-
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 Microbiology 2017is an opportunistic pathogen particularly dangerous for cystic fibrosis (CF) patients. It can cause a severe decline in CF lung function possibly developing into a... (Review)
Review
is an opportunistic pathogen particularly dangerous for cystic fibrosis (CF) patients. It can cause a severe decline in CF lung function possibly developing into a life-threatening systemic infection known as cepacia syndrome. Antibiotic resistance and presence of numerous virulence determinants in the genome make extremely difficult to treat. Better understanding of its resistance profiles and mechanisms is crucial to improve management of these infections. Here, we present the clinical distribution of described in the last 6 years and methods for identification and classification of epidemic strains. We also detail new antibiotics, clinical trials, and alternative approaches reported in the literature in the last 5 years to tackle resistance issue. All together these findings point out the urgent need of new and alternative therapies to improve CF patients' life expectancy.
PubMed: 28878751
DOI: 10.3389/fmicb.2017.01592 -
Current Research in Structural Biology 2021Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes...
Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes typically utilize a pair of tyrosine residues to orient the substrate epoxide ring in the active site and stabilize the hydrolysis intermediate. A new subclass of epoxide hydrolases that utilize a histidine in place of one of the tyrosines was established with the discovery of the CFTR Inhibitory Factor (Cif) from . Although the presence of such Cif-like epoxide hydrolases was predicted in other opportunistic pathogens based on sequence analyses, only Cif and its homolog aCif from have been characterized. Here we report the biochemical and structural characteristics of Cfl1 and Cfl2, two Cif-like epoxide hydrolases from . Cfl1 is able to hydrolyze xenobiotic as well as biological epoxides that might be encountered in the environment or during infection. In contrast, Cfl2 shows very low activity against a diverse set of epoxides. The crystal structures of the two proteins reveal quaternary structures that build on the well-known dimeric assembly of the α/β hydrolase domain, but broaden our understanding of the structural diversity encoded in novel oligomer interfaces. Analysis of the interfaces reveals both similarities and key differences in sequence conservation between the two assemblies, and between the canonical dimer and the novel oligomer interfaces of each assembly. Finally, we discuss the effects of these higher-order assemblies on the intra-monomer flexibility of Cfl1 and Cfl2 and their possible roles in regulating enzymatic activity.
PubMed: 34235487
DOI: 10.1016/j.crstbi.2021.02.002 -
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 -
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 -
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 -
Viruses May 2018, is a Gram-negative opportunistic pathogen that belongs to complex (BCC) group. BCC representatives carry various pathogenicity factors and can infect humans and...
, is a Gram-negative opportunistic pathogen that belongs to complex (BCC) group. BCC representatives carry various pathogenicity factors and can infect humans and plants. Phages as bacterial viruses play a significant role in biodiversity and ecological balance in the environment. Specifically, horizontal gene transfer (HGT) and lysogenic conversion (temperate phages) influence microbial diversification and fitness. In this study, we describe the prevalence and gene content of prophages in 16 fully sequenced genomes stored in NCBI database. The analysis was conducted in silico by manual and automatic approaches. Sixty-three potential prophage regions were found and classified as intact, incomplete, questionable, and artifacts. The regions were investigated for the presence of known virulence factors, resulting in the location of sixteen potential pathogenicity mechanisms, including toxin⁻antitoxin systems (TA), Major Facilitator Superfamily (MFS) transporters and responsible for drug resistance. Investigation of the region's closest neighborhood highlighted three groups of genes with the highest occurrence-tRNA-Arg, dehydrogenase family proteins, and ABC transporter substrate-binding proteins. Searches for antiphage systems such as BacteRiophage EXclusion (BREX) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in the analyzed strains suggested 10 sequence sets of CRISPR elements. Our results suggest that intact prophages may provide an evolutionary advantage to the bacterium, while domesticated prophages may help to maintain important genes.
Topics: Burkholderia cenocepacia; Burkholderia cepacia complex; Clustered Regularly Interspaced Short Palindromic Repeats; Genome, Bacterial; Genome, Viral; Humans; Lysogeny; Prevalence; Prophages; Sequence Analysis, DNA; Toxin-Antitoxin Systems; Virulence Factors
PubMed: 29857552
DOI: 10.3390/v10060297 -
Frontiers in Cellular and Infection... 2011Burkholderia cenocepacia is an opportunistic respiratory pathogen of individuals with cystic fibrosis (CF). Some strains of B. cenocepacia are highly transmissible and... (Review)
Review
Burkholderia cenocepacia is an opportunistic respiratory pathogen of individuals with cystic fibrosis (CF). Some strains of B. cenocepacia are highly transmissible and resistant to almost all antibiotics. Approximately one-third of B. cenocepacia infected CF patients go on to develop fatal "cepacia syndrome." During the last two decades, substantial progress has been made with regards to evasion of host innate defense mechanisms by B. cenocepacia. Almost all strains of B. cenocepacia have the capacity to survive and replicate intracellularly in both airway epithelial cells and macrophages, which are primary sentinels of the lung and play a pivotal role in clearance of infecting bacteria. Those strains of B. cenocepacia, which express both cable pili and the associated 22 kDa adhesin are also capable of transmigrating across airway epithelium and persist in mouse models of infection. In this review, we will discuss how this type of interaction between B. cenocepacia and host may lead to persistence of bacteria as well as lung inflammation in CF patients.
Topics: Animals; Burkholderia Infections; Burkholderia cenocepacia; Cystic Fibrosis; Cytokines; Disease Models, Animal; Endocytosis; Host-Pathogen Interactions; Humans; Immunity, Innate; Inflammation Mediators; Lung; Mice; Opportunistic Infections; Respiratory Mucosa; Respiratory Tract Infections; Toll-Like Receptors
PubMed: 22919590
DOI: 10.3389/fcimb.2011.00025 -
Clinical Microbiology and Infection :... Jul 2010Burkholderia cepacia complex (Bcc) bacteria have gained notoriety as pathogens in cystic fibrosis (CF) because they are difficult to identify and treat, and also have... (Review)
Review
Burkholderia cepacia complex (Bcc) bacteria have gained notoriety as pathogens in cystic fibrosis (CF) because they are difficult to identify and treat, and also have the ability to spread between CF individuals. Of the 17 formally named species within the complex, Burkholderia multivorans and Burkholderia cenocepacia dominate in CF. Multilocus sequence typing has proven to be a very useful tool for tracing the global epidemiology of Bcc bacteria and has shown that B. cenocepacia strains with high transmissibility, such as the ET-12 strain (ST-28) and the Czech strain (ST-32), have spread epidemically within CF populations in Canada and Europe. The majority of research on the molecular pathogenesis of Bcc bacteria has focused on the B. cenocepacia ET-12 epidemic lineage, with gene mutation, genome sequence analysis and, most recently, global gene expression studies shedding considerable light on the virulence and antimicrobial resistance of this pathogen. These studies demonstrate that the ability of B. cenocepacia to acquire foreign DNA (genomic islands, insertion sequences and other mobile elements), regulate gene expression via quorum sensing, compete for iron during infection, and mediate antimicrobial resistance and inflammation via its membrane and surface polysaccharides are key features that underpin the virulence of different strains. With the wealth of molecular knowledge acquired in the last decade on B. cenocepacia strains, we are now in a much better position to develop strategies for the treatment of pathogenic colonization with Bcc and to answer key questions on pathogenesis concerning, for example, the factors that trigger the rapid clinical decline in CF patients.
Topics: Burkholderia Infections; Burkholderia cenocepacia; Burkholderia cepacia complex; Canada; Cystic Fibrosis; Drug Resistance, Bacterial; Europe; Gene Expression; Humans; Interspersed Repetitive Sequences; Molecular Epidemiology; Multilocus Sequence Typing; Mutation; Polysaccharides, Bacterial; Quorum Sensing; Respiratory Tract Infections; Virulence
PubMed: 20880411
DOI: 10.1111/j.1469-0691.2010.03237.x