[Show abstract][Hide abstract]ABSTRACT: Tuberculous sputum contains multiple
(Mtb) populations with different requirements for isolation
These include cells forming colonies on solid media (platable Mtb), cells requiring standard liquid medium for growth (non-platable Mtb), and cells requiring supplementation of liquid medium with culture supernatant for growth (SN-dependent Mtb). Here we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of these Mtb populations within sputum. Our results show that first line drugs achieved only modest cidal effects on all three populations over 7 days (1-2.5xlog
reductions) and SN-dependent Mtb were more tolerant to streptomycin and isoniazid compared to platable and non-platable Mtb. Susceptibility of platable Mtb to bactericidal drugs was significantly increased after passage
, thus tolerance observed in the sputum populations was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simple
system for testing drug efficacies against mycobacteria phenotypically adapted during tuberculosis infection.
Full-text · Article · Feb 2016 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract]ABSTRACT: PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.
[Show abstract][Hide abstract]ABSTRACT: We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, 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 expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added
oleic acid influences RaaS-mediated mycobacterial improvement of survival and 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 to eliminate non-growing mycobacteria.
[Show abstract][Hide abstract]ABSTRACT: Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically,
we found that under nonpermissive 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 under nonpermissive 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.
Full-text · Article · Mar 2014 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract]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.
Full-text · Article · Apr 2012 · Protein and Peptide Letters
[Show abstract][Hide abstract]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.
[Show abstract][Hide abstract]ABSTRACT: Mal aktiv, mal inaktiv: Ein Peptidsystem, das für die Regulierung antimikrobieller Wirkungen entwickelt wurde, schaltet zwischen antimikrobiellen und inaktiven Formen um. Der Regulator besteht aus zwei α-helicalen Sequenzen: Eine, das R-Knäuel, bindet an Mikroben-Membranen, fungiert als antimikrobielle Komponente und wird durch die andere, das E-Knäuel, ein membraninaktives Peptid, desaktiviert (siehe Bild).
No preview · Article · Dec 2009 · Angewandte Chemie
[Show abstract][Hide abstract]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.
Full-text · Article · Nov 2009 · Journal of bacteriology
[Show abstract][Hide abstract]ABSTRACT: Peptides that make bacteria RE coil: A peptide system designed to regulate antimicrobial intervention switches between antimicrobial and inactive forms. The regulator comprises two α-helical sequences. One of them, R coil, binds to microbial membranes and acts as an antimicrobial component, and is inactivated by the other, E coil, a membrane-inactive peptide (see picture).
Full-text · Article · Nov 2009 · Angewandte Chemie International Edition
[Show abstract][Hide abstract]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.
Full-text · Article · Oct 2009 · American Journal of Respiratory and Critical Care Medicine
[Show abstract][Hide abstract]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.
[Show abstract][Hide abstract]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.
[Show abstract][Hide abstract]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.
No preview · Article · May 2006 · Biochemistry (Moscow)