INFECTION AND IMMUNITY, May 2010, p. 2108–2116 Vol. 78, No. 50019-9567/10/$12.00 doi:10.1128/IAI.01125-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Three Surface Exoglycosidases from Streptococcus pneumoniae, NanA,BgaA, and StrH, Promote Resistance to Opsonophagocytic

Killing by Human Neutrophils�

Ankur B. Dalia,1 Alistair J. Standish,1 and Jeffrey N. Weiser1,2*Departments of Microbiology1 and Pediatrics,2 University of Pennsylvania, Philadelphia, Pennsylvania

Received 6 October 2009/Returned for modification 8 November 2009/Accepted 8 February 2010

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen and a leading cause ofinflammatory infections such as pneumonia and otitis media. An important mechanism for host defenseagainst S. pneumoniae is opsonophagocytic killing by neutrophils. To persist in the human host, thepneumococcus has developed strategies to evade opsonization and subsequent neutrophil-mediated kill-ing. Utilizing a genomic approach, we identified NanA, the major pneumococcal neuraminidase, as a factorimportant for resistance to opsonophagocytic killing in ex vivo killing assays using human neutrophils.The effect of NanA was shown using both type 4 (TIGR4) and type 6A clinical isolates. NanA promotes thisresistance by acting in conjunction with two other surface-associated exoglycosidases, BgaA, a �-galac-tosidase, and StrH, an N-acetylglucosaminidase. Experiments using human serum showed that theseexoglycosidases reduced deposition of complement component C3 on the pneumococcal surface, providinga mechanism for this resistance. Additionally, we have shown that antibodies in human serum do notcontribute to this phenotype. These results demonstrate that deglycosylation of a human serum glyco-conjugate(s) by the combined effects of NanA, BgaA, and StrH, is important for resistance to complementdeposition and subsequent phagocytic killing of S. pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is a leadinghuman pathogen that is responsible for over a million deathsannually (36). The organism resides in the nasopharynx, andalthough colonization is asymptomatic, it can spread from thissite to cause diseases such as otitis media, pneumonia, andsepticemia. During infection, the pneumococcus elicits anacute inflammatory response, characterized by an influx ofphagocytic cells consisting primarily of neutrophils (58). Op-sonophagocytic killing by neutrophils and other professionalphagocytes is believed to be a major mechanism for host de-fense against pneumococcal infection. This is a multistep pro-cess in which bacteria must first be opsonized. A major mech-anism for opsonization is via the complement system, whichresults in covalent deposition of C3b onto the bacterial surface(30, 41, 61). C3b can then be further cleaved to iC3b forrecognition by complement receptor 3 (CR3). On neutrophils,this receptor binds complement-opsonized bacteria and stim-ulates phagocytosis, after which neutrophils efficiently kill thepneumococcus (8, 14, 31, 49).

Evading opsonophagocytosis is essential for persistence ofthis pathogen in the human host. This is evidenced by anincreased prevalence of pneumococcal infection in patientswith deficiencies in complement components (12, 45, 63). Also,mice that are rendered neutropenic are more susceptible toinvasive pneumococcal infection (34). Recently it has beenshown that during colonization there is a correlation betweenresistance to neutrophil-mediated killing and carriage of pneu-mococcal serotypes, where serotypes more resistant to killing

have a higher prevalence (60). Like many successful extracel-lular pathogens, the pneumococcus is encapsulated by a thickcoat of polysaccharide, which aids in evasion of phagocytickilling by masking underlying structures on the bacterial sur-face and reducing opsonization (18, 60). Capsular polysaccha-ride is the immunodominant antigen on the pneumococcus andis the basis for distinguishing strains, among 91 different sero-types. This antiphagocytic factor is crucial for pathogenesis,since unencapsulated strains rarely cause invasive disease andare severely attenuated in models of infection (35, 59). Wehave observed that even in the absence of capsule, however, S.pneumoniae retains some resistance to neutrophil killing.Therefore, we hypothesized that in addition to capsule, thepneumococcus expresses other factors that promote resistanceto opsonophagocytic killing. To identify these factors, we tooka whole-genome approach with a library of mutants generatedwith the mariner transposon and used ex vivo human neutro-phil killing assays to screen for mutants that were more sus-ceptible to neutrophil-mediated opsonophagocytic killing. Oneof the first genes identified by this screen encodes pneumococ-cal neuraminidase A (NanA), which catalyzes the release ofterminally linked �2-3 and �2-6-linked sialic acid residues(7, 26).

MATERIALS AND METHODS

Bacterial strains and growth conditions. The S. pneumoniae strains used inthis study are described in Table 1. Strains were routinely grown at 37°C eitherin C medium supplemented with 5% yeast extract (C�Y medium) at pH 6.8 orin tryptic soy (TS) broth (Becton, Dickinson, & Co., Sparks, MD). Bacteria werealso grown overnight at 37°C with 5% CO2 on TS plates containing 1.5% agarand 5,000 U of catalase (Worthington Biochemical Corporation, Freehold, NJ).When necessary, mutants were selected on TS that contained chloramphenicol(Cm) (2.5 �g/ml), spectinomycin (Sp) (200 �g/ml), kanamycin (Km) (500 �g/ml),or erythromycin (Erm) (1 �g/ml) as appropriate.

* Corresponding author. Mailing address: 402A Johnson Pavillion,Philadelphia, PA 19104-6076. Phone: (215) 573-3511. Fax: (215) 573-4856. E-mail: weiser@mail.med.upenn.edu.

� Published ahead of print on 16 February 2010.

2108

on October 4, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Mutation of exoglycosidases and creation of the NanA revertant strain. In-sertion-deletion mutants were created for the genes encoding NanA, BgaA, andStrH using the constructs described by King et al. (26, 27). Mariner mutants ofstrain TIGR4 were created by in vitro transposon mutagenesis as previouslydescribed (16).

To create the nanA revertant strain, the nanA gene plus 1 kb of flankinggenomic DNA from TIGR4 was PCR amplified using the primers nanAF3(5�-ATG CTA CAG TTG TGG TAA CGA TTA C-3�) and nanAR2 (5�-CATCAA CCA AAA AAT TGC TCA AAA G-3�). This product was then used totransform the T4 nanA strain of the pneumococcus to replace the insertion-deletion mutation with the wild-type (WT) copy of the gene. Transformationreaction mixtures were plated on TS without selection. The revertant strain wasthen negatively selected for by patching colonies onto TS plates with and withoutCm to screen for transformants that have lost the insertion-deletion mutation. ACm-sensitive transformant [T4 nanA(�nanA)] was then confirmed to have theWT copy of nanA by PCR and sequencing.

Isolation of neutrophils from human whole blood. Human neutrophils wereisolated as previously described (49). Briefly, heparinized whole blood fromhealthy human donors was run on a Polymorphprep gradient according to themanufacturer’s instructions (Axis-Shield, Oslo, Norway). The neutrophil-en-riched layer was collected and washed in Hanks buffer without Ca2� and Mg2�

(GIBCO, Auckland, New Zealand) plus 0.1% (wt/vol) gelatin (��� buffer).Contaminating red blood cells (RBCs) were removed by hypotonic lysis in 0.83%NH4Cl. Cells were counted using trypan blue staining and adjusted to a concen-tration of 7 � 106 cells/ml in Hanks buffer containing Ca2� and Mg2� (GIBCO)plus 0.1% gelatin (��� buffer) immediately before use.

Complement sources. Three- to 4-week baby rabbit serum (BRS) was ali-quoted and frozen at �80°C until use (Pel-Freez Biologicals, Rogers, AR).Normal human serum (NHS) was isolated from human whole blood. Blood wasallowed to clot at 37°C for 30 min and then centrifuged at 1,000 � g for 20 minat 4°C. The serum layer was collected, aliquoted, and frozen at �80°C until use.Where indicated, serum was incubated at 56°C for 30 min to inactivate comple-ment activity.

Opsonophagocytic killing assays. Neutrophil killing assays were performedessentially as previously described by Davis et al. (10). Briefly, 103 PBS-washedlate-log-phase bacteria (in 10 �l) were preopsonized in 20 �l of a complementsource (66% BRS or 10% NHS), followed by incubation with 105 neutrophils (40�l) in ��� buffer (130 �l). Reaction mixtures were then incubated at 37°C for45 min with rotation. Opsonophagocytic killing assays were stopped by incuba-tion in ice, and viable counts of bacteria were determined by dilution plating.Percent survival was determined relative to control reactions where no neutro-phils were added. For experiments performed on different days, the absolutelevel of killing varied but relative differences between strains did not. Therefore,to compile data from different experiments, results are shown relative to killingof the WT strain in each independent experiment. Where indicated, data areshown as log10 CFU/ml for both the reactions where neutrophils were added andthe control reactions where neutrophils were not added.

When specified, killing assays were modified. To complement the nanA mu-tant, 0.008 U of neuraminidase from Clostridium perfringens (Sigma Aldrich, St.Louis, MO) was added to serum immediately before addition of bacteria. Todetermine the role of complement receptor 3 in killing assays, a blocking anti-body to this receptor (CBRM1/5) or an isotype control antibody (MOPC21)(Biolegend, San Diego, CA) was added to neutrophils at a concentration of 25�g/106 neutrophils and incubated at 37°C for 30 min in ��� buffer prior to use

in killing assays. To assess the role of NanA in the absence of IgG, opsonizationreactions were performed with 20% IgG-depleted NHS. IgG was depleted fromNHS using Hi-Trap protein G columns (GE Healthcare, Uppsala, Sweden),which resulted in removal of �95% of IgG. To assess the effect of NanA on thealternative pathway of complement activation, we performed opsonization reac-tions in the presence of MgEGTA to inhibit the classical pathway as previouslydescribed (13, 42). Bacteria were opsonized in 30% IgG-depleted NHS in gelatinveronal buffer containing MgEGTA (GVB-MgEGTA) (Boston Bioproducts,Worcester, MA). Since MgEGTA buffer would interfere with neutrophil-medi-ated killing, opsonized bacteria were then removed from serum and GVB-MgEGTA by centrifugation and resuspended in ��� buffer prior to addition ofhuman neutrophils. IgG was depleted from NHS in these experiments usingHi-Trap protein G columns because agglutination of bacteria by IgG preventedcentrifugation of cells from opsonization reactions.

Cell association assays. Bacterial uptake assays were performed as previouslydescribed (32). Briefly, 107 PBS-washed bacteria grown to late log phase werelabeled with fluorescein isothiocyanate (FITC) (0.2 mg/ml) for 30 min at 37°C.Unbound FITC was removed by washing in ��� buffer, and 4 � 105 FITC-labeled bacteria were then opsonized in 10% NHS and incubated with 2 � 105

neutrophils at 37°C for 30 min. Reactions were stopped by placing samples on iceand fixed by adding an equal volume of a freshly made paraformaldehyde solu-tion (2% [wt/vol] in PBS). Uptake was assessed on a FACSCalibur flow cytom-eter, and at least 10,000 events were analyzed per sample. Percent cell associationwas determined relative to that in reactions where bacteria were preopsonized in10% heat-inactivated NHS (HI NHS). Controls using bacteria that were notopsonized or FITC stained showed results similar to those when bacteria wereopsonized in heat-inactivated serum.

Complement deposition assays. C3 deposition was analyzed essentially aspreviously described by Brown et al. (5). Briefly, 106 PBS-washed bacteria grownto late log phase were incubated in 100 �l of 100% BRS or 10% NHS for 30 minat 37°C. Reactions were stopped by placing samples on ice. Opsonized cells werethen washed and resuspended in Hanks balanced salt solution (HBSS) (GIBCO)containing 5% fetal calf serum (Sigma Aldrich). For samples opsonized in BRS,reaction mixtures were then incubated with a 1:100 dilution of an FITC-conju-gated polyclonal goat anti-rabbit C3 antibody (MP Biomedical Cappel, Irvine,CA) for 30 min on ice. For samples opsonized in NHS, reaction mixtures wereincubated with a 1:100 dilution of monoclonal antibody (MAb) 130.1, a mousemonoclonal antibody specific for human iC3b/C3b (50), for 30 min on ice,followed by incubation with an FITC anti-mouse IgG secondary antibody for 30min on ice (Sigma Aldrich). After incubation with antibodies, samples werewashed in PBS and fixed by resuspension in a freshly made paraformaldehydesolution (2% in PBS). C3 deposition was assessed on a FACSCalibur cell cy-tometer, and at least 10,000 events were analyzed per sample. C3 deposition wasdetermined relative to that in reactions where bacteria were opsonized in heat-inactivated serum. As for opsonophagocytic killing assays, results were maderelative to killing of the WT strain to compile data from different experiments.Controls where bacteria were not opsonized showed results similar to those forbacteria incubated in heat-inactivated serum.

Antibody binding assays. Antibody binding assays were performed essentiallyas previously described (65). Briefly, 107 CFU of PBS-washed bacteria wereopsonized in 1% NHS or 1% IgG-depleted NHS for 30 min on ice. Subsequently,bound IgG, IgA, and IgM were detected by incubating cells with FITC-conju-gated secondary antibodies that recognized each antibody isotype, i.e., anti-human IgG whole molecule, anti-human IgA �-chain specific, and anti-human

TABLE 1. S. pneumoniae strains used in this study

Strain Serotype Description Reference or source

T4 4 Clinical isolate TIGR4 51T4 cap Una �cps locus (Kmr) 55T4 nanA 4 �nanA (Cmr) This studyT4 bgaA 4 �bgaA (Ermr) This studyT4 strH 4 �strH (Spr) This studyT4 nanA(�nanA) 4 �nanA mutation replaced with WT copy of nanA from TIGR4 This studyT4 nanA nanB 4 �nanA (Cmr) �nanB rpsL (K56T) (Smr) 6T4 nanA::mar 4 nanA disrupted by the mariner Tn This studyT4 nanA bgaA strH 4 �nanA (Cmr) �bgaA (Ermr) �strH (Spr) This study6Atr 6A Transparent variant of clinical isolate P303 256Atr nanA 6A �nanA (Cmr) This study

a Un, unencapsulated.

VOL. 78, 2010 EXOGLYCOSIDASES INHIBIT NEUTROPHIL-MEDIATED KILLING 2109

on October 4, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

IgM �-chain specific (Sigma Aldrich), for 1 h on ice. Cells were washed withHBSS containing 5% fetal calf serum, pelleted, and resuspended in a freshlymade paraformaldehyde solution (2%). Antibody binding was analyzed on aFACSCalibur cell cytometer, and at least 10,000 events were analyzed per sam-ple. Antibody binding was determined relative to that in reactions where unop-sonized bacteria were incubated with the appropriate FITC-conjugated second-ary antibodies.

Western blot analysis. NHS or IgG-depleted NHS was diluted 1:10 in SDSsample buffer, and 20 �l per sample was loaded and separated by SDS-poly-acrylamide gel electrophoresis (PAGE) on a 10% polyacrylamide gel. Proteinswere then transferred to polyvinylidene difluoride (PVDF) transfer membranes(Thermo Scientific). Membranes were then probed with an alkaline phos-phatase-conjugated anti-human IgG Fc-specific secondary antibody (SigmaAldrich) diluted 1:10,000 for 1 h at room temperature. The presence of IgGin sera was detected with 4-nitroblue tetrazolium chloride–5-bromo-4-chloro-3-indolylphosphate.

Statistical analysis. Statistical differences between groups were assessed by anunpaired Student’s two-tailed t test (GraphPad PRISM 4; GraphPad Software).

RESULTS

NanA promotes resistance to opsonophagocytic killing. Toidentify novel bacterial factors that promote resistance to op-sonophagocytic killing, we screened mariner transposon mu-tants of the TIGR4 strain of S. pneumoniae (T4) for increasedsusceptibility to killing by human neutrophils. This was as-sessed using established ex vivo assays where bacteria werepreopsonized in serum and then incubated with human neu-trophils. During the initial screen, we identified that nanA,encoding the major pneumococcal neuraminidase, was impor-tant for promoting resistance to opsonophagocytic killing.

This phenotype was studied using two sources of serum toopsonize bacteria. The initial identification of the nanA mutantwas performed using baby rabbit serum (BRS), as this was asource of complement without specific antipneumococcal an-tibodies (Fig. 1A). These results were then confirmed andfurther analyzed using normal human serum (NHS) to opson-ize bacteria (Fig. 1B).

The role of NanA in promoting resistance to opsonophago-

cytic killing was shown with the mariner mutant identified inthe screen (T4 nanA::mar), as well as with a previously char-acterized insertion-deletion mutant (T4 nanA) (Fig. 1A) (6).The TIGR4 strain of the pneumococcus has an authenticframeshift resulting in secretion of NanA in this strain (51). Toconfirm that secretion of NanA was not required for this phe-notype, a 6A strain that expresses NanA with an intact cellwall-anchoring domain was also studied (26). As in TIGR4,NanA promoted resistance to opsonophagocytic killing in the6A strain (Fig. 1A and B, white bars).

The neuraminidase activity of NanA was required for thisphenotype, because the addition of purified neuraminidasefrom Clostridium perfringens to killing assay mixtures comple-mented a nanA mutant (Fig. 1A, bars labeled �Nan). Addi-tional evidence that the mutation in nanA is responsible forthis phenotype was provided by testing a revertant strain [T4nanA(�nanA)]. Correction of the mutation in nanA restoredWT levels of resistance to opsonophagocytic killing (Fig. 1B).The phenotype of a nanA mutant is not due to differences ingrowth rate, as growth was similar to that of the WT in bothserum and nutrient media (data not shown).

An unencapsulated mutant, T4 cap, is shown as a positivecontrol for killing by human neutrophils to highlight thecontribution of NanA to resistance relative to this majorantiphagocytic factor (Fig. 1A). To ensure that differencesin amounts of capsular polysaccharide/cell were not respon-sible for the phenotype seen in a nanA mutant, we per-formed a quantitative capture enzyme-linked immunosor-bent assay (ELISA) and found equivalent levels of capsularpolysaccharide in the nanA mutant and WT bacteria (datanot shown) (25).

TIGR4 expresses two other neuraminidases, NanB andNanC (51). NanB is present in 96% of S. pneumoniae strains,while NanC is expressed in fewer than 51% of strains (6, 39).NanC is expressed by TIGR4 but not by the 6A strain, sug-gesting that NanC is not required for this phenotype, since astrain lacking it can still be resistant to opsonophagocytic kill-ing (1A and B, white bars). Additionally, NanB does not sig-nificantly contribute to resistance to opsonophagocytic killing,since a nanA nanB double mutant is not more susceptible toneutrophil killing than a nanA mutant (Fig. 1B).

Killing of S. pneumoniae is dependent on opsonization bycomplement. To further elucidate the mechanism for the effecton opsonophagocytic killing by NanA, we looked at bacterialuptake by neutrophils. Assaying for both adherent and in-gested bacteria, we found that the nanA mutant was signifi-cantly more neutrophil associated than the WT strain (Fig.2A). Therefore, NanA acts upstream of uptake by humanneutrophils, possibly at the level of opsonization.

Opsonization by complement is a major mechanism for bac-terial uptake. Complement components are heat labile, so therole of complement in opsonophagocytic killing of S. pneu-moniae was first assessed using heat-inactivated human serum(HI NHS). When HI NHS was used to opsonize bacteria,killing of S. pneumoniae strains was almost completely abro-gated (Fig. 2B). The role of complement in neutrophil-medi-ated opsonophagocytic killing of S. pneumoniae was also as-sessed using an antibody that blocks the major complementreceptor responsible for phagocytosis, complement receptor 3(CR3). As for heat-inactivated serum, killing of S. pneumoniae

FIG. 1. Survival of S. pneumoniae in neutrophil killing assays,showing comparisons of WT and nanA mutant strains in two indepen-dent backgrounds (TIGR4 and 6Atr). (A) Opsonophagocytic killing ofS. pneumoniae by human neutrophils after bacteria were preopsonizedin 66% BRS. Biochemical complementation (�Nan) was performedby addition of 0.008 U of C. perfringens neuraminidase per reaction. Allresults are relative to killing of the WT strain (T4 [black bars] or 6Atr[white bars]). (B) Opsonophagocytic killing of S. pneumoniae by hu-man neutrophils after bacteria were preopsonized in 10% NHS. Dataare the means from at least three independent experiments performedin duplicate standard errors of the means (SEM). *, P 0.05; ***,P 0.001; NS, not significant (compared to WT or as indicated).

2110 DALIA ET AL. INFECT. IMMUN.

on October 4, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

by human neutrophils was almost completely inhibited in thepresence of the CR3-blocking antibody. This suggested thatcomplement component C3 was the major opsonin in NHSresponsible for killing by human neutrophils (Fig. 2C).

NanA promotes resistance to complement deposition. Thelevel of C3b deposition on S. pneumoniae strains was assessedfollowing opsonization by BRS or NHS using flow cytometry.As previously described, complement deposition on S. pneu-moniae has a bimodal distribution (5), where cells either haveC3 deposited or do not (Fig. 3A and C). In both BRS andNHS, a nanA mutant had a greater proportion of cells thatwere C3 positive than the WT (Fig. 3A to D).

NanA acts on the alternative pathway of complement acti-vation. C3 deposition on the pneumococcus occurs throughactivation of both the classical and alternative pathways ofcomplement (4, 5). To identify whether NanA acts on thealternative pathway, we performed opsonophagocytic killingassays using serum that was treated to prevent the activation ofthe classical pathway, as described in Materials and Methods.When the classical pathway was inhibited, there was still asignificant effect of nanA, suggesting that NanA acts to preventC3 deposition by the alternative pathway of complement acti-vation (Fig. 4).

NanA does not act on antibodies in NHS to promote resis-tance to opsonophagocytic killing. Interestingly, the phenotypeof a nanA mutant in NHS is more dramatic than that seen inBRS. One of the major differences between these serum

FIG. 2. NanA acts upstream of phagocytosis, and complement isthe predominant opsonin responsible for opsonophagocytic killing ofS. pneumoniae. (A) Uptake of S. pneumoniae strains by human neu-trophils. FITC-labeled bacteria were preopsonized in 10% NHS, fol-lowed by incubation with human neutrophils. The proportion of ad-herent and ingested pneumococci was assessed by flow cytometry. (Band C) The contribution of complement in the killing of S. pneumoniaewas shown by performing opsonophagocytic killing assays using heat-inactivated NHS (HI NHS) to opsonize bacteria (B) and by performingassays in the presence of a blocking antibody to CR3 (CBRM1/5) or acontrol antibody (Ct Ab) (C). Gray boxes in panels B and C representreactions where neutrophils were added, while white boxes representcontrol reactions where no neutrophils were added. Box-and-whiskersplots indicate high and low values, median, and interquartile ranges.Data are the results of at least two independent experiments per-formed in duplicate. *, P 0.05; **, P 0.01; ***, P 0.001; NS, notsignificant.

FIG. 3. C3 deposition on S. pneumoniae in NHS and BRS. Com-plement deposition was analyzed by flow cytometry. (A and B) Bacte-ria were preopsonized in 10% NHS, and C3 deposition was assessedusing MAb 130.1, a monoclonal antibody specific for C3 breakdownproducts. All results are relative to bacteria preopsonized in 10%heat-inactivated NHS. (C and D) Bacteria were preopsonized in 20%BRS, and C3 deposition was assessed using a polyclonal antibody toC3. All results are relative to bacteria preopsonized in 20% heat-inactivated BRS. (A and C) Means from three independent experi-ments SEM. **, P 0.01 compared to T4. (B and D) Representa-tive histograms of the data comparing complement deposition on T4(black lines), T4 nanA (gray lines), and T4 opsonized in heat-inacti-vated serum (gray fill).

VOL. 78, 2010 EXOGLYCOSIDASES INHIBIT NEUTROPHIL-MEDIATED KILLING 2111

on October 4, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

sources is the presence of specific antipneumococcal antibod-ies, which are potent activators of the complement system.NHS contains specific antipneumococcal IgG �� IgA � IgM,and bacterial recognition by these antibodies is not affected by

NanA (Fig. 5A to C). Under similar conditions, no antipneu-mococcal immunoglobulins were detected in BRS (data notshown). As NanA displayed a significant effect on complementdeposition in NHS (Fig. 3A and B), we hypothesized that onepotential mechanism for this was via deglycosylation of anti-bodies. To test the contribution of IgG in our assays, we de-pleted IgG from NHS (Fig. 5D). However, even using IgG-depleted NHS we still saw a significant effect of NanA inopsonophagocytic killing assays (Fig. 5E). Next, we wanted todetermine if any of the other antibody isotypes in NHS couldaccount for the difference in phenotype observed in NHS ver-sus BRS. This was assessed by adding heat-inactivated NHS asa source of antibodies to BRS. The phenotype of a nanAmutant in this mixture of heat-inactivated NHS and BRS wassimilar to that observed in BRS alone (Fig. 5F). This suggeststhat a heat-labile component of NHS is responsible for theincreased phenotype of a nanA mutant in this serum source.

NanA acts with two other exoglycosidases from S. pneu-moniae to promote resistance to opsonophagocytic killing andcomplement deposition. It has previously been demonstratedthat NanA can act with a �-galactosidase, BgaA, and an N-acetylglucosaminidase, StrH, to sequentially remove sugarscommonly found on the N-linked glycans of human glycocon-jugates (the prototypic structure is shown in Fig. 6A) (6, 27).

FIG. 4. NanA has an effect on the alternative pathway of complementactivation. To assess killing by the alternative pathway, opsonization wascarried out with 30% IgG-depleted NHS in GVB-MgEGTA buffer. Dataare the means from at least three independent experiments performed induplicate SEM. ***, P 0.001 compared to WT.

FIG. 5. Contribution of antibodies on the effect of NanA in NHS. (A to C) Levels of IgG (A), IgA (B), and IgM (C) in NHS were assessedby antibody binding assays on both T4 (black lines) and T4 nanA (gray lines) cells relative to unopsonized cells (gray fill). (D) Western blotdetecting IgG heavy chain presence in NHS (1) and IgG-depleted NHS (2). (E) IgG-depleted serum was used to opsonize bacteria in opsonophago-cytic killing assays. (F) Opsonophagocytic killing assays where bacteria were opsonized in either 10% NHS, 66% BRS, or a mixture of 10% BRSand 10% HI NHS (as a source of antibodies) to determine the role of antibodies in mediating the difference in phenotype observed in NHS versusBRS. Data are the means from at least two independent experiments performed in duplicate SEM. ***, P 0.001 compared to WT.

2112 DALIA ET AL. INFECT. IMMUN.

on October 4, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Therefore, we examined whether these other exoglycosidasesalso played a role in promoting resistance to opsonophagocytickilling. bgaA and strH mutants both showed a phenotype sim-ilar to that of a nanA mutant in opsonophagocytic killing andcomplement deposition assays (Fig. 6B and C). Additionally, atriple mutant with mutations in all three exoglycosidase genesshowed a phenotype similar to that of each single mutant inboth assays, supporting the hypothesis that these three exogly-cosidases work on the same pathway/target (Fig. 6B and C).

These three enzymes act exclusively on terminally linked sub-strates to sequentially remove sialic acid that is �2-3 or �2-6linked to galactose (NanA), galactose that is �1-4 linked toN-acetylglucosamine (BgaA), and N-acetylglucosamine that is�1 linked to mannose (StrH) on human glycoconjugates (Fig.6A) (27). Therefore, we conclude that the sequential activity ofthe three pneumococcal exoglycosidases promotes resistanceto opsonophagocytic killing by deglycosylating a human glyco-protein(s) important for complement deposition.

DISCUSSION

Opsonophagocytic killing by neutrophils represents an im-portant mechanism for clearance of pneumococcal infection(34). While some pneumococcal factors are known to inhibitopsonophagocytosis, a comprehensive search to identify allfactors involved in this resistance has not been performed (20).Using a whole-genome approach, we identified that neuramin-idase A (NanA) is important for resistance to opsonophago-cytic killing by human neutrophils. Furthermore, we identifiedthat NanA promotes resistance in conjunction with two otherexoglycosidases from the pneumococcus, BgaA and StrH, byreducing complement deposition on the bacterial surface.

NanA is a well-characterized and ubiquitously expressedvirulence factor in S. pneumoniae strains, and its role in thepathogenesis of the pneumococcus has been studied exten-sively. The effect of NanA on pathogenesis in vivo has beenattributed to its various roles observed in vitro. NanA is able toremove sialic acid to expose receptors to aid pneumococcaladherence, directly bind epithelial cells via a lectin domain, aidin formation of biofilms, desialylate the surface of its compet-ing nasopharyngeal flora, deglycosylate human glycoconju-gates, and liberate carbohydrates to aid metabolic fitness of theorganism (6, 27, 38, 48, 53, 54, 57). In vivo, a role for NanA incolonization and sepsis is less clear and is dependent upon theanimal model employed (26, 52, 54). In contrast, during in-flammatory diseases, such as pneumonia and otitis media,NanA has a more clear-cut role in the pathogenesis of thisorganism (33, 37, 52). These infections result in a robust influxof neutrophils and the serum components necessary for opson-ization (2, 3, 15, 22, 23, 34, 58). Therefore, the effect of NanAin promoting resistance to opsonophagocytic killing demon-strated in this study could help explain the effect of this viru-lence factor observed in vivo.

NanA does not, however, appear to be acting alone to pro-mote resistance to opsonophagocytic killing. NanA has previ-ously been shown to act together with BgaA and StrH, twoother surface-anchored exoglycosidases in the pneumococcus,which remove galactose that is �1-4 linked to N-acetylglu-cosamine (GlcNAc) and GlcNAc that is �1 linked to mannose,respectively (6, 9, 27, 28). All three enzymes act exclusively onterminally linked substrates, and recently it was shown thatthese exoglycosidases could sequentially remove sugars fromcomplex N-linked glycans (27). Additionally, these enzymesare surface associated and have been shown to be more effec-tive at deglycosylating a substrate when bound to the bacterialsurface (7, 9, 24, 26, 27, 64). Glycosylation of host proteins canaffect their stability, resistance to proteolysis, and functionalactivity (43). Therefore, it is possible that deglycosylation of ahost glycoconjugate by the action of these three exoglycosi-

FIG. 6. Role of two other surface-exposed exoglycosidases of S.pneumoniae, BgaA and StrH, in resistance to opsonophagocytic killingby neutrophils. (A) Prototypic structure of complex N-linked bianten-nary glycans present on human glycoconjugates. Cleavage sites for thethree pneumococcal exoglycosidases are indicated by arrows. (B) Neu-trophil killing assays of S. pneumoniae, using 10% NHS to preopsonizebacteria. (C) C3 deposition assays of S. pneumoniae preopsonized in10% NHS. Data are the means from three independent experiments SEM. *, P 0.05; **, P 0.01; ***, P 0.001 (compared to T4).

VOL. 78, 2010 EXOGLYCOSIDASES INHIBIT NEUTROPHIL-MEDIATED KILLING 2113

on October 4, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

dases is important for pneumococcal virulence in vivo. This issupported by the fact that these exoglycosidases deglycosylatehuman secretory component, immunoglobulin A, and lactofer-rin, three human glycoconjugates thought to be important forpneumococcal clearance (27). The functional consequence ofthe deglycosylation of these host proteins or of other hostproteins by these exoglycosidases, however, has not been ex-amined.

In this study, we show that NanA, BgaA, and StrH work onthe same pathway/target to promote resistance to opsono-phagocytic killing by reducing complement deposition on thepneumococcus. As these enzymes function as exoglycosidases,we concluded that deglycosylation of a host glycoconjugate(s)promotes this resistance.

Using antibody binding assays and blocking antibodies tocomplement receptor 3, we showed that NHS is a source ofboth complement and antipneumococcal antibodies. Glycosyl-ation of antibodies is known to be critical for their ability to fixcomplement (1, 21, 62); therefore, we wanted to test the hy-pothesis that deglycosylation of antibodies could help promoteresistance to complement deposition. Using serum depleted ofIgG, the predominant antibody isotype in NHS, we still saw asignificant effect of NanA in opsonophagocytic killing assays.This suggests that IgG is not necessary for the effect of NanAin NHS. Additionally, using heat-inactivated NHS as a sourceof antibodies, we did not see an increase in phenotype for ananA mutant when added to BRS. This suggests that a heat-labile component in NHS is responsible for promoting thegreater effect of nanA in this serum source. Therefore, thedifference between NHS and BRS could be due to increasedexoglycosidase substrate specificity, since S. pneumoniae is apathogen adapted to humans and glycosylation patterns canvary between species (17, 29, 40). Therefore, we believe thatantibodies in NHS contribute to the deposition of complementon the pneumococcal surface but are not being acted upon bypneumococcal exoglycosidases. This is further supported bythe fact that we do not see deglycosylation of human IgG usinglectins that bind specifically to terminal mannose (data notshown). Thus, we conclude that deglycosylation of a serumcomponent in NHS downstream of antibody binding and sub-sequent complement activation is important for resistance tocomplement deposition on the pneumococcus.

There has been a recent interest in studying glycosylation ofcomplement components, and in some instances glycosylationis important for the function of these proteins (11, 19, 44, 46,56). Most complement components are synthesized predomi-nantly in the liver and contain complex biantennary glycans, asdisplayed in Fig. 6A (44). Therefore, deglycosylation of a com-plement component by NanA, BgaA, and StrH could providea direct mechanism to promote resistance to complement de-position. Reducing the function of complement components,however, is only one mechanism whereby deglycosylation candirectly affect complement deposition. Glycosylation can alsobe important for resistance to proteolysis; therefore, deglyco-sylation of a complement component(s) could increase its turn-over by increasing its susceptibility to serum proteases (47).Opsonophagocytic killing assays looking at the effect of NanAon the alternative pathway suggest that deglycosylation affectsat least this pathway of complement activation (Fig. 4). Thisdoes not, however, rule out a role for pneumococcal exogly-

cosidases in the classical pathway. In fact, since the effect ofNanA on the alternative pathway was not as dramatic as thatseen in complete NHS (containing both classical and alterna-tive pathways intact) (Fig. 4), this suggests that NanA may havean effect on the classical pathway as well. Both the classical andalternative pathways of complement activation converge at theformation of the C3 convertase and deposition of C3 onto thebacterial surface. As C3 is shared by both complement path-ways, it would seem to be a likely target for these exoglycosi-dases; however, this complement component does not havecomplex-type N-linked glycans (44). Therefore, the pneumo-coccal exoglycosidases could act upstream on a regulatorycomponent of complement that affects C3 deposition. Alter-natively, this resistance could require deglycosylation of mul-tiple complement components or have an indirect role in pro-moting resistance to opsonophagocytic killing. With over 30serum proteins involved in the complement cascade, however,pinpointing the factor reducing complement deposition is com-plex.

In summary, we have shown that NanA, BgaA, and StrHpromote resistance to opsonophagocytic killing by increasingthe ability of the pneumococcus to evade complement deposi-tion and subsequent phagocytic killing. As complement con-tributes to both antibody-dependent and -independent clear-ance, this may be a mechanism to limit the effectiveness of bothinnate and adaptive immunity. Since glycoside hydrolases arenot a unique feature of S. pneumoniae, deglycosylation may bea conserved strategy to evade the immune response.

ACKNOWLEDGMENTS

We gratefully acknowledge Samantha King for providing the exo-glycosidase mutants, John Lambris for providing MAb 130.1, andErnesto Munoz-Elías and Ramkumar Iyer for providing technical as-sistance and reagents for mariner transposon mutagenesis. We alsothank Samantha King and John Lambris for helpful discussions.

This work was supported by Public Health Service grants AI44231and AI38446 to J.N.W.

REFERENCES

1. Arnold, J. N., M. R. Wormald, R. B. Sim, P. M. Rudd, and R. A. Dwek. 2007.The impact of glycosylation on the biological function and structure ofhuman immunoglobulins. Annu. Rev. Immunol. 25:21–50.

2. Bolger, M. S., D. S. Ross, H. Jiang, M. M. Frank, A. J. Ghio, D. A. Schwartz,and J. R. Wright. 2007. Complement levels and activity in the normal andLPS-injured lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 292:L748–L759.

3. Broides, A., E. Leibovitz, R. Dagan, J. Press, S. Raiz, M. Kafka, A. Leiber-man, and T. Yermiahu. 2002. Cytology of middle ear fluid during acute otitismedia. Pediatr. Infect. Dis. J. 21:57–61.

4. Brouwer, N., K. M. Dolman, M. van Houdt, M. Sta, D. Roos, and T. W.Kuijpers. 2008. Mannose-binding lectin (MBL) facilitates opsonophagocy-tosis of yeasts but not of bacteria despite MBL binding. J. Immunol. 180:4124–4132.

5. Brown, J. S., T. Hussell, S. M. Gilliland, D. W. Holden, J. C. Paton, M. R.Ehrenstein, M. J. Walport, and M. Botto. 2002. The classical pathway is thedominant complement pathway required for innate immunity to Streptococ-cus pneumoniae infection in mice. Proc. Natl. Acad. Sci. U. S. A. 99:16969–16974.

6. 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.

7. Camara, M., G. J. Boulnois, P. W. Andrew, and T. J. Mitchell. 1994. Aneuraminidase from Streptococcus pneumoniae has the features of a surfaceprotein. Infect. Immun. 62:3688–3695.

8. Caron, E., and A. Hall. 1998. Identification of two distinct mechanisms ofphagocytosis controlled by different Rho GTPases. Science 282:1717–1721.

9. Clarke, V. A., N. Platt, and T. D. Butters. 1995. Cloning and expression of thebeta-N-acetylglucosaminidase gene from Streptococcus pneumoniae. Gen-eration of truncated enzymes with modified aglycon specificity. J. Biol.Chem. 270:8805–8814.

2114 DALIA ET AL. INFECT. IMMUN.

on October 4, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

10. Davis, K. M., H. T. Akinbi, A. J. Standish, and J. N. Weiser. 2008. Resistanceto mucosal lysozyme compensates for the fitness deficit of peptidoglycanmodifications by Streptococcus pneumoniae. PLoS Pathog. 4:e1000241.

11. Fenaille, F., M. Le Mignon, C. Groseil, C. Ramon, S. Riande, L. Siret, andN. Bihoreau. 2007. Site-specific N-glycan characterization of human comple-ment factor H. Glycobiology 17:932–944.

12. Figueroa, J. E., and P. Densen. 1991. Infectious diseases associated withcomplement deficiencies. Clin. Microbiol. Rev. 4:359–395.

13. Fine, D. P. 1977. Comparison of ethyleneglycoltetraacetic acid and its mag-nesium salt as reagents for studying alternative complement pathway func-tion. Infect. Immun. 16:124–128.

14. Gordon, D. L., G. M. Johnson, and M. K. Hostetter. 1986. Ligand-receptorinteractions in the phagocytosis of virulent Streptococcus pneumoniae bypolymorphonuclear leukocytes. J. Infect. Dis. 154:619–626.

15. Gross, G. N., S. R. Rehm, and A. K. Pierce. 1978. The effect of complementdepletion on lung clearance of bacteria. J. Clin. Invest. 62:373–378.

16. Hava, D. L., and A. Camilli. 2002. Large-scale identification of serotype 4Streptococcus pneumoniae virulence factors. Mol. Microbiol. 45:1389–1406.

17. Hironaka, T., K. Furukawa, P. C. Esmon, T. Yokota, J. E. Brown, S. Sawada,M. A. Fournel, M. Kato, T. Minaga, and A. Kobata. 1993. Structural study ofthe sugar chains of porcine factor VIII: tissue- and species-specific glyco-sylation of factor VIII. Arch. Biochem. Biophys. 307:316–330.

18. Hostetter, M. K. 1986. Serotypic variations among virulent pneumococci indeposition and degradation of covalently bound C3b: implications for phago-cytosis and antibody production. J. Infect. Dis. 153:682–693.

19. Inforzato, A., G. Peri, A. Doni, C. Garlanda, A. Mantovani, A. Bastone, A.Carpentieri, A. Amoresano, P. Pucci, A. Roos, M. R. Daha, S. Vincenti, G.Gallo, P. Carminati, R. De Santis, and G. Salvatori. 2006. Structure andfunction of the long pentraxin PTX3 glycosidic moiety: fine-tuning of theinteraction with C1q and complement activation. Biochemistry 45:11540–11551.

20. Jarva, H., T. S. Jokiranta, R. Wurzner, and S. Meri. 2003. Complementresistance mechanisms of streptococci. Mol. Immunol. 40:95–107.

21. Jefferis, R., and J. Lund. 1997. Glycosylation of antibody molecules: struc-tural and functional significance. Chem. Immunol. 65:111–128.

22. Kawana, M., C. Kawana, T. Yokoo, P. G. Quie, and G. S. Giebink. 1991.Oxidative metabolic products released from polymorphonuclear leukocytesin middle ear fluid during experimental pneumococcal otitis media. Infect.Immun. 59:4084–4088.

23. Kerr, A. R., G. K. Paterson, A. Riboldi-Tunnicliffe, and T. J. Mitchell. 2005.Innate immune defense against pneumococcal pneumonia requires pulmo-nary complement component C3. Infect. Immun. 73:4245–4252.

24. Kharat, A. S., and A. Tomasz. 2003. Inactivation of the srtA gene affectslocalization of surface proteins and decreases adhesion of Streptococcuspneumoniae to human pharyngeal cells in vitro. Infect. Immun. 71:2758–2765.

25. Kim, J. O., and J. N. Weiser. 1998. Association of intrastrain phase variationin quantity of capsular polysaccharide and teichoic acid with the virulence ofStreptococcus pneumoniae. J. Infect. Dis. 177:368–377.

26. 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 protectthe airway. Mol. Microbiol. 54:159–171.

27. 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.

28. Kojima, K., M. Iwamori, S. Takasaki, K. Kubushiro, S. Nozawa, R. Iizuka,and Y. Nagai. 1987. Diplococcal beta-galactosidase with a specificity reactingto beta 1-4 linkage but not to beta 1-3 linkage as a useful exoglycosidase forthe structural elucidation of glycolipids. Anal. Biochem. 165:465–469.

29. Kuster, B., A. P. Hunter, S. F. Wheeler, R. A. Dwek, and D. J. Harvey. 1998.Structural determination of N-linked carbohydrates by matrix-assisted laserdesorption/ionization-mass spectrometry following enzymatic release withinsodium dodecyl sulphate-polyacrylamide electrophoresis gels: application tospecies-specific glycosylation of alpha1-acid glycoprotein. Electrophoresis19:1950–1959.

30. Lambris, J. D., D. Ricklin, and B. V. Geisbrecht. 2008. Complement evasionby human pathogens. Nat. Rev. Microbiol. 6:132–142.

31. Le Cabec, V., S. Carreno, A. Moisand, C. Bordier, and I. Maridonneau-Parini. 2002. Complement receptor 3 (CD11b/CD18) mediates type I andtype II phagocytosis during nonopsonic and opsonic phagocytosis, respec-tively. J. Immunol. 169:2003–2009.

32. Lu, L., Z. Ma, T. S. Jokiranta, A. R. Whitney, F. R. DeLeo, and J. R. Zhang.2008. Species-specific interaction of Streptococcus pneumoniae with humancomplement factor H. J. Immunol. 181:7138–7146.

33. 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.

34. Matthias, K. A., A. M. Roche, A. J. Standish, M. Shchepetov, and J. N.Weiser. 2008. Neutrophil-toxin interactions promote antigen delivery and

mucosal clearance of Streptococcus pneumoniae. J. Immunol. 180:6246–6254.

35. Nelson, A. L., A. M. Roche, J. M. Gould, K. Chim, A. J. Ratner, and J. N.Weiser. 2007. Capsule enhances pneumococcal colonization by limiting mu-cus-mediated clearance. Infect. Immun. 75:83–90.

36. O’Brien, K. L., L. J. Wolfson, J. P. Watt, E. Henkle, M. Deloria-Knoll, N.McCall, E. Lee, K. Mulholland, O. S. Levine, and T. Cherian. 2009. Burdenof disease caused by Streptococcus pneumoniae in children younger than 5years: global estimates. Lancet 374:893–902.

37. 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.

38. Parker, D., G. Soong, P. Planet, J. Brower, A. J. Ratner, and A. Prince. 2009.The NanA neuraminidase of Streptococcus pneumoniae is involved in bio-film formation. Infect. Immun. 77:3722–3730.

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

40. Raju, T. S., J. B. Briggs, S. M. Borge, and A. J. Jones. 2000. Species-specificvariation in glycosylation of IgG: evidence for the species-specific sialylationand branch-specific galactosylation and importance for engineering recom-binant glycoprotein therapeutics. Glycobiology 10:477–486.

41. Rambach, G., R. Wurzner, and C. Speth. 2008. Complement: an efficientsword of innate immunity. Contrib. Microbiol. 15:78–100.

42. Ren, B., A. J. Szalai, S. K. Hollingshead, and D. E. Briles. 2004. Effects ofPspA and antibodies to PspA on activation and deposition of complement onthe pneumococcal surface. Infect. Immun. 72:114–122.

43. Reuter, G., and H. J. Gabius. 1999. Eukaryotic glycosylation: whim of natureor multipurpose tool? Cell Mol. Life Sci. 55:368–422.

44. Ritchie, G. E., B. E. Moffatt, R. B. Sim, B. P. Morgan, R. A. Dwek, and P. M.Rudd. 2002. Glycosylation and the complement system. Chem. Rev. 102:305–320-319.

45. Ross, S. C., and P. Densen. 1984. Complement deficiency states and infec-tion: epidemiology, pathogenesis and consequences of neisserial and otherinfections in an immune deficiency. Medicine (Baltimore) 63:243–273.

46. Rudd, P. M., T. Elliott, P. Cresswell, I. A. Wilson, and R. A. Dwek. 2001.Glycosylation and the immune system. Science 291:2370–2376.

47. Rudd, P. M., H. C. Joao, E. Coghill, P. Fiten, M. R. Saunders, G. Opdenak-ker, and R. A. Dwek. 1994. Glycoforms modify the dynamic stability andfunctional activity of an enzyme. Biochemistry 33:17–22.

48. Shakhnovich, E. A., S. J. King, and J. N. Weiser. 2002. Neuraminidaseexpressed by Streptococcus pneumoniae desialylates the lipopolysaccharideof Neisseria meningitidis and Haemophilus influenzae: a paradigm for in-terbacterial competition among pathogens of the human respiratory tract.Infect. Immun. 70:7161–7164.

49. Standish, A. J., and J. N. Weiser. 2009. Human neutrophils kill Streptococ-cus pneumoniae via serine proteases. J. Immunol. 183:2602–2609.

50. Tamerius, J. D., M. K. Pangburn, and H. J. Muller-Eberhard. 1985. Detec-tion of a neoantigen on human C3bi and C3d by monoclonal antibody.J. Immunol. 135:2015–2019.

51. Tettelin, H., K. E. Nelson, I. T. Paulsen, J. A. Eisen, T. D. Read, S. Peterson,J. Heidelberg, R. T. DeBoy, D. H. Haft, R. J. Dodson, A. S. Durkin, M.Gwinn, J. F. Kolonay, W. C. Nelson, J. D. Peterson, L. A. Umayam, O. White,S. L. Salzberg, M. R. Lewis, D. Radune, E. Holtzapple, H. Khouri, A. M.Wolf, T. R. Utterback, C. L. Hansen, L. A. McDonald, T. V. Feldblyum, S.Angiuoli, T. Dickinson, E. K. Hickey, I. E. Holt, B. J. Loftus, F. Yang, H. O.Smith, J. C. Venter, B. A. Dougherty, D. A. Morrison, S. K. Hollingshead,and C. M. Fraser. 2001. Complete genome sequence of a virulent isolate ofStreptococcus pneumoniae. Science 293:498–506.

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

53. Tong, H. H., M. James, I. Grants, X. Liu, G. Shi, and T. F. DeMaria. 2001.Comparison of structural changes of cell surface carbohydrates in theeustachian tube epithelium of chinchillas infected with a Streptococcuspneumoniae neuraminidase-deficient mutant or its isogenic parent strain.Microb. Pathog. 31:309–317.

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. Trzcinski, K., C. M. Thompson, and M. Lipsitch. 2003. Construction ofotherwise isogenic serotype 6B, 7F, 14, and 19F capsular variants of Strep-tococcus pneumoniae strain TIGR4. Appl. Environ. Microbiol. 69:7364–7370.

56. Tsiftsoglou, S. A., J. N. Arnold, P. Roversi, M. D. Crispin, C. Radcliffe, S. M.Lea, R. A. Dwek, P. M. Rudd, and R. B. Sim. 2006. Human complementfactor I glycosylation: structural and functional characterisation of the N-linked oligosaccharides. Biochim. Biophys. Acta 1764:1757–1766.

57. Uchiyama, S., A. F. Carlin, A. Khosravi, S. Weiman, A. Banerjee, D. Quach,G. Hightower, T. J. Mitchell, K. S. Doran, and V. Nizet. 2009. The surface-

VOL. 78, 2010 EXOGLYCOSIDASES INHIBIT NEUTROPHIL-MEDIATED KILLING 2115

on October 4, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

anchored NanA protein promotes pneumococcal brain endothelial cell in-vasion. J. Exp. Med. 206:1845–1852.

58. van Rossum, A. M., E. S. Lysenko, and J. N. Weiser. 2005. Host and bacterialfactors contributing to the clearance of colonization by Streptococcus pneu-moniae in a murine model. Infect. Immun. 73:7718–7726.

59. Watson, D. A., and D. M. Musher. 1990. Interruption of capsule productionin Streptococcus pneumoniae serotype 3 by insertion of transposon Tn916.Infect. Immun. 58:3135–3138.

60. Weinberger, D. M., K. Trzcinski, Y. J. Lu, D. Bogaert, A. Brandes, J.Galagan, P. W. Anderson, R. Malley, and M. Lipsitch. 2009. Pneumo-coccal capsular polysaccharide structure predicts serotype prevalence.PLoS Pathog. 5:e1000476.

61. Winkelstein, J. A., A. S. Abramovitz, and A. Tomasz. 1980. Activation of C3via the alternative complement pathway results in fixation of C3b to thepneumococcal cell wall. J. Immunol. 124:2502–2506.

62. Wright, A., and S. L. Morrison. 1998. Effect of C2-associated carbohydratestructure on Ig effector function: studies with chimeric mouse-human IgG1antibodies in glycosylation mutants of Chinese hamster ovary cells. J. Im-munol. 160:3393–3402.

63. Yuste, J., A. Sen, L. Truedsson, G. Jonsson, L. S. Tay, C. Hyams, H. E.Baxendale, F. Goldblatt, M. Botto, and J. S. Brown. 2008. Impaired op-sonization with C3b and phagocytosis of Streptococcus pneumoniae in serafrom subjects with defects in the classical complement pathway. Infect.Immun. 76:3761–3770.

64. Zahner, D., and R. Hakenbeck. 2000. The Streptococcus pneumoniae beta-galactosidase is a surface protein. J. Bacteriol. 182:5919–5921.

65. Zola, T. A., E. S. Lysenko, and J. N. Weiser. 2009. Natural antibody toconserved targets of Haemophilus influenzae limits colonization of the mu-rine nasopharynx. Infect. Immun. 77:3458–3465.

Editor: A. Camilli

2116 DALIA ET AL. INFECT. IMMUN.

on October 4, 2020 by guest

http://iai.asm.org/

Dow

nloaded from