- The influence of cationic dendrimers on antibacterial activity of phage endolysin against P. aeruginosa cells. [Journal Article]
- BCBioorg Chem 2019 Jul 11; 91:103121
- Nowadays, the researchers make a big effort to find new alternatives to overcome bacterial drug resistance. One option is the application of bacteriophage endolysins enable to degrade peptidoglycan (…
Nowadays, the researchers make a big effort to find new alternatives to overcome bacterial drug resistance. One option is the application of bacteriophage endolysins enable to degrade peptidoglycan (PG) what in consequence leads to bacterial cell lysis. In this study we examine phage KP27 endolysin mixed with poly(propyleneimine) dendrimers to evaluate an antimicrobial effect against Pseudomonas aeruginosa. Polycationic compounds destabilize bacterial outer membrane (OM) helping endolysins to gain access to PG. We found out that not only bacterial lipopolysaccharide (LPS) is the main hindrance for highly charged cationic dendrimers to disrupt OM and make endolysin reaching the target but also the dendrimer surface modification. The reduction of a positive charge and concentration in maltose poly(propyleneimine) dendrimers significantly increased an antibacterial effect of endolysin. The application of recombinant lysins against Gram-negative bacteria is one of the future therapy options, thus OM permeabilizers such as cationic dendrimers may be of high interest to be combined with PG-degrading enzymes.
- Analysis of genetic recombination and the pan-genome of a highly recombinogenic bacteriophage species. [Journal Article]
- MGMicrob Genom 2019 Jul 16
- Bacteriophages are the most prevalent biological entities impacting on the ecosystem and are characterized by their extensive diversity. However, there are two aspects of phages that have remained la…
Bacteriophages are the most prevalent biological entities impacting on the ecosystem and are characterized by their extensive diversity. However, there are two aspects of phages that have remained largely unexplored: genetic flux by recombination between phage populations and characterization of specific phages in terms of the pan-genome. Here, we examined the recombination and pan-genome in Helicobacter pylori prophages at both the genome and gene level. In the genome-level analysis, we applied, for the first time, chromosome painting and fineSTRUCTURE algorithms to a phage species, and showed novel trends in inter-population genetic flux. Notably, hpEastAsia is a phage population that imported a higher proportion of DNA fragments from other phages, whereas the hpSWEurope phages showed weaker signatures of inter-population recombination, suggesting genetic isolation. The gene-level analysis showed that, after parameter tuning of the prokaryote pan-genome analysis program, H. pylori phages have a pan-genome consisting of 75 genes and a soft-core genome of 10 genes, which includes genes involved in the lytic and lysogenic life cycles. Quantitative analysis of recombination events of the soft-core genes showed no substantial variation in the intensity of recombination across the genes, but rather equally frequent recombination among housekeeping genes that were previously reported to be less prone to recombination. The signature of frequent recombination appears to reflect the host-phage evolutionary arms race, either by contributing to escape from bacterial immunity or by protecting the host by producing defective phages.
- Isolation and Purification of Proteus mirabilis Bacteriophage. [Journal Article]
- MMMethods Mol Biol 2019; 2021:231-240
- Bacteriophages specifically targeting different strains of bacteria can be isolated from urban sewage using properly modified enrichment techniques. This chapter provides a detailed protocol for isol…
Bacteriophages specifically targeting different strains of bacteria can be isolated from urban sewage using properly modified enrichment techniques. This chapter provides a detailed protocol for isolation of Proteus mirabilis-specific bacteriophages. Briefly, prefiltered sewage is mixed with double-concentrated tryptic soy broth containing the target strain and incubated. Subsequently, the suspension is spread on phage nutrient agar, and if needed, supplemented with swarming motility inhibitor, for the induction of bacterial growth and phage multiplication. Phages infecting bacteria are identified by plaques (patches of dead bacteria) in the confluent bacterial lawn. A pure phage preparation is obtained by cutting out a single plaque from a double-layer agar plate and subsequent virus propagation five times on a given P. mirabilis strain.
- External lysis of Escherichia coli by a bacteriophage endolysin modified with hydrophobic amino acids. [Journal Article]
- AEAMB Express 2019 Jul 15; 9(1):106
- Drug-resistant bacteria are a serious threat to global public health. Gram-positive bacterial endolysin preparations have been successfully used to fight Gram-positive bacteria as a novel antimicrobi…
Drug-resistant bacteria are a serious threat to global public health. Gram-positive bacterial endolysin preparations have been successfully used to fight Gram-positive bacteria as a novel antimicrobial replacement strategy. However, Gram-negative bacterial phage endolysins cannot be applied directly to destroy Gram-negative strains due to the externally inaccessible peptidoglycan layer of the cell wall; this has seriously hampered the development of endolysin-like antibiotics against Gram-negative bacteria. In this study, 3-12 hydrophobic amino acids were successively added to the C-terminus of Escherichia coli phage endolysin Lysep3 to create five different hydrophobic-modified endolysins. Compared with endogenous Lysep3, endolysins modified with hydrophobic amino acids surprisingly could kill E. coli from outside of the cell at the appropriate pH and endolysin concentration. The lysis ability of modified endolysins were enhanced with increasing numbers of hydrophobic amino acids at the C-terminus of endolysin. Thus, these findings demonstrate that the enhancement of hydrophobicity at the C-terminus enables the endolysin to act upon E. coli from the outside, representing a novel method of lysing Gram-negative antibiotic-resistant bacteria.
- Identification of serum biomarkers for Systemic Lupus Erythematosus using a library of phage displayed random peptides and deep sequencing. [Journal Article]
- MCMol Cell Proteomics 2019 Jul 15
- Systemic lupus erythematosus (SLE) is one of the most serious autoimmune diseases, characterized by highly diverse clinical manifestations. Biomarker is still needed for the accurate diagnostics. SLE…
Systemic lupus erythematosus (SLE) is one of the most serious autoimmune diseases, characterized by highly diverse clinical manifestations. Biomarker is still needed for the accurate diagnostics. SLE serum autoantibodies were discovered and validated using serum samples from independent sample cohorts encompassing 306 participants divided into three groups, i.e., healthy, SLE patients, and other autoimmune related diseases. To discover biomarkers for SLE, a phage displayed random peptide library (Ph.D. 12) and deep sequencing were applied to screen specific autoantibodies in a total of 100 serum samples from 50 SLE patients and 50 healthy controls. A statistical analysis protocol was setup for the identification of peptides as potential biomarkers. For validation, ten peptides were analyzed using enzyme linked immunosorbent assays (ELISA). As a result, four peptides (SLE2018Val001, SLE2018Val002, SLE2018Val006 and SLE2018Val008) were discovered with high diagnostic power to differentiate SLE patients from health controls. Among them, two peptides, i.e., SLE2018Val001 and SLE2018Val002 were confirmed between SLE with other autoimmune patients. The procedure we established could be easily adopted for the identification of autoantibodies as biomarkers for many other diseases.
- Nitrogen sourcing during viral infection of marine cyanobacteria. [Journal Article]
- PNProc Natl Acad Sci U S A 2019 Jul 15
- The building blocks of a virus derived from de novo biosynthesis during infection and/or catabolism of preexisting host cell biomass, and the relative contribution of these 2 sources has important co…
The building blocks of a virus derived from de novo biosynthesis during infection and/or catabolism of preexisting host cell biomass, and the relative contribution of these 2 sources has important consequences for understanding viral biogeochemistry. We determined the uptake of extracellular nitrogen (N) and its biosynthetic incorporation into both virus and host proteins using an isotope-labeling proteomics approach in a model marine cyanobacterium Synechococcus WH8102 infected by a lytic cyanophage S-SM1. By supplying dissolved N as 15N postinfection, we found that proteins in progeny phage particles were composed of up to 41% extracellularly derived N, while proteins of the infected host cell showed almost no isotope incorporation, demonstrating that de novo amino acid synthesis continues during infection and contributes specifically and substantially to phage replication. The source of N for phage protein synthesis shifted over the course of infection from mostly host derived in the early stages to more medium derived later on. We show that the photosystem II reaction center proteins D1 and D2, which are auxiliary metabolic genes (AMGs) in the S-SM1 genome, are made de novo during infection in an apparently light-dependent manner. We also identified a small set of host proteins that continue to be produced during infection; the majority are homologs of AMGs in S-SM1 or other viruses, suggesting selective continuation of host protein production during infection. The continued acquisition of nutrients by the infected cell and their utilization for phage replication are significant for both evolution and biogeochemical impact of viruses.
- Phage N15 protelomerase resolves its tos recognition site into hairpin telomeres within mammalian cells. [Journal Article]
- ABAnal Biochem 2019 Jul 12; :113361
- Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia …
Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human β-globin gene expressed for at least 120 h in non-β-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.
- Mimicking the endothelial glycocalyx through the supramolecular presentation of hyaluronan on patterned surfaces. [Journal Article]
- FDFaraday Discuss 2019 Jul 15
- The glycocalyx is the immediate pericellular matrix that surrounds many cell types, including endothelial cells (ECs), and is typically composed of glycans (glycosaminoglycans, proteoglycans, and gly…
The glycocalyx is the immediate pericellular matrix that surrounds many cell types, including endothelial cells (ECs), and is typically composed of glycans (glycosaminoglycans, proteoglycans, and glycoproteins). The endothelial glycocalyx is rich in hyaluronic acid (HA), which plays an important role in the maintenance of vascular integrity, although fundamental questions about the precise molecular regulation mechanisms remain unanswered. Here, we investigate the contribution of HA to the regulation of endothelial function using model surfaces. The peptide sequence GAHWQFNALTVR, previously identified by phage display with strong binding affinity for HA and named Pep-1, was thiolated at the N-terminal to form self-assembled monolayers (SAMs) on gold (Au) substrates, and microcontact printing (μCP) was used to develop patterned surfaces for the controlled spatial presentation of HA. Acetylated Pep-1 and a scrambled sequence of Pep-1 were used as controls. The SAMs and HA-coated surfaces were characterized by X-ray photoelectron spectroscopy (XPS), contact angle measurements, and quartz crystal microbalance with dissipation (QCM-D) monitoring, which confirmed the binding and presence of thiolated peptides on the Au surfaces and the deposition of HA. Fluorescence microscopy showed the localization of fluorescently labelled HA only on areas printed with Pep-1 SAMs. Cell culture studies demonstrated that low molecular weight HA improved the adhesion of human umbilical vein endothelial cells (HUVECs) to the substrate and also stimulated their migration. This research provides insight into the use of SAMs for the controlled presentation of HA with defined size in cultures of HUVECs to study their functions.
- Porphyromonas gingivalis and its CRISPR-Cas system. [Review]
- JOJ Oral Microbiol 2019; 11(1):1638196
- The clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated proteins (Cas) are immune systems in prokaryotes present in most Bacteria and Archaea. They provide adapti…
The clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated proteins (Cas) are immune systems in prokaryotes present in most Bacteria and Archaea. They provide adaptive immunity against foreign elements such as bacteriophages/viruses, plasmids and transposons. During immunization a small sequence of foreign DNA, a so-called spacer is integrated into the CRISPR locus in the host cell. Spacers are then transcribed into small RNA guides that direct cleavage of foreign DNA by Cas nucleases. Immunization through spacer acquisition is transferred vertically to the progeny. It is possible that this genetic immune system of bacteria participates in modulating the microbiome of 'chronic' periodontitis, in which Porphyromonas gingivalis has been identified as a keystone pathogen causing microbial dysbiosis. An in-depth review of our current knowledge on the CRISPR-Cas systems in P. gingivalis is given in this paper with the attempt to understand how this anaerobic bacterium may protect itself in the periodontal pocket where bacteriophages are abundant and even out-number bacteria.
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- Identification and Characterization of Mutations in Ubiquitin Required for Non-covalent Dimer Formation. [Journal Article]
- SStructure 2019 Jul 05
- Ubiquitin (Ub) is a small protein that post-translationally modifies a variety of substrates in eukaryotic cells to modulate substrate function. The ability of Ub to interact with numerous protein do…
Ubiquitin (Ub) is a small protein that post-translationally modifies a variety of substrates in eukaryotic cells to modulate substrate function. The ability of Ub to interact with numerous protein domains makes Ub an attractive scaffold for engineering ubiquitin variants (UbVs) with high target specificity. Previously, we identified a UbV that formed a non-covalent stable dimer via a β-strand exchange, and in the current work we identified and characterized the minimal substitutions in the primary sequence of Ub required to form a higher ordered complex. Using solution angle scattering and X-ray crystallography, we show that a single substitution of residue Gly10 to either Ala or Val is sufficient to convert Ub from a monomer to a dimer. We also investigate contributions to dimer formation by the residues in the surrounding sequence. These results can be used to develop next-generation phage-display libraries of UbVs to engineer new interfaces for protein recognition.