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Cell May 2022Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these...
Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.
Topics: Animals; CRISPR-Cas Systems; Cytosine; DNA, Mitochondrial; Escherichia coli; Gene Editing; Humans; Mitochondria; Mitochondrial Diseases; Purines
PubMed: 35472302
DOI: 10.1016/j.cell.2022.03.039 -
Nature Jul 2020Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted...
Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques. Because previously described cytidine deaminases operate on single-stranded nucleic acids, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.
Topics: Bacterial Toxins; Base Sequence; Burkholderia cenocepacia; Cell Respiration; Cytidine; Cytidine Deaminase; DNA, Mitochondrial; Gene Editing; Genes, Mitochondrial; Genome, Mitochondrial; HEK293 Cells; Humans; Mitochondria; Mitochondrial Diseases; Mutation; Oxidative Phosphorylation; Protein Engineering; RNA, Guide, CRISPR-Cas Systems; Substrate Specificity; Type VI Secretion Systems
PubMed: 32641830
DOI: 10.1038/s41586-020-2477-4 -
Indian Journal of Ophthalmology Nov 2020A 33-year-old lady with history of failed keratoplasty for decompensated cornea due to childhood trauma and secondary glaucoma, post glaucoma drainage implant, with...
A 33-year-old lady with history of failed keratoplasty for decompensated cornea due to childhood trauma and secondary glaucoma, post glaucoma drainage implant, with pseudophakia in the right eye, developed bacterial keratitis following foreign body trauma to corneal graft. Corneal cultures yielded Burkholderia cenocepacia identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF- MS, bioMerieux, France). She healed with topical antibiotics (moxifloxacin 0.5%) in 1 month. Ours is the first report of ocular Burkholderia cenocepacia infection, possibly an under reported, aerobic, organism.
Topics: Adult; Anti-Bacterial Agents; Burkholderia cenocepacia; Eye Infections, Bacterial; Female; Humans; Keratitis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 33120688
DOI: 10.4103/ijo.IJO_1905_20 -
Frontiers in Cellular and Infection... 2021Cystic fibrosis (CF) is a genetic disease affecting more than 70,000 people worldwide. It is caused by a mutation in the gene, a chloride ion transporter localized in... (Review)
Review
Cystic fibrosis (CF) is a genetic disease affecting more than 70,000 people worldwide. It is caused by a mutation in the gene, a chloride ion transporter localized in the plasma membrane of lung epithelial cells and other organs. The loss of CFTR function alters chloride, bicarbonate, and water transport through the plasma membrane, promoting the production of a thick and sticky mucus in which bacteria including and can produce chronic infections that eventually decrease the lung function and increase the risk of mortality. Autophagy is a well-conserved lysosomal degradation pathway that mediates pathogen clearance and plays an important role in the control of bacterial infections. In this mini-review, we describe the principal strategies used by and to survive and avoid microbicidal mechanisms within the autophagic pathway leading to the establishment of chronic inflammatory immune responses that gradually compromise the lung function and the life of CF patients.
Topics: Autophagy; Burkholderia cenocepacia; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Pseudomonas Infections; Pseudomonas aeruginosa
PubMed: 34692569
DOI: 10.3389/fcimb.2021.760922 -
Nucleic Acids Research Dec 2020Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are...
Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are plasmid-borne ORFs, carried by pBCA072 for B. cenocepacia J2315 and pESBL for E. coli O104:H4. Pacific Biosciences SMRT sequencing was used to investigate DNA methyltransferases M.BceJIII and M.EcoGIX, using artificial constructs. Mating properties of engineered pESBL derivatives were also investigated. Both MTases yield promiscuous m6A modification of single strands, in the context SAY (where S = C or G and Y = C or T). Strikingly, this methylation is asymmetric in vivo, detected almost exclusively on one DNA strand, and is incomplete: typically, around 40% of susceptible motifs are modified. Genetic and biochemical studies suggest that enzyme action depends on replication mode: DNA Polymerase I (PolI)-dependent ColE1 and p15A origins support asymmetric modification, while the PolI-independent pSC101 origin does not. An MTase-PolI complex may enable discrimination of PolI-dependent and independent plasmid origins. M.EcoGIX helps to establish pESBL in new hosts by blocking the action of restriction enzymes, in an orientation-dependent fashion. Expression and action appear to occur on the entering single strand in the recipient, early in conjugal transfer, until lagging-strand replication creates the double-stranded form.
Topics: Bacterial Proteins; Burkholderia cenocepacia; DNA Methylation; DNA Polymerase I; DNA Replication; DNA, Single-Stranded; Escherichia coli O104; Escherichia coli Proteins; Genome, Bacterial; Methyltransferases; Plasmids; Ribosomal Proteins
PubMed: 33270887
DOI: 10.1093/nar/gkaa1163 -
BMC Microbiology Jan 2023Burkholderia cenocepacia is an opportunistic pathogen that can cause acute and chronic infections in patients with weakened immune systems and in patients with cystic...
BACKGROUND
Burkholderia cenocepacia is an opportunistic pathogen that can cause acute and chronic infections in patients with weakened immune systems and in patients with cystic fibrosis. B. cenocepacia is resistant to many antibiotics making treatment challenging. Consequently, there is a critical need for alternative strategies to treat B. cenocepacia infections such as using bacteriophages and/or bacteriophages with subinhibitory doses of antibiotic called phage-antibiotic synergy.
RESULTS
We isolated a bacteriophage, KP1, from raw sewage that infects B. cenocepacia. Its morphological characteristics indicate it belongs in the family Siphoviridae, it has a 52 Kb ds DNA genome, and it has a narrow host range. We determined it rescued infections in Lemna minor (duckweed) and moderately reduced bacterial populations in our artificial sputum medium model.
CONCLUSION
These results suggest that KP1 phage alone in the duckweed model or in combination with antibiotics in the ASMDM model improves the efficacy of reducing B. cenocepacia populations.
Topics: Humans; Burkholderia cenocepacia; Bacteriophages; Anti-Bacterial Agents; Burkholderia Infections
PubMed: 36600213
DOI: 10.1186/s12866-022-02738-0 -
Scientific Reports Jan 2021Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including...
Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1β) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.
Topics: Animals; Autophagy; Burkholderia Infections; Burkholderia cenocepacia; Caspases, Initiator; Cell Death; Female; Inflammasomes; Intracellular Signaling Peptides and Proteins; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Phosphate-Binding Proteins; Reactive Oxygen Species
PubMed: 33441602
DOI: 10.1038/s41598-020-79201-5 -
Frontiers in Molecular Biosciences 2020The genus sensu lato is composed of a diverse and metabolically versatile group of bacterial species. One characteristic thought to be unique for the genus is the...
The genus sensu lato is composed of a diverse and metabolically versatile group of bacterial species. One characteristic thought to be unique for the genus is the presence of two forms each (with and without 2-hydroxylation) of the membrane lipids phosphatidylethanolamine (PE) and ornithine lipids (OLs). Here, we show that only sensu stricto strains constitutively form OLs, whereas all other analyzed strains belonging to the sensu lato group constitutively form the two forms of PE, but no OLs. We selected two model bacteria to study the function of OL in sensu lato: (1) wild-type which constitutively forms OLs and its mutant deficient in the formation of OLs and (2) (formerly ) which does not form OL constitutively, and a derived strain constitutively forming OLs. Both were characterized under free-living conditions and during pathogenic interactions with their respective hosts. The absence of OLs in slightly affected bacterial growth under specific abiotic stress conditions such as high temperature and low pH. lacking OLs caused lower mortality in larvae while constitutively forming OLs triggers an increased formation of reactive oxygen species immediately after infection of maize leaves, suggesting that OLs can have an important role during the activation of the innate immune response of eukaryotes.
PubMed: 33469548
DOI: 10.3389/fmolb.2020.610932 -
Microbiology Spectrum Aug 2022Burkholderia cenocepacia is a human opportunistic pathogen that mostly employs two types of quorum-sensing (QS) systems to regulate its various biological functions and...
Burkholderia cenocepacia is a human opportunistic pathogen that mostly employs two types of quorum-sensing (QS) systems to regulate its various biological functions and pathogenicity: the -2-dodecenoic acid (BDSF) system and the -acyl homoserine lactone (AHL) system. In this study, we reported that oridonin, which was screened from a collection of natural products, disrupted important B. cenocepacia phenotypes, including motility, biofilm formation, protease production, and virulence. Genetic and biochemical analyses showed that oridonin inhibited the production of BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Furthermore, we revealed that oridonin directly binds to the regulator RqpR of the two-component system RqpSR that dominates the above-mentioned QS systems to inhibit the expression of the BDSF and AHL signal synthase-encoding genes. Oridonin also binds to the transcriptional regulator CepR of the AHL system to inhibit its binding to the promoter of . These findings suggest that oridonin could potentially be developed as a new QS inhibitor against pathogenic B. cenocepacia. Burkholderia cenocepacia is an important human opportunistic pathogen that can cause life-threatening infections in susceptible individuals. It employs quorum-sensing (QS) systems to regulate biological functions and virulence. In this study, we have identified a lead compound, oridonin, that is capable of interfering with B. cenocepacia QS signaling and physiology. We demonstrate that oridonin suppressed -2-dodecenoic acid (BDSF) and -acyl homoserine lactone (AHL) signal production and attenuated virulence in B. cenocepacia. Oridonin also impaired QS-regulated phenotypes in various Burkholderia species. These results suggest that oridonin could interfere with QS signaling in many Burkholderia species and might be developed as a new antibacterial agent.
Topics: Acyl-Butyrolactones; Bacterial Proteins; Burkholderia cenocepacia; Diterpenes, Kaurane; Gene Expression Regulation, Bacterial; Humans; Quorum Sensing; Virulence
PubMed: 35856676
DOI: 10.1128/spectrum.01787-22 -
Microbiological Research Mar 2020Plant-growth promoting rhizobacteria benefit crop health and growth through various mechanisms including phosphate and potassium solubilisation, and antimicrobial...
Plant-growth promoting rhizobacteria benefit crop health and growth through various mechanisms including phosphate and potassium solubilisation, and antimicrobial activity. Previously, we sequenced the genome of bacterial strain Burkholderia cenocepacia CR318, which was isolated from the roots of the starch corn (Zea mays L.) in London, Ontario, Canada. In this work, the species identity of this isolate is confirmed by recA phylogeny and in silico DNA-DNA hybridization (isDDH), and its plant-growth promoting characteristics are described. B. cenocepacia CR318 exhibited strong activity of inorganic phosphate and potassium solubilization. It significantly promoted the growth of corn plants and roots by solubilizing inorganic tricalcium phosphate under greenhouse conditions. Functional analysis of the complete B. cenocepacia CR318 genome revealed genes associated with phosphate metabolism such as pstSCAB encoding a high affinity inorganic phosphate-specific transporter, and the pqqABCDE gene cluster involved in the biosynthesis of pyrroloquinoline quinone (PQQ), which is a required cofactor for quinoprotein glucose dehydrogenase (Gdh). However, it appears that B. cenocepacia CR318 lacks the quinoprotein Gdh which can produce gluconic acid to solubilize inorganic phosphate. Overall, these findings provide an important step in understanding the molecular mechanisms underlying the plant growth promotion trait of B. cenocepacia CR318.
Topics: Burkholderia cenocepacia; DNA, Bacterial; Genome, Bacterial; Glucose 1-Dehydrogenase; Ontario; PQQ Cofactor; Phosphates; Phylogeny; Plant Development; Plant Roots; Rhizosphere; Soil Microbiology; Solubility; Zea mays
PubMed: 31865096
DOI: 10.1016/j.micres.2019.126395