The efflux pumps Rv1877 and Rv0191 play differential roles in the protection of Mycobacterium tuberculosis against chemical stress

BackgroundIt was previously shown that GlnA3sc enabled Streptomyces coelicolor to survive in excess polyamines. However, subsequent studies revealed that Rv1878, the corresponding Mycobacterium tuberculosis (M.tb) ortholog, was not essential for the detoxification of spermine (Spm), in M.tb. On the other hand, the multi-drug efflux pump Rv1877 was previously shown to enable export of a wide range of compounds, while Rv0191 was shown to be more specific to chloramphenicol.RationaleTherefore, we first wanted to determine if detoxification of Spm by efflux can be achieved by any efflux pump, or if that was dependent upon the function of the pump. Next, since Rv1878 was found not to be essential for the detoxification of Spm, we sought to follow-up on the investigation of the physiological role of Rv1878 along with Rv1877 and Rv0191.ApproachTo evaluate the specificity of efflux pumps in the mycobacterial tolerance to Spm, we generated unmarked ∆rv1877 and ∆rv0191 M.tb mutants and evaluated their susceptibility to Spm. To follow up on the investigation of any other physiological roles they may have, we characterized them along with the ∆rv1878 M.tb mutant.ResultsThe ∆rv1877 mutant was sensitive to Spm stress, while the ∆rv0191 mutant was not. On the other hand, the ∆rv1878 mutant grew better than the wild-type during iron starvation yet was sensitive to cell wall stress. The proteins Rv1877 and Rv1878 seemed to play physiological roles during hypoxia and acidic stress. Lastly, the ∆rv0191 mutant was the only mutant that was sensitive to oxidative stress.ConclusionThe multidrug MFS-type efflux pump Rv1877 is required for Spm detoxification, as opposed to Rv0191 which seems to play a more specific role. Moreover, Rv1878 seems to play a role in the regulation of iron homeostasis and the reconstitution of the cell wall of M.tb. On the other hand, the sensitivity of the ∆rv0191 mutant to oxidative stress, suggests that Rv0191 may be responsible for the transport of low molecular weight thiols

Dually acting nonclassical 1,4-dihydropyridines promote the anti-tuberculosis (Tb) activities of clofazimine

The number of effective antituberculotic drugs is strongly limited to four first-line drugs in standard therapy. In case of resistances second-line antibiotics are used with a poor efficacy and tolerability. Therefore, novel antituberculotic drugs are urgently needed. We synthesized novel nonclassical 1,4-dihydropyridines and evaluated their antituberculotic properties depending on substituent effects. Preferred substituents could be identified. As related classical 1,4-dihydropyridines are known as inhibitors of the transmembrane efflux pump ABCB1 in cancer cells, we wondered whether a use of our compounds may be of favour to enhance the antituberculotic drug efficacy of the second-line antituberculotic drug clofazimine, which is a known substrate of ABCB1 by a suggested inhibition of a corresponding efflux pump in Mycobacterium tuberculosis (Mtb). For this, we determined the ABCB1 inhibiting properties of our compounds in a mouse T-lymphoma cell line model and then evaluated the drug-enhancing properties of selected compounds in a co-application with clofazimine in our Mtb strain. We identified novel enhancers of clofazimine toxicity which could prevent clofazimine resistance development mediated by an efflux pump activity.Publikationsfond ML

Synthesis and antimycobacterial assays of some new ethambutol analogs

Ethambutol (EMB) is a first-line anti-tuberculosis drug that is also considered in treatment regimens for infections caused by non-tuberculous mycobacteria (NTM). EMB targets the arabinosyl transferases EmbCAB, which are important for the synthesis of cell wall constituents. To further explore and narrow down the structural variability of EMB, we synthesized three series of new EMB analogs. We tested their activity against Mycobacterium tuberculosis, Mycobacterium smegmatis, Mycobacterium abscessus and Mycobacterium intracellulare. Only analogs that very closely resembled EMB showed comparable antimycobacterial activity

Design, synthesis and evaluation of biological activities of some novel anti-TB agents with bio-reducible functional group

Introduction: With regard to the anti-mycobacterial activity of 2-pyrazinoic acid esters (POEs), recent studies have shown that both pyrazine core and alkyl part of POE interact with the fatty acid synthase type (I) (FAS (I)) precluding a complex formation between NADPH and FAS (I). Methods: Considering this interaction at the reductase site of FAS (I) responsible for reduction of β-ketoacyl-CoA to β-hydroxyacyl-CoA, we hypothesized that POE containing a bioreducible center in its alkyl part might show an increased anti-tubercular activity due to the involvement of FAS (I) in extra bio-reduction reaction. Thus, we synthesized novel POEs, confirmed their structures by spectral data, and subsequently evaluated their anti-mycobacterial activity against Mycobacterium tuberculosis (Mtb) (H37Rv) strain at 10 μg/mL concentration. Results: Compounds 3c, 3j, and 3m showed higher activity with regard to the inhibition of Mtb growth by 45.4, 45.7, and 51.2% respectively. Unexpectedly, the maltol derived POE 3l having the lowest log p value among the POEs indicated the highest anti-mycobacterial growth activity with 56% prevention. Compounds 3c and 3l showed no remarkable cytotoxicity on human macrophages at 10 μg/mL concentration as analyzed by xCELLigence real-time cell analysis. In further experiments, some of the tested POEs, unlike pyrazinamide (PZA), exhibited significant antibacterial and also anti-fungal activities. POEs showed an enhanced bactericidal activity on gram-positive bacteria as shown for Staphylococcus aureus, e.g. compound 3b with a MIC value of 125 μg/mL but not E. coli as a gram-negative bacteria, except for maltol derived POE (3l) that showed an inverse activity in the susceptibility test. In the anticancer activity test against the human leukemia K562 cell lines using MTT assay, compounds 3e and 3j showed the highest cytotoxic effect with IC50 values of 25±8.0 μΜ and 25±5.0 μΜ, respectively. Conclusion: It was found that the majority of POEs containing a bioreducible center showed higher inhibitory activities on Mtb growth when compared to the similar compounds without a bio-reducible functional group

Inhibitors of Aspartate Transcarbamoylase Inhibit Mycobacterium tuberculosis Growth

Aspartate transcarbamoylase (ATCase) plays a key role in the second step of de novo pyrimidine biosynthesis in eukaryotes and has been proposed to be a target to suppress cell proliferation in E. coli, human cells and the malarial parasite. We hypothesized that a library of ATCase inhibitors developed for malarial ATCase (PfATCase) may also contain inhibitors of the tubercular ATCase and provide a similar inhibition of cellular proliferation. Of the 70 compounds screened, 10 showed single-digit micromolar inhibition in an in vitro activity assay and were tested for their effect on M. tuberculosis cell growth in culture. The most promising compound demonstrated a MIC 90 of 4 μM. A model of MtbATCase was generated using the experimental coordinates of PfATCase. In silico docking experiments showed this compound can occupy a similar allosteric pocket on MtbATCase to that seen on PfATCase, explaining the observed species selectivity seen for this compound series. </p

Development of a Spectral Library for the Discovery of Altered Genomic Events in Mycobacterium avium Associated With Virulence Using Mass Spectrometry-Based Proteogenomic Analysis

Mycobacterium avium is one of the prominent disease-causing bacteria in humans. It causes lymphadenitis, chronic and extrapulmonary, and disseminated infections in adults, children, and immunocompromised patients. M. avium has ∼4500 predicted protein-coding regions on average, which can help discover several variants at the proteome level. Many of them are potentially associated with virulence; thus, identifying such proteins can be a helpful feature in developing panel-based theranostics. In line with such a long-term goal, we carried out an in-depth proteomic analysis of M. avium with both data-dependent and data-independent acquisition methods. Further, a set of proteogenomic investigations were carried out using (i) a protein database for Mycobacterium tuberculosis, (ii) an M. avium genome six-frame–translated database, and (iii) a variant protein database of M. avium. A search of mass spectrometry data against M. avium protein database resulted in identifying 2954 proteins. Further, proteogenomic analyses aided in identifying 1301 novel peptide sequences and correcting translation start sites for 15 proteins. Ultimately, we created a spectral library of M. avium proteins, including novel genome search-specific peptides and variant peptides detected in this study. We validated the spectral library by a data-independent acquisition of the M. avium proteome. Thus, we present an M. avium spectral library of 29,033 peptide precursors supported by 0.4 million fragment ions for further use by the biomedical community.publishedVersio

Not Every Hit-Identification Technique Works on 1-Deoxy-D-Xylulose 5-Phosphate Synthase (DXPS):Making the Most of a Virtual Screening Campaign

In this work, we demonstrate how important it is to investigate not only on-target activity but to keep antibiotic activity against critical pathogens in mind. Since antimicrobial resistance is spreading in bacteria such as Mycobacterium tuberculosis, investigations into new targets are urgently needed. One promising new target is 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. We have recently solved the crystal structure of truncated M. tuberculosis DXPS and used it to perform a virtual screening in collaboration with Atomwise Inc. using their deep convolutional neural network-based AtomNet® platform. Of 94 virtual hit compounds only one showed interesting results in binding and activity studies. We synthesized 30 close derivatives using a straightforward synthetic route that allowed for easy derivatization. However, no improvement in activity was observed for any of the derivatives. Therefore, we tested them against a variety of pathogens and found them to be good inhibitors against Escherichia coli.</p

Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages

notes: PMCID: PMC4006850types: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov'tCandida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.Deutsche ForschungsgemeinschaftNational Institutes for HealthWellcome TrustBBSR

Tuberculostearic Acid-Containing Phosphatidylinositols as Markers of Bacterial Burden in Tuberculosis

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼10$^{2}$ colony forming units (CFUs) and ∼10$^{3}$ CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation─roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity