-
EBioMedicine Mar 2022Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly...
BACKGROUND
Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs.
METHODS
Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs).
FINDINGS
Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals.
INTERPRETATION
This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19.
FUNDING
This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).
Topics: Brevibacillus; COVID-19; Humans; RNA, Guide, CRISPR-Cas Systems; SARS-CoV-2
PubMed: 35290826
DOI: 10.1016/j.ebiom.2022.103926 -
Journal of Chemical Theory and... Jan 2021We investigated gramicidin A (gA) subunit dimerization in lipid bilayers using microsecond-long replica-exchange umbrella sampling simulations, millisecond-long unbiased...
We investigated gramicidin A (gA) subunit dimerization in lipid bilayers using microsecond-long replica-exchange umbrella sampling simulations, millisecond-long unbiased molecular dynamics simulations, and machine learning. Our simulations led to a dimer structure that is indistinguishable from the experimentally determined gA channel structures, with the two gA subunits joined by six hydrogen bonds (6HB). The simulations also uncovered two additional dimer structures, with different gA-gA stacking orientations that were stabilized by four or two hydrogen bonds (4HB or 2HB). When examining the temporal evolution of the dimerization, we found that two bilayer-inserted gA subunits can form the 6HB dimer directly, with no discernible intermediate states, as well as through paths that involve the 2HB and 4HB dimers.
Topics: Bacterial Proteins; Brevibacillus; Gramicidin; Hydrogen Bonding; Lipid Bilayers; Molecular Dynamics Simulation; Protein Conformation; Protein Multimerization; Protein Subunits; Thermodynamics
PubMed: 33378617
DOI: 10.1021/acs.jctc.0c00989 -
Microorganisms Mar 2024() is widely distributed in nature and demonstrates significant potential for applications in biological control, environmental protection, agricultural production, and... (Review)
Review
() is widely distributed in nature and demonstrates significant potential for applications in biological control, environmental protection, agricultural production, and clinical medicine. This review provides a comprehensive overview of the applications of in crop cultivation and animal feeding, as well as an examination of the antimicrobial peptides produced by and their antibacterial mechanisms. enhances crop cultivation by secreting hydrolases to improve nutrient absorption capabilities, synthesizing hormones to promote crop growth, and producing proteins to inhibit the reproduction of harmful organisms. has been used to improve animal production by regulating the structure of the intestinal microbiota and inhibiting the growth of pathogenic bacteria through the secretion of various antimicrobial peptides. The bactericidal activity of Brevilaterins secreted by is attributed to their ability to bind to lipopolysaccharide/lipid II molecules on the cell membrane, thereby altering permeability. Brevilaterins also inhibit bacterial reproduction by affecting relevant gene pathways in the cell membranes of pathogenic bacteria. These pathways include ATP synthesis, peptidoglycan biosynthesis, membrane transport, and cellular metabolism. In conclusion, exhibits substantial potential as a probiotic activity in crop and animal production. However, applications of in animal production could be improved, necessitating further research to elucidate the underlying probiotic mechanisms.
PubMed: 38543615
DOI: 10.3390/microorganisms12030564 -
Environmental Research Mar 2023While an increasing number of studies have evaluated tobacco microbiomes, comparative microbiome analyses across diverse tobacco products are non-existent. Moreover, to...
While an increasing number of studies have evaluated tobacco microbiomes, comparative microbiome analyses across diverse tobacco products are non-existent. Moreover, to our knowledge, no previous studies have characterized the metabolically-active (live) fraction of tobacco bacterial communities and compared them across products. To address these knowledge gaps, we compared bacterial communities across four commercial products (cigarettes, little cigars, cigarillos and hookah) and one research cigarette product. After total DNA extraction (n = 414) from all samples, the V3V4 region of the 16S rRNA gene was sequenced on the Illumina HiSeq platform. To identify metabolically-active bacterial communities within these products, we applied a coupled 5-bromo-2'-deoxyuridine labeling and sequencing approach to a subset of samples (n = 56). Each tobacco product was characterized by its signature microbiome, along with a shared microbiome across all tobacco products consisting of Pseudomonas aeruginosa, P. putida, P. alcaligenes, Bacillus subtilis, and Klebsiella pneumoniae. Comparing across products (using Linear discriminant analysis Effect Size (LEfSe)), a significantly higher (p < 0.05) relative abundance of Klebsiella and Acinetobacter was observed in commercial cigarettes, while a higher relative abundance of Pseudomonas and Pantoea was observed in research cigarettes. Methylorubrum and Paenibacillus were higher in hookah, and Brevibacillus, Lactobacillus, Bacillus, Lysinibacillus, and Staphylococcus were higher in little cigars and cigarillos. Across all products, the majority of the metabolically-active bacterial communities belonged to the genus Pseudomonas, followed by several genera within the Firmicutes phylum (Bacillus, Terribacillus, and Oceanobacillus). Identification of some metabolically-active pathogens such as Bacillus cereus and Haemophilus parainfluenzae in commercial products is of concern because of the potential for these microorganisms to be transferred to users' respiratory tracts via mainstream smoke. Future work is warranted to evaluate the potential impact of these tobacco bacterial communities on users' oral and lung microbiomes, which play such an important role on the spectrum from health to disease.
Topics: Nicotiana; Smoking; RNA, Ribosomal, 16S; Tobacco Products; Bacteria; Microbiota; Pseudomonas; Electronic Nicotine Delivery Systems
PubMed: 36592812
DOI: 10.1016/j.envres.2022.115205 -
Applied and Environmental Microbiology Nov 2020Nonribosomal peptides (NRPs) are a class of secondary metabolites usually produced by microorganisms. They are of paramount importance in different applications,...
Nonribosomal peptides (NRPs) are a class of secondary metabolites usually produced by microorganisms. They are of paramount importance in different applications, including biocontrol and pharmacy. spp. are a rich source of NRPs yet have received little attention. In this study, we characterize four novel bogorol variants (bogorols I to L, cationic linear lipopeptides) and four succilins (succilins I to L, containing a succinyl group that is attached to the Orn/Lys in bogorols I to L) from the biocontrol strain MG64. Further investigation revealed that the bogorol family of peptides employs an adenylation pathway for lipoinitiation, different from the usual pattern, which is based on an external ligase and coenzyme A. Moreover, the formation of valinol was proven to be mediated by a terminal reductase domain and a reductase encoded by the gene. Furthermore, succinylation, which is a novel type of modification in the family of bogorols, was discovered. Its occurrence requires a high concentration of the substrate (bogorols), but its responsible enzyme remains unknown. Bogorols display potent activity against both Gram-positive and Gram-negative bacteria. Investigation of their mode of action reveals that bogorols form pores in the cell membrane of both Gram-positive and Gram-negative bacteria. The combination of bogorols and relacidines, another class of NRPs produced by MG64, displays a synergistic effect on different pathogens, suggesting the great potential of both peptides as well as their producer MG64 for broad applications. Our study provides a further understanding of the bogorol family of peptides as well as their applications. NRPs form a class of secondary metabolites with biocontrol and pharmaceutical potential. This work describes the identification of novel bogorol variants and succinylated bogorols (namely, succilins) and further investigates their biosynthetic pathway and mode of action. Adenylation domain-mediated lipoinitiation of bogorols represents a novel pathway by which NRPs incorporate fatty acid tails. This pathway provides the possibility to engineer the lipid tail of NRPs without identifying a fatty acid coenzyme ligase, which is usually not present in the biosynthetic gene cluster. The terminal reductase domain (TD) and BogI-mediated valinol formation and their effect on the biological activity of bogorols are revealed. Succinylation, which is rarely reported in NRPs, was discovered in the bogorol family of peptides. We demonstrate that bogorols combat bacterial pathogens by forming pores in the cell membrane. We also report the synergistic effect of two natural products (relacidine B and bogorol K) produced by the same strain, which is relevant for competition for a niche.
Topics: Anti-Bacterial Agents; Brevibacillus; Lipopeptides; Microbial Sensitivity Tests
PubMed: 32978140
DOI: 10.1128/AEM.01981-20 -
Molecules (Basel, Switzerland) Dec 2022Methicillin-resistant (MRSA) is listed as a high-priority pathogen because its infection is associated with a high mortality rate. It is urgent to search for new agents...
Methicillin-resistant (MRSA) is listed as a high-priority pathogen because its infection is associated with a high mortality rate. It is urgent to search for new agents to treat such an infection. Our previous study isolated a soil bacterium ( sp. SPR-20), showing the highest antimicrobial activity against TISTR 517 and MRSA strains. The present study aimed to purify and characterize anti-MRSA substances produced by SPR-20. The result showed that five active substances (P1-P5) were found, and they were identified by LC-MS/MS that provided the peptide sequences of 14-15 residues. Circular dichroism showed that all peptides contained β-strand and disordered conformations as the major secondary structures. Only P1-P4 adopted more α-helix conformations when incubated with 50 mM SDS. These anti-MRSA peptides could inhibit and MRSA in concentrations of 2-32 μg/mL. P1 (NH-VVVNVLVKVLPPPVV-COOH) had the highest activity and was identified as a novel antimicrobial peptide (AMP). The stability study revealed that P1 was stable in response to temperature, proteolytic enzymes, surfactant, and pH. The electron micrograph showed that P1 induced bacterial membrane damage when treated at 1× MIC in the first hour of incubation. The killing kinetics of P1 was dependent on concentration and time. Mechanisms of P1 on tested pathogens involved membrane permeability, leakage of genetic material, and cell lysis. The P1 peptide at a concentration up to 32 μg/mL showed hemolysis of less than 10%, supporting its safety for human erythrocytes. This study provides promising anti-MRSA peptides that might be developed for effective antibiotics in the post-antibiotic era.
Topics: Humans; Methicillin-Resistant Staphylococcus aureus; Staphylococcus aureus; Microbial Sensitivity Tests; Brevibacillus; Chromatography, Liquid; Tandem Mass Spectrometry; Anti-Bacterial Agents; Peptides
PubMed: 36500545
DOI: 10.3390/molecules27238452 -
Medicine Sep 2022Esophageal microbiota plays important roles in esophageal squamous cell carcinoma (ESCC). The aims of this study were to clarify the changes in the bacterial community...
Esophageal microbiota plays important roles in esophageal squamous cell carcinoma (ESCC). The aims of this study were to clarify the changes in the bacterial community during ESCC development and identify latent pathogenic bacteria which may contribute to esophageal carcinogenesis and progression. Fresh tumor and nontumor esophageal mucosal samples were collected from 31 men with ESCC. High-throughput 16s rRNA sequencing was performed, and the operational taxonomic unit data and bacterial classification annotation were obtained and analyzed. The Ace, Chao, Shannon, Simpson indexes, and operational taxonomic unit numbers were higher in nontumor tissues than in tumor tissues, although without statistical significance. There were 4 phyla and 28 genera found to show significant differences between tumor and nontumor samples. The general probiotic Lactobacillus was 1.98-fold higher in nontumor tissues, while the general pathogenic genera Fusobacterium was 4.35-fold higher in tumor tissues. For tumor tissue samples, the genera Treponema and Brevibacillus were significantly higher in N1 and N2 stages, respectively, and Acinetobacter was significantly higher in T3 stage. For nontumor tissues, the genus Fusicatenibacter was significantly higher in T2 stage, and Corynebacterium, Aggregatibacter, Saccharimonadaceae-TM7x, and Cupriavidus were significantly higher in T4 stage. Additionally, bacteria related to nitrotoluene degradation were enriched in nontumor tissues, while bacteria related to base excision repair were enriched in tumor tissues. The relative abundance of several phyla and genera are different between tumor and nontumor tissue samples. The altered bacterial microbiota is correlated with different tumor stages and some microbes may take part in the carcinogenesis and development of ESCC.
Topics: Bacteria; Carcinogenesis; Carcinoma, Squamous Cell; Esophageal Mucosa; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Humans; Male; Microbiota; RNA, Ribosomal, 16S
PubMed: 36123940
DOI: 10.1097/MD.0000000000030483 -
Microbial Biotechnology Feb 2022Edeines, a group of cationic antimicrobial peptides produced by the soil bacterium Brevibacillus, have broad biological effects, such as antimicrobial, anticancer and...
Edeines, a group of cationic antimicrobial peptides produced by the soil bacterium Brevibacillus, have broad biological effects, such as antimicrobial, anticancer and immunosuppressive activities. However, the yield of edeines in wild-type (WT) Brevibacillus is extremely low, and chemical synthesis of edeines is a time-consuming process. Genetic engineering has proven to be an effective approach to produce antibiotics with high yield. In this study, the edeine biosynthetic gene cluster (ede BGC), which is involved in edeine production, was identified and characterized in Brevibacillus brevis X23. To improve edeine production in B. brevis X23, the ede BGC promoter was replaced with six different promoters, P , P , P , P , P or P , through double-crossover homologous recombination. The new promoters significantly increased the expression of the ede BGC as well as edeine production by 2.9 ± 0.4 to 20.5 ± 1.2-fold and 3.6 ± 0.1to 8.7 ± 0.7-fold respectively. The highest yield of edeines (83.6 mg l ) was obtained in B. brevis X23 with the P promoter. This study provides a practical approach for producing high yields of edeines in B. brevis.
Topics: Anti-Bacterial Agents; Bacillus; Brevibacillus; Edeine
PubMed: 34310825
DOI: 10.1111/1751-7915.13825 -
Antibiotics (Basel, Switzerland) Jun 2022Previous research found that the six major cyclodecapeptides from the tyrothricin complex, produced by , showed potent activity against chloroquine sensitive (CQS) . The...
Previous research found that the six major cyclodecapeptides from the tyrothricin complex, produced by , showed potent activity against chloroquine sensitive (CQS) . The identity of the aromatic residues in the aromatic dipeptide unit in cyclo-(D-Phe-Pro-(Phe/Trp)-D-Phe/D-Trp)-Asn-Gln-(Tyr/Phe/Trp)-Val-(Orn/Lys)-Leu was proposed to have an important role in activity. CQS and resistant (CQR) strains were challenged with three representative cyclodecapeptides. Our results confirmed that cyclodecapeptides from tyrothricin had significantly higher antiplasmodial activity than the analogous gramicidin S, rivaling that of CQ. However, the previously hypothesized size and hydrophobicity dependent activity for these peptides did not hold true for strains, other than for the CQS 3D7 strain. The Tyr in tyrocidine A (TrcA) with Phe-D-Phe seem to be related with loss in activity correlating with CQ antagonism and resistance, indicating a shared target and/or resistance mechanism in which the phenolic groups play a role. Phe in phenycidine A, the second peptide containing Phe-D-Phe, also showed CQ antagonism. Conversely, Trp in tryptocidine C (TpcC) with Trp-D-Trp showed improved peptide selectivity and activity towards the more resistant strains, without overt antagonism towards CQ. However, TpcC lead to similar parasite stage inhibition and parasite morphology changes than previously observed for TrcA. The disorganization of chromatin packing and neutral lipid structures, combined with amorphous hemozoin crystals, could account for halted growth in late trophozoite/early schizont stage and the nanomolar non-lytic activity of these peptides. These targets related to CQ antagonism, changes in neural lipid distribution, leading to hemozoin malformation, indicate that the tyrothricin cyclodecapeptides and CQ share a target in the malaria parasite. The differing activities of these cyclic peptides towards CQS and CQR strains could be due to variable target interaction in multiple modes of activity. This indicated that the cyclodecapeptide activity and parasite resistance response depended on the aromatic residues in positions 3, 4 and 7. This new insight on these natural cyclic decapeptides could also benefit the design of unique small peptidomimetics in which activity and resistance can be modulated.
PubMed: 35740207
DOI: 10.3390/antibiotics11060801 -
Applied and Environmental Microbiology Nov 2018and are two bacteria that are members of the family. Both are commonly found in beehives and have historically been difficult to distinguish from each other due to...
and are two bacteria that are members of the family. Both are commonly found in beehives and have historically been difficult to distinguish from each other due to related genetic and phenotypic characteristics and a shared ecological niche. Here, we discuss the likely mischaracterization of three 16S rRNA sequences previously published as and provide the phylogenetic evidence that supported the GenBank reassignment of the sequences as We explore the issues that arise by using only 16S rRNA or other single-gene analyses to distinguish between these bacteria. We also present three sets of molecular markers, two sets that distinguish from and other closely related species within the genus and a third set that distinguishes from and other closely related species within the genus. These molecular markers provide a tool for proper identification of these oft-mistaken species. 16S rRNA gene sequencing in bacteria has long been held as the gold standard for typing bacteria and, for the most part, is an excellent method of taxonomically identifying different bacterial species. However, the high level of 16S rRNA sequence similarity of some published strains of and , as well as possible horizontal gene transfer events within their shared ecological niche, complicates the use of 16S rRNA sequence as an effective molecular marker for differentiating these two species. Additionally, shared characteristics of these bacteria limit the effectiveness of using traditional phenotypic identification assays, such as the catalase test. The results from this study provide PCR methods to quickly differentiate between these two genera and will be useful when studying , , and other disease-relevant bacteria commonly found in beehives.
Topics: Animals; Bacterial Typing Techniques; Bees; Brevibacillus; DNA, Bacterial; DNA, Ribosomal; Paenibacillus larvae; Phylogeny; Polymerase Chain Reaction; RNA, Ribosomal, 16S
PubMed: 30217838
DOI: 10.1128/AEM.01886-18