Journal ofAPPLIEDBIOMEDICINE

J Appl Biomed 8:131–140, 2010DOI 10.2478/v10136-009-0016-6

ISSN 1214-0287

REVIEW

Streptococcus pneumoniae: from molecular biology tohost-pathogen interactions*

Pedro García, Miriam Moscoso, Violeta Rodríguez-Cerrato, José Yuste, Ernesto García

Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas(CSIC), and CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain

Received 23rd February 2010.Revised 8th April 2010.Published online 6th May 2010.

SummaryStreptococcus pneumoniae is the main cause of community acquired pneumonia and also produces meningitis,bacteremia, and otitis media, among others. Worldwide, these infections are the cause of substantial morbidityand mortality. Many different virulence factors have been described and most of them are surface-locatedmacromolecules, namely, the capsular polysaccharide and various pneumococcal proteins. Cell wallhydrolases (CWHs) specifically cleave covalent bonds of the peptidoglycan and associated polymers: mostCWHs are choline-binding proteins (CBPs) and are among the most well-known surface proteins.Pneumococcal CBPs have been investigated due to their role in pathogenesis and as candidate antigens forimproved vaccines. Among the complex host-parasite interactions characteristic of pneumococcal disease,nasopharyngeal colonization is the first step. CBPs appear to play a central role in the development of thecarrier state, possibly by affecting biofilm formation and development. Although the role of biofilms in thepathogenesis of some chronic human infections is currently widely accepted, the molecular bases underlyingthe formation of pneumococcal biofilms are only recently being studied. Among therapeutic strategies tocombat multidrug-resistant pneumococcal infections, the use of purified phage- or bacteria-encoded CWHsboth in vitro and in animal models is under investigation.

Key words: Pneumococcus; cell wall hydrolases; choline; phage therapy; biofilm; enzybiotics

*This review is dedicated to Prof. Rubens López García, our friend and former mentor.

INTRODUCTION

Streptococcus pneumoniae, a Gram-positivefacultative anaerobe, is currently one of the mostimportant human pathogens and a worldwide leading

cause of bacterial pneumonia, meningitis, and sepsis.The bacterium is carried asymptomatically in thenasopharynx of healthy children and, less frequently,of healthy adults, with colonization beginning shortlyafter birth. In the industrialized countries, seriouspneumococcal disease occurs mainly in childrenbelow the age of five, the elderly, andimmunocompromised patients. In developingcountries, these diseases are common in childrenunder five, including newborn infants; but the rates ofthe disease in the elderly population are largelyunknown. Respiratory infections are responsible forthe death of 4 million persons each year, and S.pneumoniae is the predominant species in these

Ernesto García, Centro de InvestigacionesBiológicas, CSIC, Ramiro de Maeztu, 9,28040 Madrid, Spain

e.garcia@cib.csic.es 34 91 837 3112 34 91 536 0432

García et al.: Streptococcus pneumoniae: from molecular biology to host-pathogen interactions

132

infections. Although in developed countries asubstantial reduction in pneumonia mortality duringthe 20th century has been observed, in the low-incomecountries of Asia and Africa, pneumonia is still themain cause of child death. In developing countries,over one-quarter of children have an episode ofclinical pneumonia each year throughout the first5 years of their lives (Scott et al. 2008). Recentestimates in children aged 1–59 months reportedabout 14.5 million episodes of serious pneumococcaldisease and deaths ranging from 700,000 to 1 millionevery year worldwide (O’Brien et al. 2009). Of the14.5 million pneumococcal cases, 95% wereattributable to pneumonia.

Even though S. pneumoniae mutants withincreased resistance to penicillin had been isolated invitro and in vivo from experimental animals in the1940s, clinical pneumococcal isolates were uniformlysusceptible to low concentrations of this antibioticuntil the mid-sixties. Two decades were to passbefore reports of resistant isolates from humans weredescribed in the United States and Australia.Gradually, the spread of penicillin andmultiple-antibiotic resistant pneumococcal strainsbecame worldwide. In the United States, about 44%of all pneumococcal strains are now resistant toβ-lactam antibiotics; in Spain, about 41% areresistant. This situation has prompted thedevelopment of new anti-infectives for the treatmentof pneumococcal infections, particularly thoseproduced by multi-drug resistant pneumococci(Maestro and Sanz 2007).

Growing resistance of S. pneumoniae toconventional antibiotics emphasizes the urgent needfor new antipneumococcal drugs and vaccines tocontrol pneumococcal disease. The pneumococcalcapsular polysaccharide (CPS) is immunogenic andinduces type-specific protective immunity (López2006). Although a 23-valent non-conjugated CPSvaccine for use in adults and children aged $5 yearsand a heptavalent protein-polysaccharide conjugatevaccine (designed for pediatric use) are currentlyavailable, they are far from satisfactory. Pronouncedherd immunity has resulted in a decrease in invasivepneumococcal diseases in vaccinees andnon-vaccinees and reduced antibiotic resistance rates.However, recent studies report that serotypeseradicated by the vaccines are being replaced bynon-vaccine pneumococcal serotypes. This so-called‘serotype replacement’ might soon threaten thesuccess of vaccine use (Dagan 2009).

Our group has been working during more than30 years on the molecular biology of S. pneumoniae,namely in surface proteins involved in virulence (e.g.,cell wall hydrolases; CWHs), CPS biosynthesis and

regulation, and its bacteriophages. As most of theresults obtained up to 2004 have been reviewed(López and García 2004, López et al. 2004a, López etal. 2004b, García et al. 2005, Hermoso et al. 2007), inthe present review we will update our current researchefforts aimed at further insight into thestructure-function relationships of pneumococcalCWHs and their role in virulence. Furthermore, thestate-of-the-art of biofilm development and regulationas well as the therapeutic value of bacterium- andphage-encoded CWHs will be discussed in theframework of host-S. pneumoniae interplay.

CHOLINE AND CELL WALL HYDROLASES

S. pneumoniae has a unique physiological trait amongprokaryotes: it exhibits an absolute nutritionalrequirement for choline. This aminoalcoholincorporates as phosphorylcholine (PC) in the cellwall teichoic acid (TA) and membrane lipoteichoicacid. Depending on the particular strain, each repeatunit of TA contains one or two PC residues. We havestudied some aspects of this auxotrophy andconfirmed the key role of TacF protein (a TA repeatunit transporter) in the choline-dependent phenotype.Besides this, we have also demonstrated that at leasttwo tacF mutations are required to confer animproved fitness to the choline-independentpneumococcal strains when growing in mediumlacking any aminoalcohol (González et al. 2008).

S. pneumoniae contains several CWHs, enzymesthat degrade specific linkages of the cell wall. PC actsas an anchor for a special class of surface-locatedproteins, referred to as choline-binding proteins(CBPs), through non-covalent interactions. Mostpneumococcal CWHs are CBPs and harbor twofunctional modules: one is responsible of the substratebinding and the other determines the site of action.S. pneumoniae produces 13 to 16 CBPs depending onthe particular strain. The choline-binding module(CBM) is formed by several repeats (choline-bindingrepeats; CBRs), with the consensus motifGWXK-X4-5-WYY-φ-X3-5-GXMX2-3, where X is anyresidue and φ is a hydrophobic residue(http://pfam.sanger.ac.uk/family?PF01473). Therelevant characteristics of the pneumococcal CBPsmentioned in this review are summarized in Table 1.The first crystal structure of a CBM showed that theCOOH– (C–)terminal module of the LytA majorpneumococcal autolysin, an N-acetylmuramoyl-L-alanine amidase (NAM-amidase), adopts a peculiarβ-solenoid structure (Fernández-Tornero et al. 2001).Each CBR comprises a β-hairpin followed

García et al.: Streptococcus pneumoniae: from molecular biology to host-pathogen interactions

133

Table 1. Characteristics of selected pneumococcal and phage cell wall hydrolases.

Protein Gene Signalpeptide

Number ofamino acidresiduesa

Molecularmass (kDa)b

Catalyticactivity

Numberof CBRsc

S. pneumoniaed

LytA spr1754 No 318 36.6 NAM-amidase 7 (C)

LytB spr0867 Yes 679 (702) 79.3 (81.9) endo-β-N-acetylglucosaminidase

18 (N)

LytC spr1431 Yes 468 (490) 55.2 (57.4) Lysozyme 11 (N)

Pce(CbpE)

spr0831 Yes 602 (627) 69.4 (72.1) Phosphorylcholineesterase

10 (C)

CbpF(CbpC)

spr0337 Yes 311 (338) 36.3 (39.3) NDe 5 (C)

CbpD spr2006 Yes 408 (448) 46.0 (50.4) Peptidase, putative 4 (C)

Spr1274 spr1274 No 129 14.6 NDe 4 (C)

Phage Cp-1

Cpl-1 cpl1 No 339 39.2 Lysozyme 6 (C)

Phage Dp-1

Pal pal No 296 34.4 NAM-amidase 7 (C)

a The number of residues of the unprocessed protein is indicated in parentheses.b The molecular mass of the unprocessed protein is indicated in parentheses.c The numbers correspond to consensus and non-consensus choline-binding repeats (CBRs). N and C indicate, respectively, whether the CBRs are located at the N– or C– terminal region of the protein.d The data correspond to the genome of the common laboratory strain R6. Alternative protein designations are shown in parentheses.e ND, not determined.

by a loop and a coiled region (Fig. 1a).Choline-binding sites, as described for other CBPs,are located at the interface of two consecutive CBRs,where three structurally conserved aromatic residuesform a cavity in which the choline quaternaryammonium moiety is stabilized primarily by cation-πinteractions.

The crystal structure elucidation of CBPs is acrucial step to gain insight in the structural andfunctional knowledge of these proteins. Fig. 1 showsthe crystal structures of host and phage-encodedCBPs that have been solved to date. Relevant aspectsof the four CBPs most recently crystallized are thefollowing:

a) Pce, a phosphorylcholinesterase, removes a limitednumber of PC residues from the cell wall TAs. Also,Pce is able to hydrolyze the platelet-activating factor(PAF; 1-alkyl-2-acetoyl-hexadecyl-glycero-3-phosphoryl choline), which suggests that this enzymehas other functions during infection, such asdegrading host PC-containing compounds. The cellsurface localization of Pce could facilitate thehydrolysis of PAF from the bloodstream and mightplay a role in the mechanism of pneumococcaladherence and invasiveness (see below). Pce has aglobular NH2– (N–) terminal module containing abinuclear Zn2+ catalytic center, and an elongated CBMjoined by a short linker of 12 amino acids (Fig. 1c).

García et al.: Streptococcus pneumoniae: from molecular biology to host-pathogen interactions

134

This structure has revealed that the removal of PCresidues is limited by the configuration of the activesite and, thus, only residues located at the end of theTA chains are accessible to the catalytic center(Hermoso et al. 2005).

b) CbpF is not a CWH but presents uniquefeatures compared with other CBPs, as it is assembledentirely by CBRs composed both of consensus andnon-consensus repeats distributed along its length,which markedly alter its shape, charge distributionand binding ability, and organizing the protein intotwo well-defined modules (Fig. 1e). Sequencedivergence observed in CbpF is generated bymutations of the consensus amino acid residues of therepeat, by amino acid additions in the β-hairpin turnor the loop, or by a combination of both. Biochemicalstudies have shown that CbpF selectively modulatesthe autolytic function of the LytC lysozyme and thedivergent CBRs that build its N–terminal modulehave a crucial role in this effect. It is worth notingthat analyses of known pneumococcal genomes showthe existence of several proteins that are likely todisplay the same architecture as CbpF, which mightconstitute a new subfamily within the CBPs (Molinaet al. 2009).

c) The structure of the autolysin LytC presents anunusual hook-shaped conformation with a catalyticmodule resembling a flattened ellipsoid folding intoan irregular (β/α)5β3 barrel, and eleven CBRs in thesubstrate recognition module (Fig. 1f). Structural andbiochemical data allow an explanation of thecoordinated role of LytC and CbpD (another CBPwith CWH activi ty) in fratr icide, acompetence-programmed mechanism of predation ofnoncompetent sister cells (Claverys and Håvarstein2007). LytC is only able to hydrolyze non-crosslinkedpeptidoglycan chains, a property probably connectedto a new type of choline-binding site with acharacteristic GYMA motif at the end of the thirdrepeat. Docking experiments have indicated that thepresence of these GYMA sites on the CBM couldenhance the affinity for the PC andN-acetylgalactosamine sugars of the TAs byproviding a strong multivalent recognition andattachment to the cell wall. The configuration aroundthe active site imposed limitations on hydrolysis ofpeptidoglycan, which means that only when specificcuts are introduced in the peptide stems ofpeptidoglycan in the target cells, do glycan chainsbecome fully predisposed to the LytC activity.Therefore, prior cleavage of the peptide stemsperformed by CbpD should facilitate hydrolysis of thenon-crosslinked peptidoglycan chains by LytC(Pérez-Dorado et al. 2010).

d) Spr1274 is a putative CBP of unknownfunction (Fig. 1d). The crystal structure of its CBM

(residues 44–129) has been solved with threemolecules in the asymmetric unit, forming apseudo-trimer. Chains A and C are antiparallel andinteract with the two termini of chain B. No commonthreefold axis can be found in the pseudo-trimer, buttwo fold axes are present between chains A and B andbetween chains A and C. The overall shape is like theletter V, with an angle of ~60° between the twosuperhelices (Zhang et al. 2009).

Taking advantage of the specific binding of theaminoalcohol choline and its analogs to the CBPs, wehave tested a highly promising new approach for thedevelopment of drugs to treat pneumococcalinfections. It is is based on dendrimers (tree-likebranched molecules) used as a scaffold for theattachment of choline groups to the tip of thebranches. Thus, the choline ends can simultaneouslyoccupy multiple choline-binding sites of the CBPs.Exogenously choline-charged dendrimerscompetitively inhibit the binding of CBPs to the cellwall, blocking cell separation and the characteristicautolysis at the end of the stationary phase of growth,inducing instead the formation of long chains or evenpreventing growth. These effects were observed atlow micromolar concentrations of thehigher-generation dendrimers, which represents a103–104-fold increase of apparent affinity comparedto monovalent choline (Hernández-Rocamora et al.2009). A further development of this dendrimerapproach is currently under study using severalcholine analogs that display a higher binding affinityto CBPs than choline. In fact, inhibition ofpneumococcal CBPs and cell growth by esters ofbicyclic amines has already been demonstrated(Maestro et al. 2007).

The CBM of LytA is essential to guarantee fullenzymatic activity through dimerization. In this sense,analysis of a collection of 21 mutated LytANAM-amidases indicated that Ile-315, located in thelast CBR, is a key amino acid residue in bothenzymatic activity and folding (Romero et al. 2007).Moreover, it is worth to mention that selectiveinteraction of the CBM of LytA with choline or itsanalogs (tertiary or quaternary amines) has allowed itsuse as a tag to construct a variety of fusion proteinsthat may be purified using DEAE-cellulose, whichacts as an affinity matrix for CBM-containingproteins (Sánchez-Puelles et al. 1992, Moldes et al.2004). In this sense, a commercial kit based in thissystem is already available (C-LYTAG;http://www.biomedal.com). In addition toamine-containing solid supports, aqueous two-phasesolutions containing polyethylene glycol have alsobeen used for purification of proteins tagged withCBMs (Maestro et al. 2008).

García et al.: Streptococcus pneumoniae: from molecular biology to host-pathogen interactions

135

Fig. 1. Three-dimensional structures of several choline-binding proteins of S. pneumoniae. The stereo diagrams werevisualized with FISTGLANCE in JMOL (http://molvis.sdsc.edu/fgij/index.htm) or PyMOL (http://www.pymol.org/). For C-lytAand Spr1274 only one monomer is shown for clarity. Rockets (or helices in panel f) and planks represent, respectively, α-helicesand β-strands. N and C indicate the NH2– and the COOH– termini of the protein, respectively. PC molecules are depicted in CPKmode. The PDB identification numbers are indicated in parentheses.

PNEUMOCOCCAL BIOFILMS

A biofilm is a sessile microbial community wherestructured populations of microorganisms are adheredto a surface or interface and embedded in anextracellular matrix of polymeric substances. Theimportance of biofilms in medical microbiology liesin that more than 60% of human infections are relatedto growth in biofilms and to the inherent tolerance ofthese communities to antimicrobials and host immunedefense system. Biofilms have been associated withseveral chronic infections (up to 80% of these)including chronic otitis media. Formation of

pneumococcal biofilms on the mucosal epithelial cellsfrom children with recurrent or chronic middle earinfections has been recently observed (Hoa et al.2009). However, until five years ago no informationon pneumococcal biofilms at the structural or geneticlevel was reported. Our research group has developedthe set-up of a suitable biofilm model forS. pneumoniae in microtiter plates or glass-bottomdishes to analyze the influence of severalenvironmental factors and to study its peculiarstructure (Moscoso et al. 2006, 2009).

Environmental changes analogous to those foundin the nasopharynx, such as nutrient content of the

García et al.: Streptococcus pneumoniae: from molecular biology to host-pathogen interactions

136

medium, pH, and osmolarity, influence biofilmformation in vitro. Nonencapsulated pneumococcalstrains generate a three-dimensional structure wherethe adherent cells formed a mat of cells about 25 to30 μm deep, as revealed by confocal laser scanningmicroscopy (Moscoso et al. 2006). Also, usinglow-temperature scanning electron microscopy, it wasfound that pneumococcal cells growing in biofilmsare interconnected by small, thin filaments andadopted regular shapes to form honeycomb-likestructures (Fig. 2).

Fig. 2. Low-temperature scanning electron micrographof a pneumococcal biofilm formed on the surface of aglass coverslip (g). Arrows indicate filamentous materiallinking pneumococci to each other and to the intercellularmatrix.

Extracellular DNA as well as extracytoplasmicand/or surface-exposed proteins appear to beimportant components of the biofilm matrix and arerequired for pneumococcal biofilm formation andmaintenance (Moscoso et al. 2006). Extracellularpolysaccharides often determine the biofilmarchitecture and provide the structural integrity for thethree-dimensional biofilm matrix. CPS, which isabsolutely required for pneumococcal virulence,

represents a partial physical barrier in the first stepsof biofilm formation since only reduced biofilmformation was found when encapsulated, clinicalisolates or isogenic transformants were tested(Moscoso et al. 2006). However, a minimum amountof CPS is absolutely required for efficientnasopharyngeal colonization in mice, although itplays an essential protective role against phagocytosisduring invasive infection. It has been shown thatbiofilm development may select nonencapsulatedphenotypic variants (Domenech et al. 2009). Severaldifferent mutations were found among the type 3capsular mutants that appeared in biofilms formed onpolystyrene plates. Most strains contained singlenucleotide polymorphisms in cap3A, the first gene ofthe type 3 capsular operon (Moscoso and García2009) and encoding a UDP-glucose dehydrogenase;one had a mutated –10 promoter hexamer(CATAAT), and three had large deletions affectingcap3A and, in one case, also cap3B. We haveproposed that non-encapsulated mutants ofpneumococcal type 3 strains are essentially involvedin the initial stages (the attachment stage) of biofilmformation during colonization/pathogenesis(Domenech et al. 2009).

The fact that pneumococcal biofilm isCPS-independent indicates that one or moreextracellular polysaccharides other than CPS, areinvolved in structuring of the biofilm matrix. Stainingof pneumococcal biofilms with various lectins hasprovided evidence of the presence of an extracellularpolysaccharide (unpublished data).

Several genes have been reported to be importantfor biofilm formation of pneumococci. Geneexpression profiles of pneumococci recovered fromthe lungs and brains of infected mice have beenreported to be similar to those of pneumococci grownin biofilms in vitro. Proteomic analysis confirmed thatthe biofilm development process exhibited byS. pneumoniae is correlated not only with differentialproduction of proteins but also with a dramaticincrease in the number of detectable proteins involvedin adhesion, resistance and virulence (Allegrucci et al.2006). Mutations in genes encoding CBPs have beenshown to decrease the capacity of pneumococci toform biofilms. Thus, CBPs such as the CWHs (e.g.,the NAM-amidase LytA, the LytC lysozyme, or theLytB glucosaminidase), the pneumococcal surfaceprotein A and the CbpA adhesin, were shown tocontribute to S. pneumoniae biofilm formation(Moscoso et al. 2006). More recently, a large numberof transposon insertion strains were generated as anapproach to the identification of genes involved inpneumococcal biofilm formation (Muñoz-Elías et al.2008). Such collection of mutants included strains

García et al.: Streptococcus pneumoniae: from molecular biology to host-pathogen interactions

137

with genes encoding CBPs, adhesins, extracellularproteases, efflux pumps, transporters, andtranscriptional regulators.

HOST-PATHOGEN INTERACTIONS

Pneumococcal disease is preceded by the colonizationof the nasopharynx, which is particularly common inchildren (see above) and involves binding of thebacterium to cell-surface carbohydrates. This processis mediated by cell-wall-associated surface proteins(Bogaert et al. 2004). Using CWH-deficient strainsand a human nasopharyngeal cell line we have foundthat LytB and LytC play a key role in the colonizationof the upper respiratory tract and the establishment ofnasopharyngeal carriage. LytB or LytC deficientstrains displayed a reduced ability to colonize thenasopharynx of infected mice when compared to thewild-type strain. Our studies have demonstrated thatthese CWHs are important surface-exposed proteinsinvolved in the adhesion and colonization of themucosal surfaces of the nasopharynx.

The transition from the carrier state to invasioninvolves genotypic and phenotypic changes, many ofwhich lead to enhanced bacterial adherence to hostcells. Activation of endothelial or epithelial cellsresults in up-regulation of the PAF receptor (PAFr) attheir surface, which in turn, promotes the recognitionof PAFr by PC, thus facilitating invasion byS. pneumoniae throughout this receptor (Cundell et al.1995). Choline is a key component of the bacterialenvelope of S. pneumoniae (see above) and it isinvolved in the adhesion to nasopharyngeal epithelialcells and the establishment of pneumococcal disease(Kharat and Tomasz 2006). The hydrolysis of PC andPAF by the Pce enzyme suggests that S. pneumoniaehas developed complex virulence mechanisms thatare involved in the processing of PC and host celladhesion as previously proposed (Hermoso et al.2005). To establish the infection, a microorganismmust first overcome the host’s innate immunity and,in the case of pneumococcal infection, a key bacterialelement recognized by the innate immune system isthe cell wall. Cell wall fragments released in vivoduring bacterial autolysis mediated by pneumococcalCWHs, contribute to inflammation and cell damage(López and García 2004). Some of these componentsinteract with membrane-bound Toll like receptors(TLRs), particularly TLR2, and induce aproinflammatory response. Furthermore, it isproposed that peptidoglycan degradation productsinteract with Nod proteins that are intracellularsurveillance proteins that recognize subcomponents of

peptidoglycan (van der Poll and Opal 2008).Therefore, CWHs might be involved in triggeringsome of these physiological events.

Descriptions of virulence characteristics ofS. pneumoniae generally focus on its ability to escapeunharmed from the immune system and breach hostbarriers leading to a widespread invasion. Acutephase proteins such as the C-reactive protein (CRP)or the serum amyloid P component (SAP) recognizePC on the bacterial surface and activatecomplement-mediated immunity, which is one of thefirst defence lines against invading pathogens such asS. pneumoniae, controlling bacterial replication in thelungs and in the systemic circulation (Yuste et al.2007). Limiting the content of PC by Pce mightimpair the activation of the classical pathwaymediated by CRP or SAP, and therefore, Pce couldplay an important role in pneumococcal pathogenesis.

To avoid complement immunity and phagocytosis,S. pneumoniae has developed a wide arsenal ofbacterial virulence factors. Among them, PspA andpneumolysin, which is released to the medium aftercell wall lysis mediated by LytA, contributesynergistically to the innate immune diversion bytargeting complement immunity at different levels.All these factors increase the ability of S. pneumoniaeto cause invasive disease (Yuste et al. 2005).

ENZYBIOTICS AND PHAGE THERAPY

The emergence and steady spread ofantibiotic-resistant S. pneumoniae infections havedetermined the investigation of alternative treatmentstrategies. A novel therapeutic approach is based onthe administration of purified recombinant CWHsencoded either by bacteriophages (López et al. 2004a,Hermoso et al. 2007, Romero et al. 2009 a, b) or bythe bacterium itself (Rodríguez-Cerrato et al. 2007b)to specifically kill bacterial species in whichenzyme-mediated lysis naturally occurs.

Bacteriophages have been used for years in theprevention and/or treatment of bacterial infections insome parts of the world (for a recent review, seeO’Flaherty et al. 2009). Several in vivo and in vitrostudies have showed the efficacy of phage therapy asan alternative (or complement) to conventionalantibiotic therapy. Most bacteriophages encode a lyticsystem containing a holin that causes membranedamage, and at least one CWH (endolysin) thatdestroy the bacterial cell wall and thus allow thephage to disseminate its progeny.

Recently, the use of purified phage-encodedendolysins as enzybiotics to control pathogenic

García et al.: Streptococcus pneumoniae: from molecular biology to host-pathogen interactions

138

bacteria has been tested. Two purified pneumophagelytic enzymes, the lysozyme Cpl-1 and theNAM-amidase Pal, have been used with promisingefficacy in a murine sepsis model of pneumococcalinfection. This study demonstrated that Cpl-1, aloneor combined with Pal, afforded protection frompneumococcal bacteremia and death (López et al.2004a). Therapy with Cpl-1 has also been tested inother experimental models of S. pneumoniaeinfections, including endocarditis, meningitis andpneumonia, with promising results (O’Flaherty et al.2009, Witzenrath et al. 2009). Although not tested yetin vivo, a remarkable phage endolysin is the CHAPdomain-containing NAM-amidase Skl that is encodedby a prophage from Streptococcus mitis SK137 strain(Llull et al. 2006).

In an innovative approach in the field ofenzybiotic therapy, we have recently demonstratedthe antipneumococcal efficacy of recombinant LytAand compared such effect with that of phage lysinsboth in vitro and in a murine sepsis model(Rodríguez-Cerrato et al. 2007a, b). The rationale forthe exogenous administration of purified LytA asantibacterial agent was based on the role of LytA inpneumococcal physiology, i.e., LytA causes bacterialcell lysis during the late stationary phase of growth byspecifically breaking the amidase bonds between theglycan strands and the peptide moieties of thepeptidoglycan.

Two serotype 3, penicillin-susceptible strains andtwo penicillin- and cefotaxime-resistant (serotypes19F and 19A, respectively) S. pneumoniae clinicalisolates were exposed to several combinations ofCpl-1, Pal, and/or LytA, and antibiotics (cefotaximeand moxifloxacin) using chequerboard and time-killassays (Rodríguez-Cerrato et al. 2007a). By thechequerboard technique, although synergy betweenLytA and Pal was not found for the four strains, thecombination of LytA and cefotaxime was synergisticfor one of the two cefotaxime-resistant strainsstudied. The combined use of Cpl-1 and Pal wassynergistic for three of the four strains, as was Cpl-1with antibiotics for two of the three strains studied.

In our study of mice infected with amultidrug-resistant, meningeal pneumococcal strain,intraperitoneal or intravenous therapy with purifiedLytA and intraperitoneal therapy with high-doseCpl-1 achieved significant declines in bacterial counts(ca. 3–5 log10 colony-forming units ml–1) inperitoneum and blood, compared to those ofnon-treated animals (Rodríguez-Cerrato et al. 2007b).Further research using these and new lytic enzymes aspotential antipneumococcal agents is ongoing in ourlaboratory.

ACKNOWLEDGMENTS

We are indebted to J. A. Hermoso for his help in thepreparation of Fig. 1. This review was preparedwithin the outlined actions of the ProjectsSAF2006-00390 and SAF2009-10824. CIBER deEnfermedades Respiratorias (CIBERES) is aninitiative of ISCIII. VR-C is supported by theSubprograma Juan de la Cierva (JCI-2008-02690;Ministerio de Ciencia e Innovación).

REFERENCES

Allegrucci M, Hu FZ, Shen K, Hayes J, Ehrlich GD,Post JC, Sauer K: Phenotypic characterization ofStreptococcus pneumoniae biofilm development.J Bacteriol 188:2325–2335, 2006.

Bogaert D, de Groot R, Hermans PWM:Streptococcus pneumoniae colonisation: the keyto pneumococcal disease. Lancet Infect Dis4:144–154, 2004.

Claverys J-P, Håvarstein LS: Cannibalism andfratricide: mechanisms and raisons d’être. NatRev Microbiol 5:219–229, 2007.

Cundel l DR, Gerard NP, Gerard C,Idänpään-Heikkilä I, Tuomanen EI: Streptococcuspneumoniae anchor to activated human cells bythe receptor for platelet-activating factor. Nature377:435–438, 1995.

Dagan R: Serotype replacement in perspective.Vaccine 27:C22–C24, 2009.

Domenech M, García E, Moscoso M: Versatility ofthe capsular genes during biofilm formation byStreptococcus pneumoniae. Environ Microbiol11:2542–2555, 2009.

Fernández-Tornero C, López R, García E,Giménez-Gallego G, Romero A: A novel solenoidfold in the cell wall anchoring domain of thepneumococcal virulence factor LytA. Nat StructBiol 8:1020–1024, 2001.

García P, García JL, López R, García E:Pneumococcal phages. In Waldor MK, FriedmanDLI, Adhya S (ed.): Phages: Their Role inBacterial Pathogenesis and Biotechnology, ASMPress, Washington, D.C. 2005, pp. 335–361.

González A, Llull D, Morales M, García P, García E:Mutations in the tacF gene of clinical strains andlaboratory transformants of Streptococcuspneumoniae: impact on choline auxotrophy andgrowth rate. J Bacteriol 190:4129–4138, 2008.

Hermoso JA, Lagartera L, González A, Stelter M,García P, Martínez-Ripoll M, García JL,Menéndez M: Insights into pneumococcal

García et al.: Streptococcus pneumoniae: from molecular biology to host-pathogen interactions

139

pathogenesis from the crystal structure of themodular teichoic acid phosphorylcholine esterasePce. Nat Struct Mol Biol 12:533–538, 2005.

Hermoso JA, García JL, García P: Taking aim onbacterial pathogens: from phage therapy toenzybiotics. Curr Opin Microbiol 10:461–472,2007.

Hernández-Rocamora VM, Maestro B, de Waal B,Morales M, García P, Meijer EW, Merkx M, SanzJM: Multivalent choline dendrimers as potentinhibitors of pneumococcal cell-wall hydrolysis.Angew Chem Int Ed Engl 48:948–951, 2009.

Hoa M, Tomovic S, Nistico L, Hall-Stoodley L,Stoodley P, Sachdeva L, Berk R, Coticchia JM:Identification of adenoid biofilms with middle earpathogens in otitis-prone children utilizing SEMand FISH. Int J Pediatr Otorhinolaryngol73:1242–1248, 2009.

Kharat AS, Tomasz A: Drastic reduction in thevirulence of Streptococcus pneumoniaeexpressing type 2 capsular polysaccharide butlacking choline residues in the cell wall. MolMicrobiol 60:93–107, 2006.

Llull D, López R, García E: Skl, a novelcholine-binding N-acteylmuramoyl-L-alanineamidase of Streptococcus mitis SK137 containinga CHAP domain. FEBS Lett 580:1959–1964,2006.

López R: Pneumococcus: the sugar-coated bacteria.Int Microbiol 9:179–190, 2006.

López R, García E: Recent trends on the molecularbiology of pneumococcal capsules, lytic enzymes,and bacteriophage. FEMS Microbiol Rev28:553–580, 2004.

López R, García E, García P: Enzymes foranti-infective therapy: phage lysins. Drug DiscovToday Ther Strateg 1:469–474, 2004a.

López R, García E, García P, García JL: Cell wallhydrolases. In Tuomanen EI, Mitchell TJ,Morrison DA, Spratt BG (eds.): ThePneumococcus, ASM Press, Washington, D.C.2004b, pp. 75–88.

Maestro B, Sanz JM: Novel approaches to fightStreptococcus pneumoniae. Recent Pat AntiinfectDrug Discov 2:188–196, 2007.

Maestro B, González A, García P, Sanz JM:Inhibition of pneumococcal choline-bindingproteins and cell growth by esters of bicyclicamines. FEBS J 274:364–376, 2007.

Maestro B, Velasco I, Castillejo I, Arévalo-RodríguezM, Cebolla A, Sanz JM: Affinity partitioning ofproteins tagged with choline-binding modules inaqueous two-phase systems. J Chromatogr A1208:189–196, 2008.

Moldes C, García JL, García P: Construction of achimeric thermostable pyrophosphatase tofacilitate its purification and immobilization byusing the choline-binding tag. Appl EnvironMicrobiol 70:4642–4647, 2004.

Molina R, González A, Stelter M, Pérez-Dorado I,Kahn R, Morales M, Moscoso M, Campuzano S,Campillo NE, Mobashery S, García JL, García P,Hermoso JA: Crystal structure of CbpF, abifunctional choline-binding protein and autolysisregulator from Streptococcus pneumoniae. EMBORep 10:246–251, 2009.

Moscoso M, García E: Transcriptional regulation ofthe capsular polysaccharide biosynthesis locus ofStreptococcus pneumoniae: a bioinformaticanalysis. DNA Res 16:177–186, 2009.

Moscoso M, García E, López R: Biofilm formationby Streptococcus pneumoniae: role of choline,extracellular DNA, and capsular polysaccharide inmicrobial accretion. J Bacteriol 188:7785–7795,2006.

Moscoso M, García E, López R: Pneumococcalbiofilms. Int Microbiol 12:77–85, 2009.

Muñoz-Elías EJ, Marcano J, Camilli A: Isolation ofStreptococcus pneumoniae biofilm mutants andtheir characterization during nasopharyngealcolonization. Infect Immun 76:5049–5061, 2008.

O’Brien KL, Wolfson LJ, Watt JP, Henkle E,Deloria-Knoll M, McCall N, Lee E, MulhollandK, Levine OS, Cherian T, for the Hib andPneumococcal Global Burden of Disease StudyTeam: Burden of disease caused by Streptococcuspneumoniae in children younger than 5 years:global estimates. Lancet 374:893–902, 2009.

O’Flaherty S, Ross RP, Coffey A: Bacteriophage andtheir lysins for elimination of infectious bacteria.FEMS Microbiol Rev 33:801–819, 2009.

Pérez-Dorado I, González A, Morales M, Sanles R,Striker W, Vollmer W, Mobashery S, García JL,Martínez-Ripoll M, García P, Hermoso JA:Insights into pneumococcal fratricide from crystalstructure of the modular killing factor LytC. NatStruct Mol Biol 17:576–581, 2010.

Rodríguez-Cerrato V, García P, del Prado G, GarcíaE, Gracia M, Huelves L, Ponte C, López R,Soriano F: In vitro interactions of LytA, the majorpneumococcal autolysin, with two bacteriophagelytic enzymes (Cpl-1 and Pal), cefotaxime andmoxifloxacin against antibiotic-susceptible and –resistant Streptococcus pneumoniae strains. JAntimicrob Chemother 60:1159–1162, 2007a.

Rodríguez-Cerrato V, García P, Huelves L, García E,del Prado G, Gracia M, Ponte C, López R,Soriano F: Pneumococcal LytA autolysin, a potenttherapeutic agent in experimental peritonitis-

García et al.: Streptococcus pneumoniae: from molecular biology to host-pathogen interactions

140

sepsis caused by highly β-lactam-resistantStreptococcus pneumoniae. Antimicrob AgentsChemother 51:3371–3373, 2007b.

Romero P, López R, García E: Key role of aminoacid residues in the dimerization and catalyticactivation of the autolysin LytA, an importantvirulence factor in Streptococcus pneumoniae. JBiol Chem 282:17729–17737, 2007.

Romero P, García E, Mitchell TJ: Development of aprophage typing system and analysis of prophagecarriage in Streptococcus pneumoniae. ApplEnviron Microbiol 75:1643–1649, 2009a.

Romero P, Croucher NJ, Hiller L, Hu FZ, EhrlichGD, Bentley SD, García E, Mitchell TJ:Comparative genomic analysis of 10Streptococcus pneumoniae temperatebacteriophages. J Bacteriol 191:4854–4862,2009b.

Sánchez-Puelles JM, Sanz JM, García JL, García E:Immobilization and single-step purification offusion proteins using DEAE-cellulose. Eur JBiochem 203:153–159, 1992.

Scott JAG, Brooks WA, Peiris JSM, Holtzman D,Mulhollan EK: Pneumonia research to reducechildhood mortality in the developing world. JClin Invest 118:1291–1300, 2008.

van der Poll T, Opal SM: Host-pathogen interactionsin sepsis. Lancet Infect Dis 8:32–43, 2008.

Witzenrath M, Schmeck B, Doehn JM, Tschernig T,Zahlten J, Loeffler JM, Zemlin M, Müller H,Gutbier B, Schütte H, Hippenstiel S, Fischetti VA,Suttorp N, Rosseau S: Systemic use of theendolysin Cpl-1 rescues mice with fatalpneumococcal pneumonia. Crit Care Med37:642–649, 2009.

Yuste J, Botto M, Paton JC, Holden DW, Brown JS:Additive inhibition of complement deposition bypneumolysin and PspA facilitates Streptococcuspneumoniae sept icemia. J Immunol175:1813–1819, 2005.

Yuste J, Botto M, Bottoms SE, Brown JS: Serumamyloid P aids complement-mediated immunity toStreptococcus pneumoniae. PLoS Pathog 3:e120,2007.

Zhang Z, Li W, Frolet C, Bao R, di Guilmi A-M,Vernet T, Chen Y: Structure of thecholine-binding domain of Spr1274 inStreptococcus pneumoniae. Acta CrystallogrSection F Struct Biol Crystall Commun65:757–761, 2009.