Identification of new molecular targets and antibiotics as novel strategies against filaria infections: characterization of lipid II biosynthesis in Wolbachia endobacteria
Final Report Abstract
Wolbachia, obligate intracellular bacteria of arthropods and filarial nematodes, are essential for nematode development and survival and, thus, are targets for treating human filarial infections with antibiotics. Doxycycline depletes Wolbachia from nematodes. Antibiotics targeting cell wall biosynthesis are bactericidal and work more quickly than bacteriostatic drugs, e.g. doxycycline. Thorough understanding of cell-wall targets and Wolbachia biology is essential for better progress in antibiotics as filarial treatments and should provide, at the same time, insights about the essential components and functions of cell wall biology in an endosymbiont with a reduced genome. Only penicillin binding protein (PBP) 2 and PBP6a in Wolbachia of nematodes and arthropods, and PBP 3 in arthropod Wolbachia are conserved that could act on the peptide side chains of lipid II in the endobacteria. Wolbachia of arthropods have also retained AmiD needed for processing of lipid II. Despite high sequence similarity in the binding site, Wolbachia appear to be resistant to penicillin (the Wolbachia Paradox). We examined the function of these enzymes in vitro and in vivo to elucidate their role in Wolbachia and clarify the structure of lipid II of the endobacteria. PBP2 and PBP6a from Wolbachia endosymbionts of Brugia malayi and PBP3 from Wolbachia endosymbionts of Drosophila melanogaster could rescue cell division phenotypes in conditional knockouts in E. coli. PBP3 and PBP6 had activity in vitro. However, wolbachial PBPs were β-lactam-resistant in vitro and in vivo. Wolbachia AmiD has Zn2+-dependent amidase activity and cleaves intact peptidoglycan, monomeric lipid II and anhydromuropeptides, substrates generated during bacterial growth. AmiD may be maintained in arthropod Wolbachia to avoid host immune recognition by degrading cell wall fragments in the periplasm. Lipid II and wolbachial PBP2 both interact with the outer membrane protein Pal suggesting that lipid II and PBP2 are present in the Wolbachia periplasmic space. We labeled Wolbachia peptidoglycan in a C6/36 insect cell line infected with Wolbachia from Aedes albopictus using D-alanyl-D-alanine dipeptide analogues and Click-chemistry. Fluorescent labeling of D-alanyl- D-alanine dipeptides provided visual evidence for the existence of a PG-like structure in Wolbachia. NOD 1 and NOD 2 reporter cells support the existence of PG-like structures from Wolbachia, as protein extracts depleted of Wolbachia do not stimulate the cells. Because NOD 1 was activated, we have also confirmed the presence of diaminopimelic acid in lipid II. The results from the first funding period with Corallopyronin A were instrumental in forming the German Center for Infection Research (DZIF) as we develop this antibiotic for human use. The results of the second period helped form the basis for a project in a trans-regional SFB (TR26). Our understanding of Wolbachia biology has grown, pointing out just how different these endosymbiotic intracellular bacteria are from other intracellular bacteria and Gram-negative bacteria, achieving cell division in via a slightly different route.
Publications
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(2009). Functional conservation of the lipid II biosynthesis pathway in the cell wall-less bacteria Chlamydia and Wolbachia: why is lipid II needed? Mol Microbiol. 73: 913-923
Henrichfreise B, Schiefer A, Schneider T, Nzukou E, Poellinger C, Hoffmann TJ, Johnston KL, Moelleken K, Wiedemann I, Pfarr K, Hoerauf A, Sahl HG
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(2012). Corallopyronin A specifically targets and depletes essential obligate Wolbachia endobacteria from filarial nematodes in vivo. J Infect Dis. 206: 249-57
Schiefer A, Schmitz A, TF, Specht S, Lämmer C, Johnston KL, Vassylyev DG, König GM, Hoerauf A, Pfarr K
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(2013). Requirement of lipid II biosynthesis for cell division in cell wall-less Wolbachia, endobacteria of arthropods and filarial nematodes. Int J Med Microbiol. 303: 140-149
Vollmer J, Schiefer A, Schneider T, Jülicher K, Johnston KL, Taylor MJ, Sahl HG, Hoerauf A, Pfarr K
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(2013).The Clp peptidase of Wolbachia endobacteria is a novel target for drug development against filarial infections. J Antimicrob Chemother. 68: 1790-800
Schiefer A, Vollmer J, Lämmer C, Specht S, Lentz C, Ruebsamen-Schaeff H, Brötz-Oesterhelt H, Hoerauf A, Pfarr K
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(2014). Corallopyronin A - A promising antibiotic for treatment of filariasis. Int J Med Microbiol. 304:72-8
Schäberle TF, Schiefer A, Schmitz A, König GM, Hoerauf A, Pfarr K
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(2014). Repurposing of approved drugs from the human pharmacopoeia to target Wolbachia endosymbionts of onchocerciasis and lymphatic filariasis. Int J Parasitol Drugs Drug Resist. 4:278-86
Johnston KL, Ford L, Umareddy I, Townson S, Specht S, Pfarr K, Hoerauf A, Altmeyer R, Taylor MJ
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(2015). Insights into structure−activity relationships of bacterial RNA polymerase inhibiting Corallopyronin derivatives. J Nat Prod. 78:2505-9
Schäberle TF, Schmitz A, Zocher G, Schiefer A, Kehraus S, Neu E, Roth M, Vassylyev DG, Stehle T, Bierbaum G, Hoerauf A, Pfarr K, König GM
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US 9168244 B2 Compounds for use in the treatment of filariasis (Therapeutic treatment of filariasis/dirofilariasis in humans/animals)
Kenneth Michael Pfarr, Achim Hoerauf, Gabriele Maria Koenig, Sabine Specht, Andrea Schiefer, Till Friedrich Schaeberle, Alexander Schmitz, Stefan Kehraus
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(2016). NOD2 dependent neutrophil recruitment is required for early protective immune responses against infectious Litomosoides sigmodontis L3 larvae. Sci Rep. 6:39648
Ajendra J, Specht S, Ziewer S, Schiefer A, Pfarr K, Parcina M, Kufer TA, Hoerauf A, Hubner MP
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(2017). AmiD is a novel peptidoglycan amidase in Wolbachia endosymbionts of Drosophila melanogaster. Front Cell Infect Microbiol. 7:353
Wilmes M, Meier K, Schiefer A, Josten M, Otten CF, Klöckner A, Henrichfreise B, Vollmer W, Hoerauf A, Pfarr K
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EP 2704708 B1 Compounds for use in the treatment of filariasis (Prevention and treatment of filariasis/dirofilariasis in humans/animals)
Kenneth Michael Pfarr, Achim Hoerauf, Gabriele Maria Koenig, Sabine Specht, Andrea Schiefer, Till Friedrich Schaeberle, Alexander Schmitz, Stefan Kehraus
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US 9687470 B2 Compounds for use in the treatment of filariasis (Prevention of filariasis/dirofilariasis in dogs and humans)
Kenneth Michael Pfarr, Achim Hoerauf, Gabriele Maria Koenig, Sabine Specht, Andrea Schiefer, Till Friedrich Schaeberle, Alexander Schmitz, Stefan Kehraus
