Nicole Scherr

Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.

The pathogenicity of mycobacteria such as Mycobacterium tuberculosis is closely associated with their capacity to survive within host macrophages. A crucial virulence factor for intracellular mycobacterial survival is protein kinase G (PknG), a eukaryotic-like serine/threonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular 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 compounds 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.

Antigen 84, an Effector of Pleiomorphism in Mycobacterium smegmatis

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.

Survival of pathogenic mycobacteria in macrophages is mediated through autophosphorylation of protein kinase G

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.

A ring-closing metathesis (RCM)-based approach to mycolactones A/B

The total synthesis of the mycobacterial toxins mycolactones A/B (1 a/b) has been accomplished based on a strategy built around the construction of the mycolactone core through ring-closing metathesis. By employing the Grubbs second-generation catalyst, the 12-membered core macrocycle of mycolactones, with a functionalized C2 handle attached to C11, was obtained in 60-80 % yield. The C-linked upper side chain (comprising C12-C20) was completed by a highly efficient modified Suzuki coupling between C13 and C14, while the attachment of the C5-O-linked polyunsaturated acyl side chain was achieved by Yamaguchi esterification. Surprisingly, a diene containing a simple isopropyl group attached to C11 could not be induced to undergo ring-closing metathesis. By employing fluorescence microscopy and flow cytometry techniques, the synthetic mycolactones A/B (1 a/b) were demonstrated to display similar apoptosis-inducing and cytopathic effects as mycolactones A/B extracted from Mycobacterium ulcerans. In contrast, a simplified analogue with truncated upper and lower side chains was found to be inactive.

Structure-Activity Relationship Studies on the Macrolide Exotoxin Mycolactone of Mycobacterium ulcerans

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.

The macrolide toxin mycolactone promotes bim-dependent apoptosis in Buruli ulcer through inhibition of mTOR

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.

Elimination of intracellularly residing Mycobacterium tuberculosis through targeting of host and bacterial signaling mechanisms

With more than 2 billion latently infected people, TB continues to represent a serious threat to human health. According to the WHO, 1.1 million people died from TB in 2010, which is equal to approximately 3000 deaths per day. The causative agent of the disease, Mycobacterium tuberculosis, is a highly successful pathogen having evolved remarkable strategies to persist within the host. Although normally, upon phagocytosis by macrophages, bacteria are readily eliminated by lysosomes, pathogenic mycobacteria actively prevent destruction within macrophages. The strategies that pathogenic mycobacteria apply range from releasing virulence factors to manipulating host molecules resulting in the modulation of host signal transduction pathways in order to sustain their viability within the infected host. Here, we analyze the current status of how a better understanding of both the bacterial and host factors involved in virulence can be used to develop drugs that may be helpful to curb the TB epidemic.

Identification of the Mycobacterium ulcerans protein MUL_3720 as a promising target for the development of a diagnostic test for Buruli ulcer

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.

Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by Mycobacterium ulcerans.

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.

Localization of Mycobacterial Antigens by Immunofluorescence Staining of Agarose Embedded Cells

Here we report the adaptation of an immunofluorescence staining method for mycobacterial antigens. Our approach is based on agarose embedding followed by thin sectioning of the bacterial samples. We demonstrate that this technique has great potential for protein localization studies, without the use of fluorescence tagged fusion proteins, which is of special interest for slow growing mycobacterial species.