International Journal of Medical Microbiology 304 (2014) 142– 149
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International Journal of Medical Microbiology
j ourna l ho me p age: www.elsev ier .com/ locate / i jmm
ini Review
lp chaperones and proteases are central in stress survival, virulencend antibiotic resistance of Staphylococcus aureus
orte Freesa, Ulf Gerthb, Hanne Ingmera,∗
Department of Veterinary Disease Biology, Faculty of Health and Medical Science, University of Copenhagen, Stigbøjlen 4, 1870 Frederiksberg C, DenmarkInstitute of Microbiology, Ernst-Moritz-Arndt-University Greifswald, D-17487 Greifswald, Germany
Intracellular proteolysis carried out by energy-dependent proteases is one of the most conserved bio-logical processes. In all cells proteolysis maintains and shapes the cellular proteome by ridding the cellof 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 andmitochondria of eukaryotic cells. In the serious human pathogen, Staphylococcus aureus inactivation ofclpP rendered the bacterium avirulent emphasizing the central role of proteolysis in virulence. The con-tribution of the Clp proteins to virulence is likely to occur at multiple levels. First of all, both Clp ATPasesand the Clp protease are central players in stress responses required to cope with the adverse conditionsmet 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 processesas virulence gene expression, cell wall metabolism, survival in stationary phase, and cell division. Theseobservations together with recent findings that Clp proteins contribute to adaptation to antibiotics high-lights the importance of this interesting proteolytic machinery both for understanding pathogenicity ofthe organism and for treating staphylococcal infections.
ntroduction
Staphylococcus aureus is a serious human pathogen that can giveise to a variety of infections ranging from harmless wound infec-ions to life-threatening conditions like bacteremia, osteomyelitisnd heart valve infections (Lowy, 1998). Antibiotic resistance is anncreasing problem with the spread of methicillin resistant strainsMRSAs) both in the hospitals and in the community (Otto, 2012).et, for the majority of time S. aureus is colonizing harmlesslyarm-blooded animals and humans and for the latter approxi-ately 30% are permanently colonized by the organism (DeLeo
t al., 2010). In the balance between harmless symbiosis andevastating infection, S. aureus tightly controls production of vir-lence and colonization factors. At the same time it relies ondvanced stress response systems that will allow survival anddaptation to changing environmental habitats. One of the molec-lar machineries that in S. aureus occupy roles in both virulence
nd environmental adaptation is the Clp proteolytic system. Clproteases are found well conserved in most bacterial species andhey are composed of a core proteolytic chamber flanked by one
of several possible ATPases that determine substrate specificity.Importantly, these ATPases also have chaperone activity that incombination with ClpP enable entry into the secluded, proteolyticchamber but in the absence of ClpP may function as indepen-dent molecular chaperones (Savijoki et al., 2006; Frees et al., 2007,2013). When originally examined in S. aureus, several studies indi-cated that the proteolytic subunit, ClpP and the ClpATPase, ClpX areessential for virulence as inactivation completely abolished abscessformation in a mouse model and eliminated expression of oneof the major staphylococcal hemolysins, �-hemolysin (Mei et al.,1997; Frees et al., 2003). Also, intracellular replication in bovinemammary 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 invirulence remain unknown, they must be related to two key bio-logical functionalities of the Clp complex namely in degradationof short-lived regulatory proteins (Elsholz et al., 2010a) or in pro-tein quality control (Frees et al., 2004). Recently, a large numberof ClpP substrates in S. aureus were identified by using catalyti-cally 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 revealedthat in S. aureus the Clp targets encompass a number of central reg-ulatory proteins (CtsR, Spx, HrcA, PerR, CodY) as well as proteins
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ig. 1. Large scale identification of substrates of the ClpP protease by using a protctive site was mutated, and the proteolytic inactive ClpP chamber now functions ahamber. Captured substrates were co-purified along with the His-tagged ClpP com
ith central physiological functions such as RecA, FtsZ, NrdE, Pmp,lmS and DnaK. Thus, with dual functionality in degrading bothon-native proteins generated during stress as well as specific keyegulatory proteins, the Clp proteolytic system appears to be a cor-erstone in processes of importance to survival and pathogenicity,nd as such, may not be an important target for design of newnterventions.
tructure
The Clp proteolytic chamber is a barrel shaped structure thats conserved among bacteria and is composed of two rings ofeptameric ClpP. Access to this secluded proteolytic chamber isestricted by pores that are too narrow to allow entry of folded pro-eins. In order to be degraded, substrates must first interact withhe Clp ATPase component that powers unfolding and subsequentranslocation of the substrate into the ClpP proteolytic chamberFrees et al., 2007). In S. aureus, the ClpC and ClpX ATPases canerform this function. Two additional Clp ATPases are encoded byhe organism, namely ClpL and ClpB, but they lack the conservedripeptide consensus sequence (IG(F/L)) required for interactionith ClpP (Martin et al., 2008). During proteolysis in S. aureus this
tructure undergoes dramatic conformational changes. In the pro-eolytic active state ClpP adopts an extended conformation with theatalytic triad (His-123, Asp-172, Ser-98) aligned in a proper geom-try for proteolytic activity. In contrast, in the closed conformationhe side-chains of the catalytic triad are flipped hampering proteo-ysis (Zhang et al., 2011; Gersch et al., 2012). Following degradationhe products are likely to be released through pores in the side of thearrel (Geiger et al., 2011). In some cases proteolysis is regulatedhrough the spatial and/or temporal use of adaptor proteins, whichre directly involved in the recognition and delivery of specific sub-trate proteins to the proteases (Dougan et al., 2002; Battesti andottesman, 2013). For example ClpXP can degrade substrates inde-endently of adaptors but the presence of the adaptor-like proteinjbH greatly enhances the proteolytic activity (Engman et al., 2012;ies and Maurizi, 2008).
Notably, ClpP-like proteins are also common among S. aureushages indicating that proteolytic control has a central function
n phage biology (Boyle-Vavra et al., 2011). An additional Clproteolytic complex baring resemblance to the eukaryotic 26S pro-easome is formed by the products of the clpY and clpQ genes (Frees
t al., 2005b). Despite the name, the proteolytic subunits ClpQnd ClpP are not related, and as no distinct phenotype has beeninked to the function of the ClpYQ it will not be discussed furtherere.
ally inactive ClpP-variant. To directly identify substrates of the ClpP protease, thepPTRAP′′ that will retain but not degrade substrates translocated into the proteolyticnd identified by mass spectrometry.
Stress tolerance
During infection bacterial pathogens are likely to encounterdramatic environmental changes and may experience shift intemperature, oxidative stress, antimicrobial peptides and otherconditions aimed at inactivating the invading microorganism. Suchstress exposures may lead to protein unfolding, and removal ofunfolded and non-native proteins is necessary for cellular func-tionality and growth (Truscott et al., 2011). In S. aureus inactivationof clpP, clpC, clpB and to a lesser extent clpL abolished or reducedgrowth at 45 ◦C (Frees et al., 2003, 2004, 2012). Since ClpB and ClpLare not functional ClpP partners these results suggested that ClpCPis degrading non-native proteins in S. aureus, a finding recentlyconfirmed by trapping protein substrates at high and ambient tem-perature, respectively (Feng, in preparation). The contribution ofClpB and ClpL to stress survival is likely as chaperones either toprevent protein unfolding or promote disaggregation (Glover andLindquist, 1998). This notion was supported by the finding that bothClpB and ClpL are required for thermoinduced thermotolerancewhere pre-exposure to intermediate high temperature improvessurvival at high temperatures (Frees et al., 2004). Surprisingly, inac-tivation of clpX allowed growth at an even higher temperature thanthe wild type cells. Although the basis for this finding is currentlyunknown it shows that Clp proteins contribute to virulence throughstress-independent (via ClpXP) and stress-dependent (ClpCP andClpB) pathways (Frees et al., 2003, 2004).
Also in Bacillus subtilis, non-native proteins are degraded by theClpCP complex and it takes place in a process also requiring theadaptor protein, MecA. MecA is necessary not only for substraterecognition but also for the oligomerization of ClpC into a hexamerand binding to ClpP (Kirstein et al., 2006; Schlothauer et al., 2003).A MecA homologue is also found in S. aureus where it is desig-nated teicoplanin resistance factor A (TrfA, see below, Renzoni et al.,2009). TrfA is a target of the Clp protease (Feng et al., 2013) and amutant lacking the corresponding gene is temperature sensitiveindicating that TrfA may assist ClpCP mediated proteolysis of non-native proteins also in S. aureus (Feng et al., in preparation). McsBis another adaptor-like protein also found in B. subtilis that wastrapped as Clp target (Elsholz et al., 2010b; Wozniak et al., 2012;Feng et al., 2013). Inactivation of S. aureus McsB impairs growthat 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 ClpCexpression as McsB in B. subtilis is needed for the degradation of thenegative 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 proteo-ytic trap was Spx that is a positive regulator of the disulfide-stressesponse (Feng et al., 2013). Spx has been extensively characterizedn B. subtilis where it was shown to bind directly to the C-terminalomain of the RNAP �-subunit and thereby interfering with trans-riptional activation mediated by certain response regulators ofwo-component systems (Zuber, 2004). Interestingly, the cellularevel of Spx is determined by proteolytic control: Under non-stressonditions, Spx is kept at a low concentration through degradationy the ClpXP protease in a process stimulated by the adaptor, YjbH,hile upon disulfide stress Spx is stabilized and transcriptionally
ctivates a number of genes required for oxidative stress toleranceNakano et al., 2003; Zuber, 2004; Garg et al., 2009; Larsson et al.,007; Engman et al., 2012). While the basic features in Spx func-ion and control hereof seems to be conserved among S. aureus and. subtilis, it is notable that inactivation of Spx confers much moreevere growth defects to S. aureus, and that recent research suggesthat the S. aureus spx mutant is only viable because it has accu-
ulated additional suppressor mutations (Pamp et al., 2006; Billelley, personal communication). In addition to the degradation ofpx, the combined activity of YjbH and ClpX appears to degradether desiccation tolerance regulatory proteins, as both proteinsurned out to be required for long-term survival on plastic surfacesChaibenjawong and Foster, 2011).
Another category of proteins identified as targets for Clp proteo-ysis is the DNA damage repair proteins (Feng et al., 2013). Responseo DNA damaging conditions has been characterized extensively inscherichia coli. DNA damage elicits the so-called SOS response thats controlled by the transcriptional repressor, LexA (Butala et al.,009). The LexA regulon comprises error prone DNA damage repairroteins and in the absence of stress is repressed by LexA (Kelley,006). However, upon DNA damage LexA undergoes autocleavageesulting in a C-terminal and a N-terminal fragment with the latteretaining some DNA-binding activity (Neher et al., 2003). Recentlye showed that in S. aureus, the ClpXP as well as the ClpCP proteases
ontribute to degradation of the LexA autocleavage products as inhe absence of ClpP, or one or both of the Clp ATPases, ClpX or ClpC,he LexA domains were stabilized (Cohn et al., 2011). In S. aureus,he LexA regulon includes lexA itself and the divergently transcribedene, sosA. Interestingly, when astabilized mutant allele of the LexA-terminal fragment was ectopically produced, expression of sosA
emained repressed while lexA was expressed under SOS inducingonditions (Cohn et al., 2011). This result suggests that the LexA reg-lon comprises two sets of genes namely those that are unaffectedy the N-terminal domain of LexA and those that are repressed in itsresence. Importantly, the activity of the Clp proteolytic complex isequired for the induction of the latter set of genes. Future studiesill reveal the nature of these genes and the biological importance
f Clp mediated proteolysis in the SOS response.
ontrol of clp expression
In low GC Gram positive bacteria CtsR controls expression of thelp genes (Krüger and Hecker, 1998; Derré et al., 1999; Chastanett al., 2003; Elsholz et al., 2010a,b). Under standard growth condi-ions it represses transcription of S. aureus genes clpP and clpB asell as the tetracistronic clpC operon (ctsR, mcsA, mcsB, clpC) but
hortly after heat stress expression is de-repressed (Frees et al.,004; Fleury et al., 2009). S. aureus CtsR was retained by theroteolytic inactive ClpCPTRAP (Feng et al., 2013) suggesting thats in Bacillus ClpCP mediated degradation of CtsR contributes to
e-repression of the CtsR regulon (Krüger et al., 2001; Miethket al., 2006). The monocistronic genes clpL and clpX do not belongo the CtsR regulon. clpL is solely sigB-dependently transcribedGertz et al., 2000; Frees et al., 2004) and the mechanism(s) for a
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transcriptional regulation of clpX remain to be investigated (Freeset al., 2004).
In addition to the clp genes, the CtsR regulon in Staphylococcialso comprises the classical chaperone genes dnaK and groESL,which in other Gram positive organisms are controlled by anotherheat shock repressor, HrcA (Chastanet et al., 2003). In fact the entireHrcA regulon (dnaK and groESL operons) is embedded within theCtsR regulon in Staphylococci and both repressors (HrcA, CtsR) seemto act synergistically to maintain a low basal level of the dnaK andgroESL operon expression in the absence of stress (Chastanet et al.,2003). Therefore, synthesis of the classical chaperones is directlyconnected with that of the Clp proteins in Staphylococci possi-bly enhancing the adaptability both during stress and infection(Chastanet et al., 2003).
A central question in control of clp protein expression ishow CtsR monitors the temperature. Elegant studies in B. subtilisrecently showed that the protein itself “feels” the heat as a proteinthermometer and reacts intrinsically to the temperatures of thesurroundings (Elsholz et al., 2010b). The tetra-glycine loop in thewinged helix-turn-helix domain grabs into the minor groove of theCtsR operator site on the DNA (Fuhrmann et al., 2009) and was iden-tified as thermosensing site of CtsR by site-directed mutagenesis inB. subtilis, L. lactis as well as in G. stearothermophilus, which makesthe protein susceptible for heat inactivation at species-specificheat shock temperatures (Elsholz et al., 2010b). After dissocia-tion from the DNA, CtsR is immediately degraded by B. subtilisClpEP protease (Miethke et al., 2006) and later by ClpCP, whichneeds the active arginine kinase, McsB, as adaptor protein (Elsholzet al., 2010b, 2011a). Importantly this work supports the generalhypothesis for Clp degradation namely that “un-occupied” non-functional and unprotected proteins may be substrates of the Clpcomplex but when occupied by association with for example DNAbinding they are stabilized (Michalik et al., 2012). This notionimplicates that protein substrates are found in both proteolysis-proficient and deficient forms in the same cell (Feng et al.,2013).
The mechanism for the CtsR inactivation during thiol-specificstress is different and depends solely on McsB, but apparently noton its arginine kinase activity (Elsholz et al., 2011b). In B. subtilisthiol-specific stress is sensed by the thiols of the second zinc fin-ger present in McsA, which are necessary for the McsB interaction(Elsholz et al., 2011b). These thiols act as a molecular redox switchto regulate the McsB activity by dissociating it from McsA. WhenMcsB is not associated with McsA, it binds and inactivates DNA-bound 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 indicatingthat homologues of McsB and McsA may perform similar functionsalso in this organism (Wozniak et al., 2012).
Clp proteins orchestra expression of virulence genes
The pathogenicity of S. aureus relies on a wide array of surface-bound and secreted virulence factors that equip the bacterium fortissue binding, tissue destruction, and immune evasion. One ofthe most significant phenotypes of the clpP deletion in the 8325-derived strains is the dramatic effect on transcription of majorvirulence genes (Frees et al., 2003, 2005a,b; Michel et al., 2006;Feng et al., 2013; Farrand et al., 2013). As an example, transcrip-tion of genes encoding the extracellular proteases SspA, Aur, andSpl is reduced as much as 100 fold, while transcription of the hla
gene encoding alpha hemolysin is decreased 10 fold in the post-exponential growth phase. Accordingly, the clpP mutant strain wasboth non-hemolytic and non-proteolytic (Frees et al., 2003; Fenget al., 2013). The NCTC8325-derived strains are characterized by
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cell wall metabolism or cell division are substrates of ClpP inS. aureus (see Table 1). In B. subtilis the filamentous growth ofthe clpP mutant is partially caused by accumulation of MurAAwhich catalyses carboxyvinyl transfer from phosphoenolpyruvate
Table 1ClpP substrates with a predicted role in cell wall metabolism or cell division.
ery high expression of hemolysins and extracellular proteasesompared to other S. aureus strains, a trait that has been ascribedo the low activity of the alternative sigma factor, SigB, in thesetrains (Giachino et al., 2001; Horsburgh et al., 2002; Oscarssont al., 2006a).
Interestingly, recent proteomic studies emphasized that bothhe surfacome and exoproteome of clinical S. aureus isolates displayn extreme heterogeneity, and that strain dependent expression ofirulence genes has a pivotal role in generating diversity (Dreisbacht al., 2010; Ziebandt et al., 2010). The expression of virulence fac-ors is regulated by a complex network of regulatory elements thatn addition to SigB encompass at least six two-component systemsnd a number of transcriptional regulators, of which many belongo the family of SarA homologues (Novick, 2003). Global transcrip-omic and proteomic analysis of different S. aureus strains carrying
clpP mutation revealed that expression of a large number of majorirulence regulators was affected by the absence of ClpP (Freest al., 2012). While the transcription of global virulence regulatorenes RNAIII, mgrA, sarZ, sarR and arlRS was similarly affected by
clpP mutation in all the examined strains, inactivation of ClpPnfluenced expression of the virulence genes sspA, hla, and spa in
strain dependent manner. The strain dependency is puzzling butt may at least in part be determined by a strain dependent effectf ClpP on SarS expression. SarS is a transcriptional regulator thatirectly represses transcription of sspA and hla, while stimulatingxpression of spa (Oscarsson et al., 2005, 2006a,b). In SigB proficienttrains expression of sarS is high and disruption of clpP reducedarS transcription resulting in enhanced sspA and decreased sparanscription. In contrast, sarS expression is comparable low inhe SigB-deficient strain, 8325-4, and deletion of clpP in this back-round greatly increased transcription of sarS leading to repressionf sspA and hla and activation of spa (Frees et al., 2012).
The finding that inactivation of ClpP led to major perturbationsn the cellular content of global virulence regulators indicated thatlpXP controls stability of one or more transcriptional virulenceegulators and thereby impacts the entire regulatory network.owever, none of the known virulence regulators were identi-ed as ClpP substrates neither using the proteolytically inactivelpPTRAP for the identification of substrates nor from stability assaysMichalik et al., 2012; Feng et al., 2013). Presently, we cannot ruleut that this may be due to experimental challenges as regulatoryroteins are not very abundant, are small (cannot be identified withigh confidence in MS-analysis), and are often highly basic whichake them more difficult to detect by 2DE-gel electrophoresis.lternatively, the link between ClpXP and virulence gene regula-
ion is indirect in the sense that the cellular stress imposed by theack of ClpP may create some general physiological or metabolicignals that are sensed by the regulatory network controlling viru-ence determinants. Also the changes in cell wall structure observedn clpP mutant cells (see below) may impair signaling across theell wall and lead to reduced virulence gene expression as has beenbserved for antibiotic resistant variants with alter cell wall struc-ure (Rudkin et al., 2012). However, this would not explain why thelpP deletion has a strain-dependent effect on the expression of theentral virulence regulator SarS and how this is linked to the SigBtatus of the cell.
The ClpX chaperone functions independently of ClpP to con-rol expression of spa encoding the IgG binding Protein A (Freest al., 2003, 2005b). The effect of ClpX is dramatic as disruptionf clpX virtually abolished expression of Protein A in both 8325-
and the low-passage clinical strain SA564, suggesting that theffect is not strain-dependent (Jelsbak et al., 2010). Interestingly,
lpX appears to perform dual roles in controlling Protein A expres-ion (Jelsbak et al., 2010). First, ClpX stimulates transcription of spandirectly by enhancing translation of the transcriptional regulatorot. Transcription from the spa promoter is regulated by negative
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(SarA) and positive regulators (Rot and SarS) that compete for over-lapping operator sites (Gao and Stewart, 2004). By expressing rotfrom an inducible promoter we could show that transcription of sparequires a threshold level of Rot, and that in cells lacking ClpX thecellular level of Rot is decreased below this threshold level (Jelsbaket al., 2010). Additionally, ClpX has a positive effect on translationof the spa transcript (Jelsbak et al., 2010). The finding that ClpX sti-mulates translation of both the rot- and the spa-mRNAs opens upfor the intriguing possibility that ClpX has a direct role in promotingtranslation, as was shown for HSP101, an eukaryotic relative of ClpX(Wells et al., 1998; Ling et al., 2000). Secondary structure determi-nations of the spa- and rot-mRNAs revealed that the translationalsignals are partially obscured in the stem-loop structures. Hence,ClpX may promote translation by directly or indirectly facilitatinginteraction between the spa- and rot-mRNAs and the ribosomes.
Capsule formation is another factor contributing to S. aureuspathogenicity and recently a transposon screen performed in strainNewman identified ClpC as a regulator of capsule formation (Luonget al., 2011). In the Newman background ClpC turned out to impactcapsule formation by two distinct pathways involving respectivelythe SaeRS two component system and the CodY transcriptionalregulator (Luong et al., 2011). The repressor activity of CodY is reg-ulated by the metabolic status of the cell and CodY represses thecap genes either directly, or via repressing the agr quorum sensingsystem thereby linking the cells metabolic status with virulenceregulation (Majerczyk et al., 2010). ClpC promoted capsule for-mation by reducing codY expression in both strain Newman andUAMS-1 suggesting that this positive effect of ClpC on capsule for-mation is conserved among strains. In contrast, the Sae dependenteffect of ClpC on capsule formation appears to be restricted to theNewman background and is linked to the SaeR-hyper activation inthis strain (Mainiero et al., 2010). Interestingly, the data indicatedthat ClpC facilitates auto-activation of sae transcription in strainNewman (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
a GlmM was not captured by the ClpPTRAP but was shown to accumulate in clpPmutants (Frees et al., 2012).
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o UDP-N-acetylglucosamine, the first committed step in peptid-glycan biosynthesis (Kock et al., 2004). Notably, MurAA doesot appear to be a ClpP substrate in S. aureus. Instead, GlmS andlmM, two other physiological checkpoints in the synthesis ofDP-N-acetylglucosamine (the cytoplasmic precursor of peptido-lycan), are likely substrates of ClpP in S. aureus (Jolly et al., 1997;omatsuzawa et al., 2004; Frees et al., 2012; Feng et al., 2013).owever, the molecular pathways by which ClpP impacts cell walletabolism and cell division in S. aureus remain an unexplored area
f research and much remains to be discovered.Strikingly, some of the proteins that were most abundantly cap-
ured by the ClpPTRAP are proteins with autolytic activity. Examplesnclude the autolysin SleI, the Secretory antigen precursor SsaASA0620) (that has a LytM- and a CHAP domain), and the immu-odominant antigen A, IsaA. Proteomic analysis of total cellularroteins showed that these proteins accumulate strongly in cells
acking ClpP, and as transcription of the corresponding genes isot induced, this finding supports that SleI, SsaA, and IsaA are allegraded by ClpP in the wild-type cells. It is puzzling why thesenzymes that mediate their function outside the cell are subjecto intracellular degradation. As described above the clpP mutantell has a thickened cell wall, and accordingly, we did not observencreased autolytic activity of the clpP mutants (8325-4 and SA564ackground) indicating that the autolytic enzymes accumulate inn inactive form (our unpublished data). In contrast, the 8325 clpPeletion strain exhibited enhanced autolytic activity in a similarssay (Michel et al., 2006). However, we speculate that the signif-cantly increased spontaneous release of pro-phages mediated byhe inactivation of clpP in the latter strain-background may con-ribute to this phenotype (Frees et al., 2012). In conclusion, the ClpProteolytic complex controls multiple steps in cell wall metabolismnd we anticipate that future research will reveal novel insights ofhe role of Clp proteins in control and formation of the cell wall.
ultiple roles for Clp proteins during starvation and intationary phase
Proteolysis plays an important role not only when handlingtress but also during long-term starvation in non-growing cells.
hen starved for glucose, cells are forced to save energy andurvive with very limited resources. To study the role of Clproteolysis in S. aureus in these processes, a mass spectrometry-ased stable isotope labeling by amino acid in cell culture (SILAC)echnology was employed (Michalik et al., 2012). Hereby proteinate is monitored in terms of synthesis, accumulation, aggrega-ion and degradation. The experiment revealed that when starvedor glucose, proteolysis mainly affected vegetative, anabolic andelected catabolic enzymes, whereas the expression of TCA cyclend gluconeogenetic enzymes increased (Michalik et al., 2009,012). Additionally, proteins involved in growth and reproduc-ion (e.g., ribosomal, translation and cell wall synthesis proteins)ere degraded in wild type cells, but stabilized in the clpP mutant
uggesting ClpP dependent degradation. Furthermore, anabolicnzymes such as NrdEF involved in the nucleic acid biosynthesisere clearly targeted by ClpP (Michalik et al., 2012). Commonly,any proteins were prone to aggregation in clpP mutant cells and
he absence of ClpP correlated with protein denaturation and anxidative stress response (Michalik et al., 2012).
CodY is involved in the adaptive response to starvation andt represses approximately 5% of the genome when bound toTP and/or branched chain amino acids (BCAA). When nutrients
ecome limiting, a decrease in intracellular levels of GTP and BCAAauses a deactivation of CodY and decreases its affinity for DNA,eading to the induction of its regulon. In clpP mutant cells a num-er of CodY-dependent genes/operons were strongly decreased
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in amount and synthesis including purEKCSQLFMNHD, the gltBD,sucAB, pyc, oppBCDF and the codY-operon (xerC-hslUV-codY). Sev-eral CodY-dependent enzymes involved in branched-chain aminoacid biosynthesis (e.g., LeuA, LeuB, LeuC, LeuD; IlvA2, IlvB, IlvC, andIlvD) and purine nucleotide biosynthesis (e.g., PurC, PurD, PurE,PurL, PurM, PurQ, and PurS) were also down-regulated in the clpPmutant (Michalik et al., 2012). A higher GTP level in the �clpP strainmay render the CodY repressor more active, and could be respon-sible for the enhanced CodY repression in mutant cells (Michaliket al., 2012).
The ClpP interaction partner ClpC seems to play important rolesin S. aureus with respect to late stationary phase phenomena suchas 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 lossof of CitB (aconitase) activity and accumulation of acetate wasobserved in a clpC mutant suggesting a role for ClpC in TCA cycleregulation during the stationary phase (Chatterjee et al., 2005). Itwas hypothesized that a decrease of the TCA cycle activity and res-piration in a clpC mutant result in a reduced generation of ROSand oxidative stress, which enhances survival of a S. aureus clpCmutant in the stationary phase (Chatterjee et al., 2009). However,the increased survival of the clpC mutant could also be explainedby a differential effect of the protein on one of three toxin anti-toxin systems in S. aureus (Donegan et al., 2010). These systemsare characterized by stable toxins that are inactivated by anti-toxins and the turnover of the latter in known S. aureus systemsare mediated by ClpCP (Donegan et al., 2010). Moreover, a linkbetween ClpC and the thymidine-dependent small-colony variants(TD-SCVs) was proposed suggesting a reduced generation of 5, 10-methylene-THF (substrate for thyA) in a clpC mutant which wouldprevent both accumulation of this substrate as well as the con-sumption of reduced nicotinamide (Chatterjee et al., 2007, 2009).Thus, the Clp proteins and Clp dependent proteolysis affect cellularmetabolism at multiple levels.
Clp and antibiotic resistance
In recent years several studies have pointed to a link betweenClp proteins and antibiotic resistance. In an early study a randommutagenesis approach was used to identify genes that upon inac-tivation 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 cellwall synthesis by binding to the C-terminal d-alanyl–d-alanine(d-Ala–d-Ala) residues of cell wall precursors and nascent pep-tidoglycan and blocks the crosslinking (Hiramatsu, 2001, Pereiraet al., 2007). Clinical isolates with low-level glycopeptide resis-tance are referred to as GISA strains (Tenover et al., 1998; Sieradzkiet al., 2003) and they are characterized by cell wall thickening,altered cell wall composition, reduced autolysis and reduced neg-ative cell wall charge (Cui et al., 2003, Sieradzki et al., 2003).Importantly one of two genes identified as associated with reducedglycopeptide resistance was the trfA (teicoplanin resistance factor,A). TrfA is also called MecA that is required for ClpC assembly inthe ClpCP complex and for ClpCP mediated proteolysis (Renzoniet al., 2009 see above). Remarkably trfA mutant derivatives of GISAstrains displayed increased susceptibility to both teicoplanin andvancomycin as well as oxacillin and upon passage in the presenceof teicoplanin, fewer resistant mutants arose in a trfA mutant back-ground compared to wild type cells. Interestingly, trfA transcriptionis induced by cell wall active antibiotics and this activation is medi-
ated by Spx (Jousselin et al., 2013). These findings show that Spxresponds to cell wall active antibiotics and that TrfA, perhaps inconjunction with ClpCP proteolysis, is involved in the GISA pheno-type.
f Medi
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process.More recently it has become clear that Clp proteins also have
dual roles in combating antibiotic resistant staphylococci. On onehand, hyperactivation of Clp proteolysis is the killing mechanism
Fig. 2. ClpP mediated proteolysis is central to a great number of vital cellularprocesses. ClpP mediated proteolysis is required both for general maintenance of
D. Frees et al. / International Journal o
In a later study vancomycin intermediate sensitive strains (VISA)ere examined and the mutations that gave rise to the VISA pheno-
ype was determined. In addition to mutations in the well-knownraSR and graRS genes encoding cell wall stress sensing systems,he VISA phenotype was also associated with mutations in clpPShoji et al., 2011). Antibiotic susceptibility tests showed that
clpP mutant displayed increased tolerance to vancomycin andeicoplanin compared to the parent cells and that the resistanceould be increased even further by additional inactivation of the
alRK two component system (Shoji et al., 2011). The increasedesistance of the mutant strains is likely a result of the thicken-ng of the cell wall and reduced autolysis observed for the clpP andalRK mutant cells compared to wild type cells (Shoji et al., 2011).
n addition to increased tolerance of the clpP mutant to vancomycin,he mutant strain proved more susceptible than wild type cells torotein synthesis inhibitors (Shoji et al., 2011). This susceptibilityay be due to the hampered degradation of the non-native proteins
hat accumulate in the presence of protein synthesis inhibitors.Daptomycin is an extensively used anti-staphylococcal agent
gainst methicillin-resistant Staphylococcus aureus. In a recenttudy serial passage in the presence of daptomycin, yielded twoaptomycin tolerant strains. Whole genome sequencing revealedhat while both strains carried mutations in walK and the agr quo-um sensing system one of the strains also carried a mutation inlpP (Song et al., 2013). Thus, inactivation of Clp dependent pro-eolysis may modulate fitness of daptomycin tolerant strains. Thisotion is supported by another study where daptomycin toleranceas provoked in strains of both clinical and laboratory origin. Here
olerance was also associated with walK and agr mutations as wells mutations in phospholipid biosynthesis gene (Peleg et al., 2012).mportantly, one of the strains carried a deletion in clpX indicatinggain that absence of Clp proteins may be a contributing factor instablishing daptomycin resistance.
ntibiotics that target ClpP proteases
A very interesting finding in the ClpP field is the recent dis-overies of several new types of antibiotics that kill bacteria bynterfering with the function of the ClpP proteolytic complex.otably, the ClpP proteolytic complex is an unusual drug target
n that both inhibition and activation of its function are potentialethal events. The first ClpP targeting antibiotic discovered werehe Acyldepsipeptides (ADEPs) that are naturally produced by iso-ates of Streptomyces hawaiiensis, and are active against a numberf important Gram-positive pathogens that in addition to S. aureusnclude Streptococci, Enterococci, and Listeria. Interestingly, ADEPso not inhibit the function of ClpP (Brötz-Oesterhelt et al., 2005).ather binding of ADEPS converts the ClpP protease from a highlyegulated peptidase that can degrade substrates only with the helpf associated ClpATPases to an unregulated protease that degradesascent polypeptides independently of the normal ATPase part-er and in the absence of ATP (Kirstein et al., 2009). In growingells, the ADEP bound protease seems to prefer some targets overthers, as it specifically degraded the cell division master proteintsZ at an increased rate, thereby inhibiting cell division (Sass et al.,011). In non-growing cells, however, ADEP-activated ClpP became
fairly nonspecific protease that degraded over 400 proteins at anncreased rate (Conlon et al., 2013). As an intriguing consequence,DEPS is effective in killing dormant persister cells that do notespond to traditional antibiotics (Conlon et al., 2013). The crys-al structure of ClpP in complex with ADEPs has been solved both
or ClpP from B. subtilis and for ClpP from E. coli and has led to fasci-ating new insights into the mechanism by which ADEPs interfereith the function of ClpP. For more details on this subject we refer
o Lee et al. (2010), Li et al. (2010) and Frees et al. (2013).
cal Microbiology 304 (2014) 142– 149 147
By adopting a chemical proteomic strategy, �-Lactones wereidentified as potent, cell permeable inhibitors that specifically tar-get ClpP (Böttcher and Sieber, 2008). Interestingly, growing S.aureus in the presence of the �-Lactones inhibitors completely abol-ished hemolytic and proteolytic activities and additionally reducedexpression of lipases and DNases. Moreover, application of �-Lactones was capable of shutting down synthesis of the pyrogenictoxin superantigens such as the toxic shock TSST-1 and entero-toxin B and C (Böttcher and Sieber, 2009). Therefore, �-Lactonesare proposed to be of potential use in “anti-virulence therapy”,the rationale being that inhibition or “dis-arming” S. aureus byinhibiting virulence factor production will help the host immuneresponse to eliminate S. aureus (Böttcher and Sieber, 2008, Böttcherand Sieber, 2009). Future in vivo studies are needed to clarify thepotential of both �-Lactones and anti-virulence therapy. Alterna-tively, �-Lactones may be of use in combination with traditionalantibiotics, as another inhibitor of the ClpXP protease acted syner-gistically with daptomycin to kill MRSA (McGillivray et al., 2012).
Conclusion
Without doubt the Clp protease and ATPases are central to agreat number of processes ranging from general maintenance ofprotein quality to tight control of key regulatory proteins withimpact on oxidation, DNA damaging response, starvation andantimicrobial resistance (see Fig. 2). The pleiotropic phenotypesobserved for clp mutants have impaired the determination ofthe specific contribution of the ClpATPases or the Clp proteoly-tic complex to the individual processes. Recent methodologicaland technological advances such as the Clp proteolytic trap andthe SILAC technology have been useful in advancing our knowl-edge in this area. However, for most of the proteolytic substratesidentified it still remains to be determined if Clp proteolysis isregulatory and if so how proteolysis is confined to selected envi-ronmental conditions including the role of adaptor proteins in this
protein quality and for tightly controlling the cellular level of key regulatory pro-teins. For the stress-response regulators CtsR, Spx and LexA we have some insightinto the specific conditions signaling proteolysis, however, for most of the depictedprocesses the specific regulators targeted by ClpP and the conditions signaling pro-teolysis remain elusive – see text for details.
1 f Medi
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48 D. Frees et al. / International Journal o
ehind a new antimicrobial peptide produced by Streptomyces;his finding in combination with the dramatically reduced virulencebserved in the absence of Clp proteolysis support that compoundsargeting proteolysis may pave the way for new staphylococcalreatment options. On the other hand, treatment of MRSA infectionsith vancomycin or daptomycin in vivo select for non-susceptible
solates that among other mutations carry loss of function muta-ions in clpP or clpX, suggesting that in rare cases S. aureus mayctually benefit from shutting down of Clp proteolytic activity.uture studies on Clp proteins promise to reveal exciting insights ofroteolysis as a fundamental biological process in environmentaldaptation while at the same time provide novel and much neededptions for treating serious S. aureus infections.
cknowledgments
We would like to thank Cathrine Friberg and Jingyuan Feng forroviding the EM micrograph and input to the proteolytic trap fig-res, respectively, and Tim Evison for graphical assistance.
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