Show Figures

Figure 1

Figure 1
<p>The structures of the compounds in series <b>1</b>, <b>2</b> and dichloroacetic acid (DCA). The aryl substituents in series <b>1</b> are indicated in <a href="#medicines-09-00035-t001" class="html-table">Table 1</a>.</p>
Full article ">Figure 2
<p>Kinetics of cytotoxicity induction by <b>1d</b>, <b>1g,</b> and <b>1k</b> on human oral squamous cell lines (Ca9-22, HSC-2, HSC-3, and HSC-4) and human normal oral cells (HGF, HPLF, and HPC). These cells were incubated for 48 h with the indicated concentrations of <b>1d</b>, <b>1g,</b> and <b>1k</b>, and the viable cell number was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] methods. Each value represents the mean ± S.D. (standard deviation) of triplicate assays.</p>
Full article ">Figure 3
<p>The effect of <b>1d</b>,<b>e</b>,<b>k</b> on the cell cycle in Ca9-22 cells. Cells were treated without (control) or with 1 μM actinomycin D (AD) or the indicated concentrations of <b>1d</b>, <b>1e,</b> and <b>1k</b> in the presence of the DMSO vehicle (0.1%). Experimental data are presented as the mean ± standard deviation (SD) of triplicate determinations. The significance of values was examined by one-way analysis of variance (ANOVA) and appropriate Dunnett’s post-test. A value of * <span class="html-italic">p</span> &lt; 0.05 was considered to indicate statistically significant differences. The red arrows indicate the highest value of G2/M phase cells in <b>1d</b>, <b>1e</b> and <b>1k</b>.</p>
Full article ">Figure 4
<p>Increased mitosis in Ca9-22 cells after treatment with <b>1k</b>, <b>2a,</b> and actinomycin D(AD). Cells were treated without (control) or with 1 μM actinomycin D or the indicated concentrations of <b>1k</b> and <b>2a</b> in the presence of the DMSO vehicle (0.1%).</p>
Full article ">Figure 5
<p>Cleavage of PARP and procaspase 3 in Ca9-22 cells by <b>1k</b>, <b>2a,</b> and actinomycin D(AD). Cells were treated without (control) or with 1 μM actinomycin D or the indicated concentrations of <b>1k</b> and <b>2a</b> in the presence of the DMSO vehicle (0.1%).</p>
Full article ">Figure 6
<p>The structures of three lead molecules <b>1d</b> (R<sup>1</sup> = H, R<sup>2</sup> = Cl), <b>1g</b> (R<sup>1</sup> = R<sup>2</sup> = OCH<sub>3</sub>), and <b>1k</b> (R<sup>1</sup> = H, R<sup>2</sup> = NO<sub>2</sub>).</p>
Full article ">
17 pages, 2729 KiB  
Article
Accumulation of Deleterious Effects in Gastric Epithelial Cells and Vascular Endothelial Cells In Vitro in the Milieu of Helicobacter pylori Components, 7-Ketocholesterol and Acetylsalicylic Acid
by Adrian Ł. Gajewski, Mateusz Gawrysiak, Agnieszka Krupa, Tomasz Rechciński, Maciej Chałubiński, Weronika Gonciarz and Magdalena Chmiela
Int. J. Mol. Sci. 2022, 23(11), 6355; https://doi.org/10.3390/ijms23116355 - 6 Jun 2022
Cited by 8 | Viewed by 2964
Abstract
The Gastric pathogen Helicobacter pylori (HP) may influence the development of coronary heart disease (CHD). H. pylori induce reactive oxygen species (ROS), which transform cholesterol to 7-ketocholesterol (7-kCh), a CHD risk factor. Acetylsalicylic acid (ASA)—an Anti-aggregation drug used in CHD patients—may [...] Read more.
The Gastric pathogen Helicobacter pylori (HP) may influence the development of coronary heart disease (CHD). H. pylori induce reactive oxygen species (ROS), which transform cholesterol to 7-ketocholesterol (7-kCh), a CHD risk factor. Acetylsalicylic acid (ASA)—an Anti-aggregation drug used in CHD patients—may increase gastric bleeding and inflammation. We examined whether H. pylori driven ROS effects in the cell cultures of gastric epithelial cells (AGS) and vascular endothelial cells (HUVEC) progress in the milieu of 7-kCh and ASA. Cell cultures, exposed to 7-kCh or ASA alone or pulsed with the H. pylori antigenic complex—Glycine acid extract (GE), urease (UreA), cytotoxin associated gene A (CagA) protein or lipopolysaccharide (LPS), alone or with 7-kCh and ASA—were examined for ROS, apoptosis, cell integrity, interleukin (IL)-8, the activation of signal transducer, the activator of transcription 3 (STAT3), and wound healing. ASA and 7-kCh alone, and particularly in conjunction with H. pylori components, increased the ROS level and the rate of apoptosis, which was followed by cell disintegration, the activation of STAT3, and IL-8 elevation. AGS cells were unable to undergo wound healing. The cell ROS response to H. pylori components may be elevated by 7-kCh and ASA. Full article
(This article belongs to the Special Issue Helicobacter: Infection, Diagnosis and Treatment 2.0)
Show Figures

Figure 1

Figure 1
<p>Reactive oxygen species. For the estimation of reactive oxygen species (ROS), the cell suspensions of gastric epithelial AGS cells (<b>A</b>) and vascular endothelial HUVEC cells (<b>B</b>) were treated for 24 h with <span class="html-italic">H. pylori</span> compounds: glycine acid extract—GE, subunit A of urease—UreA, cytotoxin associated gene A (CagA) protein, <span class="html-italic">H. pylori</span> lipopolysaccharide (LPS) or <span class="html-italic">E. coli</span> LPS, alone or in combination with acetylsalicylic acid—ASA, and 7-ketocholesterol—7-kCh, or in medium alone. The fluorescent probe—Dihydroetidine (DHE) was added to the wells, and fluorescence was measured. The ROS ratio was calculated based on relative fluorescence units (RFU) of stimulated cells vs. RFU of control cells in culture medium alone. Results are shown as means with standard deviations (SD) of five experiments performed in triplicates for each experimental variant. Statistical significance for * <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 2
<p>Assessment of cell viability. Gastric epithelial AGS cells (<b>A</b>) or vascular endothelial HUVEC cells (<b>B</b>) were sub-cultured for 24 h in the culture medium alone or exposed to <span class="html-italic">H. pylori</span> components: glycine acid extract—GE, subunit A of urease—UreA, cytotoxin associated gene A (CagA) protein, <span class="html-italic">H. pylori</span> lipopolysaccharide (LPS) or <span class="html-italic">E. coli</span> LPS alone or with acetylsalicylic acid—ASA, and 7-ketocholesterol—7-kCh. Cell viability was evaluated using the tetrazolium yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which was reduced by living cells to yield soluble purple formazan crystals that were solubilized and detected colorimetrically. Results are presented as the percentage means ± standard deviation (SD) relative to untreated cells of at least four independent experiments performed in triplicates for each experimental variant. Statistical significance for * <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 3
<p>Assessment of cell apoptosis. Gastric epithelial AGS cells (<b>A</b>,<b>B</b>) or vascular endothelial HUVEC cells (<b>C</b>,<b>D</b>)) were sub-cultured in the culture medium alone or in the milieu of <span class="html-italic">H. pylori</span> components: glycine acid extract—GE, subunit A of urease—UreA, cytotoxin associated gene A (CagA) protein, <span class="html-italic">H. pylori</span> lipopolysaccharide (LPS) or <span class="html-italic">E. coli</span> LPS alone or with acetylsalicylic acid—ASA and 7-ketocholesterol—7-kCh. The intensity of AGS and HUVEC cell apoptosis was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Cell nuclei were counterstained with Hoechst. Cells with apoptotic changes (red) were imaged in the fluorescence microscope at magnification ×20. (<b>A</b>,<b>C</b>) The graphs indicate the percentage of apoptotic cells. (<b>B</b>,<b>D</b>) Representative images of cells stained in the TUNEL assay (red), at magnification ×20. The results of four independent experiments performed in triplicates for each experimental variant are presented. Statistical significance for * <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 4
<p>Permeability of cell monolayers—Paracellular flux assay. Gastric epithelial AGS cells (<b>A</b>) and vascular endothelial HUVEC cells (<b>B</b>) were cultured in the transwell system until they reached the confluence and then were treated with <span class="html-italic">H. pylori</span> components: glycine acid extract—GE, subunit A of urease—UreA, cytotoxin associated gene A (CagA) protein, <span class="html-italic">H. pylori</span> lipopolysaccharide (LPS) or <span class="html-italic">E. coli</span> LPS alone or with acetylsalicylic acid-ASA and 7-ketocholesterol—7-kCh. Thereafter, isothiocyanate fluorescein (FITC) dextran was added to the medium in the insert. The fluorescence of the FITC-dextran in the lower compartment was measured. The fluorescence intensity is shown as relative fluorescence units (RFU) after 120 min incubation of cells with dextran-FITC (<b>A</b>,<b>B</b> upper graphs) or at time 0 and after 30, 60, 90, 120, and 150 min of incubation (<b>C</b>,<b>D</b> lower graphs). Results are shown as means with standard deviation (SD) of four experiments performed in triplicates for each experimental variant. Statistical significance for * <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 5
<p>Effects of ASA and/or 7-kCh on gastric epithelial cells (AGS). AGS cells were treated for 24 h with acetylsalicylic acid (ASA) and/or 7-ketocholesterol (7-kCh), and then examined for: (<b>A</b>) reactive oxygen species (ROS); (<b>B</b>) cell viability; (<b>C</b>) permeability of cell monolayers for fluorescein isothiocyanate (FITC) bound dextran, expressed as relative fluorescence units (RFU); (<b>D</b>) the production of interleukin (IL)-8; (<b>E</b>) phosphorylation of signal transducer and activator of transcription 3 (STAT3). Progress of wound healing (<b>F</b>,<b>G</b>), was assessed in scratch assay. Results are shown as means with standard deviation (SD) of four experiments performed in triplicates for each experimental variant. Statistical significance for * <span class="html-italic">p</span> &lt; 0.005. Cells in complete medium were used as control for natural cells. Cells treated with H<sub>2</sub>O<sub>2</sub> (0.06%) were used as negative control in cell viability assay and paracellular flux assay.</p>
Full article ">Figure 6
<p>Effects of ASA and/or 7-kCh on vascular endothelial cells (HUVEC). HUVEC cells were treated 24 h with acetylsalicylic acid (ASA) and/or 7-ketocholesterol (7-kCh), and then examined for: (<b>A</b>) reactive oxygen species (ROS); (<b>B</b>) cell viability; (<b>C</b>) permeability of cell monolayers for fluorescein isothiocyanate (FITC) bound dextran, expressed as relative fluorescence units (RFU); (<b>D</b>) the production of interleukin (IL)-8; (<b>E</b>) phosphorylation of signal transducer and activator of transcription 3 (STAT3). Results are shown as means with standard deviation (SD) of four experiments performed in triplicates for each experimental variant. Statistical significance for * <span class="html-italic">p</span> &lt; 0.005. RFU—Relative fluorescence units. Cells in complete culture medium (medium) were used as control for natural cells. Cells treated with H<sub>2</sub>O<sub>2</sub> (0.06%) were used as negative control in cell viability assay and paracellular flux assay.</p>
Full article ">
16 pages, 600 KiB  
Article
Detection of Enterotoxigenic Psychrotrophic Presumptive Bacillus cereus and Cereulide Producers in Food Products and Ingredients
by Jelena Jovanovic, Svitlana Tretiak, Katrien Begyn and Andreja Rajkovic
Toxins 2022, 14(4), 289; https://doi.org/10.3390/toxins14040289 - 16 Apr 2022
Cited by 11 | Viewed by 4515
Abstract
In the last decade, foodborne outbreaks and individual cases caused by bacterial toxins showed an increasing trend. The major contributors are enterotoxins and cereulide produced by Bacillus cereus, which can cause a diarrheal and emetic form of the disease, respectively. These diseases [...] Read more.
In the last decade, foodborne outbreaks and individual cases caused by bacterial toxins showed an increasing trend. The major contributors are enterotoxins and cereulide produced by Bacillus cereus, which can cause a diarrheal and emetic form of the disease, respectively. These diseases usually induce relatively mild symptoms; however, fatal cases have been reported. With the aim to detected potential toxin producers that are able to grow at refrigerator temperatures and subsequently produce cereulide, we screened the prevalence of enterotoxin and cereulide toxin gene carriers and the psychrotrophic capacity of presumptive B. cereus obtained from 250 food products (cereal products, including rice and seeds/pulses, dairy-based products, dried vegetables, mixed food, herbs, and spices). Of tested food products, 226/250 (90.4%) contained presumptive B. cereus, which communities were further tested for the presence of nheA, hblA, cytK-1, and ces genes. Food products were mainly contaminated with the nheA B. cereus carriers (77.9%), followed by hblA (64.8%), ces (23.2%), and cytK-1 (4.4%). Toxigenic B. cereus communities were further subjected to refrigerated (4 and 7 °C) and mild abuse temperatures (10 °C). Overall, 77% (94/121), 86% (104/121), and 100% (121/121) were able to grow at 4, 7, and 10 °C, respectively. Enterotoxin and cereulide potential producers were detected in 81% of psychrotrophic presumptive B. cereus. Toxin encoding genes nheA, hblA, and ces gene were found in 77.2, 55, and 11.7% of tested samples, respectively. None of the psychrotrophic presumptive B. cereus were carriers of the cytotoxin K-1 encoding gene (cytK-1). Nearly half of emetic psychrotrophic B. cereus were able to produce cereulide in optimal conditions. At 4 °C none of the examined psychrotrophs produced cereulide. The results of this research highlight the high prevalence of B. cereus and the omnipresence of toxin gene harboring presumptive B. cereus that can grow at refrigerator temperatures, with a focus on cereulide producers. Full article
Show Figures

Figure 1

Figure 1
<p>Frequency Distributions of the Time to Detection (GDT, day) of Enterotoxigenic (<span class="html-italic">n</span> = 76) and Emetic (<span class="html-italic">n</span> = 11) Psychrotrophic Presumptive <span class="html-italic">B. cereus</span>. Blue columns represent the number of enterotoxigenic <span class="html-italic">B. cereus</span>, and the orange columns represent the number of emetic <span class="html-italic">B. cereus</span>.</p>
Full article ">
52 pages, 4486 KiB  
Review
Kingella kingae RtxA Cytotoxin in the Context of Other RTX Toxins
by Katerina Filipi, Waheed Ur Rahman, Adriana Osickova and Radim Osicka
Microorganisms 2022, 10(3), 518; https://doi.org/10.3390/microorganisms10030518 - 27 Feb 2022
Cited by 8 | Viewed by 3323
Abstract
The Gram-negative bacterium Kingella kingae is part of the commensal oropharyngeal flora of young children. As detection methods have improved, K. kingae has been increasingly recognized as an emerging invasive pathogen that frequently causes skeletal system infections, bacteremia, and severe forms of infective [...] Read more.
The Gram-negative bacterium Kingella kingae is part of the commensal oropharyngeal flora of young children. As detection methods have improved, K. kingae has been increasingly recognized as an emerging invasive pathogen that frequently causes skeletal system infections, bacteremia, and severe forms of infective endocarditis. K. kingae secretes an RtxA cytotoxin, which is involved in the development of clinical infection and belongs to an ever-growing family of cytolytic RTX (Repeats in ToXin) toxins secreted by Gram-negative pathogens. All RTX cytolysins share several characteristic structural features: (i) a hydrophobic pore-forming domain in the N-terminal part of the molecule; (ii) an acylated segment where the activation of the inactive protoxin to the toxin occurs by a co-expressed toxin-activating acyltransferase; (iii) a typical calcium-binding RTX domain in the C-terminal portion of the molecule with the characteristic glycine- and aspartate-rich nonapeptide repeats; and (iv) a C-proximal secretion signal recognized by the type I secretion system. RTX toxins, including RtxA from K. kingae, have been shown to act as highly efficient ‘contact weapons’ that penetrate and permeabilize host cell membranes and thus contribute to the pathogenesis of bacterial infections. RtxA was discovered relatively recently and the knowledge of its biological role remains limited. This review describes the structure and function of RtxA in the context of the most studied RTX toxins, the knowledge of which may contribute to a better understanding of the action of RtxA in the pathogenesis of K. kingae infections. Full article
(This article belongs to the Special Issue Kingella kingae: Virulence Factors, Clinical Disease, and Diagnostics)
Show Figures

Figure 1

Figure 1
<p>The structural representation of the RTX toxins RtxA, HlyA, and CyaA. The RtxA, HlyA, and CyaA toxins consist of a pore-forming domain (yellow), an acylated segment with two posttranslationally acylated lysine residues (purple), an RTX domain (blue), and a C-terminal secretion signal (red). Unlike other RTX toxins, CyaA contains a unique adenylate cyclase domain (green).</p>
Full article ">Figure 2
<p>Genetic organization of the <span class="html-italic">rtx</span> loci. The schematic representation of the <span class="html-italic">rtx</span> gene locus of <span class="html-italic">K. kingae</span> strain 269–492 (<b>a</b>), <span class="html-italic">E. coli</span> strain CFT073 (<b>b</b>), and <span class="html-italic">B. pertussis</span> strain 18323 (<b>c</b>). The colored arrows represent coding regions and transcriptional directions of the <span class="html-italic">rtx</span> genes encoding the protoxin proRTXA (yellow), the acyltransferase RTXC (gray), and the proteins of the T1SS apparatus (blue, ABC transporter; orange, MFP; green, OMF).</p>
Full article ">Figure 3
<p>The schematic representation of the <span class="html-italic">rtx</span> gene loci of different <span class="html-italic">K. kingae</span> strains. The <span class="html-italic">rtx</span> loci of <span class="html-italic">K. kingae</span> strain 269–492 (<b>a</b>), <span class="html-italic">K. kingae</span> strains ATCC 23332 and KWG1 (<b>b</b>), <span class="html-italic">K. kingae</span> strains ATCC 23331 and NCTC 10529 (<b>c</b>). The colored arrows represent coding regions and transcriptional directions of the <span class="html-italic">rtx</span> genes encoding the protoxin proRtxA (yellow), the acyltransferase RtxC (light gray, a 167 residue-long variant; dark gray, a 162 residue-long variant), and the proteins of the T1SS apparatus (blue, ABC transporter; orange, MFP; green, OMF).</p>
Full article ">Figure 4
<p>The ClustalW sequence alignment of two RtxC variants of <span class="html-italic">K. kingae</span> strain ATCC 23331. Symbols: (*) identity; (:) strongly similar; (.) weakly similar.</p>
Full article ">Figure 5
<p>The crystal structure of residues 1530–1675 of CyaA. (<b>a</b>) The parallel β-roll structure of block V of the RTX domain and the capping structure of CyaA (PDB ID 6SUS). The blue color represents the nonapeptide repeats of block V, the red color represents the capping structure, and the calcium ions are shown in light orange. (<b>b</b>) A detailed view of the calcium-binding site within block V of the RTX repeats. Each nonapeptide motif forms two half-sites for calcium binding, with each calcium ion bound at a hexa-coordinated site between two consecutive turns. The residues whose side chains directly coordinate the calcium ion are highlighted.</p>
Full article ">Figure 6
<p>Schematic representation of the T1SS assembly process. Recognition of a C-terminal secretion signal of RTX toxin by the proteins ABC and MFP is followed by recruitment of the trimeric TolC complex and formation of the transport channel. The secretion channel spans both membranes of the Gram-negative bacterium and secretes the toxin from the cytosol into the extracellular environment in a one-step mechanism. The energy for secretion is provided by ATP hydrolysis. ABC, ATP-binding cassette transporter; MFP, membrane fusion protein; OMP, outer membrane protein; LPS, lipopolysaccharide; OM, outer membrane; IM, inner membrane; PG, peptidoglycan. This image was created using <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed date: 22 February 2022).</p>
Full article ">Figure 7
<p>Schematic representation of the β<sub>2</sub> integrins. (<b>a</b>) The common CD18 (β<sub>2</sub>) subunit (green) non-covalently associates with one of the four α-subunits, including CD11a (α<sub>L</sub>; orange), CD11b (α<sub>M</sub>; yellow), CD11c (α<sub>X</sub>; gold), and CD11d (α<sub>D</sub>; lavender). (<b>b</b>) The CD11 and CD18 subunits of β<sub>2</sub> integrins consist of a long N-terminal extracellular domain, a single transmembrane α-helical segment, and a short C-terminal cytoplasmic segment. The extracellular domain of the CD11 subunits consists of an N-terminal secretion signal (navy blue), 7 β-sheet repeats (1 to 7; dark blue) that fold into a β-propeller domain, an I-domain (water blue) inserted between repeats 2 and 3 of the β-propeller domain, a thigh domain (sienna), and 2 calf domains (calf-1 in green and calf-2 in gold). The I-domain contains an Mg<sup>2+</sup>-binding site (black star) and repeats 5, 6, and 7 have Ca<sup>2+</sup>-binding EF-hand motifs (red stars). The extracellular domain of the CD18 subunit contains an N-terminal secretion signal (navy blue), an I-like domain (light pink), and a cysteine-rich region with four I-EGF-like domains (1 to 4; dark blue). Extracellular (EC) and intracellular (IC) compartments are separated by the cell membrane (red dots with short black lines).</p>
Full article ">Figure 8
<p>The proposed mechanism of action of <span class="html-italic">K. kingae</span> strain PYKK081 OMVs in human bone. OMVs released by <span class="html-italic">K. kingae</span> are internalized by human osteoblasts and synovial cells. This leads to increased production of two cytokines, GM-CSF and IL-6, which might be involved in the signaling response of infected bone and joint tissues during <span class="html-italic">K. kingae</span> infection. This image was created using <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p>
Full article ">Figure 9
<p>The pore-forming activity of RTX toxins. Although the exact mechanism of oligomerization and pore-forming activity of RTX toxins in vivo is still largely unknown, an approximate mechanism can be given. The RTX toxin interacts with a host receptor and inserts into the membrane in a monomeric form. The monomers interact with each other to form an oligomeric complex. The number of monomers that form an oligomeric structure and the mechanism of formation are still unknown. The oligomeric complex acts as a membrane pore through which calcium ions flow into the cell cytoplasm and potassium ions flow out of the cell. HlyA and LtxA also enable ATP release from host cells directly through the membrane pores formed by the toxins. This image was created using <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p>
Full article ">
14 pages, 2159 KiB  
Article
Cytotoxic Tumour-Selective 1,5-Diaryl-3-Oxo-1,4-Pentadienes Mounted on a Piperidine Ring
by Praveen K. Roayapalley, Hiroshi Sakagami, Keitaro Satoh, Shigeru Amano, Kenjiro Bandow, Renato J. Aguilera, Karla G. Cano Hernandez, Austre Y. Schiaffino Bustamante, Stephen G. Dimmock, Rajendra K. Sharma, Umashankar Das and Jonathan R. Dimmock
Medicines 2021, 8(12), 78; https://doi.org/10.3390/medicines8120078 - 16 Dec 2021
Cited by 2 | Viewed by 2799
Abstract
A series of 3,5-bis(benzylidene)-4-piperidones 2au were prepared as candidate cytotoxic agents. In general, the compounds are highly toxic to human gingival carcinoma (Ca9-22), human squamous carcinoma-2 (HSC-2) and human squamous carcinoma-4 (HSC-4) neoplasms, but less so towards non-malignant human gingival fibroblast [...] Read more.
A series of 3,5-bis(benzylidene)-4-piperidones 2au were prepared as candidate cytotoxic agents. In general, the compounds are highly toxic to human gingival carcinoma (Ca9-22), human squamous carcinoma-2 (HSC-2) and human squamous carcinoma-4 (HSC-4) neoplasms, but less so towards non-malignant human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF) and human pulp cells (HPC), thereby demonstrating tumour-selective toxicity. A further study revealed that most of the compounds in series 2 were more toxic to the human Colo-205 adenocarcinoma cell line (Colo-205), human HT29 colorectal adenocarcinoma cells (HT-29) and human CEM lymphoid cells (CEM) neoplasms than towards non-malignant human foreskin Hs27 fibroblast line (Hs27) cells. The potency of the cytotoxins towards the six malignant cell lines increased as the sigma and sigma star values of the aryl substituents rose. Attempts to condense various aryl aldehydes with 2,2,6,6-tetramethyl-4-piperidone led to the isolation of some 1,5-diaryl-1,4-pentadien-3-ones. The highest specificity for oral cancer cells was displayed by 2e and 2r. In the case of 2r, its selective toxicity exceeded that of doxorubicin and melphalan. The enones 2k, m, o have the highest SI values towards colon cancer and leukemic cells. Both 2e,r inhibited mitosis and increased the subG1 population (with a transient increase in G2/M phase cells). Slight activation of caspase-3, based on the cleavage of poly(ADP-ribose)polymerase (PARP) and procaspase 3, was detected. Full article
Show Figures

Figure 1

Figure 1
<p>Initial series of cytotoxins <b>1</b>.</p>
Full article ">Figure 2
<p>The general structure of the compounds in series <b>2.</b> The aryl substituents are indicated in Tables 1, 2 and 4.</p>
Full article ">Figure 3
<p>Products of the reaction between 2,2,6,6-tetramethyl-4-piperidone and various aryl aldehydes, <b>a:</b> R = OCH<sub>3</sub>; <b>b:</b> R = H; <b>c:</b> R = Cl; <b>d:</b> R = F.</p>
Full article ">Figure 4
<p>Morphological change induced in Ca9-22 cells after 24 h incubation without (control) (<b>A</b>); with 1 μM actinomycin D (<b>AD</b>, reference compound) (<b>B</b>); with 0.3 and 1 μM of <b>2e</b> (<b>C</b>,<b>D</b>); and with 1 and 3 μM of <b>2r</b> (<b>E</b>,<b>F</b>). Actinomycin D induced apoptotic bodies, whereas <b>2e</b> and <b>2r</b> induced cell spreading at lower concentrations and cell shrinkage at higher concentrations (The scale bar is 200 μm). This point was clarified by Western blot analysis (<a href="#medicines-08-00078-f005" class="html-fig">Figure 5</a>) and cell cycle analysis (<a href="#medicines-08-00078-t006" class="html-table">Table 6</a>).</p>
Full article ">Figure 5
<p>Western blot analysis of <b>2e</b>, <b>r</b>. Ca9-22 cells were incubated for 24 h without (control) and with the indicated concentrations of samples, and cell lysates were prepared as described in Materials and Methods. Protein(9 μg) was loaded to each lane for Western blot analysis, and images after long exposure with contrast adjustment are presented. Raw data of stained gel and images after short and long exposure (without and with contrast adjustment) are shown in <a href="#app1-medicines-08-00078" class="html-app">Supplementary Figure S1</a>.</p>
Full article ">Figure 6
<p>Comparison of SI and PSE values of 3,5-bis(benzylidene)-4-piperidones against OSCC (<b>A</b>) and colon and lymphoid leukemia cell lines (<b>B</b>). The SI and PSE values are derived from <a href="#medicines-08-00078-t001" class="html-table">Table 1</a>, <a href="#medicines-08-00078-t002" class="html-table">Table 2</a> and <a href="#medicines-08-00078-t004" class="html-table">Table 4</a>. The cytotoxicity data of doxorubicin and melphalan against colon and lymphoid leukemia cell lines are not available.</p>
Full article ">Scheme 1
<p>A possible reaction mechanism for the formation of series <b>3</b> from 2,2,6,6-tetramethyl-4-piperidone and aryl aldehydes.</p>
Full article ">
18 pages, 25375 KiB  
Article
Antimicrobial Activity and Action Mechanism of Thymoquinone against Bacillus cereus and Its Spores
by Shuo Wang, Haichao Deng, Yihong Wang, Wushuang Rui, Pengyu Zhao, Qiyao Yong, Du Guo, Jie Liu, Xinyi Guo, Yutang Wang and Chao Shi
Foods 2021, 10(12), 3048; https://doi.org/10.3390/foods10123048 - 8 Dec 2021
Cited by 25 | Viewed by 3904
Abstract
In this study, thymoquinone (TQ), a natural active substance, was investigated for its antibacterial activity against Bacillus cereus, and its inhibitory effect on B. cereus in reconstituted infant formula (RIF) was evaluated. In addition, the inhibitory effect of TQ on B. cereus [...] Read more.
In this study, thymoquinone (TQ), a natural active substance, was investigated for its antibacterial activity against Bacillus cereus, and its inhibitory effect on B. cereus in reconstituted infant formula (RIF) was evaluated. In addition, the inhibitory effect of TQ on B. cereus spore germination was explored. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of TQ against eight B. cereus strains ranged from 4.0 to 8.0 μg/mL, whereas B. cereus treated with TQ displayed a longer lag phase than the untreated control. TQ exerted a good bactericidal effect on B. cereus in Luria–Bertani broth. In addition, TQ obviously reduced the intracellular ATP concentration of B. cereus, which caused depolarization of the cell membrane, increased the intracellular reactive oxygen species level, impaired the cell morphology, and destroyed proteins or inhibited proteins synthesis. This provides a mechanism for its bacteriostatic effect. TQ also inactivated B. cereus growth in RIF. Moreover, reverse transcription–quantitative polymerase chain reaction illustrated that TQ downregulated the transcription of genes related to hemolysin, non-hemolytic enterotoxin, enterotoxin, and cytotoxin K. Meanwhile, TQ displayed the ability to inhibit the germination of B. cereus spores. These findings indicate that TQ, as an effective natural antimicrobial preservative, has potential applications in controlling food contamination and foodborne diseases caused by B. cereus. Full article
Show Figures

Figure 1

Figure 1
<p>Chemical structure of thymoquinone.</p>
Full article ">Figure 2
<p>Growth curves of <span class="html-italic">Bacillus cereus</span> ATCC 14579 cultured in Luria–Bertani broth with various concentrations of thymoquinone (TQ).</p>
Full article ">Figure 3
<p>Inactivation of <span class="html-italic">Bacillus cereus</span> by thymoquinone (TQ) in Luria–Bertani broth. ** and * indicates statistical significance at <span class="html-italic">p</span> &lt;0.01 and <span class="html-italic">p</span> &lt; 0.05 compared with the control.</p>
Full article ">Figure 4
<p>Effects of thymoquinone (TQ) on the membrane potential of <span class="html-italic">Bacillus cereus</span> ATCC 14579. ** indicates statistical significance at <span class="html-italic">p</span> &lt; 0.01 compared with the control.</p>
Full article ">Figure 5
<p>Effects of thymoquinone (TQ) on intracellular ATP concentrations in <span class="html-italic">Bacillus cereus</span> ATCC 14579. ** indicates statistical significance at <span class="html-italic">p</span> &lt; 0.01 compared with the control.</p>
Full article ">Figure 6
<p>Effect of thymoquinone (TQ) treatment for 1 and 2 h on intracellular ROS levels in <span class="html-italic">Bacillus cereus</span>. ** indicates statistical significance at <span class="html-italic">p</span> &lt; 0.01 compared with the control.</p>
Full article ">Figure 7
<p>Field emission scanning electron microscopy observations (×15,000 magnification) of <span class="html-italic">Bacillus cereus</span> ATCC 14579. Untreated for 2 (<b>A</b>) and 4 h (<b>D</b>). Treated with thymoquinone (TQ) at 2 MIC for 2 (<b>B</b>) and 4 h (<b>E</b>). Treated with TQ at 4 MIC for 2 (<b>C</b>) and 4 h (<b>F</b>).</p>
Full article ">Figure 8
<p>Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of intracellular soluble proteins from untreated and thymoquinone-treated <span class="html-italic">Bacillus cereus</span>.</p>
Full article ">Figure 9
<p>Effect of thymoquinone (TQ) on the transcription of <span class="html-italic">Bacillus cereus</span> ATCC 14579 toxin-related genes. ** and * indicates statistical significance at <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.05 compared with the control.</p>
Full article ">Figure 10
<p>Analysis of reconstituted infant formula contaminated by <span class="html-italic">Bacillus cereus</span> inactivated by different concentrations of thymoquinone (TQ). ** indicates statistical significance at <span class="html-italic">p</span> &lt; 0.01 compared with the control.</p>
Full article ">Figure 11
<p>Spore germination rate following treatment with different concentrations of thymoquinone (TQ). ** indicates statistical significance at <span class="html-italic">p</span> &lt; 0.01 compared with the control.</p>
Full article ">Figure 12
<p>Confocal laser scanning microscopy observations (×200 magnification) of untreated <span class="html-italic">Bacillus cereus</span> spores (<b>A</b>) and spores treated with thymoquinone (TQ) at the minimum inhibitory concentration (MIC, <b>B</b>), 2 MIC (<b>C</b>), and 4 MIC (<b>D</b>).</p>
Full article ">
11 pages, 1212 KiB  
Communication
New Insights into the Potential Cytotoxic Role of Bacillus cytotoxicus Cytotoxin K-1
by Klèma Marcel Koné, Pauline Hinnekens, Jelena Jovanovic, Andreja Rajkovic and Jacques Mahillon
Toxins 2021, 13(10), 698; https://doi.org/10.3390/toxins13100698 - 1 Oct 2021
Cited by 7 | Viewed by 2796
Abstract
The thermotolerant representative of the Bacillus cereus group, Bacillus cytotoxicus, reliably harbors the coding gene of cytotoxin K-1 (CytK-1). This protein is a highly cytotoxic variant of CytK toxin, initially recovered from a diarrheal foodborne outbreak that caused the death of three [...] Read more.
The thermotolerant representative of the Bacillus cereus group, Bacillus cytotoxicus, reliably harbors the coding gene of cytotoxin K-1 (CytK-1). This protein is a highly cytotoxic variant of CytK toxin, initially recovered from a diarrheal foodborne outbreak that caused the death of three people. In recent years, the cytotoxicity of B. cytotoxicus has become controversial, with some strains displaying a high cytotoxicity while others show no cytotoxicity towards cell lines. In order to better circumscribe the potential pathogenic role of CytK-1, knockout (KO) mutants were constructed in two B. cytotoxicus strains, E8.1 and E28.3. The complementation of the cytK-1 KO mutation was implemented in a mutant strain lacking in the cytK-1 gene. Using the tetrazolium salt (MTT) method, cytotoxicity tests of the cytK-1 KO and complemented mutants, as well as those of their wild-type strains, were carried out on Caco-2 cells. The results showed that cytK-1 KO mutants were significantly less cytotoxic than the parental wild-type strains. However, the complemented mutant was as cytotoxic as the wild-type, suggesting that CytK-1 is the major cytotoxicity factor in B. cytotoxicus. Full article
(This article belongs to the Special Issue The Effect of Microbial Toxins on Animal Health and Food Safety)
Show Figures

Figure 1

Figure 1
<p>PCR-based confirmation of the five <span class="html-italic">B. cytotoxicus</span> KO-mutants selected after homologous recombination. L: DNA molecular weight markers (200 bp to 10 kb); B: negative control (buffer only). Panel (<b>a</b>): PCR-screening of the Kanamycin resistance gene: lanes 1 to 6: positive control (pDG173), E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-A</sup>, E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-B</sup>, E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-C</sup>, E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-D</sup> and E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-E</sup>. Panel (<b>b</b>): Detection of the <span class="html-italic">cytK-1</span> gene: lanes 1 to 6: positive control (E28.3 wild-type), E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-A</sup>, E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-B</sup>, E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-C</sup>, E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-D</sup> and E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-E</sup>.</p>
Full article ">Figure 2
<p>RAPD pattern of <span class="html-italic">B. cytotoxicus</span> strains using the OPA9 primer. L: DNA molecular weight markers (200 bp to 10 kb). D: donor strain E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-A</sup>; R: recipient strain E8.1; C<sup>-</sup>: negative control (buffer only); Lanes 1 to 5: candidate transconjugants. Lane #2 contains a <span class="html-italic">bona fide</span> E8.1 transconjugant containing the desired genetic loci (i.e., <span class="html-italic">kan<sup>R</sup></span> gene replacing the <span class="html-italic">cytK-1</span> gene). It was named E8.1<span class="html-italic"><sup>cytK−1-</sup></span><sup>KO-B</sup>.</p>
Full article ">Figure 3
<p>Tetrazolium salt method (MTT) used to assess viability of Caco-2 cells after 12 h of exposure to <span class="html-italic">B. cytotoxicus</span> supernatants. Cells treated with supernatant of wild-type (WT) NVH 391-98 and untreated Caco-2 cells were used as positive and negative control, respectively. Mutants (E8.1<span class="html-italic"><sup>cytK−1-</sup></span><sup>KO</sup> and E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-A</sup>) lacking the <span class="html-italic">cytK-1</span> gene are less cytotoxic than the WT strains of E8.1 and E28.3 (<span class="html-italic">p</span> &lt; 0.05). The complemented mutant of E28.3<span class="html-italic"><sup>cytK−1</sup></span><sup>-KO-A</sup> is as cytotoxic as the WT E28.3.</p>
Full article ">
7 pages, 276 KiB  
Communication
Further Insights into the Toxicity of Bacillus cytotoxicus Based on Toxin Gene Profiling and Vero Cell Cytotoxicity Assays
by Johanna Burtscher, Danai Etter, Michael Biggel, Janine Schlaepfer and Sophia Johler
Toxins 2021, 13(4), 234; https://doi.org/10.3390/toxins13040234 - 24 Mar 2021
Cited by 11 | Viewed by 3372
Abstract
Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis. The first B. cytotoxicus was isolated in the context of a severe food [...] Read more.
Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis. The first B. cytotoxicus was isolated in the context of a severe food poisoning outbreak leading to fatal cases of diarrheal disease. Subsequent characterization of the outbreak strain led to the conclusion that this Bacillus strain was highly cytotoxic and eventually resulted in the description of a novel species, whose name reflects the observed toxicity: B. cytotoxicus. However, only a few isolates of this species have been characterized with regard to their cytotoxic potential and the role of B. cytotoxicus as a causative agent of food poisoning remains largely unclear. Hence, the aim of this study was to gain further insights into the toxicity of B. cytotoxicus. To this end, 19 isolates were obtained from mashed potato powders and characterized by toxin gene profiling and Vero cell cytotoxicity assays. All isolates harbored the cytK1 (cytotoxin K1) gene and species-specific variants of the nhe (non-hemolytic enterotoxin) gene. The isolates exhibited low or no toxicity towards Vero cells. Thus, this study indicates that the cytotoxic potential of B. cytotoxicus may be potentially lower than initially assumed. Full article
Back to TopTop