Accepted Manuscript

Title: Bacterial caseinolytic proteases as novel targets for

antibacterial treatment

Author: Heike Brötz-Oesterhelt Peter Sass

PII: S1438-4221(13)00138-0
DOI: http://dx.doi.org/doi:10.1016/j.ijmm.2013.09.001
Reference: IJMM 50741

To appear in:

Please cite this article as: Brötz-Oesterhelt, H., Sass, P., Bacterial caseinolytic
proteases as novel targets for antibacterial treatment, International Journal of Medical
Microbiology (2013), http://dx.doi.org/10.1016/j.ijmm.2013.09.001

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 1 Bacterial caseinolytic proteases as novel targets for antibacterial treatment

3 Heike Brötz-Oesterhelt* and Peter Sass

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5 Institute for Pharmaceutical Biology and Biotechnology, University of Düsseldorf,

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6 Universitätsstrasse 1, D-40225 Düsseldorf, Germany

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8 * Corresponding author:

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9 Prof. Dr. Heike Brötz-Oesterhelt
10 Tel: +49-211-8114180

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11 Fax: +49-211-8111923
12 email: Heike.Broetz-Oesterhelt@uni-duesseldorf.de

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14

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16
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17 Key words
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18 ClpP
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19 AAA+ chaperones / Clp-ATPases

20 drug target
21 virulence target
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22 acyldepsipeptide antibiotics (ADEPs)

23 beta-lactones

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26

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28

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29 Summary

30 Bacterial Clp proteases are important for protein turnover and homeostasis in bacteria in
31 order to maintain vital cellular functions particularly under stress conditions. Apart from their
32 crucial role in general protein quality control by degrading abnormally folded or otherwise
33 aberrant or malfunctioning proteins, their temporally and spatially precise proteolysis of key
34

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regulatory proteins additionally guides several developmental processes like cell motility,

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35 genetic competence, cell differentiation, sporulation as well as important aspects of
36 virulence. Due to their apparent relevance for many physiological processes and their

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37 conservation among diverse bacterial species including human pathogens, Clp proteases
38 have attracted considerable attention as targets for antibacterial action in recent years.

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39 Particularly a novel class of potent acyldepsipeptide antibiotics unleashes ClpP, the uniform
40 proteolytic core unit of the degradative Clp complexes, to bring about bacterial death via
41 uncontrolled proteolysis of proteins that are essential for bacterial viability. In addition,

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42 covalent inhibition of the catalytic center of ClpP by another class of small molecule inhibitors
43 is investigated in the context of virulence inhibition. Both antibacterial mechanisms constitute
44 innovative approaches with the potential to control infections caused by multi-resistant
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45 bacterial pathogens due to the lack of cross-resistance to established antibiotic classes.

46
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47 Introduction
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48 ClpP, the proteolytic core unit of several bacterial caseinolytic proteases (Clp), represents a
49 conserved protein that is present in nearly all sequenced eubacterial genomes except for
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50 mollicutes (Yu and Houry, 2007). Furthermore, ClpP orthologs have also been detected in
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51 eukaryotes, i. e. in the plastid-derived apicoplast of the human parasite Plasmodium

52 falciparum (Lin et al., 2009; Rathore et al., 2010) as well as in mitochondria and plastids of
53 plants (Yu and Houry, 2007). In this review the term “Clp proteases” refers to the consistent
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54 proteolytic core ClpP in concert with its various corresponding AAA+ chaperones of the
55 Hsp100 family (ATPases Associated with diverse cellular Activities, here referred to as Clp-
56 ATPases) such as ClpX, ClpC or ClpA, as well as associated adaptor proteins. Thus, each
57 ClpP/Clp-ATPase pair (e.g. ClpXP, ClpCP or ClpAP) is described here as a “Clp protease”.
58 Noteworthy, in eubacteria there is a second Clp protease system that comprises the
59 threonine protease ClpQ as the proteolytic core and the ATPase ClpY (Ramachandran et al.,
60 2002) which is not subject of this review.

61 A major function of the Clp system is protein stress management, i.e. in situations when
62 mistranslated, misfolded or aggregated proteins accumulate in the bacterial cell as a result of
63 e.g heat stress or antibiotic interference with the ribosomal machinery. In a first attempt, Clp-

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64 ATPases support the refolding process of aberrant proteins in an ATP-dependent manner,
65 independently of ClpP. If refolding is unsuccessful, Clp-ATPases direct the defective protein
66 to the proteolytic core ClpP, where it is unfolded and degraded (Fig. 1A). Intracellular protein
67 substrates can be N- or C-terminally flagged by specific degradation tags (degrons), 5
68 classes of which are known to date (Baker and Sauer, 2012; Flynn et al., 2003; Neher et al.,
69 2006). For instance, when ribosomes stall during translation in Escherichia coli, the 11 amino

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70 acid SsrA-tag is attached to the C-terminus of nascent polypeptides to flag the defective

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71 protein for degradation by the ClpXP pair (Gottesman et al., 1998). Signal sequences for
72 protein secretion can also serve as degrons marking the substrate as an extracellular protein

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73 that failed to be exported.

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74 Clp proteases do also control crucial developmental processes in bacteria via proteolysis of
75 regulatory key elements such as transcription factors. In this context, the regulation of
76 motility, exoenzyme synthesis, spore formation and genetic competence is driven by Clp in

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77 Bacillus subtilis (Msadek et al., 1998; Pummi et al., 2002; Turgay et al., 1998). In E. coli, a
78 Clp protease modulates the duration of the SOS response by adjusting the level of RecN
79 (Neher et al., 2006) and in Caulobacter crescentus a Clp protease governs cell differentiation
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80 (Jenal and Fuchs, 1998). In some actinomycetales, including Mycobacterium tuberculosis,
81 Corynebacterium glutamicum and Streptomyces lividans, ClpP is even essential for growth
82 under moderate conditions in vitro (Engels et al., 2004; Gominet et al., 2011; Raju et al.,
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83 2012; Sassetti et al., 2003). Moreover, ClpP can also be a crucial factor in eukaryotic cells,
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84 since it appears to be important for the development of the apicoplast of P. falciparum, a

85 specialized organelle of bacterial origin, which the parasites require for survival (Rathore et
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86 al., 2010). Due to its prominent role in protein turnover and regulated proteolysis, inactivation
87 or deletion of ClpP causes severe phenotypes and even growth inhibition under stress
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88 conditions in many bacterial species including important human pathogens. Consequently,

89 Clp proteases have attracted considerable attention to evaluate their potential as drug
90 targets.
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92 The Clp protease system – concerted function of ClpP with Clp-ATPases

93 In recent years, our understanding of the general composition and operation mode of
94 bacterial Clp proteases has greatly benefited from the elucidation of crystal structures for a
95 variety of ClpP orthologs which revealed a conserved organization of ClpP in several
96 bacterial species including E. coli, B. subtilis, Streptococcus pneumoniae, Staphylococcus
97 aureus, Listeria monocytogenes and M. tuberculosis as well as the human mitochondrial
98 ClpP (Geiger et al., 2011; Gribun et al., 2005; Ingvarsson et al., 2007; Kang et al., 2004; Lee

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 99 et al., 2011; Wang et al., 1997; Zeiler et al., 2011). ClpP is a self-compartmentalized serine
100 protease that exhibits a cylindrical structure with 14 subunits arranged into two rings of
101 seven, in most cases identical subunits, which stack vis-à-vis to form a barrel shaped
102 structure of about 90Å in both height and diameter. Inside of the barrel, a spacious chamber
103 of roughly 50Å width encloses the protease active sites comprising 14 catalytic triads with the
104 canonical residues typical for serine proteases (Ser, His, Asp), which are located close to the

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105 equatorial plane of the ClpP barrel (Fig. 1B). Due to this compartmentalized arrangement,

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106 the catalytic triads are effectively shielded from the cytoplasmic environment and potential
107 protein substrates. Entry of cytoplasmic protein substrates to the proteolytic chamber is

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108 regulated through small entrance pores at the apical and distal surfaces of the ClpP barrel
109 (Fig. 1C).

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110 Each ClpP monomer can be sub-divided into a compact body (“head region”) and a
111 distinctive protruding α/β unit (“handle region”). While the heads of seven monomers build up

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112 the heptameric rings through mostly hydrophobic interactions, the handles establish contacts
113 to the opposite heptameric ring via hydrogen bonds to form the ClpP tetradecamer (Fig. 1D).
114 As suggested by recent structural studies, the joining of the two heptameric rings appears to
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115 induce a conformational shift inside the handle region that allows the catalytic triads of the
116 resulting ClpP tetradecamer to adopt an active orientation which is mandatory for enzymatic
117 activation (Geiger et al., 2011; Kimber et al., 2010; Lee et al., 2011). By this means, it is
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118 ensured that the proteolytic capacity of the protease is only engaged when the activated
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119 catalytic triads are securely shielded from other cytoplasmic proteins in the secluded
120 compartment of the ClpP tetradecamer. This activation mechanism represents a necessary
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121 measure of safety to prevent undesired proteolysis, since ClpP itself is almost free of
122 substrate specificity and any protein or peptide substrate that gains access to the proteolytic
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123 chamber is degraded into short peptides of about 7-8 residues (Baker and Sauer, 2012).
124 Consequently, proteolysis by ClpP is controlled via restricted access of protein substrates to
125 the degradation chamber through the narrow axial entrance pores of the ClpP tetradecamer,
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126 which are generally too small for the entry of folded proteins, rather than via a preferred
127 amino acid sequence of the active sites as encountered with other proteases. Accordingly,
128 the ClpP tetradecamer alone completely lacks proteolytic activity, since only small peptides
129 that readily diffuse through the entrance pores are degraded (Thompson and Maurizi, 1994).
130 Thus, ClpP by itself is no more than a peptidase but represents the dormant core of the
131 larger Clp proteolytic system.

132 For proteolytic activity, ClpP strictly depends on corresponding Clp-ATPases, which
133 recognize and bind protein substrates in the cytoplasm and transport them to ClpP (Fig. 1A).
134 Here, the Clp-ATPases unfold their substrates and translocate them through the axial pores

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135 into the ClpP proteolytic chamber using energy from ATP-hydrolysis. For the interaction with
136 the ClpP tetradecamer, the ClpP-ATPases form hexamers that stack to the apical and/or
137 distal surface of the ClpP barrel. Specific loops, which protrude from each Clp-ATPase
138 monomer (Kim and Kim, 2003) and contain a conserved motif at their tip (i.e. IGF in E. coli
139 ClpX or IGL in E. coli ClpA), insert into large peripheral grooves in the apical and distal
140 surfaces of ClpP (Baker and Sauer, 2012; Yu and Houry, 2007). In addition, axial contacts

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141 are established near the axial pores of the ClpP barrel between so-called pore-2 loops of the

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142 Clp-ATPase ring and N-terminal stem-loops of ClpP. Since most species contain multiple
143 Clp-ATPase paralogs that usually differ in their substrate spectrum, the particularly involved

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144 Clp-ATPase serves as the first means for determining substrate specificity of the Clp
145 proteolytic machinery. For instance, proteolysis of E. coli ClpP is stimulated by either ClpA or

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146 ClpX and B. subtilis ClpP is controlled by ClpX, ClpC and ClpE. Furthermore, so-called
147 adaptor proteins interact with particular Clp-ATPases to increase their affinity for specific
148 protein substrates, introducing another level of regulation during proteolysis (Kirstein et al.,

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149 2009b). Clearly, ClpP makes important contributions to the physiological functions of the
150 bacterial cell and at the same time it represents a potentially harmful protease whose activity
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151 underlies tight regulation on several levels to control its destructive capacity.

152
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153 ClpP and Clp-ATPases as anti-virulence targets

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154 In several bacterial species, ClpP or Clp-ATPases were reported to be important for the
155 development of bacterial virulence (Frees et al., 2007; Butler et al., 2006). For instance, in L.
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156 monocytogenes, a prominent cause of foodborne disease, one of its two ClpP paralogs,
157 ClpP2, is crucial for the production and secretion of functional listeriolysin, an exotoxin
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158 required for intracellular growth of the pathogen in macrophages. Consequently, a clpP
159 deletion mutant in L. monocytogenes was avirulent in the mouse, even when applied at a
5000-fold higher infective dose than the wild-type (LD50 wt 104.8 colony forming units, cfu,
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161 versus LD50 ΔclpP2 >5x108 cfu) (Gaillot et al., 2000). Among the Clp-ATPases, ClpC
162 promotes early escape of Listeria from the phagosomal compartment in macrophages
163 (Rouquette et al., 1998) and ClpE was also shown to contribute to virulence (Nair et al.,
164 1999). In S. pneumoniae, an important causative agent of lung infection, clpP deletion led to
165 a significant virulence reduction in several mouse models. The ΔclpP strain of a highly
166 virulent wild-type strain was defective in intranasal colonization, failed to grow in the lung
167 after intratracheal instillation and led to a considerably higher overall survival rate of the mice
168 compared to the wild-type after intraperitoneal infection (Robertson et al., 2002; Kwon et al.,
169 2003; Kwon et al., 2004). Likewise, in S. aureus, the most frequent cause of nosocomial
170 infection among Gram-positive bacteria, ClpXP was shown to down-regulate the transcription

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171 of genes encoding several secreted virulence proteins, among them α-toxin, extracellular
172 proteases, lipases and further hydrolases. For the surface adhesins a more heterogeneous
173 picture emerged, with reduced expression of the ica-operon and increased expression of
174 fibronectin binding proteins (Frees et al., 2003; Michel et al., 2006). In addition, ClpX, this
175 time independent of ClpP, seems to be required for the expression of protein A, a surface
176 protein of S. aureus that prevents antibodies from opsonization by sequestering their Fc-

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177 regions (Frees et al., 2003). In vivo the clpP deletion reduced the growth of S. aureus in a

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178 skin abscess model in mice (Frees et al., 2003).

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179 Although such reports from several independent research groups on the important functions
180 of Clp proteins in the pathogenesis of different bacterial species seem promising regarding

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181 their suitability, first and foremost of ClpP, as anti-virulence targets, it should be noted that all
182 studies described above were conducted with one exemplary model strain background per
183 species only. In the few reports on comparisons of more than one strain per species the

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184 particular effects caused by the clpP deletion varied, in strength as well as in nature. For
185 instance, while a ΔclpP mutant in S. pneumoniae D39 (the strain used in the studies
186 described above) was avirulent in mice after intranasal infection, the corresponding mutant of
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187 S. pneumoniae TIGR4 still killed all animals, albeit with a significant delay of 24 h compared
188 to the wild-type (Ibrahim et al., 2005). Similarly, when the effects of a clpP deletion in S.
189 aureus were compared between the background of NCTC 8325 (a sigB deficient family of
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190 strains employed in the initial studies described above), with that of the sigB proficient clinical
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191 isolates COL, Newman and SA564 in a more recent study, profound strain-dependent
192 differences in the expression of selected virulence genes were observed (Frees et al., 2012).
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193 Hence, is seems important to confirm the effectiveness of Clp proteins as targets for a
194 potential anti-virulence treatment strategy on the basis of a broader strain panel.
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195 In addition to the specific roles in regulating the abundance of the particular virulence factors
196 mentioned above, it shall not be forgotten that Clp proteases help bacterial cells to bear
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197 diverse sorts of stress by their general activities in protein quality control and that the entry of
198 bacterial pathogens into the hostile environment of the host imposes enormous stress on the
199 bacteria.

200 When speculating about the possible use of Clp proteases as anti-virulence targets, their
201 inhibition by a small molecule inhibitor is mandatory. Here, proof-of-principle is provided by a
202 class of lactone compounds, which inactivate ClpP by forming covalent adducts with the
203 catalytic serine. The compounds were identified, when a small library of reactive β-lactone
204 probes was applied to S. aureus cells. After the cell-permeable structures had found their
205 targets, cells were lysed and the lactone compounds were labeled with a fluorophore that
206 could be coupled to an azide moiety of the lactones by click chemistry (Böttcher and Sieber,

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207 2008). SDS-PAGE of the cell extracts identified 3 lactones that had labeled ClpP with high
208 specificity, among them lactone D3 as the most affine compound (Fig. 2). The compounds
209 covalently modified isolated ClpP of S. aureus, but not a S98A mutant of ClpP that lacked the
210 active site serine, corroborating the expected interaction site. As a consequence, the
211 catalytic activity of ClpP was inhibited and as downstream effects the syntheses of
212 hemolysins and extracellular proteases (Fig. 3A). Importantly, hemolytic and proteolytic

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213 activities were not only reduced in the background of the laboratory model strain S. aureus

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214 NCTC 8325, but also in the clinical isolates Mu50 and DSM 18827 (Böttcher and Sieber,
215 2008). Structural modification yielded the lactone U1 (Fig. 2) that was 3 to 5-fold more active

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216 than lactone D3 in inhibiting the extracellular hemolytic, proteolytic, lipolytic and DNase-
217 activities of S. aureus. Furthermore, lactone U1 reduced production of the enterotoxins B and

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218 C as well as of toxic shock syndrome toxin-1 (TSST-1), although high concentrations of 150
219 to 2500 µM were needed to achieve these effects (Böttcher and Sieber, 2009b). Follow-up
220 studies revealed that lactone U1 does also inhibit the ClpP dependent production of

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221 listeriolysin O (LLO) and phospholipase C in the facultative intracellular pathogen L.
222 monocytogenes (Böttcher and Sieber, 2009a). LLO as well as phospholipase C are
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223 instrumental for Listeria during escape from the phagosome and survival in the environment
224 of the eukaryotic host cell. Consequently, lactone U1 decreased intracellular survival of L.
225 monocytogenes in mouse macrophages by 25% (Böttcher and Sieber, 2009b). It will be
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226 interesting to investigate, if improved lactones might be able to reduce intracellular growth
227 further and if such compounds will also be effective in rodent models of staphylococcal or
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228 Listeria infection.

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229
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230 ClpP and Clp-ATPases as new targets for antibiotic attack

231 As discussed above, ClpP may serve as an anti-virulence target, inhibition of which should
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232 reduce bacterial survival and spread during host infection. However, in recent years ClpP
233 has also been discovered to serve as an antibiotic target in the classical sense, meaning that
234 bacterial growth as such is inhibited upon binding of an antimicrobial agent to this target.
235 Since ClpP is not essential for the growth of most bacterial species under moderate
236 conditions in vitro, this was an unexpected finding that cannot be explained by simple
237 inhibition of the physiological functions of ClpP. Instead, it was found that new antibacterial
238 acyldepsipeptides (designated “ADEPs”) take ClpP out of its physiological context to cause
239 bacterial death.

240 In the early 1980s, the natural product complex 54556A was reported as a collection of
241 closely related acyldepsipeptides representing secondary metabolites produced by

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242 Streptomyces hawaiiensis NRRL 15010 (Michel and Kastner, 1985). Although, this early
243 patent proposed a first structure for the natural products and provided initial data on their
244 antibacterial activity in vitro, the compounds were not further explored and their antibiotic
245 mode of action remained undetermined for the next two decades. Then, the compounds
246 were revisited during a drug discovery program of so far underexplored antibiotics, the
247 previously reported structure was revised and a route for total synthesis was developed

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248 (Hinzen et al., 2006). Starting from “factor A” (which was renamed ADEP1) representing the

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249 major component of complex 54556A, new derivatives such as ADEP2 and ADEP4 (Fig. 2)
250 were synthesized that displayed significantly improved activity compared to the natural

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251 product ADEP1. The new synthetic ADEP derivatives reached minimal inhibitory
252 concentrations (MICs) in the sub-µg/ml range for the important Gram-positive species S.

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253 aureus, S. pneumoniae and Enterococcus faecium, including multi-resistant clinical isolates,
254 and in mouse models of bacterial infection their efficacy even surpassed that of the last
255 resort antibiotic linezolid (Brötz-Oesterhelt et al., 2005). Additionally, no cross-resistance to

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256 marketed antibiotics was observed, already suggesting that ADEPs might interfere with a
257 new bacterial target. This notion was further supported during initial mode of action studies
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258 which failed to identify the target in one of the classical metabolic pathways including the
259 syntheses of DNA, RNA, proteins, fatty acids, and peptidoglycan. In the end, their target was
260 identified in the course of complementary genetic and biochemical approaches. The
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261 characterization of ADEP-resistant mutants revealed mutations in the clpP gene and
262 immobilized ADEP retained ClpP selectively during affinity chromatography (Brötz-Oesterhelt
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263 et al., 2005).

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264 In depth structural and biochemical analyses on the interaction of ADEPs with ClpP revealed
265 that ADEPs deregulate ClpP in an unprecedented and multifaceted manner. In vitro studies
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266 using purified ClpP from B. subtilis as well as from E. coli showed that in the absence of
267 ClpP-ATPases, ADEPs induce the oligomerization of ClpP monomers into the
268 tetradecameric complex which, however, is no longer functional in the physiological sense
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269 (Fig. 4A). It emerged that ADEPs prevent the interaction of ClpP with its Clp-ATPases and
270 thus inhibit the degradation of natural Clp protease substrates (Kirstein et al., 2009a).
271 Moreover, preformed ClpP/Clp-ATPase complexes were even disassembled in the presence
272 of ADEP (Kirstein et al., 2009a). Instead, it turned out that ADEPs uncouple the proteolytic
273 capacity of ClpP from its natural regulatory constraints, thereby switching ClpP from a
274 regulated to an uncontrolled protease that degrades unfolded or flexible proteins like casein
275 as well as nascent polypeptides at the ribosome independently of Clp-ATPases (Fig. 4A)
276 (Brötz-Oesterhelt et al., 2005; Kirstein et al., 2009a; Leung et al., 2011).

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277 Complementary X-ray crystallography studies by two independent groups yielded structures
278 of ADEP in complex with ClpP from B. subtilis as well as from E. coli (Lee et al., 2010; Li et
279 al., 2010) which provided a rationale for the biochemical observations. The crystal structures
280 located ADEP binding at the outer rim of the apical and distal surfaces of the ClpP
281 tetradecamer in a 1:1 stoichiometry (Fig. 4B). Each ADEP molecule binds at the interface of
282 two adjacent ClpP subunits and establishes contacts to both (Lee et al., 2010; Li et al.,

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283 2010), thereby stabilizing the oligomeric ring structure which explains the observation that

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284 monomeric ClpP of B. subtilis is transferred to the tetradecameric state in the absence of
285 Clp-ATPases (Kirstein et al., 2009a). Furthermore, the ADEP binding pocket at ClpP is

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286 exactly the position that was previously suggested as contact point for the IGF/IGL loops of
287 E. coli ClpX and ClpA (Kim et al., 2001; Li et al., 2010). Thus, ADEPs compete with Clp-

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288 ATPases for the same binding site on the ClpP tetradecamer (Lee et al., 2010; Li et al.,
289 2010) and displace the Clp-ATPases resulting in the inhibition of the normal physiological
290 functions of Clp proteases. In addition, ADEP binding induces major conformational changes

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291 in the N-terminal part of each ClpP subunit, thereby triggering a closed- to open-gate
292 structural transition of the ClpP N-terminal segments that opens the substrate entrance pore
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293 to the degradation chamber of the ClpP tetradecameric complex, which now allows the entry
294 of poorly structured regions of non-native protein substrates (Fig. 4B) (Lee et al., 2010; Li et
295 al., 2010). Considering this destructive capacity of ADEP-activated ClpP towards
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296 unstructured polypeptides or proteins, it appears likely that important proteins in the bacterial
297 cell are depleted in vital processes of the bacterial metabolism, thus already providing a
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298 plausible explanation for the inhibition of growth and bacterial killing.
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299 Although it is reasonable to assume that ADEP-activated ClpP depletes bacterial cells of
300 more than one essential protein, at least at higher ADEP concentrations, follow-up studies
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301 revealed that a particular protein seems to be preferentially degraded. At low inhibitory ADEP
302 levels close to the MIC, the syntheses of macromolecules including DNA, RNA, proteins,
303 fatty acids or peptidoglycan are not affected and the bacteria are still able to produce
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304 biomass in considerable amounts, although the cells are already unable to divide. Actually, at
305 these ADEP concentrations bacterial cell division is stalled due to the spatial mislocalization
306 of central divisome proteins (Fig. 3B) as a direct consequence of the rapid degradation of the
307 essential cell division pacemaker protein FtsZ by ADEP-activated ClpP (Sass et al., 2011).
308 Interestingly, FtsZ is neither flexible nor unstructured. This unexpected finding suggests that
309 although released from all natural regulatory controls ADEP-activated ClpP appears to attack
310 at least one natively folded protein with substantial preference. Whether additional
311 preferential target proteins exist and which structural features they harbor is not yet known,
312 but is seems that another level of complexity to the mode of action of these antibiotics still
313 remains to be discovered. Noteworthy, by using this mechanism of action ADEPs were the

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314 first antibiotics to cause bacterial death by over-activating rather than simply inhibiting their
315 target.

316 Stimulated by these findings highlighting ClpP as a putative novel therapeutic target, further
317 studies contributed to the evaluation of the ADEP class i.e. by exploring the activity of
318 structurally modified ADEP derivatives against enterococci (Socha et al., 2010) or by
319

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preliminary evaluations of ADEPs as antitubercular drugs (Ollinger et al., 2012). In addition,

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320 structurally unrelated compounds were identified that interfere with ClpP by a similar mode of
321 action. Although the most advanced of these compounds, ACP1b (Fig. 2), deregulated ClpP

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322 similarly to ADEPs, it was significantly less effective than ADEP1 and showed off-target
323 effects most probably as a result of insufficient target specificity (Leung et al., 2011).

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324 Thinking along the lines of protease over-activation, a second component of the Clp
325 machinery was recently discovered as an antibiotic target. The cyclopeptide cyclomarin A
326 (Fig. 2) was identified to be bactericidal against a panel of multidrug-resistant clinical isolates
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328
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of M. tuberculosis at concentrations of 0.3 to 2.5 µM in a natural product whole-cell screen
(Schmitt et al., 2011). Cyclomarin A was active against growing as well as hypoxic
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329 nonreplicating M. tuberculosis cells, however, it lacked activity against other tested Gram-
330 positive or Gram-negative species including S. aureus and Pseudomonas aeruginosa. Due
331 to its significant species specificity and since initial attempts failed to select for spontaneously
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332 resistant mutants, it was proposed that cyclomarin A functions by a novel mechanism of
333 action involving a target that is either unique to M. tuberculosis or uncommonly essential in
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334 this species. An affinity chromatography approach using an immobilized derivative of

335 cyclomarin A selectively trapped the mycobacterial ATPase ClpC1 and further experiments
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336 showed that cyclomarins interact with ClpC1 with high affinity in a 1:1 stoichiometry (Schmitt
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337 et al., 2011). Cyclomarin congeners lacking antibacterial activity were unable to interact with
338 ClpC1, corroborating the interaction with ClpC1 as the basis for the antibiotic activity. Whole
339 cell fluorescence-based protein degradation assays indicated that cyclomarins deregulate
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340 ClpC1 functions with the fatal consequence of over-activating the proteolytic activity of the
341 ClpP/ClpC1 complex in mycobacterial cells. By this means, cyclomarin displayed effective
342 killing of M. tuberculosis growing in culture broth as well as in macrophages (Schmitt et al.,
343 2011), and due to the essentiality of ClpC1 in mycobacteria the development of resistance to
344 cyclomarins may not be expected to occur at elevated frequencies, making it an interesting
345 compound for antimicrobial drug discovery.

346 In terms of Clp protease inhibition, the small molecule inhibitor F2 (Fig. 2) was recently
347 reported to suppress ClpXP protease activity by a yet unknown mechanism thereby
348 exhibiting synergistic antibiotic activity with cathelicidin antimicrobial peptides as well as
349 antibiotics that target the bacterial cell envelope against Bacillus anthracis and drug resistant

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350 S. aureus. In this context it is important to note that F2 alone lacked significant antibacterial
351 activity and that a clpX knockout was also tolerated by these species under the in vitro
352 conditions applied (McGillivray et al., 2012). Although the underlying principle of the
353 synergistic activity is not yet understood, the general result implies that the potential of Clp
354 protease inhibitors extends beyond virulence inhibition to direct inhibition of bacterial growth,
355 if combination approaches with suitable classical antibiotics are considered.

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356

357 Conclusions

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358 The global spread of antibiotic resistance among important human pathogens emphasizes

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359 the need to find new antibiotics with novel modes of action. All antibiotics in therapeutic
360 application or development pipelines to date inhibit bacterial growth via the principle of target
361 inactivation. More precisely, the compounds inhibit the functions of essential enzymes or

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362 molecular machines, sequester precursors of indispensable biosynthetic processes or
363 interfere with membrane integrity. The identification of bacterial Clp proteases as a new
364 antimicrobial targets and the remarkable antibacterial activity of the respective
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365 inhibitors/activators strikingly show that nature equips us with more options than mere
366 inhibition of essential protein targets and metabolic processes to combat bacterial infections.
367 At least in some species, inhibition of ClpP permits effective suppression of bacterial
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368 virulence, which points to a combination strategy to counteract pathogenic bacteria with a
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369 reduced risk of selecting for resistance. However, due to the observed species dependency,
370 more in vivo data are needed for a broader set of strains per species to grant final
p

371 conclusions. Concerning the ADEPs, their unprecedented role in ClpP deregulation expands
372 the common antibacterial strategy of target inhibition by adding the innovative concept of
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373 target over-activation to perturb the regular bacterial lifestyle and to finally cause deleterious
374 effects in the bacterial cell. The activity of the cyclomarins suggests that nature has used the
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375 concept of target over-activation more often and in different ways which proves this principle
376 as a valid strategy in antibacterial drug discovery.

377

378 References

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526 2011. Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the cell
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540 Turgay, K., Hahn, J., Burghoorn, J., Dubnau, D., 1998. Competence in Bacillus subtilis is
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542 Wang, J., Hartling, J.A., Flanagan, J.M., 1997. The structure of ClpP at 2.3 A resolution
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544 Yu, A.Y., Houry, W.A., 2007. ClpP: a distinctive family of cylindrical energy-dependent serine
545 proteases. FEBS Lett. 581, 3749-3757.

546
547
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Zeiler, E., Braun, N., Böttcher, T., Kastenmüller, A., Weinkauf, S., Sieber, S.A., 2011.
Vibralactone as a tool to study the activity and structure of the ClpP1P2 complex from
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548 Listeria monocytogenes. Angew. Chem. Int. Ed Engl. 50, 11001-11004.
549
550
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551 Acknowledgements
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552 The authors acknowledge financial support from the German Research Foundation
553 (FOR854). We would also like to thank all research colleagues who have added to our
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554 current understanding of ClpP function and the mode of action of ADEPs and other ClpP
555 inhibitors and/or activators.
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556
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557 Legends to illustrations

558 Figure 1

559 Model of the composition and operation mode of bacterial ClpP containing proteases. (A)
560 The controlled proteolysis of substrate proteins by ClpP strictly depends on associated Clp-
561 ATPases and corresponding adaptors which recognize and bind the specific substrates and
562 bring them to ClpP. Here, Clp-ATPases support the oligomerization of ClpP into the
563 tetradecameric complex, bind the protein substrates, unfold them and translocate them
564 through the apical and/or distal entrance pores into the proteolytic chamber of ClpP to allow
565 substrate proteolysis. (B) Cross section of a ClpP tetradecamer: The active site catalytic
566 triads (rectangles) are located within the degradation chamber of the barrel-shaped ClpP

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567 tetradecamer and thus are effectively shielded from the surrounding cytoplasm. (C) Top view
568 of a ClpP tetradecamer: The access of substrates to the catalytic triads is restricted by the
569 small apical and distal entrance pores of the ClpP tetradecamer. (D) Crystal structure of a
570 ClpP monomer from Bacillus subtilis indicating the head and handle region of the protein
571 (adapted with kind permission from Alexopoulos et al. (2012)).

572

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573 Figure 2

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574 Structures of Clp inhibitors / activators discussed in this review.

575

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576 Figure 3

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577 ClpP as anti-virulence and antibiotic target. (A) Inhibition of ClpP by β-lactones leads to
578 reduced production of hemolysins and proteases by S. aureus, as shown by the lack of the
579 corresponding halos around the bacterial colonies. Images are adapted with kind permission
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580 from: Böttcher and Sieber (2008), Copyright 2008 American Chemical Society. (B) ADEP-
581 treatment of S. aureus results in the inhibition of cell division and finally bacterial death.
582 Fluorescence images (upper image section) visualize the bacterial membrane (red) and the
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583 divisome protein PBP2 (green); the latter commonly localizes to the division site at the
584 equatorial plane of dividing bacteria (left panel, arrow). The absence of PBP2 from the
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585 division site after 300 min of ADEP treatment (right panel) is representative for the
586 delocalization of important cell division proteins during ADEP stress. Scale bar is 2.5 µm.
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587 Here, cell division inhibition is due to the rapid degradation of the cell division initiator protein
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588 FtsZ by ADEP-activated ClpP (lower image section).

589
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590 Figure 4

591 ADEPs unleash the proteolytic activity of ClpP. (A) Model on the mode of action of ADEPs.
592 ADEPs deregulate ClpP in a multi-layered fashion: ADEPs support the oligomerization
593 process of ClpP monomers into the tetradecameric complex and compete with Clp-ATPases
594 for the same binding site on ClpP. As a result of ADEP binding, the interaction of ClpP with
595 corresponding Clp-ATPases is prevented leading to the inhibition of the natural functions of
596 Clp in protein turnover (a). Moreover, ADEPs unleash the degradative capacity of ClpP from
597 strict regulation by Clp-ATPases to permit the proteolysis of unstructured or flexible proteins
598 or nascent polypeptides at the ribosome (b). (B) Top view of a ClpP tetradecamer cartoon
599 (upper section) and cryo-electron microscopic images (lower section): ADEPs (orange

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600 trapezes) bind to the outer rim of the apical and distal surfaces of ClpP in a 1:1 stoichiometry,
601 thereby inducing a conformational shift in the N-terminal region of ClpP that results in the
602 enlargement of the entrance pores to the degradation chamber of the ClpP tetradecamer.
603 Cryo-electron microscopic images are adapted with kind permission from Lee et al. (2010).

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