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PLoS Biology Jun 2024CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new...
CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new Cas12a nucleases for mammalian genome editing, successfully identifying 9 active ones. Notably, these Cas12a nucleases prefer pyrimidine-rich PAMs. Among these nucleases, we extensively characterized Mb4Cas12a obtained from Moraxella bovis CCUG 2133, which recognizes a YYN PAM (Y = C or T). Our biochemical analysis demonstrates that Mb4Cas12a can cleave double-strand DNA across a wide temperature range. To improve specificity, we constructed a SWISS-MODEL of Mb4Cas12a based on the FnCas12a crystal structure and identified 8 amino acids potentially forming hydrogen bonds at the target DNA-crRNA interface. By replacing these amino acids with alanine to disrupt the hydrogen bond, we tested the influence of each mutation on Mb4Cas12a specificity. Interestingly, the F370A mutation improved specificity with minimal influence on activity. Further study showed that Mb4Cas12a-F370A is capable of discriminating single-nucleotide polymorphisms. These new Cas12a orthologs and high-fidelity variants hold substantial promise for therapeutic applications.
Topics: Gene Editing; CRISPR-Cas Systems; CRISPR-Associated Proteins; Alleles; Humans; Endodeoxyribonucleases; Animals; Protein Engineering; Bacterial Proteins; Polymorphism, Single Nucleotide; Mutation; DNA; HEK293 Cells
PubMed: 38865309
DOI: 10.1371/journal.pbio.3002680 -
PNAS Nexus Apr 2024Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly...
Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly killing bacterial invaders; however, many pathogens have evolved resistance evasion mechanisms such as cell surface modification, CAP sequestration, degradation, or efflux. We have discovered that several pathogenic and commensal proteobacteria, including the urgent human threat , secrete a protein (lactoferrin-binding protein B, LbpB) that contains a low-complexity anionic domain capable of inhibiting the antimicrobial activity of host CAPs. This study focuses on a cattle pathogen, , that expresses the largest anionic domain of the LbpB homologs. We used an exhaustive biophysical approach employing circular dichroism, biolayer interferometry, cross-linking mass spectrometry, microscopy, size-exclusion chromatography with multi-angle light scattering coupled to small-angle X-ray scattering (SEC-MALS-SAXS), and NMR to understand the mechanisms of LbpB-mediated protection against CAPs. We found that the anionic domain of this LbpB displays an α-helical secondary structure but lacks a rigid tertiary fold. The addition of antimicrobial peptides derived from lactoferrin (i.e. lactoferricin) to the anionic domain of LbpB or full-length LbpB results in the formation of phase-separated droplets of LbpB together with the antimicrobial peptides. The droplets displayed a low rate of diffusion, suggesting that CAPs become trapped inside and are no longer able to kill bacteria. Our data suggest that pathogens, like , leverage anionic intrinsically disordered domains for the broad recognition and neutralization of antimicrobials via the formation of biomolecular condensates.
PubMed: 38633880
DOI: 10.1093/pnasnexus/pgae139 -
Journal of Dairy Science Oct 2016Kelp (Ascophyllum nodosum) is rich in iodine and often fed by organic dairy producers as a mineral supplement to support animal health. A commonly held belief is that...
Kelp (Ascophyllum nodosum) is rich in iodine and often fed by organic dairy producers as a mineral supplement to support animal health. A commonly held belief is that kelp supplementation decreases susceptibility to infectious bovine keratoconjunctivitis due to increased iodine concentrations in tears. Whereas serum and milk iodine concentrations are positively correlated and modulated by oral iodine supplementation, nothing is known about the iodine concentration of tears. Therefore, the 3 objectives of this pilot study were to determine (1) the iodine content of tears, milk, and serum of cows after being fed kelp for 30d; (2) the trace mineral and thyroid status of cows before (d 0) and after being fed kelp for 30d; and (3) the in vitro growth rate of bacteria in tears (Moraxella bovis) or milk (Staphylococcus aureus, Escherichia coli, Streptococcus uberis) collected from cows fed no kelp (d 0) or kelp (d 30). Cows (n=3/treatment) were individually fed 56g of kelp per day (n=3/treatment) or not (n=3/no treatment) for 30 d. Daily feed intake of the TMR was recorded and weekly TMR, kelp, milk, blood and tear samples were collected and analyzed for iodine. The feed samples were pooled and further analyzed for other minerals. On d 0 and 30, liver biopsies and blood samples were collected and analyzed for mineral content and thyroid hormone concentrations, respectively. An inhibition test used milk and tear-soaked plates from kelp-fed cows (d 0 and 30) as well as 1 and 7.5% iodine as positive and distilled water as negative control. As expected, serum iodine concentrations were positively correlated with milk and tear iodine concentrations. Whereas the iodine concentrations in serum increased significantly in the kelp-fed cows during the 30-d study, milk and tear iodine concentrations increased only numerically in these cows compared with the control group. Liver mineral profiles were comparable between groups and generally did not change over the course of the study. Thyroid hormones remained overall within the reference range throughout the trial. Neither milk nor tears from kelp-fed cows inhibited in vitro growth of any of the plated bacteria. In summary, serum iodine concentration was correlated with the iodine concentration in milk and tears and feeding kelp increased only the serum iodine levels of cows in this trial. Bacterial growth was not inhibited in milk and tears of kelp-fed cattle in vitro, and prevention of infectious bovine keratoconjunctivitis would not be based solely on increased iodine concentrations in tears.
Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Ascophyllum; Cattle; Diet; Escherichia coli; Female; Iodine; Milk; Moraxella bovis; Pilot Projects; Staphylococcus aureus; Streptococcus; Tears
PubMed: 27448858
DOI: 10.3168/jds.2015-10810 -
Veterinary Microbiology Aug 2017Infectious bovine keratoconjunctivitis (IBK) is an important production limiting disease in cattle. Moraxella bovis has historically been considered the primary causal...
Infectious bovine keratoconjunctivitis (IBK) is an important production limiting disease in cattle. Moraxella bovis has historically been considered the primary causal agent; however, vaccines have not been consistently shown as effective in controlling disease incidence. The purpose of this study was to examine the bacterial community of calf eyes prior to disease onset using high-throughput sequencing of 16S ribosomal RNA and determine if it was associated with IBK occurrence. The study was designed as a case-control nested within a randomized controlled trial (RCT). Eye swabs were collected from all spring-born calves without clinical signs of IBK (t swabs) on a research farm with a previous history of IBK disease outbreaks. At follow-up or weaning, calves were diagnosed as IBK positive or negative. The lag time between enrollment swabs (t) and IBK diagnosis ranged from approximately one to three months. Cases were randomly selected from IBK positive calves and controls were selected from IBK negative calves (i.e. calves that did not exhibit clinical signs of IBK throughout the course of the RCT). Analysis of the fold-change differences between cases and controls did not reveal large-scale distinctions in bacterial composition. However, principal component analysis suggested bacterial composition differences between calf management groups, which were based on dam parity. Moraxella was found to be among the top ten most abundant genera in our population; however, the difference in abundance was not significant between the cases and controls. No large-scale differences in the bacterial communities of calves that did or did not develop IBK were observed in our population. Nevertheless, it remains unclear whether the "natural" bacterial population of the calf might ultimately impact disease status. Further study is warranted to examine bacterial taxa that were observed to be significantly more abundant in the cases or controls as potential vaccines/therapeutic targets.
Topics: Animals; Bacteria; Cattle; Cattle Diseases; Conjunctivitis, Bacterial; Eye; Keratoconjunctivitis, Infectious
PubMed: 28757034
DOI: 10.1016/j.vetmic.2017.07.003 -
Nucleic Acids Research Apr 2015DNA-dependent T7 RNA polymerase (T7 RNAP) is the most powerful tool for both gene expression and in vitro transcription. By using a Next Generation Sequencing (NGS)...
DNA-dependent T7 RNA polymerase (T7 RNAP) is the most powerful tool for both gene expression and in vitro transcription. By using a Next Generation Sequencing (NGS) approach we have analyzed the polymorphism of a T7 RNAP-generated mRNA pool of the mboIIM2 gene. We find that the enzyme displays a relatively high level of template-dependent transcriptional infidelity. The nucleotide misincorporations and multiple insertions in A/T-rich tracts of homopolymers in mRNA (0.20 and 0.089%, respectively) cause epigenetic effects with significant impact on gene expression that is disproportionally high to their frequency of appearance. The sequence-dependent rescue of single and even double InDel frameshifting mutants and wild-type phenotype recovery is observed as a result. As a consequence, a heterogeneous pool of functional and non-functional proteins of almost the same molecular mass is produced where the proteins are indistinguishable from each other upon ordinary analysis. We suggest that transcriptional infidelity as a general feature of the most effective RNAPs may serve to repair and/or modify a protein function, thus increasing the repertoire of phenotypic variants, which in turn has a high evolutionary potential.
Topics: Amino Acids; DNA Repair; DNA-Directed RNA Polymerases; Epigenesis, Genetic; Frameshifting, Ribosomal; INDEL Mutation; Methyltransferases; Moraxella bovis; Phenotype; RNA Editing; RNA, Messenger; Viral Proteins
PubMed: 25824942
DOI: 10.1093/nar/gkv269 -
Veterinary Microbiology Apr 2019The role of the respiratory bacterial microbiota in the pathogenesis of bovine respiratory disease (BRD) is still not well defined, limiting our understanding of the... (Comparative Study)
Comparative Study
Comparison of the nasopharyngeal bacterial microbiota of beef calves raised without the use of antimicrobials between healthy calves and those diagnosed with bovine respiratory disease.
The role of the respiratory bacterial microbiota in the pathogenesis of bovine respiratory disease (BRD) is still not well defined, limiting our understanding of the disease. Specifically, there is no information on the nasopharyngeal bacterial microbiota of cattle raised without antimicrobials. The objective was to characterize and compare the nasopharyngeal bacterial microbiota in feedlot cattle raised without antimicrobials that were healthy or diagnosed with BRD. Newly-received feedlot cattle (arrival bodyweight ± SD = 218 ± 37 kg) with BRD (n = 82) and pen-matched controls (n = 82) were clinically examined and sampled by deep nasopharyngeal swab (DNS). DNA was extracted from each DNS and the 16S rRNA gene (V4) was sequenced. Alpha and beta diversity were compared between health groups and among 3 days-on-feed (DOF) groups (group A = 3-12 DOF; group B = 13-20 DOF; group C = 21-44 DOF). Observed species richness was lower (P = 0.031) in cattle with BRD compared to healthy ones. Both health status (P = 0.007) and DOF groups (P < 0.001) were sources of variation in microbiota composition. Differences between health groups were driven by multiple sequence variants, including Mycoplasma bovis, Histophilus somni, and several Moraxella spp. Notably, M. bovis was more frequently identified in cattle with BRD. M. bovis identification was also higher in cattle sampled at later DOF. The increased identification of M. bovis in cattle with BRD reaffirms a potentially significant role for this bacterium in respiratory health.
Topics: Animals; Anti-Bacterial Agents; Bovine Respiratory Disease Complex; Cattle; Cattle Diseases; DNA, Bacterial; Housing, Animal; Male; Microbiota; Moraxella; Mycoplasma bovis; Nasopharynx; RNA, Ribosomal, 16S; Red Meat
PubMed: 30955824
DOI: 10.1016/j.vetmic.2019.02.030 -
Acta Crystallographica. Section F,... Oct 2014Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the genera Neisseria...
Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the genera Neisseria and Moraxella have evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogen Neisseria meningitidis is reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogen Moraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin.
Topics: Animals; Bacterial Proteins; Carrier Proteins; Cattle; Crystallography, X-Ray; Host-Pathogen Interactions; Humans; Hydrogen Bonding; Iron; Lactoferrin; Models, Molecular; Neisseria meningitidis; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Quaternary; Protein Structure, Secondary
PubMed: 25286931
DOI: 10.1107/S2053230X14019372 -
Microbiology Resource Announcements Aug 2018We report here the complete closed genome sequence of Moraxella bovis strain Epp-63 (300) (Epp63). This strain was isolated from an infectious bovine...
We report here the complete closed genome sequence of Moraxella bovis strain Epp-63 (300) (Epp63). This strain was isolated from an infectious bovine keratoconjunctivitis (IBK) case in 1963. Since then, Epp63 has been used extensively for IBK research. Consequently, the genome sequence of Epp63 should help elucidate IBK host-pathogen interactions.
PubMed: 30533917
DOI: 10.1128/MRA.01004-18 -
The Analyst Jan 2016Abnormal DNA methylation patterns caused by altered DNA methyltransferase (MTase) activity are closely associated with cancer. Herein, using DNA adenine methylation...
Abnormal DNA methylation patterns caused by altered DNA methyltransferase (MTase) activity are closely associated with cancer. Herein, using DNA adenine methylation methyltransferase (Dam MTase) as a model analyte, we designed an allosteric molecular beacon (aMB) for sensitive detection of Dam MTase activity. When the specific site in an aMB is methylated by Dam MTase, the probe can be cut by the restriction nuclease DpnI to release a fluorophore labeled aptamer specific for streptavidin (SA) which will bind to SA beads to generate highly fluorescent beads for easy signal readout by a microscope or flow cytometer. However, aMBs maintain a hairpin structure without the binding ability to SA beads in the absence of Dam MTase, leading to weakly fluorescent SA beads. Unlike the existing signal amplified assays, our method is simpler and more convenient. The high performance of the aptamer and the easy bead separation process make this probe superior to other methods for the detection of MTase in complex biological systems. Overall, the proposed method with a detection limit of 0.57 U mL(-1) for Dam MTase shows great potential for further applications in the detection of other MTases, screening of MTase inhibitors, and early diagnosis of cancer.
Topics: Allosteric Regulation; Drug Evaluation, Preclinical; Enzyme Assays; Fluorouracil; Moraxella bovis; Oligonucleotide Probes; Site-Specific DNA-Methyltransferase (Adenine-Specific)
PubMed: 26478921
DOI: 10.1039/c5an01763g