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Data on translatome analysis of Mycoplasma gallisepticum

Fisunov GY, Evsyutina DV, Govorun VM

Data Brief, 2016 Dec;9:422-424.
PMID: 27699194

Mycoplasma gallisepticum is a bacterium of class Mollicutes which encompasses wall-less bacteria with significantly reduced genomes. Due to their overall reduction and simplicity mycoplasmas serve as a model of minimal cell and are used for systems biology studies. Here we present raw data on translatome (ribosome-bound mRNA) analysis of Mycoplasma gallisepticum under logarithm growth and heat stress. The data supports the publication of "Ribosomal profiling of Mycoplasma gallisepticum" (G. Y. Fisunov, D. V Evsyutina, A. A. Arzamasov, I. O. Butenko, V. M. Govorun, 2015) [1].
The use of the resonant mirror biosensor to detect point mutations, as demonstrated with synthetic oligonucleotides

Momynaliev KT, Govorun VM, Gnedenko O, Ivanov YD, Archakov AI

J. Mol. Recognit., 2003 Jan-Feb;16(1):1-8.
PMID: 12557232

The possibility of using the resonant mirror biosensor to detect point substitutions in oligonucleotides was demonstrated with a fragment of the Helicobacter pylori 23S rRNA gene, point mutations in which are responsible for clarythromycin resistance. Conditions were optimized for the interaction of a probe immobilized on the sensing surface with targets containing various nucleotide substitutions. A probe allowing reliable discrimination of mutant targets was selected. The mismatch position in the probe was shown to affect the kinetic parameters (response) of hybridization with mutant targets, reporting not only the position, but also the character (G or C) of a substitution.
A MALDI TOF MS-based minisequencing method for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains of Enterobacteriaceae

Ikryannikova LN, Shitikov EA, Zhivankova DG, Il'ina EN, Edelstein MV, Govorun VM

J Microbiol Methods, 2008 Dec;75(3):385-91.
PMID: 18694787 DOI: 10.1016/j.mimet.2008.07.005

A minisequencing method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was developed for rapid identification of single nucleotide polymorphisms at bla(TEM) gene codons 104, 164 and 238 associated with extended-spectrum activity on TEM-type beta-lactamases. The method was validated by testing the Escherichia coli and Klebsiella pneumoniae strains possessing the known bla(TEM) gene sequences.
Effect of induced expression of an antimicrobial peptide melittin on Chlamydia trachomatis and Mycoplasma hominis infections in vivo

Lazarev VN, Shkarupeta MM, Titova GA, Kostrjukova ES, Akopian TA, Govorun VM

Biochem Biophys Res Commun, 2005 Dec 16;338(2):946-50.
PMID: 16246304

A plasmid construct was designed in which the gene of antimicrobial peptide melittin is controlled by the tetracycline-responsive promoter of human cytomegalovirus, aided by a constitutively expressed trans-activator protein gene. Its vaginal administration and induction of melittin gene transcription with doxycycline markedly suppressed subsequent genital tract infection of mice by Mycoplasma hominis and Chlamydia trachomatis. At least half of the melittin-protected animals proved free of either pathogen within 3-4 weeks. Recombinant plasmids expressing genes of antimicrobial peptides hold much promise as agents for prevention and control of urogenital latent infections.
Induced expression of melittin, an antimicrobial peptide, inhibits infection by Chlamydia trachomatis and Mycoplasma hominis in a HeLa cell line

Lazarev VN, Parfenova TM, Gularyan SK, Misyurina OY, Akopian TA, Govorun VM

Int J Antimicrob Agents, 2002 Feb;19(2):133-7.
PMID: 11850166

As the number of pathogenic microbial strains resistant to different antibiotics increases, amphipathic peptides with antimicrobial activity are promising agents for the therapy of infectious diseases. This work deals with the effect of an amphipathic antimicrobial peptide, melittin, expressed within recombinant plasmid vectors, on infection with urogenital pathogens Chlamydia trachomatis and Mycoplasma hominis in HeLa cell culture. Recombinant plasmid constructs with the melittin gene under the control of the tetracycline-responsive promoter of human cytomegalovirus were obtained. We showed inhibition of C. trachomatis and M. hominis infection after the introduction of recombinant plasmid vectors expressing the melittin gene into the infected cell culture.
Purification and functional analysis of recombinant Acholeplasma laidlawii histone-like HU protein

Levitskiy SA, Sycheva AM, Kharlampieva DD, Oberto J, Kamashev DE, Serebryakova MV, et al.

Biochimie, 2011 Jul;93(7):1102-9.
PMID: 21443922 DOI: 10.1016/j.biochi.2011.03.005

HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3'-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA-binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.
Mass-spectrometry based minisequencing method for the rapid detection of drug resistance in Mycobacterium tuberculosis

Ikryannikova LN, Afanas'ev MV, Akopian TA, Il'ina EN, Kuz'min AV, Larionova EE, et al.

J Microbiol Methods, 2007 Sep;70(3):395-405.
PMID: 17602768

A MALDI TOF MS based minisequencing method has been developed and applied for the analysis of rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains. Eight genetic markers of RIF resistance-nucleotide polymorphisms located in RRDR of rpoB gene, and three of INH resistance including codon 315 of katG gene and -8 and -15 positions of the promoter region of fabG1-inhA operon were worked out. Based on the analysis of 100 M. tuberculosis strains collected from the Moscow region in 1997-2005 we deduced that 91% of RIF-resistant and 94% of INH-resistant strains can be identified using the technique suggested. The approach is rapid, reliable and allows to reveal the drug resistance of M. tuberculosis strains within 12 h after sample isolation.
Induced expression of the antimicrobial peptide melittin inhibits experimental infection by Mycoplasma gallisepticum in chickens

Lazarev VN, Stipkovits L, Biro J, Miklodi D, Shkarupeta MM, Titova GA, et al.

Microbes Infect., 2004 May;6(6):536-41.
PMID: 15158186

The in vivo action of the antimicrobial peptide melittin, expressed from a recombinant plasmid vector, on chickens experimentally infected with Mycoplasma gallisepticum was studied. The plasmid vector pBI/mel2/rtTA includes the melittin gene under the control of an inducible tetracycline-dependent human cytomegalovirus promoter and the gene coding for the trans-activation protein rtTA. Aerosol administration of the vector, followed by infecting the chickens with M. gallisepticum 1226, is shown to inhibit development of infection. The inhibitory action was confirmed by a complex of clinical, pathomorphological, histological and serological studies, and also by comparing the M. gallisepticum reisolation frequency from the respiratory tract and internal organs. The data suggest that plasmid vectors expressing genes of antimicrobial peptides can be considered as potential agents for the prevention and treatment of mycoplasma infections in poultry farming.
Fulltext Spider venom peptides for gene therapy of Chlamydia infection

Lazarev VN, Polina NF, Shkarupeta MM, Kostrjukova ES, Vassilevski AA, Kozlov SA, et al.

Antimicrob Agents Chemother, 2011 Nov;55(11):5367-9.
PMID: 21876050 DOI: 10.1128/AAC.00449-11

Spider venoms are vast natural pharmacopoeias selected by evolution. The venom of the ant spider Lachesana tarabaevi contains a wide variety of antimicrobial peptides. We tested six of them (latarcins 1, 2a, 3a, 4b, 5, and cytoinsectotoxin 1a) for their ability to suppress Chlamydia trachomatis infection. HEK293 cells were transfected with plasmid vectors harboring the genes of the selected peptides. Controlled expression of the transgenes led to a significant decrease of C. trachomatis viability inside the infected cells.
Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the study of Helicobacter pylori

Ilina EN, Borovskaya AD, Serebryakova MV, Chelysheva VV, Momynaliev KT, Maier T, et al.

Rapid Commun Mass Spectrom, 2010 Feb;24(3):328-34.
PMID: 20049887 DOI: 10.1002/rcm.4394

The characteristics of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry based investigation of extremely variable bacteria such as Helicobacter pylori were studied. H. pylori possesses a very high natural variability. Accurate tools for species identification and epidemiological characterization could help the scientific community to better understand the transmission pathways and virulence mechanisms of these bacteria. Seventeen clinical as well as two laboratory strains of H. pylori were analyzed by the MALDI Biotyper method for rapid species identification. Mass spectra collected were found containing 7-13 significant peaks per sample, and only six protein signals were identical for more than half of the strains. Four of them could be assigned to ribosomal proteins RL32, RL33, RL34, and RL36. The reproducible peak with m/z 6948 was identified as a histidine-rich metal-binding polypeptide by tandem mass spectrometry (MS/MS). In spite of the evident protein heterogeneity of H. pylori the mass spectra collected for a particular strain under several cultivations were highly reproducible. Moreover, all clinical strains were perfectly identified as H. pylori species through comparative analysis using the MALDI Biotyper software (Bruker Daltonics, Germany) by pattern matching against a database containing mass spectra from different microbial strains (n = 3287) including H. pylori 26695 and J99. The results of this study allow the conclusion that the MALDI-TOF direct bacterial profiling is suited for H. pylori identification and could be supported by mass spectra fragmentation of the observed polypeptide if necessary.
Fulltext Comprehensive analysis of the venom gland transcriptome of the spider Dolomedes fimbriatus

Kozlov SA, Lazarev VN, Kostryukova ES, Selezneva OV, Ospanova EA, Alexeev DG, et al.

Sci Data, 2014;1:140023.
PMID: 25977780 DOI: 10.1038/sdata.2014.23

A comprehensive transcriptome analysis of an expressed sequence tag (EST) database of the spider Dolomedes fimbriatus venom glands using single-residue distribution analysis (SRDA) identified 7,169 unique sequences. Mature chains of 163 different toxin-like polypeptides were predicted on the basis of well-established methodology. The number of protein precursors of these polypeptides was appreciably numerous than the number of mature polypeptides. A total of 451 different polypeptide precursors, translated from 795 unique nucleotide sequences, were deduced. A homology search divided the 163 mature polypeptide sequences into 16 superfamilies and 19 singletons. The number of mature toxins in a superfamily ranged from 2 to 49, whereas the diversity of the original nucleotide sequences was greater (2-261 variants). We observed a predominance of inhibitor cysteine knot toxin-like polypeptides among the cysteine-containing structures in the analyzed transcriptome bank. Uncommon spatial folds were also found.
Fulltext The Pathogenesis of the Demyelinating Form of Guillain-Barre Syndrome (GBS): Proteo-peptidomic and Immunological Profiling of Physiological Fluids

Ziganshin RH, Ivanova OM, Lomakin YA, Belogurov AA, Kovalchuk SI, Azarkin IV, et al.

Mol Cell Proteomics, 2016 Jul;15(7):2366-78.
PMID: 27143409 DOI: 10.1074/mcp.M115.056036

Acute inflammatory demyelinating polyneuropathy (AIDP) - the main form of Guillain-Barre syndrome-is a rare and severe disorder of the peripheral nervous system with an unknown etiology. One of the hallmarks of the AIDP pathogenesis is a significantly elevated cerebrospinal fluid (CSF) protein level. In this paper CSF peptidome and proteome in AIDP were analyzed and compared with multiple sclerosis and control patients. A total protein concentration increase was shown to be because of even changes in all proteins rather than some specific response, supporting the hypothesis of protein leakage from blood through the blood-nerve barrier. The elevated CSF protein level in AIDP was complemented by activization of protein degradation and much higher peptidome diversity. Because of the studies of the acute motor axonal form, Guillain-Barre syndrome as a whole is thought to be associated with autoimmune response against neurospecific molecules. Thus, in AIDP, autoantibodies against cell adhesion proteins localized at Ranvier's nodes were suggested as possible targets in AIDP. Indeed, AIDP CSF peptidome analysis revealed cell adhesion proteins degradation, however no reliable dependence on the corresponding autoantibodies levels was found. Proteome analysis revealed overrepresentation of Gene Ontology groups related to responses to bacteria and virus infections, which were earlier suggested as possible AIDP triggers. Immunoglobulin blood serum analysis against most common neuronal viruses did not reveal any specific pathogen; however, AIDP patients were more immunopositive in average and often had polyinfections. Cytokine analysis of both AIDP CSF and blood did not show a systemic adaptive immune response or general inflammation, whereas innate immunity cytokines were up-regulated. To supplement the widely-accepted though still unproven autoimmunity-based AIDP mechanism we propose a hypothesis of the primary peripheral nervous system damaging initiated as an innate immunity-associated local inflammation following neurotropic viruses egress, whereas the autoantibody production might be an optional complementary secondary process.
Snapper: high-sensitive detection of methylation motifs based on Oxford Nanopore reads

Konanov DN, Babenko VV, Belova AM, Madan AG, Boldyreva DI, Glushenko OE, et al.

Bioinformatics, 2023 Nov 20.
PMID: 37982752 DOI: 10.1093/bioinformatics/btad702

MOTIVATION: The Oxford Nanopore technology has a great potential for the analysis of methylated motifs in genomes, including whole genome methylome profiling. However, we found that there are no methylation motifs detection algorithms which would be sensitive enough and return deterministic results. Thus, the MEME suit does not extract all H. pylori methylation sites de novo even using the iterative manually controlled approach implemented in the most up-to-date methylation analysis tool Nanodisco.
RESULTS: We present Snapper, a new highly-sensitive approach to extract methylation motif sequences based on a greedy motif selection algorithm. Snapper does not require manual control during the enrichment process and has enrichment sensitivity higher than MEME coupled with Tombo or Nanodisco instruments that was demonstrated on H. pylori strain J99 studied earlier by the PacBio technology and on four external datasets representing different bacterial species. We used Snapper to characterize the total methylome of a new H.pylori strain A45. At least four methylation sites that have not been described for H. pylori earlier were revealed. We experimentally confirmed the presence of a new CCAG-specific methyltransferase and inferred a gene encoding a new CCAAK-specific methyltransferase.
AVAILABILITY: Snapper is implemented using Python and freely available as a pip package named 'snapper-ont'. Also, Snapper and the demo dataset are available in Zenodo (10.5281/zenodo.10117651).
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Fulltext Complete genome and proteome of Acholeplasma laidlawii

Lazarev VN, Levitskii SA, Basovskii YI, Chukin MM, Akopian TA, Vereshchagin VV, et al.

J Bacteriol, 2011 Sep;193(18):4943-53.
PMID: 21784942 DOI: 10.1128/JB.05059-11

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.

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