Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, is central to the pathogenesis of the chronic necrotizing skin disease Buruli ulcer (BU). Here we show that mycolactone acts as ...an inhibitor of the mechanistic Target of Rapamycin (mTOR) signaling pathway by interfering with the assembly of the two distinct mTOR protein complexes mTORC1 and mTORC2, which regulate different cellular processes. Inhibition of the assembly of the rictor containing mTORC2 complex by mycolactone prevents phosphorylation of the serine/threonine protein kinase Akt. The associated inactivation of Akt leads to the dephosphorylation and activation of the Akt-targeted transcription factor FoxO3. Subsequent up-regulation of the FoxO3 target gene BCL2L11 (Bim) increases expression of the pro-apoptotic regulator Bim, driving mycolactone treated mammalian cells into apoptosis. The central role of Bim-dependent apoptosis in BU pathogenesis deduced from our experiments with cultured mammalian cells was further verified in an experimental M. ulcerans infection model. As predicted by the model, M. ulcerans infected Bim knockout mice did not develop necrotic BU lesions with large clusters of extracellular bacteria, but were able to contain the mycobacterial multiplication. Our findings provide a new coherent and comprehensive concept of BU pathogenesis.
Abstract
Mycolactone is a cytotoxin responsible for most of the chronic necrotizing pathology of Mycobacterium ulcerans disease (Buruli ulcer). The polyketide toxin consists of a 12-membered lactone ...ring with a lower O-linked polyunsaturated acyl side chain and an upper C-linked side chain. Mycolactone is unique to M. ulcerans and an immunological Ag capture assay would represent an important tool for the study of Buruli ulcer pathogenesis and for laboratory diagnosis. When testing sets of mycolactone-specific mouse mAbs, we found that Abs against the hydrophobic lower side chain only bind mycolactone immobilized on a solid support but not when present in solution. This observation supports previous findings that mycolactone forms micellar structures in aqueous solution with the hydrophobic region sequestered into the inner core of the aggregates. Although an Ag capture assay typically requires two Abs that recognize nonoverlapping epitopes, our search for matching pairs of mAbs showed that the same mAb could be used both as capture and as detecting reagent for the detection of the mycolactone aggregates. However, the combination of a core-specific and a core/upper side chain–specific mAb constituted the most sensitive ELISA with a sensitivity in the low nanogram range. The results of a pilot experiment showed that the sensitivity of the assay is sufficient to detect mycolactone in swab samples from Buruli ulcer lesions. Although the described capture ELISA can serve as a tool for research on the biology of mycolactone, the assay system will have to be adapted for use as a diagnostic tool.
BackgroundThe Kassena-Nankana District (KND) of northern Ghana lies in the African meningitis belt, where epidemics of bacterial meningitis have been reoccurring every 8–12 years. These epidemics are ...generally caused by Neisseria meningitidis an organism that is considered to be uniquely capable of causing meningitis epidemics MethodsWe recruited all patients with suspected meningitis in the KND between 1998 and 2003. Cerebrospinal fluid samples were collected and analyzed by standard microbiological techniques. Bacterial isolates were subjected to serotyping, multilocus sequence typing (MLST), and antibiotic-resistance testing ResultsA continual increase in the incidence of pneumococcal meningitis was observed from 2000 to 2003. This outbreak exhibited strong seasonality, a broad host age range, and clonal dominance, all of which are characteristic of meningococcal meningitis epidemics in the African meningitis belt. The case-fatality rate for pneumococcal meningitis was 44.4%; the majority of pneumococcal isolates were antibiotic sensitive and expressed the serotype 1 capsule. MLST revealed that these isolates belonged to a clonal complex dominated by sequence type (ST) 217 and its 2 single-locus variants, ST303 and ST612 ConclusionsThe S. pneumoniae ST217 clonal complex represents a hypervirulent lineage with a high propensity to cause meningitis, and our results suggest that this lineage might have the potential to cause an epidemic. Serotype 1 is not included in the currently licensed pediatric heptavalent pneumococcal vaccine. Mass vaccination with a less complex conjugate vaccine that targets hypervirulent serotypes should, therefore, be considered
Mycolactones A/B (1a/b) are exotoxins of Mycobacterium ulcerans that are the molecular cause of Buruli ulcer. 1a/b represent a rapidly equilibrating mixture of Z/E isomers about the C4′C5′ double ...bond of the C5-side chain. Here, we describe the syntheses of mycolactone analogs with configurationally stable C5-side chains (2a, E mimetic; 2b/c, Z mimetics). Based on the cytotoxicity of 2a–c, the Δ4′,5′-trans isomer of mycolactones A/B (1b) appears to be the major virulence factor.
Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein ...essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.
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•Plasmodium falciparum CyRPA binds to carbohydrates with terminal 2-6-linked Neu5Ac•Lectin activity of Plasmodium falciparum CyRPA contributes to erythrocyte invasion•Targeting lectin activity with drugs and antibodies is a promising anti-malarial strategy
Day et al. show that the essential Plasmodium falciparum invasion protein PfCyRPA is a lectin targeting 2-6-linked Neu5Ac. Molecular modeling, mutagenesis, and transgenic parasite studies show that PfCyRPA lectin activity is required for erythrocyte invasion. Drug and antibody inhibitors validate this activity as a therapeutic target to prevent and treat malaria.
Background. The brain’s inflammatory response to the infecting pathogen determines the outcome of bacterial meningitis (BM), for example, the associated mortality and the extent of brain injury. The ...inflammatory cascade is initiated by the presence of bacteria in the cerebrospinal fluid (CSF) activating resident immune cells and leading to the influx of blood derived leukocytes. To elucidate the pathomechanisms behind the observed difference in outcome between different pathogens, we compared the inflammatory profile in the CSF of patients with BM caused by Streptococcus pneumonia (n=14), Neisseria meningitidis (n=22), and Haemophilus influenza (n=9). Methods. CSF inflammatory parameters, including cytokines and chemokines, MMP-9, and nitric oxide synthase activity, were assessed in a cohort of patients with BM from Burkina Faso. Results. Pneumococcal meningitis was associated with significantly higher CSF concentrations of IFN-γ, MCP-1, and the matrix-metalloproteinase (MMP-) 9. In patients with a fatal outcome, levels of TNF-α, IL-1β, IL-1RA, IL-6, and TGF-α were significantly higher. Conclusion. The signature of pro- and anti-inflammatory mediators and the intensity of inflammatory processes in CSF are determined by the bacterial pathogen causing bacterial meningitis with pneumococcal meningitis being associated with a higher case fatality rate than meningitis caused by N. meningitidis or H. influenzae.
Mycolactones, macrolide cytotoxins, are key virulence factors of Mycobacterium ulcerans, the etiological agent of the chronic necrotizing skin disease Buruli ulcer. There is urgent need for a simple ...point-of-care laboratory test for Buruli ulcer and mycolactone represents a promising target for the development of an immunological assay. However, for a long time, all efforts to generate mycolactone-specific antibodies have failed. By using a protein conjugate of a truncated non-toxic synthetic mycolactone derivative, we recently described generation of a set of mycolactone-specific monoclonal antibodies. Using the first mycolactone-specific monoclonal antibodies that we have described before, we were able to develop an antigen competition assay that detects mycolactones. By the systematic selection of a capturing antibody and a reporter molecule, and the optimization of assay conditions, we developed an ELISA that detects common natural variants of mycolactone with a limit of detection in the low nanomolar range. The mycolactone-specific ELISA described here will be a very useful tool for research on the biology of this macrolide toxin. After conversion into a simple point-of-care test format, the competition assay may have great potential as laboratory assay for both the diagnosis of Buruli ulcer and for the monitoring of treatment efficacy.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Surveillance programmes often use malaria rapid diagnostic tests (RDTs) to determine the proportion of the population carrying parasites in their peripheral blood to assess the malaria transmission ...intensity. Despite an increasing number of reports on false-negative and false-positive RDT results, there is a lack of systematic quality control activities for RDTs deployed in malaria surveillance programmes.
The diagnostic performance of field-deployed RDTs used for malaria surveys was assessed by retrospective molecular analysis of the blood retained on the tests.
Of the 2865 RDTs that were collected in 2018 on Bioko Island and analysed in this study, 4.7% had a false-negative result. These false-negative RDTs were associated with low parasite density infections. In 16.6% of analysed samples, masked pfhrp2 and pfhrp3 gene deletions were identified, in which at least one Plasmodium falciparum strain carried a gene deletion. Among all positive RDTs analysed, 28.4% were tested negative by qPCR and therefore considered to be false-positive. Analysing the questionnaire data collected from the participants, this high proportion of false-positive RDTs could be explained by P. falciparum histidine rich protein 2 (PfHRP2) antigen persistence after recent malaria treatment.
Malaria surveillance depending solely on RDTs needs well-integrated quality control procedures to assess the extent and impact of reduced sensitivity and specificity of RDTs on malaria control programmes.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mycolactones are a family of polyketide-derived macrolide exotoxins produced by Mycobacterium ulcerans, the causative agent of the chronic necrotizing skin disease Buruli ulcer. The toxin is ...synthesized by polyketide synthases encoded by the virulence plasmid pMUM. The apoptotic, necrotic and immunosuppressive properties of mycolactones play a central role in the pathogenesis of M. ulcerans.
We have synthesized and tested a series of mycolactone derivatives to conduct structure-activity relationship studies. Flow cytometry, fluorescence microscopy and Alamar Blue-based metabolic assays were used to assess activities of mycolactones on the murine L929 fibroblast cell line. Modifications of the C-linked upper side chain (comprising C12-C20) caused less pronounced changes in cytotoxicity than modifications in the lower C5-O-linked polyunsaturated acyl side chain. A derivative with a truncated lower side chain was unique in having strong inhibitory effects on fibroblast metabolism and cell proliferation at non-cytotoxic concentrations. We also tested whether mycolactones have antimicrobial activity and found no activity against representatives of Gram-positive (Streptococcus pneumoniae) or Gram-negative bacteria (Neisseria meningitis and Escherichia coli), the fungus Saccharomyces cerevisae or the amoeba Dictyostelium discoideum.
Highly defined synthetic compounds allowed to unambiguously compare biological activities of mycolactones expressed by different M. ulcerans lineages and may help identifying target structures and triggering pathways.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of ...antibodies that could neutralize the cytotoxic activities of mycolactone.
Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone.
The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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