Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

Background: Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new antituberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs. Methodology/Principal Findings: Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC50 1–7 mg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC50. The candidate compounds suppressed resuscitation of dormant (‘‘non-culturable’’) cells of M. smegmatis at 1 mg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 mg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations. Conclusions/Significance: NPT are the first example of low molecular weight compounds that inhibit the enzymatic an

The Effect of Antimicrobial Photodynamic Inactivation on the Protein Profile of Dormant <i>Mycolicibacterium smegmatis</i> Containing Endogenous Porphyrins

During transition into a dormant state, Mycolicibacterium (Mycobacterium) smegmatis cells are able to accumulate free porphyrins that makes them sensitive to photodynamic inactivation (PDI). The formation of dormant cells in a liquid medium with an increased concentration of magnesium (up to 25 mM) and zinc (up to 62 µM) resulted in an increase in the total amount of endogenous porphyrins in dormant M. smegmatis cells and their photosensitivity, especially for bacteria phagocytosed by macrophages. To gain insight into possible targets for PDI in bacterial dormant mycobacterial cells, a proteomic profiling with SDS gel electrophoresis and mass spectrometry analysis were conducted. Illumination of dormant forms of M. smegmatis resulted in the disappearance of proteins in the separating SDS gel. Dormant cells obtained under an elevated concentration of metal ions were more sensitive to PDI. Differential analysis of proteins with their identification with MALDI-TOF revealed that 45.2% and 63.9% of individual proteins disappeared from the separating gel after illumination for 5 and 15 min, respectively. Light-sensitive proteins include enzymes belonging to the glycolytic pathway, TCA cycle, pentose phosphate pathway, oxidative phosphorylation and energy production. Several proteins involved in protecting against oxygen stress and protein aggregation were found to be sensitive to light. This makes dormant cells highly vulnerable to harmful factors during a long stay in a non-replicative state. PDI caused inhibition of the respiratory chain activity and destroyed enzymes involved in the synthesis of proteins and nucleic acids, the processes which are necessary for dormant cell reactivation and their transition to multiplying bacteria. Because of such multiple targeting, PDI action via endogenous porphyrins could be considered as an effective approach for killing dormant bacteria and a perspective to inactivate dormant mycobacteria and combat the latent form of mycobacteriosis, first of all, with surface localization

Benzoylphenyl thiocyanates are new, effective inhibitors of the mycobacterial resuscitation promoting factor B protein

Abstract Background Resuscitation promoting factors (Rpfs) are the proteins involved in the process of reactivation of the dormant cells of mycobacteria. Recently a new class of nitrophenylthiocyanates (NPTs), capable of inhibiting the biological and enzymatic activities of Rpfs has been discovered. In the current study the inhibitory properties of the compounds containing both nitro and thiocyanate groups alongside with the compounds with the modified number and different spatial location of the substituents are compared. Methods New benzoylphenyl thiocyanates alongside with nitrophenylthiocyanates were tested in the enzymatic assay of bacterial peptidoglycan hydrolysis as well as against strains of several actinobacteria (Mycobacterium smegmatis, Mycobacterium tuberculosis) on in-lab developed models of resuscitation of the dormant forms. Results Introduction of the additional nitro and thiocyanate groups to the benzophenone scaffold did not influence the inhibitory activity of the compounds. Removal of the nitro groups analogously did not impair the functional properties of the molecules. Among the tested compounds two molecules without nitro group: 3-benzoylphenyl thiocyanate and 4-benzoylphenyl thiocyanate demonstrated the maximum activity in both enzymatic assay (inhibition of the Rpf-mediated peptidoglycan hydrolysis) and in the resuscitation assay of the dormant M. tuberculosis cells. Conclusions The current study demonstrates dispensability of the nitro group in the NPT’s structure for inhibition of the enzymatic and biological activities of the Rpf protein molecules. These findings provide new prospects in anti-TB drug discovery especially in finding of molecular scaffolds effective for the latent infection treatment

Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice

Earlier we demonstrated that the adenylyl cyclase (AC) encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis (Mtb), the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro, the AC-overexpressing pMindRv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo, AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence) in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMindRv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo

Measurement of pseudorapidity distributions of charged particles in proton-proton collisions at sqrt(s) = 8 TeV by the CMS and TOTEM experiments

Pseudorapidity ( $\eta$ ) distributions of charged particles produced in proton–proton collisions at a centre-of-mass energy of 8 $~\text {TeV}$ are measured in the ranges $|\eta | < 2.2$ and $5.3 < |\eta | < 6.4$ covered by the CMS and TOTEM detectors, respectively. The data correspond to an integrated luminosity of $\mathcal {L} = 45 \mu {\mathrm {b}}^{-1}$ . Measurements are presented for three event categories. The most inclusive category is sensitive to 91–96 % of the total inelastic proton–proton cross section. The other two categories are disjoint subsets of the inclusive sample that are either enhanced or depleted in single diffractive dissociation events. The data are compared to models used to describe high-energy hadronic interactions. None of the models considered provide a consistent description of the measured distributions

The very forward CASTOR calorimeter of the CMS experiment

International audienceThe physics motivation, detector design, triggers, calibration, alignment, simulation, and overall performance of the very forward CASTOR calorimeter of the CMS experiment are reviewed. The CASTOR Cherenkov sampling calorimeter is located very close to the LHC beam line, at a radial distance of about 1 cm from the beam pipe, and at 14.4 m from the CMS interaction point, covering the pseudorapidity range of $-$6.6 $\lt\eta\lt$ $-$5.2. It was designed to withstand high ambient radiation and strong magnetic fields. The performance of the detector in measurements of forward energy density, jets, and processes characterized by rapidity gaps, is reviewed using data collected in proton and nuclear collisions at the LHC