A CASE STUDY INTO PHAGE GENOME ASSEMBLY ERWINIA AMYLOVORA PHAGE KEY

A CASE STUDY INTO PHAGE GENOME ASSEMBLY ERWINIA AMYLOVORA PHAGE KEY

Zlatohurska M.

D.K. Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine,

Department of bacteriophage molecular genetics

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Phage genomes contain complex genomic structures (long direct or inverted repeats, terminal redundancies) that are problematic for assembly of the whole-genome sequence from the reads (Klumpp et al., 2012). In the present paper, the genome assembly steps are described on the example of Erwinia amylovora phage KEY. The purpose was to obtain the most reliable genome assembly that will serve as the basis for downstream genome analyses.

Phage KEY was isolated from quince with symptoms of fire blight. Virion DNA was obtained by the phenol-chloroform extraction method. Sequencing was performed using the Illumina HiSeq 2500 platform at The Centre for Applied Genomics in the Hospital for Sick Children, Toronto, Canada.

Quality control of the raw Illumina data was performed using FastQC v0.11.9 (Andrews, 2010). The sequencing run yielded a total of 14,790,000 raw reads with the length of 126 bp. The FastQC results indicate that the minimum quality value of reads is above Q30 (error probability 0.001). Additionally, a significant sequence duplication level (between 10 and 100 repeats) was detected for almost 80% of the sequences. There are also several overrepresented sequences. Next, the set of pair-end reads was processed by Trimmomatic (Bolger et al., 2014) to remove unpaired reads.

De novo assembly was performed using Velvet v7.0.4 (Zerbino et al., 2008), Unicycler v0.4.9b (Wick et al., 2017) and SPAdes v3.13.1 (Bankevich et al., 2012). Assembly statistics were assessed using QUAST v5.0.2 (Gurevich et al., 2013) at the contig level. Assembler outputs were compared with regards to total assembly length, the number of contigs, N50, NG50, and others. None of the assemblers generated a single contig. SPAdes output were considered as the best choice for downstream manipulation.

At graph assembly level, the topological structure of assembly was analyzed by Bandage v.8.1 (Wick et al., 2015). Many pairs of contigs with high identity level and different coverage values were found at the same time. Together with overrepresented and duplicated sequences this fact indicates that the mix of highly related genomes was sequenced.

Using BLASTn, Pantoea phage vB_PagS_AAS21 (MK770119.1) was identified as the closest genome to KEY. SPAdes contigs were mapped unto the AAS21 genome as a reference sequence to determine the low-coverage regions and possible sites of misassemble. As a result, an 118,944 bp long final contig suitable for further genome analyses was obtained.

ANTIHERPETIC ACTIVITY OF TRIVALENT AND TETRAVALENT CERIUM SALTS

ANTIHERPETIC ACTIVITY OF TRIVALENT AND TETRAVALENT CERIUM SALTS

Zholudenko Y, Zholobak N.

D.K. Zabolotny Institute of Microbiology and Virology NAS of Ukraine,

Department of interferon and immunomodulator problems

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Compounds based on cerium are highly promising objects in biotechnology regarding their high biological activities such as antiviral, antibacterial, antifungal, neuro- and radioprotective action, antioxidant activity, as well these compounds can also increase the lifespan of micro- and macroorganisms (Shcherbakov, 2020).

On their basis it is possible to develop compositions capable of activating the systems of cellular and humoral immune defense and used for the prevention and therapy of viral diseases, which makes it achievable to use them for the development of potential antiherpetic agents (Thakur, 2019).

Thus, cerium-based compounds have a range of unique properties, but despite the success of their application in biotechnological fields, the mechanism of their action on biological objects requires detailed research. Therefore, the aim of the work was to verify in vitro antiherpetic activity of trivalent and tetravalent cerium salts.

In this research we used the salts CeCl3·7Н2О and (NH4)2Ce(NO3)6 (Sigma, USA), where cerium has valency (III) and (IV) respectively, and also an African green monkey kidney cell culture MA-104, herpes simplex virus (HSV-1/2), isolate "GMM".

To determine antiviral activity of cerium salts preventive and therapeutic regimens were used. According to the preventive regimen, cerium salt samples were added to the MA-104 cell culture 24 h before the infection with HSV-1/2. The antiviral effect of the researched compounds was determined in the tenfold concentration range from 100 μM to 0.01 nM. According to the treatment regimen, salt samples were added to MA-104 cells 60 min after infection with HSV-1/2. In this regimen, the antiviral effect of cerium salts was determined in the tenfold concentration range an order of magnitude higher than in the preventive regimen from 1.0 mM to 0.1 nM. HSV-1/2 with the multiplicity of infection 1 × 103 TCD50 in 100 μL of medium 199 (Sigma, USA) was used. Crystal violet staining was used to determine the total number of adherent viable cells.

We have shown that cerium salts are capable of providing the formation of a state of antiviral resistance against HSV-1/2, provided that they are present for 24 h in the culture medium. The therapeutic treatment regimen in the same salt concentrations as the preventive regimen (100 μM 0.01 nM) has no antiviral effectiveness. Salt (NH4)2Ce(NO3)6 in vitro provides the formation of an effective state of antiviral resistance, while the salt CeCl3·7Н2О forms a non-linear, sinusoidal-like concentration-dependent anti-HSV-1/2 response of cells.

An antiviral effect of CeCl3·7Н2О and (NH4)2Ce(NO3)6 salts at various concentrations was studied in vitro in MA-104 cell culture at a high multiplicity of HSV-1/2 infection. Cerium salts were found to be capable of providing anti herpetic activity.

APOPTOSIS-INDUCING ACTIVITY OF POTENTIAL INHIBITORS OF EPSHTEIN-BARR VIRUS PROTEIN – BHRF1

APOPTOSIS-INDUCING ACTIVITY OF POTENTIAL INHIBITORS OF EPSHTEIN-BARR VIRUS PROTEIN – BHRF1

Zaremba P1,2 Zaremba A1,2 Naumenko K2 Platonov M3 Zagorodnya S.2

1ESC “Institute of Biology and Medicine” Taras Shevchenko National University

2D.K. Zabolotny Institute of Microbiology and Virology of NASU,

Department of virus reproduction

3Enamine Ltd.

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Epstein-Barr virus (EBV), which is a member of the Herpesvirus family, in addition to its wide distribution among the world's population, has the ability to transform cells and induce cancer. This is facilitated by latency programs and other defense mechanisms to avoid the body's immune response. One of these mechanisms is the inhibition of apoptosis in an infected cell by the viral protein BHRF1, which is a complete homologue of cellular Bcl-2. And since EBV is able to prevent apoptosis, the search for substances that can induce this process by suppressing viral effects is one step towards developing effective antiviral therapy.

The aim of this study was to determine the cytotoxicity and apoptosis-inducing activity of pre-selected in silico 11 chemically different compounds (Z-1 – Z-11), which are potential inhibitors of viral protein BHRF1. The studies were performed on virus-containing cell culture B95-8 (lymphocytes of cotton-top tamarin) and two reference cultures of different origin that do not contain EBV: Wish (human amnion cells) and MDCK (Madin-Darby canine kidney cells). Cytotoxicity was determined using the MTT assay, which is based on the determination of metabolic activity of cells. Apoptosis-inducing activity was determined by incubating B95-8 cells for 4 and 24 hours with selected compounds. Hoechst 33342 dye was used to visualize the results.

The CC50 values for some compounds differed significantly depending on cell culture. So the CC50 for Z-1 were: B95-8 - 48.88 μg/ml, Wish - 288.66 μg/mL, MDCK - 289.62 μg/mL. Substance Z-2 also showed a similar pattern: B95-8 - 47.49 μg/mL, Wish - 227.49 μg/mL, MDCK - 224.61 μg/mL. The CC50 of compound Z-6 were low for all cells: B95-8 - 48.81 μg/mL, Wish - 68.16 μg/mL, MDCK - 97.03 μg/mL, which may be associated with total toxicity of the compound.

Counting B95-8 in a fluorescent microscope (350 nm) showed that after incubation with compounds Z-1, Z-2, Z-6 the number of apoptotic cells increased. For substance Z-1 at a concentration of 80 μg/mL this increase occurred 2 times after 4 h of incubation and 1.8 times after 24 h compared to control cells. After incubation of B95-8 with Z-2 at the same concentration, the number of apoptotic cells increased in 2 (4 h) and 3 times (24 h). Compound Z-6 at a concentration of 60 μg/mL gave a significant increase in cells at the stage of apoptosis: 13.75 times after 4 h and 5.83 times after the day of incubation.

Thus, it can be concluded that of the 11 potential inhibitors of BHRF1 protein, three Z-1, Z-2, Z-6 - do show specific activity on EBV-containing culture. The substances are all of different origin: Z-1 is the derivative of tetrahydrobenzothiophen, Z-2 is the derivative of naphthalene‐2‐sulfonamide and Z-6 is the derivative of 4‐(benzyloxy)phenol. Among them, the compound Z-6 in addition to the specific effect on Epstein-Barr virus has a non-specific effect on virus-negative cultures. We plan to investigate the nature of this impact in more detail.

THE MECHANISM OF FORMALDEHYDE DEHYDROHENASE DEGRADATION IN METHYLOTROPHIC YEAST KOMAGATAELLA PHAFFII

THE MECHANISM OF FORMALDEHYDE DEHYDROHENASE DEGRADATION IN METHYLOTROPHIC YEAST KOMAGATAELLA PHAFFII

Yanchuk L1,2 Dmytruk O2 Dmytruk K2 Sibirny A.2,3

1Ivan Franko National University of Lviv,

Department of Microbiology, Faculty of Biology

2Institute of Cell Biology, National Academy of Science of Ukraine,

Department of Molecular Genetics and Biotechnology

3University of Rzeszow, Department of Biotechnology and Microbiology, Rzeszow, Poland

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Today, the mechanisms of degradation of cytosolic proteins of their own, as well as of recombinant foreign proteins of biotechnological significance with cytosolic localization in methylotrophic yeast, remain unclear. Autophagy dysfunction is associated with cancer, neurodegeneration, microbial infection and aging, therefore, the study of various aspects of autophagy on a model object (methylotrophic yeast) and extrapolation of the obtained data to other eukaryotic organisms may be important for medicine.

Many enzymes of methanol metabolism of methylotrophic yeasts are located in peroxisomes whereas some of them have the cytosolic localization. During shift of methanol grown cells of methylotrophic yeasts to glucose medium, a decrease in the activity of cytosolic (formaldehyde dehydrogenase (FLD), formate dehydrogenase (FDH), fructose-1,6-bisphosphatase (FBP)) enzymes of methanol metabolism is observed. Inactivation of peroxisomal enzymes occurs due to the autophagic degradation (pexophagy) whereas mechanisms of inactivation of cytosolic enzymes remain unknown. We aimed to study the mechanisms of FLD degradation in methylotrophic yeasts Komagataella phaffii.

The changes of the specific activity of FLD in the wild type strain GS200 and strain defected in autophagy pathway SMD1163 of K. phaffii in short-term and long-term induction with methanol, and with or without the addition of the MG132 (proteasome degradation inhibitor) was investigated. To confirm FLD degradation pathway the recombinant strains with GFP-labeled Fld of K. phaffii were constructed on the background of GS200 and SMD1163. Degradation of Fld by the Western blot analysis in GS200 and SMD1163 strains with GFP-labeled Fld was studied. The fluorescent microscopy analysis of the constructed strains was made. It was shown that the effect of the proteasome inhibitor MG132 was insignificant. FLD degrades by a vacuolar pathway, regardless of the duration of methanol induction, which correlates with the activity data of this enzyme.

SENSITIVITY TO DISINFECTANTS OF BIOFILM-FORMING HOSPITAL-ACQUIRED STRAINS OF BACTERIA

SENSITIVITY TO DISINFECTANTS OF BIOFILM-FORMING HOSPITAL-ACQUIRED STRAINS OF BACTERIA

Venzheha O, Bahrova A, Priyan I, Lavrentieva K, Sklyar T.

Oles Honchar Dnipro National University

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Nosocomial infections are a global medical, social, and economic problem. The most important role in their occurrence, according to world statistics, belongs to the species Pseudomonas aeruginosa and Staphylococcus aureus. Recently, there have been increasing reports of appearance of cross-resistance to antibacterial preparations among pathogens: multiple antibiotic resistance due to adaptation to disinfectants.

The aim of the study was to assess the level of resistance to disinfectants of biofilm-forming and nonbiofilm-forming clinical strains of P. aeruginosa and S. aureus – the causative agents of nosocomial infections.

Samples of detachable wounds of patients with nosocomial infections were taken in accordance with the guidelines for compliance with the sanitary-epidemiological regime in health care facilities of Ukraine. The ability to form biofilms was detected by using a modified method (O'Toole et al., 2000). The sensitivity of isolated strains – pathogens of nosocomial infections to disinfectants (0.1 and 0.2% solutions of «Desactin», 0.03 and 0.1% solutions of «Neochlor tabs») was determined by the method of batiste test objects.

Among the clinical samples, 43 strains of S. aureus were isolated, of which 72.1% had the biofilm-forming ability, and 39 strains of P. aeruginosa, of which 61.5% were biofilm-forming. Of the two types of chlorine-containing disinfectants, «Desactin» was the most effective against all isolates of S. aureus. In 90.3% of cases, the tested solutions of both concentrations of «Desactin» inhibited the growth of biofilm-forming isolates of S. aureus. All 12 nonbiofilm-forming variants of S. aureus were sensitive to this disinfectant. «Neochlor tabs» proved to be a less effective disinfectant. The number of susceptible strains of S.aureus, not capable of biofilm formation, was less than 75.0% and in the case of biofilm-forming isolates – even less (61.3%). For P. aeruginosa strains, in 100% of cases, the growth of nonbiofilm-forming isolates was inhibited by «Desactin» (0.1 or 0.2% solution). The same effect was observed using 0.1% solution of «Neochlor tabs». Biofilm-forming strains of P. aeruginosa had a higher degree of resistance to the action of both types of disinfectants: bactericidal effect of 0.1% solution of «Neochlor tabs» was observed in 75.0%, and solutions of «Desactin» – in 62.5% of cases.

According to the results of determining the effectiveness of chlorine-containing disinfectants «Dezactin» and «Neochlor tabs» against isolated agents of nosocomial infections, the strains of S. aureus and P. aeruginosa, capable of biofilm formation, were found to be characterized by a higher degree of resistance to both types of disinfectants. The most effective disinfectant against isolated clinical nonbiofilm-forming strains of S. aureus and P. aeruginosa was «Desactin» in concentration of 0.1% or 0.2%. For S. aureus isolates capable of biofilm formation, its efficiency decreased by 9.7%. The best bactericidal effect against biofilm-forming strains of P. aeruginosa showed 0.1% solution of «Neochlor tabs». It inhibited the growth of 75.0% of the tested cultures.