Antibiotics of the ADEP and empedopeptin class - Molecular modes of action and studies on bacterial resistance
Final Report Abstract
ADEPs (an acronym for a novel class of acyldepsipeptides) and empedopeptin are natural products or derivatives thereof with promising antibacterial activity. One work package within the project reported here was concerned with detailed mode of action studies on empedopeptin. From our preliminary work, we already knew that empedopeptin binds to several lipid-bound peptidoglycan precursors and prevents the activity of distinct late-stage peptidoglycan synthesizing enzymes in vitro. Here, were elucidated the specific region of the peptidoglycan precursors recognized by empedopeptin. The primary physiological target of empedopeptin is undecaprenyl pyrophosphate-N-acetylmuramic acid(pentapeptide)-N- acetylglucosamine (lipid II), which is readily accessible at the outside of the cell and which forms a complex with the antibiotic in a 1:2 molar stoichiometry. Empedopeptin recognizes a region that involves the pyrophosphate group, the first sugar, and the proximal parts of both, the stem peptide and the undecaprenyl chain. Regarding ADEP, our previous studies had revealed that the antibiotics bind to ClpP, the proteolytic core of the bacterial caseinolytic protease. ADEP unleashes ClpP, which is normally tightly controlled by cognate Clp-ATPases, to act independently. The unrestrained ClpP no longer degrades the proteins that it is supposed to digest but other essential proteins including the cell division protein FtsZ, which emerged as a preferred target for ADEP-deregulated ClpP. In the current project, we investigated the reason for the particularly high sensitivity of FtsZ to ADEP-activated ClpP, and by employing a library of mutant FtsZ proteins. Thereby, we could identify the structural feature of FtsZ that is responsible for the protein’s high sensitivity. Another project part was concerned with in depth mechanistic studies on the mode of activation of ClpP by ADEP. It turned out that ADEP and chaperones exert extensive conformational control over ClpP in a process affecting the entire ClpP tetradecamer. In addition to opening the axial pore for facilitated substrate passage, ADEPs increase catalytic activity of ClpP through cooperative, allosteric binding to an important regulative site. ClpP activation thus reaches beyond pore opening and also involves conformational control of the catalytic residues. While our previous studies with ADEP concentrated on firmicutes, we studied its effects on distinct bacterial species in the current project. We observed that, in contrast to B. subtilis or S. aureus, mycobacteria are killed by ADEPs through inhibition, as opposed to activation, of ClpP function. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP. This result indicates that ADEP inhibits the growth of mycobacteria by preventing interaction of ClpP1P2 with the regulatory Clp-ATPases, thus inhibiting essential ATP-dependent protein degradation. Studies with Wolbachia showed that ADEP also has the capacity to dysregulate ClpP in these bacterial endobacteria. Like in firmicutes, FtsZ is degraded and Wolbachia are killed by ClpP activation. ADEP-mediated depletion of Wolbachia from filarial worms inhibited growth of these parasites, thereby validating Wolbachia ClpP as a new anti-filarial target.
Publications
- (2011). Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the cell division protein FtsZ. Proc. Natl. Acad. Sci. USA 108, 17474-17479
Sass P, Josten M, Famulla K, Schiffer G, Sahl HG, Hamoen L, Brötz-Oesterhelt H
(See online at https://doi.org/10.1073/pnas.1110385108) - (2012). Substrate recognition and processing by a Walker B mutant of the human mitochondrial AAA+ protein ClpX. J. Struct. Biol. 179, 193-201
Lowth BR Kirstein-Miles J, Saiyet T, Brötz-Oesterhelt H, Morimoto RI, Truscott KN, Dougan DA
(See online at https://doi.org/10.1016/j.jsb.2012.06.001) - (2012). The lipodepsipeptide empedopeptin inhibits cell wall biosynthesis through Ca2+-dependent complex formation with peptidoglycan precursors. J Biol Chem. 287, 20270-20280
Müller A, Münch D, Schmidt Y, Reder-Christ K, Schiffer G, Bendas G, Gross H, Sahl HG, Schneider T, Brötz-Oesterhelt H
(See online at https://doi.org/10.1074/jbc.M112.369561) - (2013). The peptidase ClpP of Wolbachia endobacteria is a novel target for drug development against filarial infections. J. Antimicrob. Chemother. 68, 1790-1800
Schiefer A, Vollmer J, Lämmer C, Specht S, Lentz C, Ruebsamen-Schaeff H, Brötz-Oesterhelt H, Hoerauf A, Pfarr K
(See online at https://doi.org/10.1093/jac/dkt105) - (2015). AAA+ chaperones and acyldepsipeptides activate ClpP via conformational control. Nat. Commun. 6:6320
Gersch M, Famulla K, Dahmen M, Göbl C, Malik I, Richter K, Korotkoc VS, Sass P, Rübsamen- Schaeff H, Madl T, Brötz-Oesterhelt H, Sieber S
(See online at https://doi.org/10.1038/ncomms7320) - (2016). Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease. Mol Microbiol. 101(2), 194-209
Famulla K, Sass P, Malik I, Akopian T, Kandror O, Alber M, Hinzen B, Ruebsamen-Schaeff H, Kalscheuer R, Goldberg AL, Brötz-Oesterhelt H
(See online at https://doi.org/10.1111/mmi.13362)
