Novel targets for antibiotics in Staphylococcus aureus

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10.2217/17460913.2.6.655 © 2007 Future Medicine Ltd ISSN 1746-0913

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Future Microbiol. (2007) 2(6), 655–666 655

Novel targets for antibiotics in Staphylococcus aureusKnut Ohlsen† & Udo Lorenz†Author for correspondenceUniversity of Würzburg,Institute for Molecular Infection Biology,Röntgenring 11,97070 Würzburg, GermanyTel.: +49 931 312 155;Fax: +49 931 312 578;[email protected]

Keywords: antibiotic resistance, Gram-positive pathogens, immunotherapy, MRSA, new targets, novel antibacterials, Staphylococcus aureus

Multiple resistant staphylococci that cause significant morbidity and mortality are the leading cause of nosocomial infections. Meanwhile, methicillin-resistant Staphylococcus aureus (MRSA) also spreads in the community, where highly virulent strains infect children and young adults who have no predisposing risk factors. Although some treatment options remain, the search for new antibacterial targets and lead compounds is urgently required to ensure that staphylococcal infections can be effectively treated in the future. Promising targets for new antibacterials are gene products that are involved in essential cell functions. In addition to antibacterials, active and passive immunization strategies are being developed that target surface components of staphylococci such as cell wall-linked adhesins, teichoic acids and capsule or immunodominant antigens.

In the past decade, a notable rise in the numberof infections caused by antibiotic resistantGram-positive pathogens such as methi-cillin-resistant Staphylococcus aureus (MRSA),vancomycin-resistant enterococci (VRE) andpenicillin-resistant Streptococcus pneumoniae(PRSP) pose specific therapeutic challenges. Spe-cifically, MRSA is now the most common causeof hospital-acquired infections worldwide. Thespectrum of diseases caused by S. aureus canrange from mild superficial lesions to life-threat-ening infections including bacteremia, pneumo-nia and endocarditis. Newborns, patients withchronic bronchopulmonary disorders, those whohave undergone surgical procedures, patientswith diabetes mellitus, indwelling intravascularcatheters and immunocompromised individuals,are especially susceptible to S. aureus. Accordingto the National Nosocomial Infections Surveil-lance (NNIS) system, MRSA strains accountedfor more than 50% of all S. aureus infections inintensive care units in the USA [1]. High MRSArates have also been reported in Europe, increas-ing most rapidly in the UK (now up to 45%) [2].This situation is further complicated by therecent spread of community acquired (CA)-MRSA [3]. Although almost all of the MRSA iso-lates are susceptible to vancomycin, and also tothe newly introduced antibiotics linezolid anddaptomycin, there is an urgent need to discoverantibiotics targeting novel cellular functionsnot yet targeted by current antibacterial agents.This is especially important as S. aureus isolateswith intermediate and high resistance to vanco-mycin (as well as to all other antibiotics) havenow been reported [4–6]. There is substantial fearin the medical community that a ‘superbug’

will emerge that is resistant to all antibiotics. Inthis review, the recent development of newstrategies to combat infections caused byS. aureus are summarized and trends for thefuture are discussed.

Search for essential gene productsThe search of new targets for novel antibacterialsrepresents a challenging task. In principle, eachessential protein can serve as a target for anti-microbials. However, further criteria have to bemet, including selectivity (functional and/orstructural differences between human and bacte-rial target molecules), distribution (which micro-organisms are susceptible), ease of assay (can anassay for high-throughput screening be devel-oped?), intracellular concentration of the poten-tial target, mutational potential (development ofresistance), bacteriocidal versus bacteriostaticaction (in vivo efficiency) and drugability (likeli-hood of being able to modulate a target with asmall-molecule drug), before a target is selectedfor lead development. Several attempts for defin-ing essential factors of bacteria have recently beenpublished, including theoretical calculations onthe minimal genome of bacteria and extensivemutational analysis [7–9]. For example, in Bacillussubtilis, 271 genes were predicted to be essential[10]. The vast majority of essential genes encodefactors that are involved in synthesis of the cellenvelope, information processing, the determina-tion of cell shape and division and generation ofenergy. In S. aureus various essential proteinshave been defined by antisense-RNA interferenceand mutagenesis studies [11,12]. These studiesgenerated lists of putative novel targets coveringall known key pathways, for example translation,

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transcription, cell division and metabolism.Importantly, for approximately 30% of the puta-tive essential genes the function remains unclear.Within the group of proteins with no knownfunction some attractive novel targets have beenidentified. For example, in an attempt to evaluatethe potential of proteins of unknown functionfor target search, Zalacain et al. investigated 144open reading frames (ORFs) without significanthomologies to proteins with known functions inS. pneumoniae, 36 of which were essential inS. pneumoniae and 14 of these were also essentialin S. aureus and Haemophilus influenzae [13]. Inaddition to a number of previously validated orsuggested targets, in vivo essentiality in S. aureushas been proven for five ORFs: spoIIIJ2, trmU,ydiC, ydiE and yneS. Interestingly, by testing ofnonessential in vitro genes, in vivo, four out of 17genes were also essential, suggesting a broaderselection of bacterial targets [13]. Consequently,essentiality of a given gene product is not onlyspecies dependent but is also dependent on theenvironmental test conditions. For example, theavailability of specific macromolecules or nutri-ents has a pivotal impact on the growth of thebacteria. Thus, every novel target has to be vali-dated in vitro and also in vivo in different patho-gens under various test conditions. This isespecially of importance for the development ofbroad-spectrum antibiotics.

In S. aureus particularly, three systems havebeen applied for conditional gene expression tovalidate putative essential genes. The Pxyl–tetO-system contains the xylose promoter controlledby tetracycline or anhydrotetracycline, thePxyl–xylR system can be induced by xylose andrepressed by glucose, and the Pspac–lacI system,including the Pspac hybrid promoter, is controlledby the lac-operator inducible by isopropyl β-D-1-thiogalactopyranoside (IPTG) [14–16]. Systemsusing tetracycline as the inducer are commonlyapplied for in vivo gene expression as tetracycline,or its nontoxic derivative anhydrotetracycline, iseasily distributed in the body and also penetratesintracellularly [12,17]. Recently, an alternative con-ditional in vivo expression system has beendeveloped based on the Pspac-lacI-regulatablepromoter element (Figure 1) [14,18].

Novel targets for antibacterials in Staphylococcus aureusThe investigation of novel targets in new path-ways has been suggested to be especially promis-ing as it is regarded that antimicrobials targetingnew pathways will most likely exhibit no

cross-resistance to commonly used antimicrobialsand, therefore, it should take more time for resist-ant strains to arise. However, a recent study inves-tigating Salmonella metabolic pathways suggesteda shortage of new metabolic targets for broad-spectrum antibiotics. In a comprehensive in vitroand in vivo approach, 155 promising targets totreat Salmonella infections were identified, 64 ofwhich were also conserved in other importantpathogens such as S. aureus, Enterococcus faecalis,S. pneumoniae and H. influenzae. Almost all ofthese targets belong to pathways already inhibitedby current antibiotics (peptidoglycan biosynthe-sis, folate biosynthesis, isoprenoid biosynthesis,fatty acid biosynthesis and tRNA synthases) orpathways previously considered for antimicrobialdevelopment [19]. Consequently, novel targets inalready approved pathways are being investigated.However, the discovery of new drugs againstolder targets, based on new structural informa-tion, represents an additional promising strategythat will be discussed in the following sections.

Cell division (FtsZ)FtsZ is a tubulin-like GTPase that plays anessential role in bacterial cell division. Itshomologs are present in almost all eubacteriaand archaea. During cell division, FtsZ formspolymers in the presence of GTP, which recruitother proteins implicated in division to form thecell-division apparatus. Inhibition of FtsZpolymerization prevents cells from dividing,leading to cell death [20,21]. Recently, a com-pound named viriditoxin has been identifiedthat blocked FtsZ polymerization. Viriditoxinexhibited broad-spectrum antibacterial activityagainst clinically relevant Gram-positive patho-gens, including MRSA and VRE, withoutaffecting the viability of eukaryotic cells [21].

Deformylase (Pdf1) Peptide deformylase (PDF) is an essential bac-terial metalloenzyme which deformylates theN-formylmethionine of newly synthesizedpolypeptides. In prokaryotes, the amino groupof the methionyl moiety carried by the initiatortRNAfMet is N-formylated by formyltransferaseprior to its incorporation into a polypeptide.Consequently, N-formylmethionine is alwayspresent at the N-terminus of a nascent bacterialpolypeptide. To further proceed translation, theformyl group is hydrolyzed by peptide deformy-lase. PDF is essential for bacterial growth andrepresents an attractive novel target [22,23]. Thegene encoding PDF (def ) is present in all

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sequenced pathogenic bacterial genomes. Inter-estingly, a human mitochondrial peptidedeformylase has been reported recently thatmay provide a new anticancer target of actin-onin-based antibiotics [24]. The x-ray structureof PDF has been resolved, defining PDF as anew class of metalloproteases [25]. Severalhydroxamic acid derivatives have been reportedto be potent PDF inhibitors with in vitro anti-bacterial activity [26,27]. A number of relatedcompounds inhibited the growth of severalclinically relevant bacterial pathogens. Impor-tantly, one compound termed BB-83698 wasalso active in systemic models of S. aureus infec-tion in the mouse and is the first compound inthis novel class to enter clinical trials [28].

Teichoic acid synthesisTeichoic acid (TA) synthesis has been regarded asthe target for antimicrobials as its essentiality hasbeen postulated for B. subtilis and Listeria mono-cytogenes [29,30]. However, more recent work dem-onstrated that wall teichoic acids (WTAs) aredispensible both in B. subtilis and S. aureus. Para-doxically, the gene catalyzing the first step inWTA synthesis, tagO in B. subtilis and tarO inS. aureus, could be deleted to yield viable mutantslacking TA in the cell wall while later-actingenzymes of the pathway display an essential phe-notype [31,32]. Possibly, an accumulation of unde-caprenyl-linked precursors in the WTA-synthesispathway results in cessation of the bacterial cellwall synthesis due to depletion of the small

Figure 1. Use of in vivo imaging technology to assess essential gene function of ligA.

The time course of bioluminescence levels was monitored for seven consecutive days (from 2 h until day 7) after subdermal infection of the lower back area of mice with 1 x 108 colony forming units of Staphylococcus aureus Xen29-Pspac:ligA. Conditional expression of essential ligA depends (in this strain) on the presence of the inducer IPTG [14]. S. aureus infection has been established in mice that have received IPTG (upper row). In animals that had not received IPTG the bacteria were not able to cause infection (lower row). Signal intensity is indicated by a pseudocolor scale. IPTG: Isopropyl β-d-1-thiogalactopyranoside; p.i.: Post-infection.

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bactoprenyl-phosphate pool. Results from theauthors’ laboratory support an essential pheno-type of one of the downstream genes. By use of aconditional mutant, in vitro essentiality ofSA0245 (tarF ) has been demonstrated [Unpublished

Data]. Moreover, TagO has been demonstrated toplay a critical role in nasal colonization, which is animportant risk factor for nosocomial infections [33].Furthermore, alanylation of TAs undertaken byfactors encoded by the dlt operon play an essentialrole in resistance to cationic antimicrobialpeptides (CAMPs) of the host innate immunesystem [34]. More work has to be done to definethe exact function of TAs for cell viability andvirulence of Gram-positive pathogens.

Other essential targetsA number of components have been suggestedto be attractive targets in S. aureus based onin vitro essentiality data (Table 1), such as thetwo-component systems YycFG and YhcSR,type I signal peptidase (SPase) SpsB, the ABCtransporter, a glycoprotease Gcp, thioredoxinTrxA, thioredoxin reductase TrxB, DNApolymerase III DnaE, DNA ligase LigA andribonuclease P [35–42]. For most of these targetsessentiality has been investigated only in vitroand in vivo validation is still lacking. Neverthe-less, for some of these targets (e.g., SpsB),inhibitors have been discovered that arecurrently under investigation [43].

Table 1. Promising targets for novel antibacterials against Staphylococcus aureus.

Pathway/target Function Ref.

Fatty acid synthesis

FabI Enoyl-acyl carrier protein reductase [44]

FabF/H β-ketoacyl-(acyl carrier protein) synthase I/II [46]

DNA replication

GyrA DNA-gyrase [57,58]

ParE Topoisomerase IV

Protein modification

Pdf Peptide deformylase [23,28]

Protein elongation

tRNA synthetases Protein biosynthesis [54]

Peptidoglycan synthesis

PBP2 Peptidoglycan glycosyltransferases [49]

MurB UDP-N-acetylglucosamine-enolpyruvyl reductase [50]

FmhB Pentaglycine interpeptide biosynthesis [16]

FemAB Pentaglycine interpeptide biosynthesis [84]

Ddl D-alanine:D-alanine ligase [85]

Regulation

YycG/YycF (VicRK) Autolysis [42]

YhcSR Unknown [40]

Gcp Glycoprotease, autolysis [41,86]

Protein secretion

SpsB Signal peptidase [43]

Cell division

FtsZ GTPase [21]

Teichoic acid biosynthesis

TarB, TarD, TarF, TarIJ, TarH Teichoic acid polymer formation [32]

Stress response

TrxA Thioredoxin [36]

TrxB Thioredoxin reductase [36]

ClpP Proteolytic component of Clp complex [87]

LigA DNA-ligase [39,88]

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Old targets: new drugsRenewed interest is directed towards the discoveryof new compounds against old targets or previouslytargeted pathways. For example, new structuraldata on target proteins will help to define novelinhibitors based on computer-aided modeling.

Type II fatty acid-synthesis pathwayType II fatty acid synthesis is essential for bacte-rial cell viability. In bacteria, FabH catalyzes thefirst condensation reaction using acetyl-CoA andmalonyl-acetyl carrier protein (ACP) to produceacetoacetyl-ACP. This product is reduced byFabG, dehydrated by FabA/Z and then reducedby FabI to generate butyryl-ACP. In the next stepof this cycle, FabF synthesizes β-ketoacyl-ACP.Several inhibitors of this pathway are knownincluding the antiseptic triclosan targeting FabI,isoniazid, which also targets this enzyme inMycobacterium tuberculosis and cerulenin, whichis a selective FabF/B inhibitor. Novel com-pounds have been isolated including platensi-mycin acting on FabF/B or platencin (acting onFabF/B and FabH), or naphthyridin acrylamidederivatives targeting FabI [44–46]. The main prob-lem with FabI inhibitors is the lack of broad-spectrum activity as, for example, S. pneumoniaepossesses FabK as enoyl-ACP reductase [47]. Nev-ertheless, potent FabI inhibitors may be usedagainst S. aureus and other staphylococci. Therecently described FabF and FabF/FabH inhibi-tors platensimycin and platensin, isolated fromStreptomyces platensis, are promising candidatesfor novel antibacterial drugs as these compoundsdemonstrated broad-spectrum Gram-positiveactivity and, importantly, have demonstratedin vivo efficacy [45,46].

Cell wall synthesisNovel, effective compounds against targets of thecell wall machinery are excellent candidates fornew antibacterials. Several potential targets of cellwall-synthesis pathways have been postulated,including penicillin-binding proteins (PBPs),UDP-N-acetylglucosamine-enolpyruvyl reductase(MurB), UDP-N-acetylglucosamine-enolpyruvyltransferase (MurA), UDP-N-acetyl muramylL-alanine ligase (MurC), N-acetylglucosamine-1-Pacetyl transferase (GlmU) and D-alanine:D-alanineligase (Ddl) or, specifically for S. aureus, enzymeswhich are involved in formation of the penta-glycine cross-bridge such as FemAB or FemX[16,48–51]. Inhibitor design should be facilitated bythe recent resolution of 3D structures of, forexample, MurB and PBP2 [49].

There are several compounds in preclinicaland clinical development targeting lipid II,which include glycopeptides (dalbavancin, orita-vancin and telavancin), lantibiotics (e.g. nisinand mersacidin), mannopeptimycins and ramo-planin [52]. Lipid II is a membrane-anchored cell-wall precursor that is essential for bacterial cell-wall biosynthesis. The effectiveness of inhibitinglipid II-derived pathways as an antibacterialstrategy is highlighted by the fact that this mole-cule is the target for at least four different classesof antibiotics, including the clinically importantglycopeptide antibiotic vancomycin. The spec-trum of activity of novel lipid II inhibitorsencompasses mostly Gram-positive pathogensincluding problematic resistant types such asMRSA, VRSA, VRE and pneumococci. Struc-tural modifications of peptide antibiotics alsoincreased activity against some Gram-negativebacteria such as Helicobacter pylori, Campylo-bacter jejuni, Neisseria spp., Salmonella spp.,Pseudomonas aeruginosa and Shigella spp.

tRNA synthetasestRNA synthetases meet many of the criteria forantibacterial targets. These enzymes are essen-tial for growth of all pathogenic species and cat-alyze a limiting step in a vital bacterial function.Several aminoacyl-tRNA synthetases have beenvalidated as potential drug targets, and leadcompounds with potent inhibitory activitieshave been generated to block, for example, Ile-,Phe-, Met-, Tyr-, Pro- and Trp-tRNA syn-thetase [35,53,54]. Although theoretically all 20amino acid-tRNA-synthetases represent attrac-tive targets and a large number of promisinglead compounds have been identified, only afew agents have moved forward into clinicaldevelopment [35,55]. Mupirocin is the onlyexample of an inhibitor of amino acid-tRNA-synthetases in clinical use. The drug is apotent inhibitor of isoleucyl-tRNA synthetaseand has a specific importance for control ofMRSA [56]. This topical antibiotic, mostly usedas a cream to eradicate MRSA from the nose, ishighly active against S. aureus and Streptococcuspyogenes, it also active against H. influenzae,Neisseria spp. and Bordetella pertussis, but isrelatively inactive against anaerobic strep-tococci, enterococci and most Gram-negativebacteria [54]. Phenyl-thiazolylurea-sulfona-mides were identified as a class of inhibitors ofphenylalanyl (Phe)-tRNA synthetase by high-throughput screening and chemical variation ofthe lead structures. The compounds inhibit

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Phe-RS of E. coli, H. influenzae, S. pneumoniaeand S. aureus, with 50% inhibitory concentra-tions in the nanomolar range [53]. Further stud-ies are needed to develop active compounds thatalso act systematically against S. aureus andother pathogens.

Bacterial DNA gyrase & topoisomerase Bacterial DNA gyrase and topoisomerase IV(topo IV) are highly conserved type II topo-isomerases that play essential roles in promotingDNA replication and transcription. These mole-cules are attractive targets for antibacterial drugdiscovery. The fluoroquinolones bind to theGyrA subunit of gyrase and/or the ParC subunitof topo IV blocking essential functions. Theessentiality and evolutionary conservation ofgyrase and topo IV in bacteria suggests broad-spectrum antibacterial activity to the fluoro-quinolones. A recent advance was obtained witha structure-guided drug design approach to opti-mize a novel series of aminobenzimidazoles thatinhibit the essential ATPase activities of bacterialDNA gyrase and topoisomerase IV [57,58]. Thesestudies led to the development of compoundswith potent activities against a variety of bacterialpathogens, for example, S. aureus, H. influenzae,S. pneumoniae, E. coli and E. faecalis.

Immunotherapy: an alternative strategy to antibiotics for the treatment of infections by MRSAThe need of new therapeutic options to treatinfections due to MRSA strains has released anumber of initiatives for the development ofactive or passive immunotherapy approaches [59].There are some clearly defined at-risk populationsfor staphylococcal infections that would benefitfrom immunotherapeutics, including dialysispatients, patients with ventriculoperitonealshunts, patients at risk of infective endocarditis

and residents of nursing homes. Furthermore,increasing incidence of MRSA infections in chil-dren with no predisposing risk factors, as well asin young adults led to the discussion of whetherthere is a specific need for a general immunizationcampaign. In addition, passive immunoprophy-laxis using either polyclonal or monoclonal anti-bodies could aid immunocompromised patients,premature infants, mechanical ventilated persons,and patients who carry foreign medical devicessuch as catheters and implants to generate apotent immune response [60]. Consequently, allintensive care unit patients may benefit frompassive immunization.

The identification of effective target structuresfor antibody-based therapy is a prerequisite forthe development of immunotherapeutics. Prom-ising antigens can be selected from so-calledmicrobial surface components recognizing adhe-sive matrix molecules (MSCRAMMs). Theseproteins are located on the surface of S. aureusand play a prominent role in the process of adhe-sion to specific sites on human tissues orimplanted medical devices [61]. Antibodies recog-nizing MSCRAMMs promoted an enhancedimmune clearance and inhibited the adherenceof S. aureus bacteria to tissues in several animalmodels [62]. Several clinical trials evaluatingin vivo efficiency of immunotherapy in humanrisk populations have been published, however,most of these studies failed (Table 2). For example,a hyperimmune IgG preparation to clumpingfactor A (ClfA) called Veronate® (Inhibitex, GA,USA) has been developed by collecting sera frompatients with high titers to ClfA [63]. PositivePhase II data had indicated the potential for pro-tecting low birth weight infants from staphylo-coccal infections, but in a following Phase IIIstudy, Veronate failed to show efficiency [64,65].In another Phase III clinical trial that showed nosignificant protection, hemodialysis patients

Table 2. Antibody-based therapies against Staphylococcus aureus in clinical studies.

Drug Preparation Company Target Phase

Aurograb® Antibody fragment NeuTec Pharma (Novartis subsidiary)

ABC transporter III

Altastaph® Polyclonal antibody Nabi Capsular polysaccharide II

StaphVax® Polyclonal antibody Nabi Capsular polysaccharide III

Veronate® Polyclonal antibody Inhibitex Clumping factor A III

Aurexis® (tefibazumab) Monoclonal antibody Inhibitex Clumping factor A II

PagibaximabBSYX-A110

Humanized monoclonal antibody Biosynexus and GlaxoSmithKline

Lipoteichoic acid II

V710 Vaccine Intercell and Merck Conserved protein I

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were vaccinated with a polysaccharide conjugatevaccine containing capsule polysaccharides type 5and type 8 coupled to pseudomonas exotoxin Atoxoid (StaphVAX®) [66]. Two recent studies sug-gest that in addition to type 5 and 8 capsulepolysaccharides, the newly described type 336, apolyribitol phosphate N-acetylglucosamine,should be included into a capsular polysaccha-ride-protein conjugate vaccine as it would extendthe coverage substantially. Such a vaccine wouldtarget almost all MSSA and MRSA strains cur-rently circulating in Germany and France [67,68].Furthermore, tefibazumab (Aurexis®) a human-ized monoclonal antibody targeting clumpingfactor A, is in development as an adjunctive ther-apy for serious S. aureus infections. After aPhase I trial in 2006, additional trials provingefficacy are pending [69–71]. Another candidatefor immunotherapy that could possibly provideprotection against S. aureus is an immunodomi-nant ABC transporter [72]. NeuTec Pharma(Manchester, UK) has developed geneticallyrecombinant antibodies (Aurograb®) against thisstructure, which has intrinsic activity inS. aureus, synergistic activity with vancomycinand a broad spectrum of activity to differentstrains of S. aureus, including the currently epi-demic strains of MRSA [73]. The Phase II studywith Aurograb was completed in 2003 and PhaseIII trials have commenced. Moreover, a chimeric

antibody targeting lipoteichoic acid named Pag-ibaximab has entered Phase II studies to preventstaphylococci sepsis in the very low birth weightneonate [73]. Furthermore, recent clinical Phase Istudies were completed with a vaccine developedto target a conserved protein antigen [201].

Several other experimental vaccine candidatesthat demonstrated some efficiency in preclinicalstudies are in development. These vaccines targetmostly surface components such as the polysac-charide intercellular adhesin, alternatively desig-nated poly N-acetyl-β-1,6-glucosamine, ironsurface determinant A and B, fibrinogen-bind-ing proteins ClfA and ClfB, and serine-aspartate(SD) repeat-containing proteins SdrD, SdrE andSdrG (Table 3) [74–79]. Promising results in animalmodels justify ongoing clinical validation. How-ever, the studies also revealed that due to thegreat variety of virulence factors produced byS. aureus, a single immunologic target might notbe sufficient for complete protection. It is likelythat a cocktail containing a combination of sev-eral Staphylococcal virulence factors, as demon-strated recently by using a combination of IsdA,IsdB, SdrD and SdrE antigens, might be the bestapproach for a successful vaccine [78]. Further-more, completely unknown at present is thepotential of the so-called secretable expandedrepertoire adhesive molecules (SERAMs), suchas the extracellular adhesive protein, the

Table 3. Targets of Staphylococcus aureus for implementing antibody-based therapy.

Function Ref.

Targets with efficacy in animal models

Clumping factor A (ClfA) Adhesion [75,77]

Capsule polysaccharides type 5 and 8 Attachment to surfaces and immune evasion [89,90]

Iron-responsive surface determinant A Adhesion [78,91]

Iron-responsive surface determinant B Adhesion [76,78]

Poly-N-acetyl glucosamine Biofilm production [79,92]

Collagen-binding adhesin Adhesion [74]

Fibronectin-binding protein Adhesion [74]

Fibronectin-binding proteins Adhesion [93–95]

Serine-aspartate repeat-containing proteins SdrD, SdrE

Adhesion [78]

Targets with theoretical efficacy

Exfoliative toxin A + B Esterase/protease activity [96]

Toxic shock syndrome toxin Superantigenic activity [97]

Bone sialoprotein-binding protein Adhesion [98]

α + γ-hemolysin Cell lysis [99]

Panton–Valentine leukocidin toxin Polymorphonuclear lysis [100]

Peptidoglycan Various [101]

Immunodominant antigen A and B Lytic transglycosylase and adhesion [102]

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extracellular matrix binding protein, the coagu-lase or the extracellular fibrinogen binding pro-tein, as targets for vaccine development. Theseproteins bind to host matrix proteins and arereceiving progressively more attention becausemany of them possess immunmodulatory activi-ties [80]. Despite recent progress, more experi-mental work is necessary to developimmunotherapeutics that can protect humans atrisk of serious S. aureus infections.

Conclusion & future perspectiveThe spread of antibiotic-resistant bacteria in hos-pitals and communities raises serious concerns.However, only a handful of new antibacterialagents have been approved for therapy since1998, and only two of these, linezolid and dap-tomycin, have novel mechanisms of action.Although there is a clear need for new anti-bacterials, research and development expendi-tures for antibacterials spent by the largestpharmaceutical companies have continued todecrease [81,82]. Since 1995, when the first com-plete bacterial genome was published, scientists’and physicians’ enthusiasm has mounted and anew era for antibacterial drug discovery has beenheralded. Meanwhile, more than 500 bacterial

genomes have been sequenced and the completegenome sequences of 12 S. aureus strains areavailable in public databases. However, only ahandful of targets have been selected by highthroughput screens (HTSs) using genome-com-parision approaches to identify novel lead com-pounds. Surprisingly, the success of HTScampaigns run by several pharmaceutical com-panies was very poor. For example, GlaxoSmith-Kline ran 70 HTS campaigns during 1995 to2001 and only five leads were delivered. Like-wise, other companies failed to identify suitablecandidates. Therefore, such programmes werestopped and the focus of antibacterial researchshifted to optimization of known antibacterialmolecules, screening of small natural productlibraries and rational design initiatives [35]. Theenthusiasm of the genome era has passed, lead-ing to the realization that only a very limitednumber of proteins have the potential to be anovel target for drug development and immuno-therapy [19,83]. Further basic research is necessaryto understand the physiology and virulence ofS. aureus and other pathogens that will form thebasis for discovery of novel targets for antimicro-bial drugs and protective immunotherapeutics inthe future.

Executive summary

Methicillin-resistant Staphylococcus aureus infections

• Staphylococcus aureus is a leading cause of nosocomial and community acquired infections.• Resistance development has become a global health problem.• New resistance types such as vancomycin-resistant S. aureus have recently developed.• New pathotypes such as community-acquired methicillin-resistant S. aureus expressing Panton–Valentine leucotoxin have evolved.

Novel targets against S. aureus

• Identification of new targets addressing novel pathways is urgently needed.• In vivo validation of targets is essential to assess the role of targets for bacterial viability in vivo.• Novel targets with broad-spectrum activity are difficult to define.• New structural information may result in the design of new inhibitory molecules.• Search for novel inhibitors of old targets or pathways should be considered.

Immunotherapy

• Passive or active immunization strategies could help to protect persons at high risk.• Human antibodies in clinical trials mostly failed or provided only dissatisfying results.• Several immunotherapeutics targeting surface components with efficacy in animal models are under development.

Conclusion

• Novel targets against S. aureus have to be discovered. • The number of new targets is limited, so few antibiotics have been approved in the past 10 years. • Protection observed in experimental infections suggests that a staphylococcal immunotherapy is achievable.• More preclinical, epidemiological and clinical studies are required before an optimal approach can be developed.

Future perspective

• Exploitation of functions of proteins with unknown function will help to identify novel targets.• Structure-guided drug design will lead to the development of novel antibacterials.• Antigen combinations for active immunization and antibody cocktails for passive immunotherapy should protect persons at risk.

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AcknowledgementsWe thank U Wallner and T Schäfer for technical assistanceand support in animal experiments.

Financial & competing interests disclosure The work of Knut Ohlsen was supported by a grant of theBavarian Science Foundation (FORINGEN), the Deutsche

Forschungsgemeinschaft (SFB630, TR34), and the EUframework 6 project ‘StaphDynamics’. The authors have noother relevant affiliations or financial involvement with anyorganization or entity with a financial interest in or finan-cial conflict with the subject matter or materials discussed inthe manuscript apart from those disclosed. No writingassistance was utilized in the production of this manuscript.

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• Excellent review on current status of staphylococcal vaccines and immunotherapeutical approaches.

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Website201. Staphylococcus aureus vaccine development

on track– safe and immunogenic in Phase I clinical Trials. Intercell AG. www.intercell.com/images/content/binaries/952c2eba-0622–42e3-a7f0–5b831e83bb3b.pdf

Affiliations• Knut Ohlsen

University of Würzburg, Institute for Molecular Infection Biology, Röntgenring 11,97070 Würzburg, GermanyTel.: +49 931 312 155;Fax: +49 931 312 578;[email protected]

• Udo LorenzUniversity of Würzburg, Centre for Operative Medicine, Department of Surgery I,Oberdürrbacher Str. 6, 97080 Würzburg, GermanyTel.: +49 931 2013 8513;Fax: +49 931 2013 8609;[email protected]

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Novel targets for antibiotics in Staphylococcus aureus