Clp chaperones and proteases are central in stress survival, virulence and antibiotic resistance of Staphylococcus aureus

Clp chaperones and proteases are central in stress survival, virulence and antibiotic resistance of Staphylococcus aureus

International Journal of Medical Microbiology 304 (2014) 142–149 Contents lists available at ScienceDirect International Journal of Medical Microbio...

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International Journal of Medical Microbiology 304 (2014) 142–149

Contents lists available at ScienceDirect

International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm

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Clp chaperones and proteases are central in stress survival, virulence and antibiotic resistance of Staphylococcus aureus Dorte Frees a , Ulf Gerth b , Hanne Ingmer a,∗ a b

Department of Veterinary Disease Biology, Faculty of Health and Medical Science, University of Copenhagen, Stigbøjlen 4, 1870 Frederiksberg C, Denmark Institute of Microbiology, Ernst-Moritz-Arndt-University Greifswald, D-17487 Greifswald, Germany

a r t i c l e

i n f o

Keywords: ClpP Clp ATPase Virulence S. aureus Antibiotic resistance Proteolysis

a b s t r a c t Intracellular proteolysis carried out by energy-dependent proteases is one of the most conserved biological processes. In all cells proteolysis maintains and shapes the cellular proteome by ridding the cell of damaged proteins and by regulating abundance of functional proteins such as regulatory proteins. The ATP-dependent ClpP protease is highly conserved among eubacteria and in the chloroplasts and mitochondria of eukaryotic cells. In the serious human pathogen, Staphylococcus aureus inactivation of clpP rendered the bacterium avirulent emphasizing the central role of proteolysis in virulence. The contribution of the Clp proteins to virulence is likely to occur at multiple levels. First of all, both Clp ATPases and the Clp protease are central players in stress responses required to cope with the adverse conditions met in the host. The ClpP protease has a dual role herein, as it both eliminates stress-damaged proteins as well as ensures the timely degradation of major stress regulators such as Spx, LexA and CtsR. Additionally, as we will summarize in this review, Clp proteases and Clp chaperones impact on such central processes as virulence gene expression, cell wall metabolism, survival in stationary phase, and cell division. These observations together with recent findings that Clp proteins contribute to adaptation to antibiotics highlights the importance of this interesting proteolytic machinery both for understanding pathogenicity of the organism and for treating staphylococcal infections. © 2013 Elsevier GmbH. All rights reserved.

Introduction Staphylococcus aureus is a serious human pathogen that can give rise to a variety of infections ranging from harmless wound infections to life-threatening conditions like bacteremia, osteomyelitis and heart valve infections (Lowy, 1998). Antibiotic resistance is an increasing problem with the spread of methicillin resistant strains (MRSAs) both in the hospitals and in the community (Otto, 2012). Yet, for the majority of time S. aureus is colonizing harmlessly warm-blooded animals and humans and for the latter approximately 30% are permanently colonized by the organism (DeLeo et al., 2010). In the balance between harmless symbiosis and devastating infection, S. aureus tightly controls production of virulence and colonization factors. At the same time it relies on advanced stress response systems that will allow survival and adaptation to changing environmental habitats. One of the molecular machineries that in S. aureus occupy roles in both virulence and environmental adaptation is the Clp proteolytic system. Clp proteases are found well conserved in most bacterial species and they are composed of a core proteolytic chamber flanked by one

∗ Corresponding author. E-mail address: [email protected] (H. Ingmer). 1438-4221/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ijmm.2013.11.009

of several possible ATPases that determine substrate specificity. Importantly, these ATPases also have chaperone activity that in combination with ClpP enable entry into the secluded, proteolytic chamber but in the absence of ClpP may function as independent molecular chaperones (Savijoki et al., 2006; Frees et al., 2007, 2013). When originally examined in S. aureus, several studies indicated that the proteolytic subunit, ClpP and the ClpATPase, ClpX are essential for virulence as inactivation completely abolished abscess formation in a mouse model and eliminated expression of one of the major staphylococcal hemolysins, ␣-hemolysin (Mei et al., 1997; Frees et al., 2003). Also, intracellular replication in bovine mammary cells was eliminated for mutants lacking either clpP, clpX or clpB and was significantly reduced for clpC mutant cells (Frees et al., 2004). While the mechanisms behind the defects in virulence remain unknown, they must be related to two key biological functionalities of the Clp complex namely in degradation of short-lived regulatory proteins (Elsholz et al., 2010a) or in protein quality control (Frees et al., 2004). Recently, a large number of ClpP substrates in S. aureus were identified by using catalytically inactive ClpP or ClpC variants (“clpPTRAP ”) that will retain but not degrade substrates translocated into the proteolytic chamber (Fig. 1, Feng et al., 2013; Graham et al., 2013). This study revealed that in S. aureus the Clp targets encompass a number of central regulatory proteins (CtsR, Spx, HrcA, PerR, CodY) as well as proteins

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Fig. 1. Large scale identification of substrates of the ClpP protease by using a proteolytically inactive ClpP-variant. To directly identify substrates of the ClpP protease, the  active site was mutated, and the proteolytic inactive ClpP chamber now functions as a “ClpPTRAP that will retain but not degrade substrates translocated into the proteolytic chamber. Captured substrates were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry.

with central physiological functions such as RecA, FtsZ, NrdE, Pmp, GlmS and DnaK. Thus, with dual functionality in degrading both non-native proteins generated during stress as well as specific key regulatory proteins, the Clp proteolytic system appears to be a cornerstone in processes of importance to survival and pathogenicity, and as such, may not be an important target for design of new interventions. Structure The Clp proteolytic chamber is a barrel shaped structure that is conserved among bacteria and is composed of two rings of heptameric ClpP. Access to this secluded proteolytic chamber is restricted by pores that are too narrow to allow entry of folded proteins. In order to be degraded, substrates must first interact with the Clp ATPase component that powers unfolding and subsequent translocation of the substrate into the ClpP proteolytic chamber (Frees et al., 2007). In S. aureus, the ClpC and ClpX ATPases can perform this function. Two additional Clp ATPases are encoded by the organism, namely ClpL and ClpB, but they lack the conserved tripeptide consensus sequence (IG(F/L)) required for interaction with ClpP (Martin et al., 2008). During proteolysis in S. aureus this structure undergoes dramatic conformational changes. In the proteolytic active state ClpP adopts an extended conformation with the catalytic triad (His-123, Asp-172, Ser-98) aligned in a proper geometry for proteolytic activity. In contrast, in the closed conformation the side-chains of the catalytic triad are flipped hampering proteolysis (Zhang et al., 2011; Gersch et al., 2012). Following degradation the products are likely to be released through pores in the side of the barrel (Geiger et al., 2011). In some cases proteolysis is regulated through the spatial and/or temporal use of adaptor proteins, which are directly involved in the recognition and delivery of specific substrate proteins to the proteases (Dougan et al., 2002; Battesti and Gottesman, 2013). For example ClpXP can degrade substrates independently of adaptors but the presence of the adaptor-like protein YjbH greatly enhances the proteolytic activity (Engman et al., 2012; Lies and Maurizi, 2008). Notably, ClpP-like proteins are also common among S. aureus phages indicating that proteolytic control has a central function in phage biology (Boyle-Vavra et al., 2011). An additional Clp proteolytic complex baring resemblance to the eukaryotic 26S proteasome is formed by the products of the clpY and clpQ genes (Frees et al., 2005b). Despite the name, the proteolytic subunits ClpQ and ClpP are not related, and as no distinct phenotype has been linked to the function of the ClpYQ it will not be discussed further here.

Stress tolerance During infection bacterial pathogens are likely to encounter dramatic environmental changes and may experience shift in temperature, oxidative stress, antimicrobial peptides and other conditions aimed at inactivating the invading microorganism. Such stress exposures may lead to protein unfolding, and removal of unfolded and non-native proteins is necessary for cellular functionality and growth (Truscott et al., 2011). In S. aureus inactivation of clpP, clpC, clpB and to a lesser extent clpL abolished or reduced growth at 45 ◦ C (Frees et al., 2003, 2004, 2012). Since ClpB and ClpL are not functional ClpP partners these results suggested that ClpCP is degrading non-native proteins in S. aureus, a finding recently confirmed by trapping protein substrates at high and ambient temperature, respectively (Feng, in preparation). The contribution of ClpB and ClpL to stress survival is likely as chaperones either to prevent protein unfolding or promote disaggregation (Glover and Lindquist, 1998). This notion was supported by the finding that both ClpB and ClpL are required for thermoinduced thermotolerance where pre-exposure to intermediate high temperature improves survival at high temperatures (Frees et al., 2004). Surprisingly, inactivation of clpX allowed growth at an even higher temperature than the wild type cells. Although the basis for this finding is currently unknown it shows that Clp proteins contribute to virulence through stress-independent (via ClpXP) and stress-dependent (ClpCP and ClpB) pathways (Frees et al., 2003, 2004). Also in Bacillus subtilis, non-native proteins are degraded by the ClpCP complex and it takes place in a process also requiring the adaptor protein, MecA. MecA is necessary not only for substrate recognition but also for the oligomerization of ClpC into a hexamer and binding to ClpP (Kirstein et al., 2006; Schlothauer et al., 2003). A MecA homologue is also found in S. aureus where it is designated teicoplanin resistance factor A (TrfA, see below, Renzoni et al., 2009). TrfA is a target of the Clp protease (Feng et al., 2013) and a mutant lacking the corresponding gene is temperature sensitive indicating that TrfA may assist ClpCP mediated proteolysis of nonnative proteins also in S. aureus (Feng et al., in preparation). McsB is another adaptor-like protein also found in B. subtilis that was trapped as Clp target (Elsholz et al., 2010b; Wozniak et al., 2012; Feng et al., 2013). Inactivation of S. aureus McsB impairs growth at high temperature as well as in the presence of heavy metals, osmotic pressure, oxidative stress and at low pH (Wozniak et al., 2012). In this case the heat sensitivity may be due to lack of ClpC expression as McsB in B. subtilis is needed for the degradation of the negative heat shock regulator, CtsR that controls clpC expression (Elsholz et al., 2011a, see below).

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Among the global stress regulators identified in the ClpP proteolytic trap was Spx that is a positive regulator of the disulfide-stress response (Feng et al., 2013). Spx has been extensively characterized in B. subtilis where it was shown to bind directly to the C-terminal domain of the RNAP ␣-subunit and thereby interfering with transcriptional activation mediated by certain response regulators of two-component systems (Zuber, 2004). Interestingly, the cellular level of Spx is determined by proteolytic control: Under non-stress conditions, Spx is kept at a low concentration through degradation by the ClpXP protease in a process stimulated by the adaptor, YjbH, while upon disulfide stress Spx is stabilized and transcriptionally activates a number of genes required for oxidative stress tolerance (Nakano et al., 2003; Zuber, 2004; Garg et al., 2009; Larsson et al., 2007; Engman et al., 2012). While the basic features in Spx function and control hereof seems to be conserved among S. aureus and B. subtilis, it is notable that inactivation of Spx confers much more severe growth defects to S. aureus, and that recent research suggest that the S. aureus spx mutant is only viable because it has accumulated additional suppressor mutations (Pamp et al., 2006; Bill Kelley, personal communication). In addition to the degradation of Spx, the combined activity of YjbH and ClpX appears to degrade other desiccation tolerance regulatory proteins, as both proteins turned out to be required for long-term survival on plastic surfaces (Chaibenjawong and Foster, 2011). Another category of proteins identified as targets for Clp proteolysis is the DNA damage repair proteins (Feng et al., 2013). Response to DNA damaging conditions has been characterized extensively in Escherichia coli. DNA damage elicits the so-called SOS response that is controlled by the transcriptional repressor, LexA (Butala et al., 2009). The LexA regulon comprises error prone DNA damage repair proteins and in the absence of stress is repressed by LexA (Kelley, 2006). However, upon DNA damage LexA undergoes autocleavage resulting in a C-terminal and a N-terminal fragment with the latter retaining some DNA-binding activity (Neher et al., 2003). Recently we showed that in S. aureus, the ClpXP as well as the ClpCP proteases contribute to degradation of the LexA autocleavage products as in the absence of ClpP, or one or both of the Clp ATPases, ClpX or ClpC, the LexA domains were stabilized (Cohn et al., 2011). In S. aureus, the LexA regulon includes lexA itself and the divergently transcribed gene, sosA. Interestingly, when astabilized mutant allele of the LexA N-terminal fragment was ectopically produced, expression of sosA remained repressed while lexA was expressed under SOS inducing conditions (Cohn et al., 2011). This result suggests that the LexA regulon comprises two sets of genes namely those that are unaffected by the N-terminal domain of LexA and those that are repressed in its presence. Importantly, the activity of the Clp proteolytic complex is required for the induction of the latter set of genes. Future studies will reveal the nature of these genes and the biological importance of Clp mediated proteolysis in the SOS response.

transcriptional regulation of clpX remain to be investigated (Frees et al., 2004). In addition to the clp genes, the CtsR regulon in Staphylococci also comprises the classical chaperone genes dnaK and groESL, which in other Gram positive organisms are controlled by another heat shock repressor, HrcA (Chastanet et al., 2003). In fact the entire HrcA regulon (dnaK and groESL operons) is embedded within the CtsR regulon in Staphylococci and both repressors (HrcA, CtsR) seem to act synergistically to maintain a low basal level of the dnaK and groESL operon expression in the absence of stress (Chastanet et al., 2003). Therefore, synthesis of the classical chaperones is directly connected with that of the Clp proteins in Staphylococci possibly enhancing the adaptability both during stress and infection (Chastanet et al., 2003). A central question in control of clp protein expression is how CtsR monitors the temperature. Elegant studies in B. subtilis recently showed that the protein itself “feels” the heat as a protein thermometer and reacts intrinsically to the temperatures of the surroundings (Elsholz et al., 2010b). The tetra-glycine loop in the winged helix-turn-helix domain grabs into the minor groove of the CtsR operator site on the DNA (Fuhrmann et al., 2009) and was identified as thermosensing site of CtsR by site-directed mutagenesis in B. subtilis, L. lactis as well as in G. stearothermophilus, which makes the protein susceptible for heat inactivation at species-specific heat shock temperatures (Elsholz et al., 2010b). After dissociation from the DNA, CtsR is immediately degraded by B. subtilis ClpEP protease (Miethke et al., 2006) and later by ClpCP, which needs the active arginine kinase, McsB, as adaptor protein (Elsholz et al., 2010b, 2011a). Importantly this work supports the general hypothesis for Clp degradation namely that “un-occupied” nonfunctional and unprotected proteins may be substrates of the Clp complex but when occupied by association with for example DNA binding they are stabilized (Michalik et al., 2012). This notion implicates that protein substrates are found in both proteolysisproficient and deficient forms in the same cell (Feng et al., 2013). The mechanism for the CtsR inactivation during thiol-specific stress is different and depends solely on McsB, but apparently not on its arginine kinase activity (Elsholz et al., 2011b). In B. subtilis thiol-specific stress is sensed by the thiols of the second zinc finger present in McsA, which are necessary for the McsB interaction (Elsholz et al., 2011b). These thiols act as a molecular redox switch to regulate the McsB activity by dissociating it from McsA. When McsB is not associated with McsA, it binds and inactivates DNAbound CtsR leading to de-repression of the CtsR regulated genes. Although little is known of the role of these proteins in S. aureus, inactivation of mcsB lead to oxidative stress sensitivity indicating that homologues of McsB and McsA may perform similar functions also in this organism (Wozniak et al., 2012).

Control of clp expression

Clp proteins orchestra expression of virulence genes

In low GC Gram positive bacteria CtsR controls expression of the clp genes (Krüger and Hecker, 1998; Derré et al., 1999; Chastanet et al., 2003; Elsholz et al., 2010a,b). Under standard growth conditions it represses transcription of S. aureus genes clpP and clpB as well as the tetracistronic clpC operon (ctsR, mcsA, mcsB, clpC) but shortly after heat stress expression is de-repressed (Frees et al., 2004; Fleury et al., 2009). S. aureus CtsR was retained by the proteolytic inactive ClpCPTRAP (Feng et al., 2013) suggesting that as in Bacillus ClpCP mediated degradation of CtsR contributes to de-repression of the CtsR regulon (Krüger et al., 2001; Miethke et al., 2006). The monocistronic genes clpL and clpX do not belong to the CtsR regulon. clpL is solely sigB-dependently transcribed (Gertz et al., 2000; Frees et al., 2004) and the mechanism(s) for a

The pathogenicity of S. aureus relies on a wide array of surfacebound and secreted virulence factors that equip the bacterium for tissue binding, tissue destruction, and immune evasion. One of the most significant phenotypes of the clpP deletion in the 8325derived strains is the dramatic effect on transcription of major virulence genes (Frees et al., 2003, 2005a,b; Michel et al., 2006; Feng et al., 2013; Farrand et al., 2013). As an example, transcription of genes encoding the extracellular proteases SspA, Aur, and Spl is reduced as much as 100 fold, while transcription of the hla gene encoding alpha hemolysin is decreased 10 fold in the postexponential growth phase. Accordingly, the clpP mutant strain was both non-hemolytic and non-proteolytic (Frees et al., 2003; Feng et al., 2013). The NCTC8325-derived strains are characterized by

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very high expression of hemolysins and extracellular proteases compared to other S. aureus strains, a trait that has been ascribed to the low activity of the alternative sigma factor, SigB, in these strains (Giachino et al., 2001; Horsburgh et al., 2002; Oscarsson et al., 2006a). Interestingly, recent proteomic studies emphasized that both the surfacome and exoproteome of clinical S. aureus isolates display an extreme heterogeneity, and that strain dependent expression of virulence genes has a pivotal role in generating diversity (Dreisbach et al., 2010; Ziebandt et al., 2010). The expression of virulence factors is regulated by a complex network of regulatory elements that in addition to SigB encompass at least six two-component systems and a number of transcriptional regulators, of which many belong to the family of SarA homologues (Novick, 2003). Global transcriptomic and proteomic analysis of different S. aureus strains carrying a clpP mutation revealed that expression of a large number of major virulence regulators was affected by the absence of ClpP (Frees et al., 2012). While the transcription of global virulence regulator genes RNAIII, mgrA, sarZ, sarR and arlRS was similarly affected by a clpP mutation in all the examined strains, inactivation of ClpP influenced expression of the virulence genes sspA, hla, and spa in a strain dependent manner. The strain dependency is puzzling but it may at least in part be determined by a strain dependent effect of ClpP on SarS expression. SarS is a transcriptional regulator that directly represses transcription of sspA and hla, while stimulating expression of spa (Oscarsson et al., 2005, 2006a,b). In SigB proficient strains expression of sarS is high and disruption of clpP reduced sarS transcription resulting in enhanced sspA and decreased spa transcription. In contrast, sarS expression is comparable low in the SigB-deficient strain, 8325-4, and deletion of clpP in this background greatly increased transcription of sarS leading to repression of sspA and hla and activation of spa (Frees et al., 2012). The finding that inactivation of ClpP led to major perturbations in the cellular content of global virulence regulators indicated that ClpXP controls stability of one or more transcriptional virulence regulators and thereby impacts the entire regulatory network. However, none of the known virulence regulators were identified as ClpP substrates neither using the proteolytically inactive ClpPTRAP for the identification of substrates nor from stability assays (Michalik et al., 2012; Feng et al., 2013). Presently, we cannot rule out that this may be due to experimental challenges as regulatory proteins are not very abundant, are small (cannot be identified with high confidence in MS-analysis), and are often highly basic which make them more difficult to detect by 2DE-gel electrophoresis. Alternatively, the link between ClpXP and virulence gene regulation is indirect in the sense that the cellular stress imposed by the lack of ClpP may create some general physiological or metabolic signals that are sensed by the regulatory network controlling virulence determinants. Also the changes in cell wall structure observed in clpP mutant cells (see below) may impair signaling across the cell wall and lead to reduced virulence gene expression as has been observed for antibiotic resistant variants with alter cell wall structure (Rudkin et al., 2012). However, this would not explain why the clpP deletion has a strain-dependent effect on the expression of the central virulence regulator SarS and how this is linked to the SigB status of the cell. The ClpX chaperone functions independently of ClpP to control expression of spa encoding the IgG binding Protein A (Frees et al., 2003, 2005b). The effect of ClpX is dramatic as disruption of clpX virtually abolished expression of Protein A in both 83254 and the low-passage clinical strain SA564, suggesting that the effect is not strain-dependent (Jelsbak et al., 2010). Interestingly, ClpX appears to perform dual roles in controlling Protein A expression (Jelsbak et al., 2010). First, ClpX stimulates transcription of spa indirectly by enhancing translation of the transcriptional regulator Rot. Transcription from the spa promoter is regulated by negative

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(SarA) and positive regulators (Rot and SarS) that compete for overlapping operator sites (Gao and Stewart, 2004). By expressing rot from an inducible promoter we could show that transcription of spa requires a threshold level of Rot, and that in cells lacking ClpX the cellular level of Rot is decreased below this threshold level (Jelsbak et al., 2010). Additionally, ClpX has a positive effect on translation of the spa transcript (Jelsbak et al., 2010). The finding that ClpX stimulates translation of both the rot- and the spa-mRNAs opens up for the intriguing possibility that ClpX has a direct role in promoting translation, as was shown for HSP101, an eukaryotic relative of ClpX (Wells et al., 1998; Ling et al., 2000). Secondary structure determinations of the spa- and rot-mRNAs revealed that the translational signals are partially obscured in the stem-loop structures. Hence, ClpX may promote translation by directly or indirectly facilitating interaction between the spa- and rot-mRNAs and the ribosomes. Capsule formation is another factor contributing to S. aureus pathogenicity and recently a transposon screen performed in strain Newman identified ClpC as a regulator of capsule formation (Luong et al., 2011). In the Newman background ClpC turned out to impact capsule formation by two distinct pathways involving respectively the SaeRS two component system and the CodY transcriptional regulator (Luong et al., 2011). The repressor activity of CodY is regulated by the metabolic status of the cell and CodY represses the cap genes either directly, or via repressing the agr quorum sensing system thereby linking the cells metabolic status with virulence regulation (Majerczyk et al., 2010). ClpC promoted capsule formation by reducing codY expression in both strain Newman and UAMS-1 suggesting that this positive effect of ClpC on capsule formation is conserved among strains. In contrast, the Sae dependent effect of ClpC on capsule formation appears to be restricted to the Newman background and is linked to the SaeR-hyper activation in this strain (Mainiero et al., 2010). Interestingly, the data indicated that ClpC facilitates auto-activation of sae transcription in strain Newman (Luong et al., 2011). Clps in cell wall metabolism and cell division The large scale identification of ClpP substrates using a ClpPTRAP variant indicated that a large number of proteins involved in cell wall metabolism or cell division are substrates of ClpP in S. aureus (see Table 1). In B. subtilis the filamentous growth of the clpP mutant is partially caused by accumulation of MurAA which catalyses carboxyvinyl transfer from phosphoenolpyruvate Table 1 ClpP substrates with a predicted role in cell wall metabolism or cell division. – Sle1 GlmS FtsA IsaA FtsZ FtsL MurI FemA FemB Pbp2 SsaA MurE

Atl MurC GlmMa

Secretory antigen SsaA-like protein SAOUHSC 00671 Autolysin precursor Glucosamine–fructose-6-phosphate aminotransferase, isomerizing Cell division protein Immunodominant antigen A Cell division protein Hypothetical protein SAOUHSC 01144 Glutamate racemase Formation of the pentaglycine cross bridge (methicillin resistance factor) Formation of the pentaglycine cross bridge (methicillin resistance factor) Penicillin-binding protein 2, peptidoglycan cross linking Secretory antigen precursor SAOUHSC 02571 UDP-N-acetylmuramoylalanyl-d-glutamate–2,6diaminopimelate ligase Bifunctional autolysin precursor UDP-N-acetylmuramate–alanine ligase Phosphoglucosamine mutase (methicillin resistance factor)

a GlmM was not captured by the ClpPTRAP but was shown to accumulate in clpP mutants (Frees et al., 2012).

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to UDP-N-acetylglucosamine, the first committed step in peptidoglycan biosynthesis (Kock et al., 2004). Notably, MurAA does not appear to be a ClpP substrate in S. aureus. Instead, GlmS and GlmM, two other physiological checkpoints in the synthesis of UDP-N-acetylglucosamine (the cytoplasmic precursor of peptidoglycan), are likely substrates of ClpP in S. aureus (Jolly et al., 1997; Komatsuzawa et al., 2004; Frees et al., 2012; Feng et al., 2013). However, the molecular pathways by which ClpP impacts cell wall metabolism and cell division in S. aureus remain an unexplored area of research and much remains to be discovered. Strikingly, some of the proteins that were most abundantly captured by the ClpPTRAP are proteins with autolytic activity. Examples include the autolysin SleI, the Secretory antigen precursor SsaA (SA0620) (that has a LytM- and a CHAP domain), and the immunodominant antigen A, IsaA. Proteomic analysis of total cellular proteins showed that these proteins accumulate strongly in cells lacking ClpP, and as transcription of the corresponding genes is not induced, this finding supports that SleI, SsaA, and IsaA are all degraded by ClpP in the wild-type cells. It is puzzling why these enzymes that mediate their function outside the cell are subject to intracellular degradation. As described above the clpP mutant cell has a thickened cell wall, and accordingly, we did not observe increased autolytic activity of the clpP mutants (8325-4 and SA564 background) indicating that the autolytic enzymes accumulate in an inactive form (our unpublished data). In contrast, the 8325 clpP deletion strain exhibited enhanced autolytic activity in a similar assay (Michel et al., 2006). However, we speculate that the significantly increased spontaneous release of pro-phages mediated by the inactivation of clpP in the latter strain-background may contribute to this phenotype (Frees et al., 2012). In conclusion, the ClpP proteolytic complex controls multiple steps in cell wall metabolism and we anticipate that future research will reveal novel insights of the role of Clp proteins in control and formation of the cell wall.

in amount and synthesis including purEKCSQLFMNHD, the gltBD, sucAB, pyc, oppBCDF and the codY-operon (xerC-hslUV-codY). Several CodY-dependent enzymes involved in branched-chain amino acid biosynthesis (e.g., LeuA, LeuB, LeuC, LeuD; IlvA2, IlvB, IlvC, and IlvD) and purine nucleotide biosynthesis (e.g., PurC, PurD, PurE, PurL, PurM, PurQ, and PurS) were also down-regulated in the clpP mutant (Michalik et al., 2012). A higher GTP level in the clpP strain may render the CodY repressor more active, and could be responsible for the enhanced CodY repression in mutant cells (Michalik et al., 2012). The ClpP interaction partner ClpC seems to play important roles in S. aureus with respect to late stationary phase phenomena such as carbon metabolism, ion homeostasis, oxidative stress response, survival and programmed cell death (Chatterjee et al., 2010, 2011). A strongly reduced transcription of the TCA cycle gene citB, a loss of of CitB (aconitase) activity and accumulation of acetate was observed in a clpC mutant suggesting a role for ClpC in TCA cycle regulation during the stationary phase (Chatterjee et al., 2005). It was hypothesized that a decrease of the TCA cycle activity and respiration in a clpC mutant result in a reduced generation of ROS and oxidative stress, which enhances survival of a S. aureus clpC mutant in the stationary phase (Chatterjee et al., 2009). However, the increased survival of the clpC mutant could also be explained by a differential effect of the protein on one of three toxin antitoxin systems in S. aureus (Donegan et al., 2010). These systems are characterized by stable toxins that are inactivated by antitoxins and the turnover of the latter in known S. aureus systems are mediated by ClpCP (Donegan et al., 2010). Moreover, a link between ClpC and the thymidine-dependent small-colony variants (TD-SCVs) was proposed suggesting a reduced generation of 5, 10methylene-THF (substrate for thyA) in a clpC mutant which would prevent both accumulation of this substrate as well as the consumption of reduced nicotinamide (Chatterjee et al., 2007, 2009). Thus, the Clp proteins and Clp dependent proteolysis affect cellular metabolism at multiple levels.

Multiple roles for Clp proteins during starvation and in stationary phase

Clp and antibiotic resistance

Proteolysis plays an important role not only when handling stress but also during long-term starvation in non-growing cells. When starved for glucose, cells are forced to save energy and survive with very limited resources. To study the role of Clp proteolysis in S. aureus in these processes, a mass spectrometrybased stable isotope labeling by amino acid in cell culture (SILAC) technology was employed (Michalik et al., 2012). Hereby protein fate is monitored in terms of synthesis, accumulation, aggregation and degradation. The experiment revealed that when starved for glucose, proteolysis mainly affected vegetative, anabolic and selected catabolic enzymes, whereas the expression of TCA cycle and gluconeogenetic enzymes increased (Michalik et al., 2009, 2012). Additionally, proteins involved in growth and reproduction (e.g., ribosomal, translation and cell wall synthesis proteins) were degraded in wild type cells, but stabilized in the clpP mutant suggesting ClpP dependent degradation. Furthermore, anabolic enzymes such as NrdEF involved in the nucleic acid biosynthesis were clearly targeted by ClpP (Michalik et al., 2012). Commonly, many proteins were prone to aggregation in clpP mutant cells and the absence of ClpP correlated with protein denaturation and an oxidative stress response (Michalik et al., 2012). CodY is involved in the adaptive response to starvation and it represses approximately 5% of the genome when bound to GTP and/or branched chain amino acids (BCAA). When nutrients become limiting, a decrease in intracellular levels of GTP and BCAA causes a deactivation of CodY and decreases its affinity for DNA, leading to the induction of its regulon. In clpP mutant cells a number of CodY-dependent genes/operons were strongly decreased

In recent years several studies have pointed to a link between Clp proteins and antibiotic resistance. In an early study a random mutagenesis approach was used to identify genes that upon inactivation reduced the resistance level of the so-called GISA strains (glycopeptide intermediate sensitive strains, Renzoni et al., 2009). Glycopeptides such as vancomycin and teicoplanin inhibit cell wall synthesis by binding to the C-terminal d-alanyl–d-alanine (d-Ala–d-Ala) residues of cell wall precursors and nascent peptidoglycan and blocks the crosslinking (Hiramatsu, 2001, Pereira et al., 2007). Clinical isolates with low-level glycopeptide resistance are referred to as GISA strains (Tenover et al., 1998; Sieradzki et al., 2003) and they are characterized by cell wall thickening, altered cell wall composition, reduced autolysis and reduced negative cell wall charge (Cui et al., 2003, Sieradzki et al., 2003). Importantly one of two genes identified as associated with reduced glycopeptide resistance was the trfA (teicoplanin resistance factor, A). TrfA is also called MecA that is required for ClpC assembly in the ClpCP complex and for ClpCP mediated proteolysis (Renzoni et al., 2009 see above). Remarkably trfA mutant derivatives of GISA strains displayed increased susceptibility to both teicoplanin and vancomycin as well as oxacillin and upon passage in the presence of teicoplanin, fewer resistant mutants arose in a trfA mutant background compared to wild type cells. Interestingly, trfA transcription is induced by cell wall active antibiotics and this activation is mediated by Spx (Jousselin et al., 2013). These findings show that Spx responds to cell wall active antibiotics and that TrfA, perhaps in conjunction with ClpCP proteolysis, is involved in the GISA phenotype.

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In a later study vancomycin intermediate sensitive strains (VISA) were examined and the mutations that gave rise to the VISA phenotype was determined. In addition to mutations in the well-known vraSR and graRS genes encoding cell wall stress sensing systems, the VISA phenotype was also associated with mutations in clpP (Shoji et al., 2011). Antibiotic susceptibility tests showed that a clpP mutant displayed increased tolerance to vancomycin and teicoplanin compared to the parent cells and that the resistance could be increased even further by additional inactivation of the WalRK two component system (Shoji et al., 2011). The increased resistance of the mutant strains is likely a result of the thickening of the cell wall and reduced autolysis observed for the clpP and walRK mutant cells compared to wild type cells (Shoji et al., 2011). In addition to increased tolerance of the clpP mutant to vancomycin, the mutant strain proved more susceptible than wild type cells to protein synthesis inhibitors (Shoji et al., 2011). This susceptibility may be due to the hampered degradation of the non-native proteins that accumulate in the presence of protein synthesis inhibitors. Daptomycin is an extensively used anti-staphylococcal agent against methicillin-resistant Staphylococcus aureus. In a recent study serial passage in the presence of daptomycin, yielded two daptomycin tolerant strains. Whole genome sequencing revealed that while both strains carried mutations in walK and the agr quorum sensing system one of the strains also carried a mutation in clpP (Song et al., 2013). Thus, inactivation of Clp dependent proteolysis may modulate fitness of daptomycin tolerant strains. This notion is supported by another study where daptomycin tolerance was provoked in strains of both clinical and laboratory origin. Here tolerance was also associated with walK and agr mutations as well as mutations in phospholipid biosynthesis gene (Peleg et al., 2012). Importantly, one of the strains carried a deletion in clpX indicating again that absence of Clp proteins may be a contributing factor in establishing daptomycin resistance.

Antibiotics that target ClpP proteases A very interesting finding in the ClpP field is the recent discoveries of several new types of antibiotics that kill bacteria by interfering with the function of the ClpP proteolytic complex. Notably, the ClpP proteolytic complex is an unusual drug target in that both inhibition and activation of its function are potential lethal events. The first ClpP targeting antibiotic discovered were the Acyldepsipeptides (ADEPs) that are naturally produced by isolates of Streptomyces hawaiiensis, and are active against a number of important Gram-positive pathogens that in addition to S. aureus include Streptococci, Enterococci, and Listeria. Interestingly, ADEPs do not inhibit the function of ClpP (Brötz-Oesterhelt et al., 2005). Rather binding of ADEPS converts the ClpP protease from a highly regulated peptidase that can degrade substrates only with the help of associated ClpATPases to an unregulated protease that degrades nascent polypeptides independently of the normal ATPase partner and in the absence of ATP (Kirstein et al., 2009). In growing cells, the ADEP bound protease seems to prefer some targets over others, as it specifically degraded the cell division master protein FtsZ at an increased rate, thereby inhibiting cell division (Sass et al., 2011). In non-growing cells, however, ADEP-activated ClpP became a fairly nonspecific protease that degraded over 400 proteins at an increased rate (Conlon et al., 2013). As an intriguing consequence, ADEPS is effective in killing dormant persister cells that do not respond to traditional antibiotics (Conlon et al., 2013). The crystal structure of ClpP in complex with ADEPs has been solved both for ClpP from B. subtilis and for ClpP from E. coli and has led to fascinating new insights into the mechanism by which ADEPs interfere with the function of ClpP. For more details on this subject we refer to Lee et al. (2010), Li et al. (2010) and Frees et al. (2013).

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By adopting a chemical proteomic strategy, ␤-Lactones were identified as potent, cell permeable inhibitors that specifically target ClpP (Böttcher and Sieber, 2008). Interestingly, growing S. aureus in the presence of the ␤-Lactones inhibitors completely abolished hemolytic and proteolytic activities and additionally reduced expression of lipases and DNases. Moreover, application of ␤Lactones was capable of shutting down synthesis of the pyrogenic toxin superantigens such as the toxic shock TSST-1 and enterotoxin B and C (Böttcher and Sieber, 2009). Therefore, ␤-Lactones are proposed to be of potential use in “anti-virulence therapy”, the rationale being that inhibition or “dis-arming” S. aureus by inhibiting virulence factor production will help the host immune response to eliminate S. aureus (Böttcher and Sieber, 2008, Böttcher and Sieber, 2009). Future in vivo studies are needed to clarify the potential of both ␤-Lactones and anti-virulence therapy. Alternatively, ␤-Lactones may be of use in combination with traditional antibiotics, as another inhibitor of the ClpXP protease acted synergistically with daptomycin to kill MRSA (McGillivray et al., 2012). Conclusion Without doubt the Clp protease and ATPases are central to a great number of processes ranging from general maintenance of protein quality to tight control of key regulatory proteins with impact on oxidation, DNA damaging response, starvation and antimicrobial resistance (see Fig. 2). The pleiotropic phenotypes observed for clp mutants have impaired the determination of the specific contribution of the ClpATPases or the Clp proteolytic complex to the individual processes. Recent methodological and technological advances such as the Clp proteolytic trap and the SILAC technology have been useful in advancing our knowledge in this area. However, for most of the proteolytic substrates identified it still remains to be determined if Clp proteolysis is regulatory and if so how proteolysis is confined to selected environmental conditions including the role of adaptor proteins in this process. More recently it has become clear that Clp proteins also have dual roles in combating antibiotic resistant staphylococci. On one hand, hyperactivation of Clp proteolysis is the killing mechanism

Fig. 2. ClpP mediated proteolysis is central to a great number of vital cellular processes. ClpP mediated proteolysis is required both for general maintenance of protein quality and for tightly controlling the cellular level of key regulatory proteins. For the stress-response regulators CtsR, Spx and LexA we have some insight into the specific conditions signaling proteolysis, however, for most of the depicted processes the specific regulators targeted by ClpP and the conditions signaling proteolysis remain elusive – see text for details.

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behind a new antimicrobial peptide produced by Streptomyces; this finding in combination with the dramatically reduced virulence observed in the absence of Clp proteolysis support that compounds targeting proteolysis may pave the way for new staphylococcal treatment options. On the other hand, treatment of MRSA infections with vancomycin or daptomycin in vivo select for non-susceptible isolates that among other mutations carry loss of function mutations in clpP or clpX, suggesting that in rare cases S. aureus may actually benefit from shutting down of Clp proteolytic activity. Future studies on Clp proteins promise to reveal exciting insights of proteolysis as a fundamental biological process in environmental adaptation while at the same time provide novel and much needed options for treating serious S. aureus infections.

Acknowledgments We would like to thank Cathrine Friberg and Jingyuan Feng for providing the EM micrograph and input to the proteolytic trap figures, respectively, and Tim Evison for graphical assistance.

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