The NanA Neuraminidase of Streptococcus pneumoniae Is ... · NanA is the neuraminidase that is most...

INFECTION AND IMMUNITY, Sept. 2009, p. 3722–3730 Vol. 77, No. 90019-9567/09/$08.00�0 doi:10.1128/IAI.00228-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The NanA Neuraminidase of Streptococcus pneumoniae Is Involved inBiofilm Formation�

Dane Parker,1 Grace Soong,1 Paul Planet,1 Jonathan Brower,1 Adam J. Ratner,2 and Alice Prince1*Department of Pediatrics and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York,1 and

Department of Pediatrics and Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York2

Received 26 February 2009/Returned for modification 5 May 2009/Accepted 23 June 2009

Streptococcus pneumoniae remains a major cause of bacteremia, pneumonia, and otitis media despite vaccinesand effective antibiotics. The neuraminidase of S. pneumoniae, which catalyzes the release of terminal sialic acidresidues from glycoconjugates, is involved in host colonization in animal models of infection and may providea novel target for preventing pneumococcal infection. We demonstrate that the S. pneumoniae neuraminidase(NanA) cleaves sialic acid and show that it is involved in biofilm formation, suggesting an additional role inpathogenesis, and that it shares this property with the neuraminidase of Pseudomonas aeruginosa even thoughwe show that the two enzymes are phylogenetically divergent. Using an in vitro model of biofilm formationincorporating human airway epithelial cells, we demonstrate that small-molecule inhibitors of NanA blockbiofilm formation and may provide a novel target for preventative therapy. This work highlights the role playedby the neuraminidase in pathogenesis and represents an important step in drug development for preventionof colonization of the respiratory tract by this important pathogen.

Neuraminidases are widespread in animals and microorgan-isms and catalyze the release of terminal sialic acid residuesfrom glycoconjugates (51). The best-characterized neuramini-dase is the influenza virus neuraminidase, which is required tofacilitate spread of this virus. Not only is the influenza virusneuraminidase a key antigen for the highly successful influenzavaccine, but it is also the target of the drugs zanamivir andoseltamivir, which have been useful for preventing and ame-liorating influenza virus infection (57). Streptococcus pneu-moniae produces at least three distinct neuraminidases (41);NanA is the neuraminidase that is most active and most highlyexpressed at the transcriptional level (5, 31), and it is conservedin all strains (21, 24, 41). Production of NanA can be detectedin vivo, and its expression is upregulated upon interaction withhost cells (27, 39, 46, 58). The pneumococcal neuraminidasemodifies host glycoconjugates, including immune defense pro-teins (22, 23), and exposes potential binding receptors (3, 26,28, 54, 55). Pneumococcal neuraminidase activity also providesa source of carbohydrates for bacterial metabolism, cleavingsugars from the mucosal surface (8, 23, 61), but whether thissignificantly contributes to bacterial growth in vivo has notbeen clearly established. Several studies have suggested thatnanA mutants colonize the rodent respiratory tract less effi-ciently than wild-type strains (31, 40, 52), and vaccination withpurified NanA affords some protection against nasopharyngealcolonization and otitis media (29, 30, 53). However, the differ-ences can be mouse strain and animal model dependent (6, 13,22, 23).

In addition to targeting host glycoconjugates, some bacterialneuraminidases have a role in biofilm formation, presumably

targeting sialylated bacterial exopolysaccharides (47). S. pneu-moniae biofilms have been characterized (1, 34, 36) and havebeen observed directly in the middle-ear mucosa from childrenwith chronic otitis media (15), contributing to the colonizationprocess (36). It is noteworthy that expression of nanA is up-regulated when S. pneumoniae is grown under biofilm condi-tions (39). There is a need for new therapeutic strategies as theprevalence of serotypes not covered by available vaccines isincreasing due to genetic recombination and strain replace-ment and these serotypes are increasingly associated with in-vasive disease (7, 20, 45, 59). We postulated that the neuramin-idase of S. pneumoniae is involved in biofilm formation andsought to identify compounds that inhibit its activity in vitro.

MATERIALS AND METHODS

Bacterial strains and media. S. pneumoniae strains D39 (4), D39 nanA (22),R6 (18), and R6 nanA (23) were grown on Trypticase soy (TS) agar or brothsupplemented with 200 U/ml of catalase (Worthington) and 1 �g/ml of chlor-amphenicol for nanA strains. Plate cultures were grown at 37°C in the presenceof carbon dioxide (5%). All chemicals were purchased from Sigma unless oth-erwise stated.

Epithelial cell culture. 16HBE14o� human bronchial epithelial cells and1HAEo� human airway cells (originally obtained from D. Gruenert, CaliforniaPacific Medical Center Research Institute, San Francisco) were grown in mini-mum essential medium with Earle’s salts (Cellgro and Gibco, respectively) sup-plemented with 10% fetal bovine serum (Cambrex and Gibco, respectively), 100U/ml penicillin, and 100 �g/ml streptomycin. The medium used for 16HBE14o�

cells was also supplemented with 2 mM glutamine (Invitrogen). The cells weregrown at 37°C with 5% CO2 in a humidified incubator.

Neuraminidase assay. NanA was purified as previously described (19). Neur-aminidase activity of NanA was detected using the fluorogenic substrate 2�-(4-methylumbelliferyl)-�-D-N-acetylneuraminic acid (Sigma). The reaction mix-tures contained 1.5 mM 2�-(4-methylumbelliferyl)-�-D-N-acetylneuraminic acidand 1 nM NanA in 2.5 mM sodium phosphate buffer (pH 5). The reactionmixtures were incubated for 2 h at 37°C before the fluorescence intensity wasmeasured using excitation and emission wavelengths of 360 nm and 465 nm,respectively, on a Tecan microplate reader (Mannedorf, Switzerland). Com-pounds were obtained from a variety of sources (Otava, Kiev, Ukraine; Inter-bioscreen, Moscow, Russia; Chembridge, San Diego, CA; Maybridge, Cornwell,United Kingdom; Sigma, St. Louis, MO; Princeton Biomolecular Research,

* Corresponding author. Mailing address: Department of Pediatrics,Columbia University, 650 W. 168th St., Black Building BB4-416, NewYork, NY 10032. Phone: (212) 305-4193. Fax: (212) 342-5728. E-mail:[email protected].

� Published ahead of print on 29 June 2009.

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Princeton, NJ; Lifechem, Burlington, Canada; Enamine, Kiev, Ukraine). Neur-aminidase assays with oseltamivir were performed using the hydrolyzed versionof the compound. Briefly, a mixture of oseltamivir (300 mg) in methanol (10 ml)was added to 5 N NaOH (3 ml). The mixture was stirred overnight and evapo-rated under reduced pressure. The residue was dissolved in 10 ml H2O and thenwashed with 5 ml of ethyl acetate. The aqueous solution was acidified with 5 NHCl to pH 2 to 3. Evaporation of water under reduced pressure resulted in thehydrolyzed product. The structure of the product was confirmed by nuclearmagnetic resonance and mass spectrometry. Divalent cations were supplied inthe form of calcium, magnesium, ferric, and copper chlorides. All neuraminidaseassays were performed at least in triplicate.

Quantification of asialo-GM1 exposure by flow cytometry. 16HBE14o� cellswere grown in 24-well plates to confluence, exposed to bacterial supernatants for3 h, and then washed three times with phosphate-buffered saline (PBS). Super-natants were concentrated approximately 30-fold (Amicon Ultra; Millipore), andthe protein quantity was adjusted. As a control, medium alone was also concen-trated. Cells were stained with rabbit polyclonal anti-asialo-GM1 antibody(Wako), followed by Alexa Fluor 488 donkey anti-rabbit immunoglobulin G(Molecular Probes). Cells were detached from the plates using 0.02% EGTA inHanks buffered saline solution, fixed with 1% paraformaldehyde, and analyzedwith a FACSCalibur using CellQuest software (version 3.3; BD). Data wereanalyzed using WinMDI (version 2.8; Joseph Trotter).

Adherence assays. Adherence assays were performed using 16HBE14o� cells.Bacterial strains were grown to mid-log phase and washed with PBS, and 0.7 �107 to 2 � 107 CFU of bacterial cells were added to confluent monolayers in24-well plates (multiplicity of infection, 30). Bacterial cells were allowed toadhere for 1 h at 37°C before three washes with PBS. Bacteria were dissociatedfrom epithelial cells using TrypLE Express (Gibco) and were serially dilutedbefore plating to determine the numbers of adherent bacteria. The assay wasperformed with three biological replicates and with duplicate technical replicatesin two separate experiments.

Biofilm assay. Bacterial strains were grown to mid-log phase before they werediluted 1:100 in TS broth with catalase. One hundred microliters of a dilutedculture was added in triplicate to 96-well flat-bottom tissue culture-treated plates(Falcon) and incubated for 18 to 24 h at 37°C in the presence of 5% CO2. Plateswere read at 600 nm to determine the levels of growth before they were washedin water. Adherent biofilm-forming cells were then stained with 125 �l of 1%crystal violet for 15 min before two washes in water and allowed to dry. Thebound crystal violet was then suspended in 200 �l of ethanol and shaken for 15min, and the results were read at 540 nm.

Biofilm formation after epithelial cell interaction. Bacterial strains weregrown and inoculated onto 1HAEo� cells as described above for the adherenceassay. After the initial PBS washes, fresh minimum essential medium was addedbefore a further 1 h of incubation. Removal and addition of fresh medium wererepeated another four times before adherent bacteria were detached usingTrypLE Express (Gibco). The detached bacteria were then diluted 1:100 in TSbroth with catalase, and assays were performed as described above for the biofilmassay. Experiments were repeated four times, each time in sextuplicate usingepithelial cells without bacteria as a negative control. When inhibitors were used,they were present during epithelial cell interaction and in microtiter trays forbiofilm formation. Inhibition with N-acetylneuraminic acid (NANA) was per-formed using 0.2% (wt/vol) NANA (Sigma) (56). Images of crystal violet-stainedmicrotiter wells were taken with a standard digital camera. Fluorescence micros-copy was performed using a Zeiss Observer Z1 inverted fluorescence microscopewith ApoTome (Zeiss) for optical sectioning and AxioVision software (version4.6.2.0; Zeiss). Microtiter wells were stained using the BacLight live/dead stainfrom Invitrogen (Carlsbad, CA).

Phylogenetic analysis. Our sampling strategy was aimed at maximizing phylo-genetic breadth in order to understand the overall pattern of evolution in theneuraminidase-sialidase gene family. We began with a list of well-known neura-minidases, including those from Vibrio cholerae, Salmonella enterica serovar Ty-phimurium, Clostridium perfringens, S. pneumoniae, Trypanosoma cruzi, andPseudomonas aeruginosa. For each sequence we performed a standard BLASTsearch and collected one sequence from each genus in the list of hits that had ane value of 1 � 10�5 or less. Duplicates were deleted. We also included sequencesthat have been included in previous studies of the evolution of sialidases (43).The GenBank (http://www.ncbi.nlm.nih.gov/Entrez/) accession numbers of thesequences utilized for the phylogenetic analysis are as follows: Verrucomicrobiumspinosum, gi 164421336:353068-354225; Blastopirellula marina, gi 87311313:89394-90503; Lentisphaera araneosa, gi 149198907:89577-90743; Propionibacte-rium acnes, gi 50841496:752060-754375; Ruminococcus lactaris, gi 197302028:32228-35857; Erysipelothrix rhusiopathiae, gi 13516389:295-3807; Pasteurella mul-tocida, gi 15601865:1176085-1179327; Actinomyces odontolyticus, gi 145845834:

308755-310992; Mannheimia haemolytica, gi 125433996:1-2376; Haemophilusparasuis, gi 167854877:54475-56886; Bacteroides fragilis, gi 53711291:4836372-4838006; Akkermansia muciniphila, gi 187734516:2229943-2231967; Capnocyto-phaga canimorsus, gi 194454827:2197-3765; Parabacteroides distasonis, gi 150006674:3525685-3527310; Shewanella pealeana, gi 157959830:1838982-1841831; Fla-vobacteriales bacterium, gi 88710837:680637-681797; Rhodopirellula baltica,gi 32470666:1724290-1725519; Opitutaceae bacterium, gi 153892517:3249-4847;Sassharopolyspora erythraea, gi 134096620:5769332-5771182; Pseudoalteromonashaloplanktis, gi 77361923:196316-197458; Chthoniobacter flavus, gi 196231426:66519-67730; Janibacter sp., gi 84494251:767782-770736; Monosiga brevicollis,gi 167534964:1-984; Strongylocentrotus purpuratus, gi 115616575:1-719; Plancto-myces maris, gi 149177549:10030-11205; Acinetobacter baumannii, gi 169632029:647539-649371; Opitutaceae bacterium, gi 153890920:5481-6641; Danio rerio,gi 148539964:69-1220; Corynebacterium diphtheriae, gi 38232642:512872-515037;Gemmata obscuriglobus, gi 163804184:63331-64515; Streptomyces coelicolor,gi 32141095:7255596-7257542; Takifugu rubripes, gi 148372013:1-87; V. cholerae,gi 12057212:1933231-1935654; C. perfringens nanI, gi 18308982:900997-903081;C. perfringens nanH, gi 18308982:904693-905499; P. aeruginosa, gi 110227054:3150886-3152202; Clostridium septicum, gi 40662; Clostridium sordellii, gi 1710442; Actinomyces viscosus, gi 39254; Trypanosoma rangeli, gi 2894809; T. cruzi,gi 162265; T. cruzi SAPA (shed acute-phase antigen), gi 10943; Micromonosportaviridifaciens, gi 216782:816-2759; Arthrobacter ureafaciens, gi 60544840; influenzavirus A H5N1, gi 108671038; Macrobdella decora, gi 1353880; S. enterica serovarTyphimurium, gi 16763390:1002088-1003326; S. pneumoniae, gi 116515308:1522475-1525468; Arcanobacterium pyogenes, gi 18146340:1026-6239; Xenopuslaevis, gi 148228846:180-1376; Trichomonas vaginalis, gi 123473002:1-1050; Rat-tus norvegicus, gi 71896601:59-1288; Bos taurus, gi 149676185:61-219, 650-842,1541-1803, 2490-2672, 2849-3071, and 3185-3411; and Monodelphis domestica,gi 126309689:1-1404. Sequences were aligned using the ClustalW algorithm asimplemented in the program BioEdit using default settings. Amino acid se-quences were aligned and then transposed to obtain the original nucleotidesequences, maintaining the gaps determined by the initial alignment (a total of5,394 characters, 4,124 parsimony-informative characters with gaps as a fifthstate, and 3,766 parsimony-informative characters with gaps treated as missing).

We performed a rigorous phylogenetic analysis using the maximum parsimonyalgorithm implemented in PAUP* (50). We used 1,000 replicates for randomaddition, followed by the tree branch reconnection algorithm using the “mul-trees” option to save more than one optimal tree if more than one optimal treewas discovered in the search. All characters and state transformations were givenequal weight. Terminal gaps were scored as missing data in all analyses. Weperformed two analyses, designating internal gaps as a fifth character state in oneanalysis and as missing data in the other analysis. Although the trees had manynodes in agreement, there were major differences between the structures of thetwo trees. Since gaps can be informative characters (14, 42), we favor the analysisin which internal gaps are counted as character states. We performed nonpara-metric bootstrap analyses with 100 iterations consisting of 100 random additionreplicates, followed by tree branch reconnection to gauge the robustness ofthe tree.

Statistics. The significance of data was determined using a Student t test and,for multivariant data, analysis of variance followed by a Dunnetti posttest usingGraphPad Prism software.

RESULTS

Biochemical properties of NanA. To better understand thebiochemical properties of NanA and to facilitate screening ofpotential inhibitory compounds, we established an assay tomeasure its neuraminidase activity. The biochemical activity ofNanA was assayed using the fluorogenic sialic acid derivative2�-(4-methylumbelliferyl)-�-D-N-acetylneuraminic acid. NanAcleaved the fluorogenic substrate significantly at low nanomo-lar and even picomolar concentrations (Fig. 1A). The Km ofNanA for this substrate is about 1.4 mM (data not shown),which is generally comparable to the Km values reported forother neuraminidases. The neuraminidase from V. choleraerequires divalent cations, specifically calcium, to be active (10,17). We investigated the effect of adding ions to the reactionmixture and observed that calcium was not essential for NanAactivity but did increase the activity by 70% at a concentration

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of 1 mM (Fig. 1B), that there was a 50% increase in the activityin the presence of magnesium ions (Fig. 1B), and that therewas decreased activity in the presence of iron and copper ions,presumably due to the higher molecular masses of these ions(Fig. 1B). The presence of either copper or ferric ions de-creased the activity by 90% or more at millimolar levels.

The ability of sialic acids to competitively inhibit NanA ac-tivity was also tested. The two sialic acids used, NANA and2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), wereboth utilized to obtain cocrystals of NanA (19). NANA caused50% inhibition at a concentration of 600 �M (Fig. 2). Weobserved greater inhibition with the transition state analogDANA than with NANA. DANA reduced the activity by 50%at a concentration of 200 �M.

Phylogeny of the neuraminidases. The neuraminidase super-family is known to be highly divergent (43, 51). We postulatedthat neuraminidases from organisms that infect the same sitemay have similar functions and hence may be evolutionarilyrelated, which might be useful in the identification of commoninhibitors. To better ascertain the evolutionary relatedness ofNanA, a phylogenetic analysis of a number of bacterial, eu-karyotic, and viral neuraminidases was conducted (Fig. 3).NanA clustered closely with the large neuraminidase of C.perfringens (38) and was more closely related to well-charac-terized bacterial neuraminidases. NanA did not cluster with

the neuraminidase from P. aeruginosa, which is also involved inrespiratory tract colonization.

Biological activity of pneumococcal NanA. Many lung patho-gens, including S. pneumoniae and P. aeruginosa, bind toasialylated ganglioside receptor GM1 (asialo-GM1) (Gal�1-3GalNAc�1-4Gal�1-4Glc�1-1Cer) (26). Either purified NanAor concentrated supernatant from wild-type S. pneumoniaestrain D39, but not concentrated supernatant from an isogenicnanA mutant of this strain, exposed asialo-GM1 on the surface

FIG. 1. Activity of S. pneumoniae neuraminidase. (A) Titration of activity using different concentrations of purified NanA. (B) Effect of divalentcations on the activity of purified NanA compared to the activity of the wild-type enzyme (control). The assay was performed using 2�-(4-methylumbelliferyl)-�-D-N-acetylneuraminic acid (MNN). *, P � 0.05.

FIG. 2. Inhibition of NanA neuraminidase activity by the sialic acidcompounds NANA and DANA. The activity is expressed as a percent-age of the activity of NanA without an inhibitor (control). The assaywas performed using 2�-(4-methylumbelliferyl)-�-D-N-acetylneura-minic acid. *, P � 0.05.

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of 16HBE14o� epithelial cells (Fig. 4). However, no effect onbacterial adherence was observed, nor was there a growthadvantage in the presence of airway epithelial cells (data notshown).

NanA is involved in biofilm formation. As S. pneumoniaenanA expression is upregulated in lung tissue and in biofilm-grown cells (39), the contribution of nanA to the formation ofbiofilms was examined (Fig. 5). The increase in expression was

FIG. 3. Phylogenetic analysis of neuraminidases: unrooted phylogenetic tree based on our broad survey of neuraminidase phylogeny. The treeis the strict consensus of three most parsimonious trees (45,885steps; consistency index, 0.326; retention index, 0.385; rescaled consistency, 0.125).Black circles indicate branches with bootstrap values greater than 80%. Gray circles indicate branches with bootstrap values between 50 and 80%.Open circles indicate nodes with bootstrap values less than 50% for agreement with the consensus bootstrap tree. Branches without an indicationof the bootstrap value are found in the maximum parsimony tree but not in the bootstrap tree.

FIG. 4. Release of sialic acid from the surface of airway epithelial cells by NanA. (A) Exposure of asialo-GM1 to concentrated supernatantfrom wild-type (WT) and nanA strains. (B) Exposure of asialo-GM1 to purified NanA. Cells were stained with antibody to asialo-GM1 andquantified by flow cytometry, and the results are expressed as the changes compared to a medium-only control. *, P � 0.05.



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exploited by initially growing the bacteria on airway epithelialcells over a day (see Materials and Methods). Adherent bac-terial cells were then recovered before the standard microtiterbiofilm assay was performed. Exposure of S. pneumoniae toairway epithelial cells before the biofilm assay was performednot only resulted in a significant increase in biofilm formationbut also showed that the nanA mutant had a significantly re-duced capacity to form biofilms. When S. pneumoniae was notexposed previously to airway epithelial cells, no difference inbiofilm formation was observed between the wild-type andnanA strains, nor was much biofilm formation observed. In anS. pneumoniae R6, unencapsulated background, significantlymore biofilm was produced, and the ability of the nanA strainto form biofilms was also significantly reduced (Fig. 5A). Con-sistent with the hypothesis that NANA acts as an inhibitor inthe neuraminidase assay (Fig. 2), adding exogenous NANA tothe biofilm assay mixture also resulted in decreased biofilmformation by the wild-type strain (Fig. 5B).

The biofilms produced by R6 were even layers of cells onmicrotiter plates, and the reduction in biofilm formation ob-served with the nanA mutant was indicated by reduced inten-sity of crystal violet staining (Fig. 6A). In the D39 background,after exposure to epithelial cells, the wild-type strain had moreintense crystal violet staining overall than the nanA strain (Fig.6A). Macroscopically, the wild-type strain had regions of in-tense crystal violet staining in clumps. When the D39 biofilmswere examined under the microscope (Fig. 6B), a lattice-likearrangement of cells was observed for both the wild-type andnanA strains. Regions of concentrated cells were observed forthe nanA strain, but no macrostructures were observed. How-ever, for the wild-type background, structures that had signif-icant height were observed (Fig. 6B and C). These structureswere approximately 70 �m high, as shown by the three-dimen-sional reconstruction (Fig. 6C). For the nanA background onlycells in the initial sections were attached to the microtiter wells(Fig. 6B and D).

Identification of small-molecule inhibitors of the neura-minidases. Virtual library screening was performed (Schro-dinger LLC, Portland, OR) (25, 44) that identified small mol-ecules that were predicted to interact with the active site of theenzyme. As a control we tested the ability of the influenza virusneuraminidase inhibitor oseltamivir to inhibit NanA (Fig. 7).

FIG. 5. S. pneumoniae biofilm formation. (A) Encapsulated (D39 background) strains were grown in microtiter trays without (filled bars) orwith (striped bars) previous exposure to epithelial cells. Unencapsulated R6 strains were grown in microtiter trays without exposure to epithelialcells. (B) Incubation with NANA results in reduced biofilm formation by wild-type strain D39 (WT). Biofilms were measured by using crystal violetstaining. Biofilm formation was normalized to growth and expressed as a percentage compared to the R6 wild-type strain. *, P � 0.05.

FIG. 6. Imaging of S. pneumoniae biofilms. (A) Images of crystalviolet-stained biofilms in microtiter wells for wild-type (WT) and nanAstrains with D39 (after exposure to epithelial cells) and R6 back-grounds. (B) Fluorescence microscopy of wild-type strain D39 andnanA biofilms grown in microtiter trays after exposure to epithelialcells and stained with BacLight live/dead stain. Magnification, �200.(C) Three-dimensional reconstruction of the biofilm structure for thewild-type strain shown in panel B. (D) Three-dimensional reconstruc-tion of cells of the nanA strain shown in panel B.



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Oseltamivir had a 50% inhibitory concentration (IC50) of 2mM. A number of compounds identified in this screen showedhigh degrees of inhibition in vitro (Fig. 8A). A lead compounddesignated XX1 (Fig. 8D) with a pyrrolidine-2,3-dione chem-ical scaffold was found to inhibit NanA over a range of con-centrations in a dose-dependent manner (Fig. 8B). An IC50 of28 �M was determined. The inhibition of NanA by XX1 wasmore than 7, 20, and 70 times more effective than the inhibition

by DANA, NANA, and oseltamivir, respectively. At a XX1concentration of 100 �M we observed a significant reduction inbiofilm formation, and at a concentration of 400 �M the levelsof biofilm formation were comparable to those of the nanAstrain (Fig. 8C). XX1 did not affect the growth of planktoniccells (data not shown).

DISCUSSION

For S. pneumoniae, the biofilm phenotype is most evident invivo (15) or, as we show, under in vitro conditions that favorneuraminidase expression. We were able to demonstrate adifference in biofilm production after growth on human airwaycells, which has been shown to induce nanA expression (27, 39,46) and reduce capsule production (16). By selecting for or-ganisms on human airway cells, we apparently mimicked alikely in vivo process in which encapsulated organisms that areable to avoid mucus entrapment and clearance gradually pro-duce less capsule to facilitate epithelial attachment (20). Thisassociation between capsule expression and biofilm formationwas evident with the R6 strain. R6 does not produce capsule,and we observed that it produced significant biofilms and thata nanA mutant of R6 displayed a reduced propensity for bio-film formation. The reduced capsule is important in initial

FIG. 7. Inhibition of NanA by oseltamivir. The activity is expressedas a percentage of the activity without inhibitor. The assay was per-formed using 2�-(4-methylumbelliferyl)-�-D-N-acetylneuraminic acid.*, P � 0.05.

FIG. 8. Inhibitory activity of potential NanA inhibitors. (A) Screening of potential inhibitors was performed with NanA and inhibitors at aconcentration of 100 �M in the neuraminidase assay. Results are percentages of the level for the control without any inhibitor. (B) Dose-responsecurve for NanA with lead compound XX1. Data were fitted with a logarithmically based trend line. The data are the percentages of activitycompared to the control with only the vehicle (dimethyl sulfoxide). (C) Biofilm formation by wild-type strain D39 (WT) grown in the presence ofXX1 during exposure to epithelial cells and growth in microtiter trays. The nanA strain was used as a reference. Biofilm formation was normalizedto growth and was expressed as a percentage compared to the results for the wild-type control. (D) Chemical structure of XX1. *, P � 0.05.



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airway colonization both for facilitating the initial adherenceand for promoting cell-cell interactions for biofilm formation(20). This conclusion is also consistent with other studies thatshowed that cells induced to form a biofilm had a greaterpropensity to cause pneumonia than planktonic cells (39).

It is likely that the role of the neuraminidase is somewherebetween the cluster formation and biofilm maturation pro-cesses (1). This conclusion is based on the fact that the wild-type strain biofilms observed resembled the mature biofilmsgrown under flow conditions (2), and while the nanA strain stillproduced regions of clustered cells, it had no large cellularstructures. It should be noted that some differences are alsoexpected when static and continuous-flow systems are com-pared. Even though the development and composition of bio-films are complex and numerous genes and proteins are dif-ferentially expressed (1, 34, 39), the high level of expression ofnanA in biofilms and other data presented here indicate thatthe neuraminidase has a crucial role in biofilm production. Arecent study by Trappetti et al. (56) demonstrated the role ofsialic acid in pneumococcal biofilm formation. Addition ofsialic acid, but not addition of other sugars, resulted in in-creased biofilm formation, as well as increases in the numberof organisms present in the nasopharynx in a murine model ofcolonization (56). We found that NANA at high concentra-tions inhibits biofilm formation by S. pneumoniae by occupyingthe binding site of NanA. Based on this study and our otherwork, the liberation of sialic acid residues from the airwayepithelium by the action of NanA appears to contribute tobiofilm formation by S. pneumoniae and hence is a desirabletarget for therapeutic intervention.

Even though nanA is the most highly expressed neuramini-dase gene, the other neuraminidases of S. pneumoniae maycontribute to biofilm formation. In the serotype 4 background(strain TIGR4) nanB is involved to a small extent in biofilmformation (36). It should be noted that there is a mutation innanA in this strain that affects its cell wall attachment. Thesestudies highlighted the important role that biofilms play inpathogenesis, establishing a link between biofilm formationand colonization of the murine nasopharynx. Our data areconsistent with these observations, suggesting that a reducedcapacity to form biofilms is behind the reduced pathogenesis ofthe neuraminidase (nanA) mutant in animal models (31, 40).Based on this work and other work, a model for the establish-ment of infection in the respiratory tract by S. pneumoniae canbe established. Encapsulated cells reach the mucus layer,where the capsule is required to avoid mucous entrapment.NanA is also able to utilize the mucin as a carbon source (61).When the respiratory epithelium is reached, capsule expres-sion is downregulated to facilitate intimate attachment. It is atthis point that neuraminidase expression is increased and cellsbegin to establish a biofilm.

Another respiratory pathogen, P. aeruginosa, also produces aneuraminidase (NanPs) that is important for biofilm formationand pathogenesis in animal models (47), indicating that therole in pathogenesis of the two neuraminidases is conserved.Desialylated glycolipids provide receptors for many of thecommon bacterial pulmonary pathogens, including both S.pneumoniae and P. aeruginosa (26), which bind to the exposedGalNAc�1-4Gal residues when terminal sialic acid is cleaved.We demonstrated that NanA, like P. aeruginosa neuraminidase

(47), was capable of exposing this receptor on human airwaycells. The neuraminidase activity associated with intact organ-isms, either S. pneumoniae or P. aeruginosa (47), was not as-sociated with increased bacterial attachment, which is an in-teresting observation given that the desialylation of airwaymucosal cells by the influenza virus neuraminidase increasessusceptibility to secondary infections often caused by S. pneu-moniae (33).

The crystal structures of both NanA and NanPs were re-cently solved (19, 60). Structural analysis indicated that whilethese two enzymes had similar overall structures, their activesites were remarkably different, indicating likely differences insubstrate specificity and biochemical function (9, 47). We alsoobserved that the two enzymes are phylogenetically distinct,with NanA clustering closer to other well-characterized canon-ical neuraminidases and NanPs located in a phylogeneticallydiverse branch of the tree. Yet despite their differences instructure and likely different substrates, these two neuramini-dases have similar functions in pathogenesis.

The neuraminidase of V. cholerae requires divalent cations,specifically calcium, to be active (10, 17), and while we ob-served an increase in activity in the presence of calcium ions,such ions were not an absolute requirement for activity. This isan observation analogous to the observation for the neuramin-idase of P. aeruginosa (9). The inhibition that we did observewith iron and copper ions is consistent with inhibition of suchenzymes by metal ions (17). The increase in activity with cal-cium might suggest the presence of a calcium binding site, likethat in V. cholerae (10) or C. perfringens (38), although this isnot a categorical feature of neuraminidases nor was such a siteidentified from the crystal structure (9, 11, 19, 48, 60). How-ever, the presence of a calcium binding site would not beincongruous given the close phylogenetic relationship betweenNanA and the other well-characterized bacterial neuramini-dases.

There is a need for new therapeutic strategies against S.pneumoniae as the prevalence of serotypes not covered byavailable vaccines is increasing due to genetic recombinationand such serotypes are increasingly associated with invasivedisease (7, 20, 45, 59); in addition, antibiotic resistance of S.pneumoniae strains is a growing problem (32). Using in silicodocking studies, we were able to identify a number of com-pounds that were significantly more inhibitory than the sialicacids NANA and DANA and the influenza virus inhibitoroseltamivir. Many neuraminidases have been crystallized in thepresence of the sialic acid DANA (11, 19, 35, 38), and weobserved that this compound inhibits the neuraminidase activ-ity of NanA better than it inhibits the activity of NANA. Al-though the inhibition by DANA observed was not as great asthe inhibition of some neuraminidases tested to date, it iswithin the observed range of inhibition (12, 48). Our leadcompound XX1 was found to inhibit the neuraminidase activ-ity of NanA at concentrations in the low-micromolar range andalso to inhibit the ability of organisms to form biofilms. Somework examining inhibition of other bacterial neuraminidaseshas been done, but most of the work to date has been donewith the trypanosome trans-sialidases (37, 48, 49). Trypano-some trans-sialidase studies have identified inhibitors withIC50s in the range from 100 to 300 �M, indicating that thestudies presented here are progressing well. We are actively



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involved in synthesizing XX1 in an attempt to examine itsability to prevent infection in vivo.

Based on structural data, it is likely that inhibitors of thepneumococcal neuraminidase will be organism specific. TheNanPs neuraminidase of P. aeruginosa has an active site that issignificantly different. The binding pocket of NanA is tight,while NanPs has an open conformation that likely requiresdifferent inhibitor structures for effective inhibition (19). Weenvision that delivery of a neuraminidase inhibitor to the lungcould be used as prophylaxis to circumvent bacterial pneumo-nia after influenza virus infection and also in at-risk popula-tions.

Despite biochemical, structural, and phylogenetic differ-ences, we demonstrate a common role for the neuraminidasesof S. pneumoniae and P. aeruginosa in biofilm formation andthe pathogenesis of respiratory tract infection. Due to theimportance of biofilms in S. pneumoniae colonization, we havebegun to identify inhibitors targeting the pneumococcal neur-aminidase to prevent infection. This study represents a startingpoint in the development of a potentially novel drug againstpneumococcal pneumonia.

ACKNOWLEDGMENTS

We thank Jeffrey Weiser for providing S. pneumoniae strains.D.P. was the recipient of an NHRMC Biomedical Overseas Fellow-

ship. This work was supported by the Thrasher Research Fund (D.P.)and by a Deans pilot grant from Columbia University (A.P.).

REFERENCES

1. Allegrucci, M., F. Z. Hu, K. Shen, J. Hayes, G. D. Ehrlich, J. C. Post, and K.Sauer. 2006. Phenotypic characterization of Streptococcus pneumoniae bio-film development. J. Bacteriol. 188:2325–2335.

2. Allegrucci, M., and K. Sauer. 2007. Characterization of colony morphologyvariants isolated from Streptococcus pneumoniae biofilms. J. Bacteriol. 189:2030–2038.

3. Andersson, B., J. Dahmen, T. Frejd, H. Leffler, G. Magnusson, G. Noori, andC. S. Eden. 1983. Identification of an active disaccharide unit of a glycocon-jugate receptor for pneumococci attaching to human pharyngeal epithelialcells. J. Exp. Med. 158:559–570.

4. Avery, O. T., C. M. MacLeod, and M. McCarty. 1944. Studies on the chem-ical nature of the substance inducing transformation of pneumococcal types:inductions of transformation by a desoxyribonucleic acid fraction isolatedfrom pneumococcus type III. J. Exp. Med. 79:137–158.

5. Berry, A. M., R. A. Lock, and J. C. Paton. 1996. Cloning and characterizationof nanB, a second Streptococcus pneumoniae neuraminidase gene, and puri-fication of the NanB enzyme from recombinant Escherichia coli. J. Bacteriol.178:4854–4860.

6. Berry, A. M., and J. C. Paton. 2000. Additive attenuation of virulence ofStreptococcus pneumoniae by mutation of the genes encoding pneumolysinand other putative pneumococcal virulence proteins. Infect. Immun. 68:133–140.

7. Brueggemann, A. B., R. Pai, D. W. Crook, and B. Beall. 2007. Vaccine escaperecombinants emerge after pneumococcal vaccination in the United States.PLoS Pathog. 3:e168.

8. Burnaugh, A. M., L. J. Frantz, and S. J. King. 2008. Growth of Streptococcuspneumoniae on human glycoconjugates is dependent upon the sequentialactivity of bacterial exoglycosidases. J. Bacteriol. 190:221–230.

9. Cacalano, G., M. Kays, L. Saiman, and A. Prince. 1992. Production of thePseudomonas aeruginosa neuraminidase is increased under hyperosmolarconditions and is regulated by genes involved in alginate expression. J. Clin.Investig. 89:1866–1874.

10. Crennell, S., E. Garman, G. Laver, E. Vimr, and G. Taylor. 1994. Crystalstructure of Vibrio cholerae neuraminidase reveals dual lectin-like domains inaddition to the catalytic domain. Structure 2:535–544.

11. Crennell, S. J., E. F. Garman, W. G. Laver, E. R. Vimr, and G. L. Taylor.1993. Crystal structure of a bacterial sialidase (from Salmonella typhimuriumLT2) shows the same fold as an influenza virus neuraminidase. Proc. Natl.Acad. Sci. USA 90:9852–9856.

12. Crennell, S. J., E. F. Garman, C. Philippon, A. Vasella, W. G. Laver, E. R.Vimr, and G. L. Taylor. 1996. The structures of Salmonella typhimurium LT2neuraminidase and its complexes with three inhibitors at high resolution. J.Mol. Biol. 259:264–280.

13. Gingles, N. A., J. E. Alexander, A. Kadioglu, P. W. Andrew, A. Kerr, T. J.Mitchell, E. Hopes, P. Denny, S. Brown, H. B. Jones, S. Little, G. C. Booth,and W. L. McPheat. 2001. Role of genetic resistance in invasive pneumo-coccal infection: identification and study of susceptibility and resistance ininbred mouse strains. Infect. Immun. 69:426–434.

14. Giribet, G., and W. C. Wheeler. 1999. On gaps. Mol. Phylogenet. Evol.13:132–143.

15. Hall-Stoodley, L., F. Z. Hu, A. Gieseke, L. Nistico, D. Nguyen, J. Hayes, M.Forbes, D. P. Greenberg, B. Dice, A. Burrows, P. A. Wackym, P. Stoodley,J. C. Post, G. D. Ehrlich, and J. E. Kerschner. 2006. Direct detection ofbacterial biofilms on the middle-ear mucosa of children with chronic otitismedia. JAMA 296:202–211.

16. Hammerschmidt, S., S. Wolff, A. Hocke, S. Rosseau, E. Muller, and M.Rohde. 2005. Illustration of pneumococcal polysaccharide capsule duringadherence and invasion of epithelial cells. Infect. Immun. 73:4653–4667.

17. Holmquist, L. 1975. Activation of Vibrio cholerae neuraminidase by divalentcations. FEBS Lett. 50:269–271.

18. Hoskins, J., W. E. Alborn, Jr., J. Arnold, L. C. Blaszczak, S. Burgett, B. S.DeHoff, S. T. Estrem, L. Fritz, D. J. Fu, W. Fuller, C. Geringer, R. Gilmour,J. S. Glass, H. Khoja, A. R. Kraft, R. E. Lagace, D. J. LeBlanc, L. N. Lee,E. J. Lefkowitz, J. Lu, P. Matsushima, S. M. McAhren, M. McHenney, K.McLeaster, C. W. Mundy, T. I. Nicas, F. H. Norris, M. O’Gara, R. B. Peery,G. T. Robertson, P. Rockey, P. M. Sun, M. E. Winkler, Y. Yang, M. Young-Bellido, G. Zhao, C. A. Zook, R. H. Baltz, S. R. Jaskunas, P. R. Rosteck, Jr.,P. L. Skatrud, and J. I. Glass. 2001. Genome of the bacterium Streptococcuspneumoniae strain R6. J. Bacteriol. 183:5709–5717.

19. Hsiao, Y. S., D. Parker, A. J. Ratner, A. Prince, and L. Tong. 2009. Crystalstructures of respiratory pathogen neuraminidases. Biochem. Biophys. Res.Commun. 380:467–471.

20. Kadioglu, A., J. N. Weiser, J. C. Paton, and P. W. Andrew. 2008. The role ofStreptococcus pneumoniae virulence factors in host respiratory colonizationand disease. Nat. Rev. Microbiol. 6:288–301.

21. Kelly, R. T., S. Farmer, and D. Greiff. 1967. Neuraminidase activities ofclinical isolates of Diplococcus pneumoniae. J. Bacteriol. 94:272–273.

22. King, S. J., K. R. Hippe, J. M. Gould, D. Bae, S. Peterson, R. T. Cline, C.Fasching, E. N. Janoff, and J. N. Weiser. 2004. Phase variable desialylationof host proteins that bind to Streptococcus pneumoniae in vivo and protect theairway. Mol. Microbiol. 54:159–171.

23. King, S. J., K. R. Hippe, and J. N. Weiser. 2006. Deglycosylation of humanglycoconjugates by the sequential activities of exoglycosidases expressed byStreptococcus pneumoniae. Mol. Microbiol. 59:961–974.

24. King, S. J., A. M. Whatmore, and C. G. Dowson. 2005. NanA, a neuramin-idase from Streptococcus pneumoniae, shows high levels of sequence diver-sity, at least in part through recombination with Streptococcus oralis. J.Bacteriol. 187:5376–5386.

25. Kontoyianni, M., L. M. McClellan, and G. S. Sokol. 2004. Evaluation ofdocking performance: comparative data on docking algorithms. J. Med.Chem. 47:558–565.

26. Krivan, H. C., D. D. Roberts, and V. Ginsburg. 1988. Many pulmonarypathogenic bacteria bind specifically to the carbohydrate sequence GalNAcbeta 1-4Gal found in some glycolipids. Proc. Natl. Acad. Sci. USA 85:6157–6161.

27. LeMessurier, K. S., A. D. Ogunniyi, and J. C. Paton. 2006. Differentialexpression of key pneumococcal virulence genes in vivo. Microbiology 152:305–311.

28. Linder, T. E., R. L. Daniels, D. J. Lim, and T. F. DeMaria. 1994. Effect ofintranasal inoculation of Streptococcus pneumoniae on the structure of thesurface carbohydrates of the chinchilla eustachian tube and middle earmucosa. Microb. Pathog. 16:435–441.

29. Lock, R. A., J. C. Paton, and D. Hansman. 1988. Comparative efficacy ofpneumococcal neuraminidase and pneumolysin as immunogens protectiveagainst Streptococcus pneumoniae. Microb. Pathog. 5:461–467.

30. Long, J. P., H. H. Tong, and T. F. DeMaria. 2004. Immunization with nativeor recombinant Streptococcus pneumoniae neuraminidase affords protectionin the chinchilla otitis media model. Infect. Immun. 72:4309–4313.

31. Manco, S., F. Hernon, H. Yesilkaya, J. C. Paton, P. W. Andrew, and A.Kadioglu. 2006. Pneumococcal neuraminidases A and B both have essentialroles during infection of the respiratory tract and sepsis. Infect. Immun.74:4014–4020.

32. McCormick, A. W., C. G. Whitney, M. M. Farley, R. Lynfield, L. H. Harrison,N. M. Bennett, W. Schaffner, A. Reingold, J. Hadler, P. Cieslak, M. H.Samore, and M. Lipsitch. 2003. Geographic diversity and temporal trends ofantimicrobial resistance in Streptococcus pneumoniae in the United States.Nat. Med. 9:424–430.

33. McCullers, J. A. 2006. Insights into the interaction between influenza virusand pneumococcus. Clin. Microbiol. Rev. 19:571–582.

34. Moscoso, M., E. Garcia, and R. Lopez. 2006. Biofilm formation by Strepto-coccus pneumoniae: role of choline, extracellular DNA, and capsular poly-saccharide in microbial accretion. J. Bacteriol. 188:7785–7795.

35. Moustafa, I., H. Connaris, M. Taylor, V. Zaitsev, J. C. Wilson, M. J. Kiefel,M. von Itzstein, and G. Taylor. 2004. Sialic acid recognition by Vibrio chol-erae neuraminidase. J. Biol. Chem. 279:40819–40826.



.org/D

ownloaded from

http://iai.asm.org/

36. Munoz-Elias, E. J., J. Marcano, and A. Camilli. 2008. Isolation of Strepto-coccus pneumoniae biofilm mutants and their characterization during naso-pharyngeal colonization. Infect. Immun. 76:5049–5061.

37. Neres, J., M. L. Brewer, L. Ratier, H. Botti, A. Buschiazzo, P. N. Edwards,P. N. Mortenson, M. H. Charlton, P. M. Alzari, A. C. Frasch, R. A. Bryce,and K. T. Douglas. 2009. Discovery of novel inhibitors of Trypanosoma cruzitrans-sialidase from in silico screening. Bioorg. Med. Chem. Lett. 19:589–596.

38. Newstead, S. L., J. A. Potter, J. C. Wilson, G. Xu, C. H. Chien, A. G. Watts,S. G. Withers, and G. L. Taylor. 2008. The structure of Clostridium perfrin-gens NanI sialidase and its catalytic intermediates. J. Biol. Chem. 283:9080–9088.

39. Oggioni, M. R., C. Trappetti, A. Kadioglu, M. Cassone, F. Iannelli, S. Ricci,P. W. Andrew, and G. Pozzi. 2006. Switch from planktonic to sessile life: amajor event in pneumococcal pathogenesis. Mol. Microbiol. 61:1196–1210.

40. Orihuela, C. J., G. Gao, K. P. Francis, J. Yu, and E. I. Tuomanen. 2004.Tissue-specific contributions of pneumococcal virulence factors to pathogen-esis. J. Infect. Dis. 190:1661–1669.

41. Pettigrew, M. M., K. P. Fennie, M. P. York, J. Daniels, and F. Ghaffar. 2006.Variation in the presence of neuraminidase genes among Streptococcus pneu-moniae isolates with identical sequence types. Infect. Immun. 74:3360–3365.

42. Phillips, A., D. Janies, and W. Wheeler. 2000. Multiple sequence alignmentin phylogenetic analysis. Mol. Phylogenet. Evol. 16:317–330.

43. Roggentin, P., R. Schauer, L. L. Hoyer, and E. R. Vimr. 1993. The sialidasesuperfamily and its spread by horizontal gene transfer. Mol. Microbiol.9:915–921.

44. Sherman, W., T. Day, M. P. Jacobson, R. A. Friesner, and R. Farid. 2006.Novel procedure for modeling ligand/receptor induced fit effects. J. Med.Chem. 49:534–553.

45. Singleton, R. J., T. W. Hennessy, L. R. Bulkow, L. L. Hammitt, T. Zulz, D. A.Hurlburt, J. C. Butler, K. Rudolph, and A. Parkinson. 2007. Invasive pneu-mococcal disease caused by nonvaccine serotypes among Alaska native chil-dren with high levels of 7-valent pneumococcal conjugate vaccine coverage.JAMA 297:1784–1792.

46. Song, X. M., W. Connor, K. Hokamp, L. A. Babiuk, and A. A. Potter. 2008.Streptococcus pneumoniae early response genes to human lung epithelialcells. BMC Res. Notes 1:64.

47. Soong, G., A. Muir, M. I. Gomez, J. Waks, B. Reddy, P. Planet, P. K. Singh,Y. Kaneko, M. C. Wolfgang, Y. S. Hsiao, L. Tong, and A. Prince. 2006.Bacterial neuraminidase facilitates mucosal infection by participating in bio-film production. J. Clin. Investig. 116:2297–2305.

48. Streicher, H. 2004. Inhibition of microbial sialidases—what has happenedbeyond the influenza virus? Curr. Med. Chem. 3:149–161.

49. Streicher, H., and H. Busse. 2006. Building a successful structural motif intosialylmimetics—cyclohexenephosphonate monoesters as pseudo-sialosideswith promising inhibitory properties. Bioorg. Med. Chem. 14:1047–1057.

50. Swofford, D. L. 1998. PAUP*: phylogenetic analysis using parsimony (*andother methods), 4.0 beta ed. Sinauer, Sunderland, MA.

51. Taylor, G. 1996. Sialidases: structures, biological significance and therapeuticpotential. Curr. Opin. Struct. Biol. 6:830–837.

52. Tong, H. H., L. E. Blue, M. A. James, and T. F. DeMaria. 2000. Evaluationof the virulence of a Streptococcus pneumoniae neuraminidase-deficient mu-tant in nasopharyngeal colonization and development of otitis media in thechinchilla model. Infect. Immun. 68:921–924.

53. Tong, H. H., D. Li, S. Chen, J. P. Long, and T. F. DeMaria. 2005. Immuni-zation with recombinant Streptococcus pneumoniae neuraminidase NanAprotects chinchillas against nasopharyngeal colonization. Infect. Immun. 73:7775–7778.

54. Tong, H. H., X. Liu, Y. Chen, M. James, and T. Demaria. 2002. Effect ofneuraminidase on receptor-mediated adherence of Streptococcus pneu-moniae to chinchilla tracheal epithelium. Acta Otolaryngol. 122:413–419.

55. Tong, H. H., M. A. McIver, L. M. Fisher, and T. F. DeMaria. 1999. Effect oflacto-N-neotetraose, asialoganglioside-GM1 and neuraminidase on adher-ence of otitis media-associated serotypes of Streptococcus pneumoniae tochinchilla tracheal epithelium. Microb. Pathog. 26:111–119.

56. Trappetti, C., A. Kadioglu, C. Carter, J. Hayre, F. Iannelli, G. Pozzi, P. W.Andrew, and M. R. Oggioni. 2009. Sialic acid: a preventable signal forpneumococcal biofilm formation, colonization, and invasion of the host.J. Infect. Dis. 199:1497–1505.

57. von Itzstein, M. 2007. The war against influenza: discovery and developmentof sialidase inhibitors. Nat. Rev. Drug Discov. 6:967–974.

58. Williamson, Y. M., R. Gowrisankar, D. L. Longo, R. Facklam, I. K. Gipson,E. P. Ades, G. M. Carlone, and J. S. Sampson. 2008. Adherence of nontype-able Streptococcus pneumoniae to human conjunctival epithelial cells. Mi-crob. Pathog. 44:175–185.

59. World Health Organization. 2007. Pneumococcal conjugate vaccine forchildhood immunization. W.H.O. position paper. Weekly epidemiologicalrecord. World Health Organization, Geneva, Switzerland.

60. Xu, G., X. Li, P. W. Andrew, and G. L. Taylor. 2008. Structure of the catalyticdomain of Streptococcus pneumoniae sialidase NanA. Acta Crystallogr. F64:772–775.

61. Yesilkaya, H., S. Manco, A. Kadioglu, V. S. Terra, and P. W. Andrew. 2008.The ability to utilize mucin affects the regulation of virulence gene expres-sion in Streptococcus pneumoniae. FEMS Microbiol. Lett. 278:231–235.

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