Galina V Mukamolova

University of Leicester, Leicester, ENG, United Kingdom

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Publications (41)117.68 Total impact

  • American Journal of Respiratory and Critical Care Medicine 12/2014; 190(12):1455-7. · 11.04 Impact Factor
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    ABSTRACT: Regulator of Antimicrobial-Assisted Survival), encoded by Rv1219c in Mycobacterium tuberculosis and bcg_1279c in Mycobacterium bovis BCG, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and to lesser extent palmitoyl-CoA) modulate RaaS binding to DNA and the expression of the genes downstream that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influenced RaaS-mediated mycobacterial improvement of survival and the expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used eliminate non-growing mycobacteria.
    The Journal of biological chemistry. 07/2014;
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    ABSTRACT: Antimicrobials targeting cell wall biosynthesis are generally considered inactive against non-replicating bacteria. Paradoxically, we found that in non-permissive growth conditions exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, Raas (for regulator of antimicrobial-assisted survival) encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death in non-permissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases such as tuberculosis.
    Antimicrobial Agents and Chemotherapy 03/2014; · 4.57 Impact Factor
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    ABSTRACT: Resuscitation promoting factors (Rpf) are a family of proteins secreted by actively growing actinobacteria, including Mycobacterium tuberculosis. Experimental evidence suggests that Rpfs play a distinct role in bacterial resuscitation and re-growth as well as reactivation of chronic tuberculosis in mice. The striking similarity of the Rpfs structure to cell wall hydrolysing enzymes has provided a basis for the development of novel low molecular weight inhibitors of Rpfs activity. In particular, recently characterised nitrophenylthiocyanate compounds could be considered as a promising scaffold for generation of therapeutic agents targeting reactivation of latent tuberculosis. This review describes recent progress in understanding of molecular mechanisms of Rpf biological activity.
    Protein and Peptide Letters 04/2012; 19(10):1026-34. · 1.99 Impact Factor
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    ABSTRACT: PknB is a transmembrane Ser/Thr protein kinase that defines and belongs to an ultraconserved kinase subfamily found in Gram-positive bacteria. Essential for Mycobacterium tuberculosis growth, its close homolog in Bacillus subtilis has been linked to exit from dormancy. The kinase possesses an extracellular region composed of a repetition of PASTA domains, believed to bind peptidoglycan fragments that might act as a signaling molecule. We report here the first solution structure of this extracellular region. Small-angle X-ray scattering and nuclear magnetic resonance studies show that the four PASTA domains display an unexpected linear organization, contrary to what is observed in the distant protein PBP2x from Streptococccus pneumoniae where two PASTA domains fold over in a compact structure. We propose a model for PknB activation based on a ligand-dependent dimerization of the extracellular PASTA domains that initiates multiple signaling pathways.
    Structure 05/2010; 18(5):606-15. · 5.99 Impact Factor
  • Angewandte Chemie 12/2009;
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    Angewandte Chemie International Edition 11/2009; 48(51):9676-9. · 11.34 Impact Factor
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    ABSTRACT: Micrococcus luteus (NCTC2665, "Fleming strain") has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to beta-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.
    Journal of bacteriology 11/2009; 192(3):841-60. · 3.94 Impact Factor
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    ABSTRACT: Resuscitation-promoting factors (Rpfs) are a family of secreted proteins produced by Mycobacterium tuberculosis (Mtb) that stimulate mycobacterial growth. Although mouse infection studies show that they support bacterial survival and disease reactivation, it is currently unknown whether Rpfs influence human infection. We hypothesized that tuberculous sputum might include a population of Rpf-dependent Mtb cells. To determine whether Rpf-dependent Mtb cells are present in human sputum and explore the impact of chemotherapy on this population. In tuberculous sputum samples we compared the number of cells detected by conventional agar colony-forming assay with that determined by limiting dilution, most-probable number assay in the presence or absence of Rpf preparations. In 20 of 25 prechemotherapy samples from separate patients, 80-99.99% of the cells demonstrated by cultivation could be detected only with Rpf stimulation. Mtb cells with this phenotype were not generated on specimen storage or by inoculating sputum samples with a selection of clinical isolates; moreover, Rpf dependency was lost after primary isolation. During chemotherapy, the proportion of Rpf-dependent cells was found to increase relative to the surviving colony-forming population. Smear-positive sputum samples are dominated by a population of Mtb cells that can be grown only in the presence of Rpfs. These intriguing proteins are therefore relevant to human infection. The Rpf-dependent population is invisible to conventional culture and is progressively enhanced in relative terms during chemotherapy, indicating a form of phenotypic resistance that may be significant for both chemotherapy and transmission.
    American Journal of Respiratory and Critical Care Medicine 10/2009; 181(2):174-80. · 11.04 Impact Factor
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    ABSTRACT: Efficient protein digestion is a critical step for successful mass spectrometry analysis. Here we describe simultaneous tryptic digestion and gradual unfolding of native proteins by application of a temperature gradient using a single cycle of 5 min or less in a PCR thermocycler. Chemicals typically used for chromatographic techniques did not affect the digestion efficiency. Tryptic digestion was performed in a small volume (3 microL) with 1.5 microg of trypsin without denaturing agents. This rapid procedure yielded more peptides than conventional methods utilizing chemical denaturation for 18 proteins out of 20. Samples were directly spotted on the MALDI-TOF target plate, without additional purification, thus reducing losses on reversed-phase resins.
    Analytical Chemistry 07/2008; 80(15):6093-9. · 5.82 Impact Factor
  • Galina Mukamolova, Elena Salina, Arseny Kaprelyants
    12/2007: pages 83-90;
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    ABSTRACT: Two-component systems are important constituents of bacterial regulatory networks. Results of this investigation into the role of the MprAB two-component system of Mycobacterium tuberculosis indicate that it is associated with the regulation of several stress-responsive regulons. Using a deletion mutant lacking portions of the response regulator, MprA, and the histidine kinase, MprB, it was demonstrated by real-time PCR, primer extension analyses and DNA microarrays that MprAB activates sigma factor genes sigE and sigB, under SDS stress and during exponential growth. SDS-inducible, MprA-dependent transcriptional start points were identified for mprA, sigE and sigB, and variations in distance between these points and MprA-binding sites suggest that MprA is involved in different mechanisms of promoter activation. Although most of the SigE regulon was downregulated in the deletion mutant, the cluster of genes Rv1129c, Rv1130 and Rv1131, which is associated with growth in monocytes, was upregulated in the deletion mutant under SDS stress, and this upregulation was dependent upon atmospheric growth conditions. Multiple stress-associated genes of the DosR, SigD and IdeR regulons were also upregulated in the deletion mutant, during exponential growth and/or in the presence of SDS. Surprisingly, the deletion mutant had increased resistance to SDS compared to the parental strain, and enhanced growth in human peripheral blood monocytes, characteristics which may result from a loss of repression of stress-associated genes.
    Microbiology 05/2007; 153(Pt 4):1229-42. · 2.85 Impact Factor
  • G. Demina, G. Mukamolova, A. Kaprelyants
    International Journal of Antimicrobial Agents - INT J ANTIMICROBIAL AGENTS. 01/2007; 29.
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    ABSTRACT: The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.
    Biochemistry (Moscow) 05/2006; 71(4):414-22. · 1.15 Impact Factor
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    ABSTRACT: The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.
    Molecular Microbiology 02/2006; 59(1):84-98. · 5.03 Impact Factor
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    ABSTRACT: Conditions were investigated that promote the formation of 'non-culturable' (NC) cells of Mycobacterium (Myc.) smegmatis in stationary phase. After cultivation in a rich medium, or under conditions that may be considered optimal for bacterial growth, or starvation for carbon, nitrogen or phosphorus, bacteria failed to enter a NC state. However, when grown under suboptimal conditions, resulting in a reduced growth rate or maximal cell concentration (e.g. in modified Hartman's-de Bont medium), bacteria adopted a stable NC state after 3-4 days incubation in stationary phase. Such conditions are not specific as purF and devR mutants of Myc. smegmatis also showed (transient) loss of culturability following growth to stationary phase in an optimized medium, but under oxygen-limited conditions. The behaviour of the same mutants in oxygen-sufficient but nutrient-inappropriate medium (modified Hartman's-de Bont medium) was similar to that of the wild-type (adoption of a stable NC state). It is hypothesized that adoption of a NC state may represent an adaptive response of the bacteria, grown under conditions when their metabolism is significantly compromised due to the simultaneous action of several factors, such as usage of inappropriate nutrients or low oxygen availability or impairment of a particular metabolic pathway. NC cells of wild-type Myc. smegmatis resume growth when transferred to a suitable resuscitation medium. Significantly, resuscitation was observed when either recombinant Rpf protein or supernatant derived from a growing bacterial culture was incorporated into the resuscitation medium. Moreover, co-culture with Micrococcus (Mcc.) luteus cells (producing and secreting Rpf) also permitted resuscitation. Isogenic strains of Myc. smegmatis harbouring plasmids containing the Mcc. luteus rpf gene also adopt a similar NC state after growth to stationary phase in modified Hartman's-de Bont medium. However, in contrast to the behaviour noted above, these strains resuscitated spontaneously when transferred to the resuscitation medium, presumably because they are able to resume endogenous synthesis of Mcc. luteus Rpf. Resuscitation was not observed in the control strain harbouring a plasmid lacking Mcc. luteus rpf. In contrast to wild-type, the NC cells of purF and devR mutants obtained under oxygen-limited conditions resuscitate spontaneously, presumably because the heterogeneous population contains some residual viable cells that continue to make Rpf-like proteins.
    Microbiology 07/2004; 150(Pt 6):1687-97. · 2.85 Impact Factor
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    ABSTRACT: Microbial culturability can be ephemeral. Cells are not merely either dead or alive but can adopt physiological states in which they appear to be (transiently) non-culturable under conditions in which they are known normally to be able to grow and divide. The reacquisition of culturability from such states is referred to as resuscitation. We here develop the idea that this "transient non-culturability" is a consequence of a special survival strategy, and summarise the morphological, physiological and genetic evidence underpinning such behaviour and its adaptive significance.
    Advances in Microbial Physiology 02/2003; 47:65-129. · 6.55 Impact Factor
  • Galina V. Mukamolova, A S Kaprelyants, Douglas B Kell, Michael Young
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    ABSTRACT: contents - Physiological diversity and niche adaptation in marine Synechococcus Adoption of the Transiently Non-culturable State - a Bacterial Survival Strategy? The Biodiversity of Microbial Cytochromes P450 The Tat protein translocation pathway and its role in microbial physiology Microbial Globins
    01/2003;
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    ABSTRACT: Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long post-stationary phase. These cells were small (0.6-0.8 micron) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosis culture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.
    Mikrobiologiia 01/2003; 72(1):76-83.
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    ABSTRACT: Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long poststationary phase. These cells were small (0.6–0.8 m) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosisculture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.
    Microbiology 12/2002; 72(1):64-70. · 0.65 Impact Factor

Publication Stats

1k Citations
117.68 Total Impact Points

Institutions

  • 2009–2010
    • University of Leicester
      • Department of Infection, Immunity and Inflammation
      Leicester, ENG, United Kingdom
    • Hebrew University of Jerusalem
      • Department of Plant and Environmental Sciences
      Yerushalayim, Jerusalem District, Israel
  • 1994–2007
    • Russian Academy of Sciences
      • A.N. Bach Institute of Biochemistry
      Moskva, Moscow, Russia
    • Innsbruck Economics
      Absam, Tyrol, Austria
  • 1995–2006
    • University of Wales
      Cardiff, Wales, United Kingdom