Bacteriophage Lambda Research Articles

Role of DNA sliding friction and nonequilibrium dynamics in viral DNA ejection and packaging.

In many viruses DNA is packaged to high densities, resulting in high “internal force” inside the capsid shell. This helps drive DNA ejection through the portal channel when viruses infect cells. Imaging of the in vitro ejection of fluorescent-labeled DNA from phages T5 and lambda found that it was initially slow despite the internal force being highest when the capsid is full. This was attributed to hydrodynamic drag on the tightly-packed DNA, but this hypothesis has not been tested. Here we introduce a new method for probing the DNA mobility by using optical tweezers to pull it out from phage phi29 capsids. This allows us to measure the dynamics with higher precision, avoid using DNA-binding dyes that alter the dynamics, and apply variable forces. We measure even slower DNA exit than reported previously, a steeper, exponential velocity increase as the first 20% of the genome length exits, and find that exit velocity is not proportional to driving force as expected for hydrodynamic friction. Rather, our findings are consistent with theoretical predictions for DNA-DNA sliding friction. We also observe large heterogeneity in the exit dynamics and stochastic pausing, indicating that ejection is a nonequilibrium process, in qualitative agreement with simulations. Features of the pausing suggest it is related to the phenomenon of “clogging” in soft-matter physics, which occurs in the flow of disordered materials through constrictions. Our results indicate that intramolecular friction and nonequilibrium pausing control the kinetics of ejection but that this friction does not significantly affect DNA packaging.

Co-infecting phages impede each other's entry into the cell.

Bacteriophage lambda decides whether to pursue the lysogenic pathway based on the number of phages co-infecting the cell (multiplicity of infection, MOI). This strategy is believed to be a way for phages to infer the abundance of available bacterial hosts. However, the single-cell assessment would reliably reflect the environment only if the intracellular number of phages is proportional to the extracellular ratio of phages to bacteria. It is unknown whether this premise is true. Here, we show that co-infecting phages impede each other's entry into the cell, thus reducing the effective MOI that drives the decision. Using fluorescent labeling of phage capsids and DNA, we found that the intracellular number of phage genomes scales sublinearly with the number of phages adsorbed to the cell. Time-lapse measurements and mathematical modeling revealed that the entry probability per phage decreases with MOI, suggesting mutual interference between co-infecting phages at the entry stage. This effect likely reflects the phage-induced perturbations to the envelope integrity, ionic concentrations in the cytoplasm, and membrane potential of the infected cell. Since these perturbations are known to be MOI dependent, we reasoned that, the more phages infect the same cell, the more impeded their entries are. The degree to which phage entry is impeded successfully accounts for the different lysogenization frequencies measured in different infection media and recapitulates the heterogeneity in infection outcomes observed in individual cells. Altogether, our findings demonstrate the role played by entry dynamics in driving cell fate choice following lambda infection.

KCK-tag-induced quantitative and qualitative property changes of DNA-binding protein ParB.

Lysine-cysteine-lysine (KCK) tags are commonly employed to label proteins with maleimide-fluorescent dye conjugates to enhance dye-labeling efficiency. In our study, we systematically quantified the actions of a DNA-binding CTPase ParB protein and its KCK-tagged versions on flow-stretched lambda DNA using an in vitro single-molecule DNA flow-stretching assay. We measured DNA compaction rates in a series of experimental conditions, including the presence of the specific binding sequence (parS), CTP, or its non-hydrolyzable analog CTPγS. We found that KCK tag (either at N- or C-terminus) artificially increased the compaction rates of flow-stretched DNAs and even reversed ParB proteins’ response to these experimental conditions. DNA flow-stretching experiments with fluorescently-labeled ParB protein showed that the appended KCK-tag enhances protein loading onto a DNA. Furthermore, DNA compaction by ParB protein with a negatively-charged glutamate-cysteine-glutamate (ECE) tag was inefficient compared with its non-tagged ParB protein. These results suggest that the quantitative and qualitative changes of the KCK-tagged proteins were, at least in part, derived from electrostatic interactions between positively-charged KCK-tag and negatively-charged DNA backbone. Interestingly, we found that the KCK-tagged ParB proteins do not lead to any noticeable changes from in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays. It implies that a lack of perturbation in in vivo assays does not necessarily guarantee intact in vitro results. Despite the small size of KCK-tag, when it is used for fluorescent-dye labeling on a protein, rigorous test must be accompanied to ensure absence of any in vitro artifacts.

Probing the bridging behaviour of the Rok protein from Bacillus subtilis with quadruple-trap optical tweezers.

Bacteria densely organize their genome in the form of a nucleoid stored in the cytoplasm. This compact, functional form of DNA is achieved by the use of nucleoid-associated proteins (NAPs), proteins that play a role in DNA organization as well as regulating gene expression. The various NAPs found across bacterial species are classed based on shared functions with well-studied NAPs (H-NS, HU, IHF and Fis). In Bacillus subtilis, the NAP Rok shares functional similarities with H-NS and is proposed to be a H-NS-like protein, however, some fundamental differences in binding activity and response to physio-chemical changes have been observed. The aim of this study is to investigate the structural, kinetic and dynamic attributes of Rok-DNA binding in order to better understand the role of Rok as a NAP in Bacillus subtilis. Dual-trap optical tweezer experiments, in which Rok was allowed to bind to one double stranded lambda DNA molecule, showed that Rok compacts DNA while no impact on the mechanical properties of DNA was observed. Using quadruple-trap optical tweezer experiments, Rok was allowed to bind to two double stranded lambda DNA molecules. In this tweezer configuration, shearing and unzipping experiments were used to probe the interaction of Rok with DNA and revealed formation of DNA-Rok-DNA bridges. We found that the Rok-mediated DNA compaction and bridging is likely not due to protein-protein interaction suggesting that one Rok protein is able to bind two DNA molecules or two sites on a single DNA molecule. Additionally, DNA-Rok bonds withstood considerably high forces before rupturing. Our results combined with recent literature suggest that Rok might not have a similar function to H-NS in Bacillus subtilis but that it rather acts as a stabilizer facilitating the function of other proteins.

A methodology for using Lambda phages as a proxy for pathogen transmission in hospitals

One major concern in hospitalized patients is acquiring infections from pathogens borne on surfaces, patients, and healthcare workers (HCWs). Fundamental to controlling healthcare-associated infections is identifying the sources of pathogens, monitoring the processes responsible for their transmission, and evaluating the efficacy of the procedures employed for restricting their transmission. To present a method using the bacteriophage Lambda (λ) to achieve these ends. Defined densities of multiple genetically marked λ phages were inoculated at known hotspots for contamination on high-fidelity mannequins. HCWs then entered a pre-sanitized simulated hospital room and performed a series of patient care tasks on the mannequins. Sampling occurred on the scrubs and hands of the HCWs, as well as previously defined high-touch surfaces in hospital rooms. Following sampling, the rooms were decontaminated using procedures demonstrated to be effective. Following the conclusion of the simulation, the samples were tested for the presence, identity, and densities of these λ phages. The data generated enabled the determination of the sources and magnitude of contamination caused by the breakdown of established infection prevention practices by HCWs. This technique enabled the standardized tracking of multiple contaminants during a single episode of patient care. Unlike other biological surrogates, λ phages are susceptible to common hospital disinfectants, and allow for a more accurate evaluation of pathogen transmission. Whereas our application of these methods focused on healthcare-associated infections and the role of HCW behaviours in their spread, these methods could be employed for identifying the sources and sites of microbial contamination in other settings.

Genome Analysis of Two Lichen Bacteriobionts, <i>Lichenibacterium</i> <i>ramalinae</i> and <i>Lichenibacterium minor</i>: Factors of Virulence and Adaptation

Successful invasion and adaptation of bacteria into lichen symbiosis requires the involvement of a number of physiological mechanisms that regulate interactions between bacteriobionts, mycobionts and algobionts and ensure the optimization of thallus growth and development. The genomes of two lichen bacteriobionts Lichenibacterium ramalinae and L. minor were analysed to identify those factors. No nodulation genes traditional for rhizobia were found. TIGR02302 protein has been suggested to have a similar function. Genes encoding proteins associated with the implementation of planktonic and biofilm phenotypes – flagellins and pilins – have been found. These genes have a high level of similarity with the genes of plant symbiont bacteria. Genetic determinants of virulence factors such as locus B invasion proteins, Integration host factor (IHF), sensory histidine kinases, siderophore receptor and transport proteins and monooxygenase of antibiotic synthesis (Abm) were found in the genomes of the studied bacteria. The listed genes have similarities with those in organisms forming associations of different degrees of association with plants. Based on the data obtained, the assumption of a priority association of bacteria of the Lichenibacteriaceae family with green lichen algae was suggested.

Thermodynamics of bacteria-phage interactions T4 and Lambda bacteriophages, and E. coli can coexist in natural ecosystems due to the ratio of their Gibbs energies of biosynthesis

The model of T4 phage, Lambda phage, and E. coli is often used in research on virus-host interactions. This paper reports for the first time the thermodynamic driving force of biosynthesis, catabolism and metabolism for the three organisms, on the M9 medium. Moreover, the influence of activities of nutrients and metabolic products is analyzed. All three organisms were found to have very similar Gibbs energies of metabolism. Moreover, since they share the same catabolism, their Gibbs energies of catabolism are identical. However, Gibbs energies of biosynthesis differ. The calculated thermodynamic properties have been used to explain the coexistence of both bacteria and phages in a dynamic equilibrium in natural ecosystems.

Integration Host Factor Binds DNA Holliday Junctions.

Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.

EGFR-targeted bacteriophage lambda penetrates model stromal and colorectal carcinoma tissues, is taken up into carcinoma cells, and interferes with 3-dimensional tumor formation.

Colorectal cancer and other adult solid cancers pose a significant challenge for successful treatment because the tumor microenvironment both hinders the action of conventional therapeutics and suppresses the immune activities of infiltrating leukocytes. The immune suppression is largely the effect of enhanced local mediators such as purine nucleosides and eicosanoids. Genetic approaches have the promise of interfering with these mechanisms of local immunosuppression to allow both intrinsic and therapeutic immunological anticancer processes. Bacterial phages offer a novel means of enabling access into tissues for therapeutic genetic manipulations. We generated spheroids of fibroblastic and CRC cancer cells to model the 3-dimensional stromal and parenchymal components of colorectal tumours. We used these to examine the access and effects of both wildtype (WT) and epidermal growth factor (EGF)-presenting bacteriophage λ (WT- λ and EGF-λ) as a means of delivery of targeted genetic interventions in solid cancers. We used both confocal microscopy of spheroids exposed to AF488-tagged phages, and the recovery of viable phages as measured by plaque-forming assays to evaluate access; and measures of mitochondrial enzyme activity and cellular ATP to evaluate the outcome on the constituent cells. Using flourescence-tagged derivatives of these bacteriophages (AF488-WT-λ and AF488-EGF-λ) we showed that phage entry into these tumour microenvironments was possible and that the EGF ligand enabled efficient and persistent uptake into the cancer cell mass. EGF-λ became localized in the intracellular portion of cancer cells and was subjected to subsequent cellular processing. The targeted λ phage had no independent effect upon mature tumour spheroids, but interfered with the early formation and growth of cancer tissues without the need for addition of a toxic payload, suggesting that it might have beneficial effects by itself in addition to any genetic intervention delivered to the tumour. Interference with spheroid formation persisted over the duration of culture. We conclude that targeted phage technology is a feasible strategy to facilitate delivery into colorectal cancer tumour tissue (and by extension other solid carcinomas) and provides an appropriate delivery vehicle for a gene therapeutic that can reduce local immunosuppression and/or deliver an additional direct anticancer activity.

The bacteriophage lambda integrase catalytic domain can be modified to act with the regulatory domain as a recombination-competent binary recombinase

Site-specific recombinase Int mediates integration of the bacteriophage λ genome into the E. coli chromosome. Integration occurs once the Int tetramer, assisted by the integration host factor IHF, forms the intasome, a higher order structure, within which Int, a heterobivalent protein, interacts with two non-homologous DNA sequences: the core recombination sites and the accessory arm sites. The binding to these sites is mediated by the catalytic C-terminal domain (CTD) and the regulatory N-terminal domain (NTD) of Int, respectively. Within Int, the NTD can activate or inhibit the recombination activity of the CTD depending on whether the NTD is bound to the arm sites. The CTD alone cannot mediate recombination, and even when the NTD and the CTD are mixed together as individual polypeptides, the NTD cannot trigger recombination in the CTD. In this work, we set to determine what modifications can unlock the recombination activity in the CTD alone and how the CTD can be modified to respond to recombination-triggering signals from the NTD. For this, we performed a series of genetic analyses which showed that a single mutation that stabilizes the CTD on DNA, E174K, allows the CTD to recombine the core DNA sequences. When the NTD is paired with the CTD(E174K) that also bears a short polypeptide from the C-terminus of the NTD, the resulting binary Int can recombine arm-bearing substrates. Our results provide insights into the molecular basis of the regulation of the Int activity and suggest how binary recombinases of the integrase type can be engineered.

Topological examination of the bacteriophage lambda S holin by EPR spectroscopy

The S protein from bacteriophage lambda is a three-helix transmembrane protein produced by the prophage which accumulates in the host membrane during late gene expression. It is responsible for the first step in lysing the host cell at the end of the viral life cycle by multimerizing together to form large pores which permeabilize the host membrane to allow the escape of virions. Several previous studies have established a model for the assembly of holin into functional holes and the manner in which they pack together, but it is still not fully understood how the very rapid transition from monomer or dimer to multimeric pore occurs with such precise timing once the requisite threshold is reached. Here, site-directed spin labeling with a nitroxide label at introduced cysteine residues is used to corroborate existing topological data from a crosslinking study of the multimerized holin by EPR spectroscopy. CW-EPR spectral lineshape analysis and power saturation data are consistent with a three-helix topology with an unstructured C-terminal domain, as well as at least one interface on transmembrane domain 1 which is exposed to the lumen of the hole, and a highly constrained steric environment suggestive of a tight helical packing interface at transmembrane domain 2.

Machine learning approach for label-free rapid detection and identification of virus using Raman spectra

ObjectiveThe objective of this study was to develop a robust method for rapid detection and identification of the virus based on Raman spectroscopy combined with machine learning approach. MethodsWe have used saliva spiked with different bacterial viruses such as P1 Phage, M13 Phage, and Lambda Phage, for demonstrating the utility of this method for virus detection. The Raman spectra collected from a large number of independent samples, each of different phages with and without saliva were used to train a supervised convolutional neural network (CNN) with its hyperparameters optimized by Bayesian optimization. The CNN method was not only able to detect the presence of a phage but was also able to identify the phage type using unprocessed Raman spectra having high noise. In addition, a semi-supervised auto-encoder was utilized for differentiating healthy saliva from saliva spiked with phages thereby making it possible to detect the presence of phages in saliva samples. ResultsThe CNN could identify the virus with an accuracy of 98.86% based on ten-fold cross-validation, precision of 98.8%, recall of 98.7%, and F1 score of 98.7%. The area under the curve of receiver operating characteristic curve was 0.99. Autoencoder was capable of differentiating healthy saliva from the virus spiked saliva with an accuracy of 99.7% in a semi-supervised manner. Thus, Raman spectroscopy coupled with machine learning approach was able to directly detect and identify the virus without consuming time for lengthy sample processing. ConclusionA robust method based on Raman spectroscopy coupled with machine learning may be capable of detection and identification of the virus even from the signal with low intensity and high noise. This label-free method is fast, sensitive, specific, and cost effective.

Chromatin Liquid-Liquid Phase Separation (LLPS) Is Regulated by Ionic Conditions and Fiber Length.

The dynamic regulation of the physical states of chromatin in the cell nucleus is crucial for maintaining cellular homeostasis. Chromatin can exist in solid- or liquid-like forms depending on the surrounding ions, binding proteins, post-translational modifications and many other factors. Several recent studies suggested that chromatin undergoes liquid–liquid phase separation (LLPS) in vitro and also in vivo; yet, controversial conclusions about the nature of chromatin LLPS were also observed from the in vitro studies. These inconsistencies are partially due to deviations in the in vitro buffer conditions that induce the condensation/aggregation of chromatin as well as to differences in chromatin (nucleosome array) constructs used in the studies. In this work, we present a detailed characterization of the effects of K+, Mg2+ and nucleosome fiber length on the physical state and property of reconstituted nucleosome arrays. LLPS was generally observed for shorter nucleosome arrays (15-197-601, reconstituted from 15 repeats of the Widom 601 DNA with 197 bp nucleosome repeat length) at physiological ion concentrations. In contrast, gel- or solid-like condensates were detected for the considerably longer 62-202-601 and lambda DNA (~48.5 kbp) nucleosome arrays under the same conditions. In addition, we demonstrated that the presence of reduced BSA and acetate buffer is not essential for the chromatin LLPS process. Overall, this study provides a comprehensive understanding of several factors regarding chromatin physical states and sheds light on the mechanism and biological relevance of chromatin phase separation in vivo.

Versatile seamless DNA vector production in E. coli using enhanced phage lambda integrase.

Seamless DNA vectors derived from bacterial plasmids are devoid of bacterial genetic elements and represent attractive alternatives for biomedical applications including DNA vaccines. Larger scale production of seamless vectors employs engineered Escherichia coli strains in order to enable tightly regulated expression of site-specific DNA recombinases which precisely delete unwanted sequences from bacterial plasmids. As a novel component of a developing lambda integrase genome editing platform, we describe here strain MG1655-ISC as a means to easily produce different scales of seamless vectors, ranging in size from a few hundred base pairs to more than ten kilo base pairs. Since we employed an engineered lambda integrase that is able to efficiently recombine pairs of DNA crossover sites that differ in sequence, the resulting seamless vectors will be useful for subsequent genome editing in higher eukaryotes to accommodate variations in target site sequences. Future inclusion of single cognate sites for other genome targeting systems could enable modularity. These features, together with the demonstrated simplicity of in vivo seamless vector production, add to their utility in the biomedical space.

Behavioral dynamics of bacteriophage gene regulatory networks.

We present hybrid system-based gene regulatory network models for lambda, HK022, and Mu bacteriophages together with dynamics analysis of the modeled networks. The proposed lambda phage model LPH2 is based on an earlier work and incorporates more recent biological assumptions about the underlying gene regulatory mechanism, HK022, and Mu phage models are new. All three models provide accurate representations of experimentally observed lytic and lysogenic behavioral cycles. Importantly, the models also imply that lysis and lysogeny are the only stable behaviors that can occur in the modeled networks. In addition, the models allow to derive switching conditions that irrevocably lead to either lytic or lysogenic behavioral cycle as well as constraints that are required for their biological feasibility. For LPH2 model the feasibility constraints place two mutually independent requirements on comparative order of cro and cI protein binding site affinities. However, HK022 model, while broadly similar, does not require any of these constraints. Biologically very different lysis-lysogeny switching mechanism of Mu phage is also accurately reproduced by its model. In general the results show that hybrid system model (HSM) hybrid system framework can be successfully applied to modeling small ([Formula: see text] gene) regulatory networks and used for comprehensive analysis of model dynamics and stable behavior regions.

Application of an Ultrasonic Nebulizer Closet in the Disinfection of Textiles and Footwear.

The emergence of the coronavirus disease 2019 (COVID-19) pandemic highlighted the importance of disinfection processes in health safety. Textiles and footwear have been identified as vectors for spreading infections. Therefore, their disinfection can be crucial to controlling pathogens’ dissemination. The present work aimed to evaluate the effectiveness of a commercial disinfectant aerosolized by an ultrasonic nebulizer closet as an effective method for disinfecting textiles and footwear. The disinfection was evaluated in three steps: suspension tests; nebulization in a 0.08 m3 closet; nebulization in the upscaled 0.58 m3 closet. The disinfection process of textiles and footwear was followed by the use of bacteriophages, bacterial spores, and bacterial cells. The disinfection in the 0.58 m3 closet was efficient for textiles (4 log reduction) when bacteriophage Lambda, Pseudomonas aeruginosa, and Bacillus subtilis were used. The footwear disinfection was achieved (4 log reduction) in the 0.08 m3 closet for Escherichia coli and Staphylococcus aureus. Disinfection in an ultrasonic nebulization closet has advantages such as being quick, not wetting, being efficient on porous surfaces, and is performed at room temperature. Ultrasonic nebulization disinfection in a closet proves to be useful in clothing and footwear stores to prevent pathogen transmission by the items’ widespread handling.

A Robust One-Step Recombineering System for Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) can cause severe diarrheic in humans. To improve therapy options, a better understanding of EHEC pathogenicity is essential. The genetic manipulation of EHEC with classical one-step methods, such as the transient overexpression of the phage lambda (λ) Red functions, is not very efficient. Here, we provide a robust and reliable method for increasing recombineering efficiency in EHEC based on the transient coexpression of recX together with gam, beta, and exo. We demonstrate that the genetic manipulation is 3–4 times more efficient in EHEC O157:H7 EDL933 Δstx1/2 with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in Escherichia coli K-12 MG1655. Coexpression of recX did not enhance the Gam-mediated inhibition of sparfloxacin-mediated SOS response. Therefore, the additional inhibition of the RecFOR pathway rather than a stronger inhibition of the RecBCD pathway of SOS response induction might have resulted in the increased recombineering efficiency by indirectly blocking phage induction. Even though additional experiments are required to unravel the precise mechanistic details of the improved recombineering efficiency, we recommend the use of our method for the robust genetic manipulation of EHEC and other prophage-carrying E. coli isolates.

Experimental evolution of Vibrio cholerae identifies hypervesiculation as a way to increase motility in the presence of polymyxin B.

Vibrio cholerae includes strains responsible for the cholera disease and is a natural inhabitant of aquatic environments. V. cholerae possesses a unique polar flagellum essential for motility, adhesion, and biofilm formation. In a previous study, we showed that motility and biofilm formation are altered in the presence of subinhibitory concentrations of polymyxin B in V. cholerae O1 and O139. In this study, we performed an experimental evolution to identify the genes restoring the motility in the presence of a subinhibitory concentration of polymyxin B. Mutations in five genes have been identified in three variants derived from two different parental strains A1552 and MO10: ihfA that encodes a subunit of the integration host factor (IHF), vacJ (mlaA) and mlaF, two genes belonging to the maintenance of the lipid asymmetry (Mla) pathway, dacB that encodes a penicillin-binding protein (PBP4) and involved in cell wall synthesis, and ccmH that encodes a c-type cytochrome maturation protein. We further demonstrated that the variants derived from MO10 containing mutations in vacJ, mlaF, and dacB secrete more and larger membrane vesicles that titer the polymyxin B, which increases the bacterial survival and is expected to limit its impact on the bacterial envelope and participate in the flagellum’s retention and motility.

Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing.

DNA methylation is the most recognized epigenetic mark that leads to a massive distortion in cancer cells. It has been observed that a large number of DNA aberrant methylation events occur simultaneously in a group of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Due to this reason, methylation profiles have been suggested as promising cancer biomarkers. Here, we designed and performed a first step of validation of a novel targeted next generation sequencing (NGS) panel for methylation analysis, which can simultaneously evaluate the methylation levels at CpG sites of multiple cancer-related genes. The OPERA_MET-A methylation panel was designed using the Ion AmpliSeq™ technology to amplify 155 regions with 125-175 bp mean length and covers a total of 1107 CpGs of 18 cancer-related genes. The performance of the panel was assessed by running commercially available fully methylated and unmethylated control human genomic DNA (gDNA) samples and a variable mixture of them. The libraries were run on Ion Torrent platform and the sequencing output was analyzed using the “methylation_analysis” plugin. DNA methylation calls on both Watson (W) and Crick (C) strands and methylated:unmethylated ratio for each CpG site were obtained. Cell lines, fresh frozen and formalin-fixed paraffin-embedded (FFPE) lung cancer tissues were tested. The OPERA_MET-A panel allows to run a minimum of 6 samples/530 chip to reach an observed mean target depth ≥2,500X (W and C strands) and an average number of mapped reads >750,000/sample. The conversion efficiency, determined by spiking-in unmethylated Lambda DNA into each sample before the bisulfite conversion process, was >97% for all samples. The observed percentage of global methylation for all CpGs was >95% and <5% for fully methylated and unmethylated gDNA samples, respectively, and the observed results for the variable mixtures were in agreement with what was expected. Methylation-specific NGS analysis represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. Moreover, it offers the opportunity to construct a more robust algorithm for disease prediction in cancer patients having a low quantity of biological material available.

An Engineered λ Phage Enables Enhanced and Strain-Specific Killing of Enterohemorrhagic Escherichia coli.

ABSTRACTBacteriophages (phages) are ideal alternatives to traditional antimicrobial agents in a world where antimicrobial resistance (AMR) is emerging and spreading at an unprecedented speed. In addition, due to their narrow host ranges, phages are also ideal tools to modulate the gut microbiota in which alterations of specific bacterial strains underlie human diseases, while dysbiosis caused by broad-spectrum antibiotics can be harmful. Here, we engineered a lambda phage (Eλ) to target enterohemorrhagic Escherichia coli (EHEC) that causes a severe, sometimes lethal intestinal infection in humans. We enhanced the killing ability of the Eλ phage by incorporating a CRISPR-Cas3 system into the wild-type λ (wtλ) and the specificity by introducing multiple EHEC-targeting CRISPR spacers while knocking out the lytic gene cro. In vitro experiments showed that the Eλ suppressed the growth of EHEC up to 18 h compared with only 6 h with the wtλ; at the multiplicity of infection (MOI) of 10, the Eλ killed the EHEC cells with ~100% efficiency and did not affect the growth of other laboratory- and human-gut isolated E. coli strains. In addition, the EHEC cells did not develop resistance to the Eλ. Mouse experiments further confirmed the enhanced and strain-specific killing of the Eλ to EHEC, while the overall mouse gut microbiota was not disturbed. Our methods can be used to target other genes that are responsible for antibiotic resistance genes and/or human toxins, engineer other phages, and support in vivo application of the engineered phages.IMPORTANCE Pathogenic strains of Escherichia coli are responsible for 0.8 million deaths per year and together ranked the first among all pathogenic species. Here, we obtained, for the first time, an engineered phage, Eλ, that could specifically and efficiently eliminate EHEC, one of the most common and often lethal pathogens that can spread from person to person. We verified the superior performance of the Eλ over the wild-type phage with in vitro and in vivo experiments and showed that the Eλ could suppress EHEC growth to nondetectable levels, fully rescue the EHEC-infected mice, and rescore disturbed mouse gut microbiota. Our results also indicated that the EHEC did not develop resistance to the Eλ, which has been the biggest challenge in phage therapy. We believe our methods can be used to target other pathogenic strains of E. coli and support in vivo application of the engineered phages.