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Background: 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. Methodology/Principal Findings: 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. Conclusions/Significance: The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target. [ABSTRACT FROM AUTHOR]

A comprehensive analysis was done to evaluate the potential use of anti-parasitic macrocyclic lactones (including avermectins and milbemycins) for Buruli ulcer (BU) therapy. A panel containing nearly all macrocyclic lactones used in human or in veterinary medicine was analyzed for activity in vitro against clinical isolates of Mycobacterium ulcerans. Milbemycin oxime and selamectin were the most active drugs against M. ulcerans with MIC values from 2 to 8 μg/mL and 2 to 4 μg/mL, respectively. In contrast, ivermectin and moxidectin, which are both in clinical use, showed no significant activity (MIC> 32 μg/mL). Time-kill kinetic assays showed bactericidal activity of selamectin and in vitro pharmacodynamic studies demonstrated exposure-dependent activity. These data together with analyses of published pharmacokinetic information strongly suggest that selamectin is the most promising macrocyclic lactone for BU treatment. [ABSTRACT FROM AUTHOR]

Background: 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. Methodology/Principal Findings: 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. Conclusion: 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. Author Summary: Buruli ulcer is a chronic necrotizing skin disease caused by Mycobacterium ulcerans. The characteristic histopathological features of Buruli ulcer, severe destruction of subcutaneous tissue with minimal inflammation in the core of the lesion, are primarily attributed to the cytotoxic activity of mycolactone, the macrolide exotoxin of M. ulcerans. Different geographical lineages of M. ulcerans produce different structural variants of mycolactone. By using highly defined synthetic mycolactones, including both naturally occurring molecular species and additional non-natural variants, we have assessed the influence of the structure of the C-linked upper side chain and the lower C5-O-linked polyunsaturated acyl side chain on biological activity. Changes in the lower side chain affected the cytotoxic activity against mammalian cells more profoundly than changes in the upper side chain. Mycolactone A/B had no antimicrobial activity against Gram-positive and Gram-negative bacteria and was also inactive against Saccharomyces and Dictyostelium. [ABSTRACT FROM AUTHOR]

Background: 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. Methodology/Principal Findings: We have synthesized and tested a series of mycolactone derivatives to conduct structureactivity 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

At first glance, bacteria that belong to the two genera Streptomyces and Mycobacterium of the phylum Actinobacteria show no sign of similarity. Whereas Streptomyces species are generally classified as spore-forming, filamentous bacteria, species of the Mycobacterium genus have been considered non-sporulating, rod-like shaped. However, recent studies in genetics and cell biology of Streptomyces and Mycobacterium have revealed striking analogies in the developmental and morphological hallmarks of their life cycles. Understanding the mechanisms underlying these similarities, as well as variations in morphogenesis and development of these two groups of bacteria may not only provide important insights in the evolution of cell shapes in Actinobacteria, but also lead to medical interventions that impact human health. [Copyright &y& Elsevier]

Pathogenic mycobacteria survive within macrophages through the inhibition of phagosome-lysosome fusion. A crucial factor for avoiding lysosomal degradation is the mycobacterial serine/threonine protein kinase G (PknG). PknG is released into the macrophage cytosol upon mycobacterial infection, suggesting that PknG might exert its activity by interfering with host signaling cascades, but the mode of action of PknG remains unknown. Here, we show that PknG undergoes autophosphorylation on threonine residues located at the N terminus. In contrast to all other mycobacterial kinases investigated thus far, autophosphorylation of PknG was not involved in the regulation of its kinase activity. However, autophosphorylation was crucial for the capacity of PknG to promote mycobacterial survival within macrophages. These results will contribute to a better understanding of the virulence mechanisms of pathogenic mycobacteria and may help to design improved inhibitors of PknG to be developed as antimycobacterial compounds. [ABSTRACT FROM AUTHOR]

The pathogenicity of mycobacteria such as Mycobacterium tuberculosis is closely associated with their capacity to survive within host macrophages. A crucial virulence factor for intra cellular mycobacterial survival is protein kinase G (PknG), a eukaryotic-like serine/threonine protein kinase expressed by pathogenic mycobacteria that blocks the intra cellular degradation of mycobacteria in lysosomes. Inhibition of PknG with the highly selective low-molecular-weight inhibitor AX20017 results in mycobacterial transfer to lysosomes and killing of the mycobacteria. Here, we report the 2.4 Å x-ray crystal structure of PknG in complex with AX20017. The unique multidomain topology of PknG reveals a central kinase domain that is flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeat domains, respectively. Directed mutagenesis suggests that the rubredoxin domain functions as a regulator of PknG kinase activity. The structure of PknG-AX20017 further reveals that the inhibitor is buried deep within the adenosine-binding site, targeting an active conformation of the kinase domain. Remarkably, although the topology of the kinase domain is reminiscent of eukaryotic kinases, the AX20017-binding pocket is shaped by a unique set of amino acid side chains that are not found in any human kinase. Directed mutagenesis of the unique set of residues resulted in a drastic loss of the compound's inhibitory potency. Our results explain the specific mode of action of AX20017 and demonstrate that virulence factors highly homologous to host molecules can be successfully targeted to block the proliferation of M. tuberculosis. [ABSTRACT FROM AUTHOR]

Nuclear migration and positioning in Aspergillus nidulans depend on microtubules, the microtubule-dependent motor protein dynein, and auxiliary proteins, two of which are ApsA and ApsB. In apsA and apsB mutants nuclei are clustered and show various kinds of nuclear navigation defects, although nuclear migration itself is still possible. We studied the role of several components involved in nuclear migration through in viva fluorescence microscopy using fluorescent-protein tagging. Because ApsA localizes to the cell cortex and mitotic spindles were immobile in apsA mutants, we suggest that astral microtubule-cortex interactions are necessary for oscillation and movement of mitotic spindles along hyphae, but not for post-mitotic nuclear migration. Mutation of apsA resulted in longer and curved microtubules and displayed synthetic lethality in combination with the conventional kinesin mutation ΔkinA. By contrast, ApsB localized to spindle-pole bodies (the fungal centrosome), to septa and to spots moving rapidly along microtubules. The number of cytoplasmic microtubules was reduced in apsB mutants in comparison to the wild type, indicating that cytoplasmic microtubule nucleation was affected, whereas mitotic spindle formation appeared normal. Mutation of apsB suppressed dynein null mutants, whereas apsA mutation had no effect. We suggest that nuclear positioning defects in the apsA and apsB mutants are due to different effects on microtubule organisation. A model of spindle-pole body led nuclear migration and the roles of dynein and microtubules are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Infectious diseases continue to represent a great burden to human society, being responsible for approximately 14 to 16 million deaths annually. Today, Mycobacterium tuberculosis, the causative agent of tuberculosis, remains one of the most important infectious agents. Moreover, with the emergence of multi drug and extensive drug resistant strains of M. tuberculosis, there is a great need for a better understanding of the virulence strategies utilized by this pathogen. In this manuscript, we discuss some of the strategies that have been followed to unravel virulence mechanisms utilized by M. tuberculosis. Knowledge of these mechanisms is important to reveal potential targets that may be useful for the development of compounds to treat tuberculosis. [Copyright &y& Elsevier]

While in most rod-shaped bacteria, morphology is based on MreB-like proteins that form an actin-like cytoskeletal scaffold for cell wall biosynthesis, the factors that determine the more flexible rod-like shape in actinobacteria such as Mycobacterium species are unknown. Here we show that a Mycobacterium smegmatis protein homologous to eubacterial DivIVA-like proteins, including M. tuberculosis antigen 84 (Ag84), localized symmetrically to centers of peptidoglycan biosynthesis at the poles and septa. Controlled gene disruption experiments indicated that the gene encoding Ag84, wag31, was essential; when overexpressed, cells became longer and wider, with Ag84 asymmetrically distributed at one pole. Many became grossly enlarged, bowling-pin-shaped cells having up to 80-fold-increased volume. In these cells, Ag84 accumulated predominantly at a bulbous pole that was apparently generated by uncontrolled cell wall expansion. In some cells, Ag84 was associated with exceptional sites of cell wall expansion (buds) that evolved into branches. M. bovis BCG Ag84 was able to form oligomers in vitro, perhaps reflecting its superstructure in vivo. These data suggested a role for Ag84 in cell division and modulating cell shape in pleiomorphic actinobacteria. [ABSTRACT FROM AUTHOR]

The emergence of antibiotic-resistant bacteria in the last century is alarming and calls for alternative, nonchemical treatment strategies. Thermal medicine uses heat for the treatment of infectious diseases but its use in facultative and obligate intracellular bacteria remains poorly studied. In this review, we summarize previous research on reducing the infectious burden of Mycobacterium ulcerans and Chlamydia trachomatis by using water-filtered infrared A-radiation (wIRA), a special form of heat radiation with high tissue penetration and low thermal load on the skin surface. Mycobacterium ulcerans is a thermosensitive bacterium causing chronic necrotizing skin disease. Therefore, previous data on wIRA-induced improvement of wound healing and reduction of wound infections is summarized first. Then, pathogenesis and treatment of infections with M. ulcerans causing Buruli ulcer and of those with C. trachomatis infecting the ocular conjunctiva and resulting in blinding trachoma are discussed. Both bacteria cause neglected tropical diseases and have similar geographical distributions. Results of previous in vitro and in vivo studies using wIRA on M. ulcerans and C. trachomatis infections are presented. Finally, technical aspects of using wIRA in patients are critically reviewed and open questions driving future research are highlighted. In conclusion, wIRA is a promising tool for reducing infectious burden due to intracellular bacteria such as M. ulcerans and C. trachomatis. [ABSTRACT FROM AUTHOR]

Dengue fever can be caused by one of four distinct dengue virus (DENV) serotypes that cocirculate in many parts of the world. Point of care serotype-specific nonstructural protein-1 (NS1) capture assays for the rapid serotyping of DENV in human sera would greatly support epidemiological surveillance and potentially also prognosis in individual patients. To ensure both serotype specificity and broad coverage of variants within serotypes' we have applied an innovative approach for the generation and selection of serotype-specific anti-NSl mAbs. To elicit mAbs against conformational epitopes, NMRI mice were immunized with living HEK 293 transfectants expressing the native target Ags in multiple display on the cell surface. For each serotype, three different NS1 sequence variants were sequentially used for immunization of mice, hybridoma selection, and capture assay development, respectively. Selection of optimal combinations of capturing and detecting mAbs yielded highly sensitive and specific NS1 serotyping ELISAs (st-ELISAs) for the four serotypes. st-ELISA testing of 41 dengue patient sera showed a 100% concordance with the serotype determined by serotype-specific reverse transcriptase real-time quantitative PCR. The respective NS1 variants could be detected for ~10 d after the onset of illness. Ab-dependent enhancement of DENV infections may be associated with a specific range of pre-existing anti-DENV serological Ab titers. Testing of patient sera with the developed st-ELISAs will not only be useful for epidemiological studies and surveillance, but it may also help to develop and validate assays that can distinguish protective versus enhancing Ab responses for risk assessment for the development of severe dengue disease in individual patients. The Journal of Immunology, 2018, 200: 3857-3866. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis is one of the most successful pathogens known, having infected more than a third of the global population. An important strategy for intracellular survival of pathogenic mycobacteria relies on their capacity to resist delivery to lysosomes, instead surviving within macrophage phagosomes. Several factors of both mycobacterial and host origin have been implicated in this process. However, whether or not this strategy is employed in vivo is not clear. Here we show that in vivo, following intravenous infection, M. tuberculosis and Mycobacterium bovis BCG initially survived by resisting lysosomal transfer. However, after prolonged infection the bacteria were transferred to lysosomes yet continued to proliferate. A M. bovis BCG mutant lacking protein kinase G (PknG), that cannot avoid lysosomal transfer and is readily cleared in vitro , was found to survive and proliferate in vivo. The ability to survive and proliferate in lysosomal organelles in vivo was found to be due to an altered host environment rather than changes in the inherent ability of the bacteria to arrest phagosome maturation. Thus, within an infected host, both M. tuberculosis and M. bovis BCG adapts to infection-specific host responses. These results are important to understand the pathology of tuberculosis and may have implications for the development of effective strategies to combat tuberculosis. [ABSTRACT FROM AUTHOR]

Buruli ulcer (BU) caused by Mycobacterium ulcerans is a devastating skin disease, occurring mainly in remote West African communities with poor access to health care. Early case detection and subsequent antibiotic treatment are essential to counteract the progression of the characteristic chronic ulcerative lesions. Since the accuracy of clinical BU diagnosis is limited, laboratory reconfirmation is crucial. However, currently available diagnostic techniques with sufficient sensitivity and specificity require infrastructure and resources only accessible at a few reference centres in the African endemic countries. Hence, the development of a simple, rapid, sensitive and specific point-of-care diagnostic tool is one of the major research priorities for BU. In this study, we have identified a previously unknown M. ulcerans protein, MUL_3720, as a promising target for antigen capture-based detection assays. We show that MUL_3720 is highly expressed by M. ulcerans and has no orthologs in other prevalent pathogenic mycobacteria. We generated a panel of anti-MUL_3720 antibodies and used them to confirm a cell wall location for MUL_3720. These antibodies could also specifically detect M. ulcerans in infected human tissue samples as well as in lysates of infected mouse footpads. A bacterial 2-hybrid screen suggested a potential role for MUL_3720 in cell wall biosynthesis pathways. Finally, we demonstrate that a combination of MUL_3720 specific antibody reagents in a sandwich-ELISA format has sufficient sensitivity to make them suitable for the development of antigen capture-based diagnostic tests for BU. Author Summary: According to the recommendations of the World Health Organization, the clinical diagnosis of BU should be reconfirmed by at least two laboratory techniques. However, out of the four currently available tests, three (PCR, histopathology and cultivation of M. ulcerans) can only be performed at centralized reference laboratories; the fourth (microscopic detection of acid fast bacilli) lacks the required sensitivity and specificity. Therefore, a simple tool for early diagnosis of the disease, which can be implemented in rural health care facilities of the endemic countries, is of urgent need. In this study we aimed at the identification of M. ulcerans proteins as potential targets for the development of a simple and rapid diagnostic antigen detection assay. Among 36 proteins, MUL_3720 best met the predefined criteria of being highly expressed by M. ulcerans and not having orthologs in other pathogenic mycobacterial species prevalent in the endemic regions. Here we generated monoclonal and polyclonal antibodies against this protein and carried out pilot studies for the development of an antigen capture-based diagnostic test. [ABSTRACT FROM AUTHOR]

Buruli ulcer (BU) caused by Mycobacterium ulcerans is a devastating skin disease, occurring mainly in remote West African communities with poor access to health care. Early case detection and subsequent antibiotic treatment are essential to counteract the progression of the characteristic chronic ulcerative lesions. Since the accuracy of clinical BU diagnosis is limited, laboratory reconfirmation is crucial. However, currently available diagnostic techniques with sufficient sensitivity and specificity require infrastructure and resources only accessible at a few reference centres in the African endemic countries. Hence, the development of a simple, rapid, sensitive and specific point-of-care diagnostic tool is one of the major research priorities for BU. In this study, we have identified a previously unknown M. ulcerans protein, MUL_3720, as a promising target for antigen capture-based detection assays. We show that MUL_3720 is highly expressed by M. ulcerans and has no orthologs in other prevalent pathogenic mycobacteria. We generated a panel of anti-MUL_3720 antibodies and used them to confirm a cell wall location for MUL_3720. These antibodies could also specifically detect M. ulcerans in infected human tissue samples as well as in lysates of infected mouse footpads. A bacterial 2-hybrid screen suggested a potential role for MUL_3720 in cell wall biosynthesis pathways. Finally, we demonstrate that a combination of MUL_3720 specific antibody reagents in a sandwich-ELISA format has sufficient sensitivity to make them suitable for the development of antigen capture-based diagnostic tests for BU. [ABSTRACT FROM AUTHOR]