AWARD LECTURE / CONFÉRENCE D'HONNEUR

Fisher Scientific Award Lecture — The capsularpolysaccharides of Group B Streptococcus andStreptococcus suis differently modulate bacterialinteractions with dendritic cells1

Mariela Segura

Abstract: Infections with encapsulated bacteria cause serious clinical problems. Besides being poorly immunogenic, thebacterial capsular polysaccharide (CPS) cloaks antigenic proteins, allowing bacterial evasion of the host immune system.Despite the clinical significance of bacterial CPS and its suggested role in the pathogenesis of the infection, the mechanismsunderlying innate and, critically, adaptive immune responses to encapsulated bacteria have not been fully elucidated. Assuch, we became interested in studying the CPS of two similar, but unique, streptococcal species: Group B Streptococcus(GBS) and Streptococcus suis. Both streptococci are well encapsulated, some capsular types are more virulent than others,and they can cause severe meningitis and septicemia. For both pathogens, the CPS is considered the major virulence factor.Finally, these two streptococci are the sole Gram-positive bacteria possessing sialic acid in their capsules. GBS type III is aleading cause of neonatal invasive infections. Streptococcus suis type 2 is an important swine and emerging zoonotic patho-gen in humans. We recently characterized the S. suis type 2 CPS. It shares common structural elements with GBS, but sialicacid is a2,6-linked to galactose rather than a2,3-linked. Differential sialic acid expression by pathogens might result in mod-ulation of immune cell activation and, consequently, may affect the immuno-pathogenesis of these bacterial infections. Here,we review and compare the interactions of these two sialylated encapsulated bacteria with dendritic cells, known as the mostpotent antigen-presenting cells linking innate and adaptive immunity. We further address differences between dendritic cellsand professional phagocytes, such as macrophages and neutrophils, in their interplay with these encapsulated pathogens.Elucidation of the molecular and cellular basis of the impact of CPS composition on bacterial interactions with immunecells is critical for mechanistic understanding of anti-CPS responses. Knowledge generated will help to advance the develop-ment of novel, more effective anti-CPS vaccines and improved immunotherapies.

Key words: Streptococcus, capsular polysaccharide, sialic acid, dendritic cells, endocytosis, lipid rafts.

Résumé : Les infections par des bactéries encapsulées causent de sérieux problèmes cliniques. En plus d’être faiblement im-munogène, le polysaccharide capsulaire (PSC) bactérien enveloppe les protéines antigéniques, ce qui permet à la bactérie des’évader du système immunitaire de l’hôte. Malgré l’importance clinique du PSC bactérien et son rôle pressenti dans la pa-thogenèse de l’infection, les mécanismes qui sous-tendent les réponses immunes innées et, de façon critique, adaptativesface aux bactéries encapsulées n’ont pas été totalement élucidés. Ainsi, nous nous sommes intéressés à l’étude du PSC dedeux espèces de streptocoques similaires mais distinctes : Streptococcus du groupe B (SGB) et Streptococcus suis. Les deuxstreptocoques sont bien encapsulés, certains types capsulaires étant plus virulents que d’autres, et ils causent des méningiteset des septicémies sévères. Chez les deux pathogènes, le PSC est considéré comme étant le principal facteur de virulence.Finalement, ces deux streptocoques sont les seules bactéries Gram positifs qui possèdent un acide sialique dans leur capsule.Le SGB de type III est la principale cause d’infections invasives néonatales. Streptococcus suis de type 2 est un pathogèneimportant chez le porc, et est considéré de plus en plus comme pathogène zoonotique chez l’humain. Nous avons récem-ment caractérisé de PSC de S. suis de type 2. Il partage certains éléments structuraux avec le SGB, mais l’acide sialique estlié au galactose en a2,6 plutôt qu’en a2,3. L’expression différentielle d’acide sialique par les pathogènes peut résulter enune modulation de l’activation des cellules immunitaires et conséquemment, affecter l’immunopathogenèse de ces infections

Received 22 December 2011. Accepted 3 January 2012. Published at www.nrcresearchpress.com/cjm on 22 February 2012.

M. Segura. Laboratory of Immunology, Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Université deMontréal, 3200 rue Sicotte, St-Hyacinthe, QC J2S 2M2, Canada.

E-mail for correspondence: mariela.segura@umontreal.ca.1This article is based on an Award Lecture by Dr. Mariela Segura at the 61st Annual Meeting of the Canadian Society of Microbiologistsin St. John’s, Newfoundland, on 23 June 2011. Dr. Segura was the recipient of the 2011 Fisher Scientific Award, a national award spon-sored by Fisher Scientific to recognize excellence in microbiology research.

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Can. J. Microbiol. 58: 249–260 (2012) doi:10.1139/W2012-003 Published by NRC Research Press

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bactériennes. Nous passons ici en revue et nous comparons les interactions de ces deux bactéries encapsulées sialylées avecles cellules dendritiques, connues pour être les cellules présentatrices d’antigène les plus puissantes qui lient l’immunité in-née et adaptative. Nous abordons de manière plus approfondie les différences entre les cellules dendritiques et les phago-cytes professionnels tels les macrophages et les neutrophiles dans leurs interactions avec ces pathogènes encapsulés.L’élucidation des bases moléculaires et cellulaires de l’impact de la composition du PSC sur les interactions bactériennesavec les cellules immunitaires est critique à la compréhension des mécanismes de la réponse anti-PSC. Les connaissancesacquises aideront à faire avancer le développement de nouveaux vaccins et plus efficaces, et à améliorer l’immunothérapie.

Mots‐clés : Streptococcus, capsule polysaccharidique, acide sialique, cellules dendritiques, endocytose, radeaux lipidiques.

[Traduit par la Rédaction]

Introduction

Despite improved antimicrobial therapy, infections with en-capsulated bacteria of importance to human and (or) veteri-nary medicine, such as Group B Streptococcus (GBS),Streptococcus pneumoniae, Haemophilus influenzae type b,Neisseria meningitidis, Escherichia coli, and Streptococcussuis, continue to cause serious clinical and economic prob-lems (Klein Klouwenberg and Bont 2008). For pathogenicbacteria, cell-associated capsular polysaccharide (CPS) playsa major role in bacterial survival and dissemination withinthe host (Corbett and Roberts 2009). This “capsule” or CPScovers antigenic proteins on the bacterial surface that wouldotherwise trigger a protective immune response, thus allow-ing bacterial evasion of the host immune system. CPS typesare water soluble, commonly acidic, with molecular massesof 100 to 1000 kDa. They consist of linear or branched re-peating subunits of one to eight monosaccharides, leading toenormous structural diversity. Despite the clinical signifi-cance of bacterial CPS and their key role in the pathogenesisof the infection, the mechanisms underlying innate and, crit-ically, adaptive immune responses to encapsulated bacteriahave not been fully elucidated. CPS are considered poorlyimmunogenic, as they generate nonlasting adaptive immuneresponses. Paradoxically, antibodies against the CPS are pro-ven essential to host defense against most infections with sys-temic encapsulated bacteria (Mond et al. 1995; Weintraub2003; Klein Klouwenberg and Bont 2008).

The unique characteristic of sialic acid-containing capsules

Sialic acid is a wide family of related nine-carbon sugaracids that feature at terminal positions of many eukaryoticglycoconjugates, conferring important properties at the cellsurface. Therefore, it is not surprising that pathogens haveevolved to express sialic acid, which grants ability to resisthost immune responses and to modulate the interactions withdifferent host cells. The most abundant sialic acid is N-acetylneuraminic acid (Neu5Ac) (Severi et al. 2007). In addi-tion, several pathogenic bacteria are known to modify sialicacid residues by O-acetylation (Lewis et al. 2004, 2006;Song et al. 2011). In other contexts, O-acetylation influenceshost physiological and immunological processes (Shi et al.1996; Malisan et al. 2002). Sialic acid itself is either synthe-sized de novo by bacteria or scavenged directly from the host(Severi et al. 2007). Albeit numerous redoubtable Gram-negative pathogens were shown to display sialic acid at their

surface lipopolysaccharides or CPS, this is a less commonfeature among their Gram-positive counterparts (Severi et al.2007). Thus, we became interested in studying the CPS oftwo similar, but unique, streptococcal species: GBS andS. suis. Both streptococci are well encapsulated, some capsu-lar types are more virulent than others, and they can causesevere meningitis and septicemia. For both pathogens, theCPS is considered the major virulence factor. Finally, thesetwo streptococci are the sole known Gram-positive bacteriapossessing sialic acid in their capsules.

Group B Streptococcus: a well-known humanpathogenGBS is a leading cause of life-threatening invasive bacte-

rial infections in pregnant women and infants, as well as theelderly and immune-compromised individuals in NorthAmerica and western Europe (Edwards and Baker 2005;Edwards 2008; Melin 2011). All nine GBS CPS types thathave been characterized (Ia, Ib, II–VIII), as well as the re-cently proposed serotype IX, can cause human infection(Slotved et al. 2007). Type III GBS is one of the three majorcapsular types associated with invasive neonatal infection andis the most common type in GBS meningitis (Koenig andKeenan 2009). Different GBS serotypes arise from the syn-thesis of distinct CPS repeat units or from differences inCPS polymerization. The cps loci are organized similarlyand comprise 16 genes involved in regulation, chain length,sialic acid synthesis, and oligosaccharide polymerization. No-tably, all characterized CPS types possess a terminal sialicacid (Neu5Ac). The structure of the GBS type III CPS isformed by the monosaccharides glucose, galactose, and N-acetylglucosamine into an unique repeating unit that containsa side chain terminated by sialic acid a2,3-linked to galactose(Chaffin et al. 2005; Cieslewicz et al. 2005). In addition, re-cent work has shown that sialic acid residue in GBS can bemodified by O-acetylation (Lewis et al. 2004), an emergingand exciting new feature of sialic acid metabolism.The GBS oligosaccharide motifs are reportedly very simi-

lar to the sialyl-Lewis and related epitopes on human glyco-proteins. Thus, GBS CPS is suggested to be involved inimmune evasion through molecular mimicry (Cieslewicz etal. 2005). The striking conservation of a-D-Neu5Ac-(2→3)-b-D-Gal among all GBS capsular types suggests that thisstructural element is central to the immune evasion functionof all GBS CPS (Cieslewicz et al. 2005). In addition, thepresence of terminal sialic acid on the GBS type III CPSwas suggested to inhibit the activation of the alternative com-

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plement pathway (Edwards et al. 1982; Marques et al. 1992;Platt et al. 1994; Takahashi et al. 1999; Weiman et al. 2009).GBS type III CPS was shown to interact with different sialicacid-recognizing Ig superfamily lectins (Siglecs), a family ofregulatory receptors expressed on the surface of leukocytes(Carlin et al. 2007). It has been shown that different levelsof O-acetylation of sialic acid are present in all GBS capsulartypes (Lewis et al. 2004, 2006). In the case of GBS type III,sialic acid O-acetylation modifies (either reduces or pro-motes) binding to different Siglecs (Carlin et al. 2007;Weiman et al. 2009). It has been suggested that productionof sialic acid-capped bacterial CPS to engage Siglecs repre-sents an example of a previously unrecognized bacterialmechanism of leukocyte manipulation. A phenomenon thatcould potentially favor pathogen or host, depending on sev-eral variables (Crocker et al. 2007; Lewis et al. 2007; Carlinet al. 2009; Weiman et al. 2010).

Streptococcus suis: a neglected, emerginghuman pathogen

Streptococcus suis is one of the most important swinepathogens worldwide and a zoonotic agent able to inducesepticemia with sudden death, meningitis, endocarditis, pneu-monia, and arthritis (Segura 2009; Gottschalk et al. 2010). Atotal of 35 capsular types of S. suis have been described,with type 2 being the most commonly isolated type from dis-eased animals and humans. Until recently, S. suis disease inhumans was considered as rare, mostly affecting people inclose contact with swine or pork by-products. However,S. suis is now emerging as an important threat to humanhealth, especially in Asian countries. In fact, S. suis has beenidentified as the leading cause of adult meningitis in Viet-nam, the second in Thailand, and the third in Hong Kong(Gottschalk et al. 2007, 2010). Moreover, in 2005, an impor-tant outbreak in China resulted in more than 200 humancases with a fatality rate nearing 20% (Ye et al. 2006). Pa-tients presented symptoms associated with streptococcaltoxic-shock-like syndrome, such as high fever, malaise, nau-sea, and vomiting, followed by subcutaneous hemorrhageand coma in severe cases (Ye et al. 2006; Yu et al. 2006).The sole known CPS structure of S. suis is that of capsular

type 2 (Van Calsteren et al. 2010). This CPS is composed ofthe monosaccharides glucose, galactose, N-acetylglucos-amine, and rhamnose arranged into a unique repeating unitthat contains a side chain terminated by sialic acid a2,6-linked to galactose. Sequencing of S. suis type 2 cps locusrevealed 17 potential genes involved in sugar transfer, polym-erization of CPS, and synthesis of sialic acid (Smith et al.2000). The presence of genes homologous to GBS neuD (O-acetyltransferase) and neuA (O-acetylesterase) in S. suis cpsloci suggests that S. suis CPS is probably O-acetylated(Song et al. 2011).Streptococcus suis type 2 CPS repeating unit structure is

similar to that of GBS in certain constituent monosaccharidesor structural motifs, yet they differ sufficiently to be antigeni-cally distinct, and more importantly, the sialic acid compo-nent is a2,6-gal-linked. Each Siglec has a distinct preference

for specific types of sialic acid and for specific types of link-age to subterminal sugars. These binding preferences arelikely related to their biological functions; for example,CD22 (Siglec-2) on B cells binds specifically to a2,6-linkedsialic acids (Crocker et al. 2007). Thus, differences in sialicacid linkage, (2→6)-b-D-Gal in S. suis versus (2→3)-b-D-Galin GBS, might differentially modulate host immune responses(Sjoberg et al. 1994; Crocker et al. 2007). Indeed, and assummarized in the sections below, their interplay with com-ponents of the immune system, including professional phago-cytes and antigen-presenting cells (APCs), seems to radicallydiffer.

GBS: a “transient” intracellular pathogenGBS is a systemic invasive pathogen, therefore, most stud-

ies have heretofore focused on phagocytosis and killing ofGBS by macrophages and neutrophils. By contrast, the inter-actions of GBS with dendritic cells (DCs), known as themost potent APCs, and the consequences of such interactionsin the development of a specific immune response, includingDC maturation and activation, have not been explored in de-tail. In a recent work, we used C57BL/6 mouse bone-marrow-derived dendritic cells (bmDCs) to evaluate GBS type IIIcapacity to modulate the functions of these importantAPCs. Phagocytosis assays and confocal and electronmicroscopy showed that bmDCs efficiently internalize en-capsulated GBS, but the latter possesses strong intracellularsurvival capacity. These results are in agreement with pre-vious studies using macrophage and (or) monocyte celllines or primary macrophages or neutrophils of either hu-man or mouse origin (Valentin-Weigand et al. 1996; Cor-nacchione et al. 1998; Segura et al. 1998; Poyart et al.2001, 2003; Henneke et al. 2002; Monteiro et al. 2004;Areschoug et al. 2008; Carlin et al. 2009; Chattopadhyay etal. 2011). Overall, the capacity of DCs to internalize encap-sulated GBS seems similar to or lower than that reportedfor these cells. It is generally accepted that different typesof leukocytes differentially perform their specialized tasks.As such, it has been shown that neutrophils and monocytesexhibit a much higher capacity to kill ingested bacteria thando DCs (Nagl et al. 2002; Netea et al. 2004). Accordingly,in a recent study, Mancuso et al. (2009) showed that encap-sulated GBS survived longer in conventional bmDCs thanin mouse macrophages.To evaluate the role of CPS in GBS interactions with DCs,

we generated a CPS-deficient mutant strain by precise in-frame deletion of cpsE gene coding for CPS biosynthesis us-ing splicing-by-overlap-extension PCR. In the absence of se-rum opsonization, GBS devoid of CPS was internalized andkilled by DCs at higher and faster rates than encapsulatedGBS early after infection (Lemire et al.2). In contrast to thisobservation, several studies reported that under non-opsonicconditions, nonencapsulated mutants were equally phagocy-tosed and survived within macrophages at levels similar tothat of the wild-type GBS type III strain (Areschoug et al.2008; Cornacchione et al. 1998; Poyart et al. 2001; Seguraet al. 1998). Nevertheless, a study suggested involvement ofsialic acid-specific receptors on murine macrophages and lec-

2P. Lemire, M. Houde, M.P. Lecours, N. Fittipaldi, and M. Segura. Role of capsular polysaccharide in Group B Streptococccus interactionswith dendritic cells. Manuscript submitted to Microbes and Infection.

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tinophagocytosis in non-opsonic interaction and survival ofGBS within these cells (Monteiro et al. 2004). Similarly, itwas recently reported that GBS sialic acid binds to Siglec-9and impairs the bactericidal functions of human neutrophils(Carlin et al. 2009). Thus, under non-opsonic conditions, theextent of GBS intracellular survival and the role of the sialy-lated CPS in this process seem to differ depending on the celltype and (or) the assay conditions.Complement opsonization has been shown to be critical

for efficient intracellular killing of GBS by monocytes and(or) macrophages and neutrophils (Edwards et al. 1980,1993; Smith et al. 1990; Noel et al. 1991; Wessels et al.1995; Albanyan and Edwards 2000; Maisey et al. 2008).However, encapsulated GBS evades complement opsoniza-tion by means of its sialylated CPS as well as other surfacecomponents, and thus avoids or shows markedly reduced op-sonophagocytic killing by professional phagocytes (Edwardset al. 1980, 1993; Marques et al. 1992; Wessels et al. 1992;Aoyagi et al. 2005; Maruvada et al. 2008, 2009). In ourstudy, serum-mediated opsonization increased GBS internal-ization by DCs but did not significantly alter intracellular sur-vival rates of either the encapsulated or the nonencapsulatedstrains. Furthermore, experiments with heat-inactivated serumsuggested that complement opsonization does not play an im-portant role in GBS interactions with DCs (Lemire et al.2).Once again, these findings suggest important differences inGBS interplay with professional phagocytes and conventionalDCs. The effect of other serum or host components, such asextracellular matrix proteins, in GBS phagocytosis and intra-cellular survival in macrophages or neutrophils has been re-ported. Nevertheless, a discrepancy seems to exist as towhether this type of opsonization confers to GBS protectionagainst the bactericidal functions of phagocytes or rather en-hances bacterial phagocytosis and (or) intracellular killing(Jacobs et al. 1985; Traore et al. 1991; Schubert et al. 2002;Harris et al. 2003; He et al. 2004; Pierno et al. 2006; Wanget al. 2008).Endocytosis pathways are divided into at least five classes

on the basis of the molecular machineries that drive the pro-cess, including clathrin-dependent endocytosis, caveolae-dependent endocytosis, clathrin- and caveolae-independentendocytosis (which can be either lipid raft-dependent or-independent), and macropinocytosis (Bonazzi and Cossart2006). A pioneering study suggested receptor-mediated endo-cytosis and intracellular survival of GBS type III in macro-phages by a mechanism partially dependent on the formationof clathrin-coated pits and the microtubulin network(Valentin-Weigand et al. 1996). By using co-localizationstudies, endocytosis inhibitors, and caveolin–/– mice, we dem-onstrated that GBS uses multiple endocytosis mechanisms toenter DCs. CPS selectively drives GBS internalization viaclathrin-mediated endocytosis and caveolae-independent butlipid raft-dependent pathways, which require an intact actinnetwork. Interestingly, nonencapsulated bacteria are unableto engage lipid rafts (Segura et al. 2011). It has been sug-gested that the strategic utilization of a specific endocyticpathway may provide the pathogen with a mechanism toavoid immunoclearance (Vieira et al. 2010). In this regard,Mancuso et al. (2009) studied the spatial relationships amongGBS and DC endosomal markers with structured illuminationfluorescence microscopy. They found GBS antigens in two

distinct DC intracellular compartments: bacterial DNA+phagosomes and DNA– phagolysosomes containing partiallydigested GBS material. These observations obtained withDCs and the reported ability of GBS to invade and survivein epithelial and endothelial cells as well as professionalphagocytes suggest that GBS, hitherto considered an extrac-ellular pathogen, has evolved some strategies to survive forsome time within various cell types as an “intracellular mi-croorganism”. This “transient” survival in host cells mightrepresent a strategy of immune evasion and dissemination inthe host, but the underlying mechanisms of GBS intracellularsurvival have not been identified.

Streptococcus suis: a “truly” extracellularpathogenIt has been suggested that S. suis infects pigs via the respi-

ratory tract and remains localized in palatine tonsils, whereasskin abrasions and oral consumption of infected pork prod-ucts represent the main route of entry in humans (Gottschalkand Segura 2000; Gottschalk et al. 2007). In certain cases,bacteria reach the bloodstream and persist causing either arapid septic shock or delayed specific infections, dependingon the targeted tissue. Different theories have been put for-ward to explain the ability of S. suis to survive within thehost. The “Trojan horse” theory (bacteria traveling insidemonocytes) has been suggested (Williams and Blakemore1990). However, this theory was withdrawn, as it has beendemonstrated that S. suis severely avoids phagocytosis bymonocytes, macrophages, microglial cells, and neutrophilsfrom mouse, human, or swine origin (Charland et al. 1998;Segura et al. 1998, 2004; Smith et al. 1999; Chabot-Roy etal. 2006; Benga et al. 2008; Domínguez-Punaro et al. 2010).Thus, in the case of S. suis, its sialylated CPS mediates bac-terial attachment to the surface of phagocytes, but this attach-ment does not progress into bacterial internalization (Seguraand Gottschalk 2002). Findings obtained with phagocyteswere recently confirmed using swine and mouse bmDCs. Weshowed that the CPS plays a very distinct role in S. suis in-teractions with DCs compared with its counterpart, GBS. En-capsulated S. suis was largely resistant to phagocytosis, andonly a few cocci were found inside cells, which were easilydigested by DCs. In contrast a nonencapsulated S. suis mu-tant was not only easily internalized by DCs but also rapidlykilled upon ingestion (Lecours et al. 2011a, 2011b). In con-clusion, encapsulated GBS is found largely inside phagocytesand DCs, and thus, its CPS cannot be considered a major“anti-phagocytic” factor. Quite the opposite, S. suis is mostlyextracellularly located, and thus, its CPS undoubtedly acts asshielding factor.These contrasting findings defeat the old dogma stating

that the anti-phagocytic effect of bacterial CPSs is due totheir net electrostatic charge, and therefore, other more dy-namic mechanisms are probably involved in CPS modulationof host cells. As mentioned above, the sialylated CPS of GBStype III uses lipid rafts as an entry port. Lipid rafts aresphingolipid- and cholesterol-rich microdomains that havebeen shown to function as signal transduction platforms(Mañes et al. 2003; Nakayama et al. 2008; Yoshizaki et al.2008). For example, lactosylceramide (LacCer), a neutral gly-cosphingolipid, forms microdomains that mediate, among

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other functions, phagocytosis by neutrophils (Nakayama et al.2008; Yoshizaki et al. 2008). We consequently decided toevaluate the capacity of S. suis CPS to interact with lipidrafts and modulate phagocytosis by macrophages and DCs.We adapted a technique commonly used to conjugate a pro-tein carrier to CPS (Guttormsen et al. 2008) replacing theprotein by a latex bead with amine groups at its surface,which covalently link purified CPS. We obtained an artificialparticle about the size of a bacterium, which allows us to dis-criminate biological effects directly related to CPS withoutinterference from other virulence factors (Houde et al. 2012).Linking S. suis type 2 CPS to latex beads is sufficient to im-pair their phagocytosis by macrophages and DCs. This couldbe due to CPS acting as a shell that prevents contact betweenthe beads and the phagocyte surface. However, our resultssuggest that CPS plays a more active and specific role at thephagocyte surface. Indeed, CPS seems to actively disrupt es-sential cellular processes involved in phagocytosis. This issupported by the fact that S. suis CPS-linked beads possessthe ability to inhibit not only their own entry into phagocytesbut also that of bystander unlinked beads. By using a nonen-capsulated S. suis mutant we showed that S. suis CPS causesdisruption of lipid microdomains, as demonstrated by confo-cal analysis of lipid raft distribution and dot-blot analysis ofultracentrifugation-fractionated cell membranes using choleratoxin to label GM1 ganglioside. The observed disruption oflipid microdomains led us to investigate specific componentsinvolved in pathogen recognition that could be inhibited byS. suis CPS. We showed that CPS-mediated destabilizationof lipid rafts prevents accumulation of LacCer at the interac-tion points between S. suis and the cell membrane with con-sequent inhibition of key signaling pathways involved inphagocytosis and cell activation, including nitric oxide pro-duction (Segura et al. 2004; Domínguez-Punaro of et al.2010; Houde et al. 2012). Phosphoinositide 3-kinase(PI3K)–Akt and p38 mitogen-activated protein kinase(MAPK) pathways are both known activators of phagocyticmechanisms and are recruited at the lipid-raft-LacCer plat-form (Yoshizaki et al. 2008). Ligand binding to LacCer inLacCer-enriched microdomains induces activation of theLyn–PI3K–p38 MAPK–protein kinase C (PKC) signal trans-duction pathway leading to phagocyte function activation(Yoshizaki et al. 2008). Previous work has shown high levelsof Akt and PKC phosphorylation after infection of macro-phages with a nonencapsulated mutant of S. suis type 2,whereas the encapsulated strain showed reduced activationof the PI3K–Akt–PKC signaling pathway (Segura et al.2004). In addition, p38 MAPK phosphorylation was impairedby the presence of CPS in S. suis-infected microglial cells(Domínguez-Punaro et al. 2010). This represents a novel de-scribed mechanism used by an encapsulated bacterial patho-gen to avoid phagocytosis by disruption of lipidmicrodomain stability and LacCer modulation in phagocytes.Altogether, findings obtained with phagocytes and DCs sug-gest that encapsulated S. suis type 2 has evolved powerfulstrategies to avoid ingestion by these immune cells to coun-teract its inefficiency to survive within them.

Which is the role of sialic acid in theinteractions of these two encapsulatedbacteria with host cells?

Our knowledge on the specific contribution of sialic acidto the aforementioned interactions of GBS type III with im-mune cells is hampered by the fact that deletion of neuA(CMP-N-acetylneuraminic acid synthetase) and cpsK (a2,3-sialyltransferase) genes (Chaffin et al. 2005; Haft et al. 1996;Wessels et al. 1989, 1992), and of the more recently reportedneuB (sialic acid synthetase) gene (Lecours et al.3), results inconsiderable loss of CPS expression at the bacterial surface(<20% production of CPS compared with the wild-typestrain). In view of these data, caution should be used in eval-uating the effect of sialic acid mutagenesis on GBS pathogen-esis and interactions with host cells, as this mutagenesis canbe confounded with an overall decrease in CPS production.Nevertheless, recently performed elegant mutagenesis studies(Carlin et al. 2007, 2009; Lewis et al. 2007; Weiman et al.2009) allowed the elucidation of the importance of sialicacid O-acetylation levels in GBS capacity to manipulate neu-trophil functions and virulence (Carlin et al. 2009; Weiman etal. 2009, 2010).In the case of S. suis, very little is known concerning the

role of its capsular sialic acid. To this aim we performed tar-geted mutation of S. suis type 2 neuC gene involved in sialicacid synthesis. Results showed a complete loss of CPS ex-pression at the bacterial surface and the mutant phenotypi-cally behaved as a nonencapsulated strain (Lecours et al.3).These results indicate that sialylation is essential for full syn-thesis of CPS by S. suis. In the case of GBS type III, it hasbeen suggested that the 80% reduction in surface-associatedCPS produced by the GBS asialo mutant strains could be re-lated to diminished production of CPS oligosaccharide pre-cursors and (or) reduced transfer of CPS precursors acrossthe bacterial membrane. If the sialyltransferase is an integralpart of a membrane-associated complex of glycosyltransfer-ases, its loss may also disrupt the functional integrity of theGBS CPS synthesis complex (Chaffin et al. 2005). This ob-servation appears to be in contrast to that found in Haemo-philus ducreyi and N. meningitidis, whereby loss ofsialyltransferase activity did not reduce production of theasialo–lipopolysaccharide backbone structure (Chaffin et al.2005). The molecular bases underlying the complete loss ofCPS expression in S. suis after sialic acid synthetase gene de-letion so far remain unknown (Lecours et al.3).Despite these technical restrictions, and in contrast with

that suggested for GBS type III, the sialic acid moiety inS. suis type 2 does not seem to play an important role in re-sistance to complement activation, and no correlation hasbeen observed between sialic acid content and virulence(Charland et al. 1996; Lecours et al. 2011a). In addition, nosignificant differences could be found in the phagocytosisrates by porcine blood monocytes of S. suis treated or notwith sialidase or with the sialic acid-binding lectin, SNA-I(Charland et al. 1996). These preliminary results suggestedthat sialic acid itself may not play an important role in

3M.P. Lecours, N. Fittipaldi, D. Takamatsu, M. Okura, M. Segura, G. Goyette-Desjardins, M.R. Van Calsteren, and M. Gottschalk. Sialylationof Streptococcus suis serotype 2 is essential for capsule expression but is not responsible for the main capsular epitope. Manuscript submittedto Microbes and Infection.

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S. suis virulence. Further, more advanced studies would berequired to dissect the contribution of S. suis sialic acid inthe pathogenesis of the disease.

DC activation and maturation: Are these twopathogens equally able to activate theseAPCs?Different types of leukocytes fulfill specialized tasks in

antigen presentation and killing of pathogens (Nagl et al.2002). The primary function of DCs is to alert the immunesystem, not to clear invading microorganisms. In fact, DCsare recognized as the most powerful APCs that initiate im-mune responses against pathogens and are considered an es-sential link between innate and adaptive immunity. DCscapture and process antigens and then undergo a maturationprocess characterized by the production of cytokines and up-regulation of co-stimulatory molecules. Afterwards, DCs mi-grate to adjacent lymphoid organs where they activate T cells(Alvarez et al. 2008). Consequently, the interactions betweenDCs and pathogens can strongly influence the outcome of adisease and, more importantly, the magnitude and phenotypeof the ensuing adaptive immune response to the invadingpathogen. In our recent work, we showed that encapsulatedGBS type III induces increased DC expression of the co-stimulatory molecules CD40, CD86, as well as MHC-II, andtriggers DC release of several cytokines, including the pro-inflammatory cytokines TNF-a, IL-1b, and IL-6; the Th1driven cytokines IL-12p70 and IL-23; as well as the regula-tory cytokine IL-10. The production of these cytokines byDCs was partially reduced by the presence of CPS (Lemireet al.2). In vivo studies demonstrated that IL-12 is importantin controlling the cytokine production that leads to the evolu-tion of GBS-induced pathology and has a major role in re-stricting bacterial growth during infection (La Pine et al.2003; Mancuso et al. 1997). Similarly, the IL-23–IL-17 path-way has been shown to mediate resistance to Klebsiellapneumoniae infection (Hunter 2005). GBS internalization islargely required for modulation of the IL-12–IL-23 and IL-10 pathways, while production of the pro-inflammatory cyto-kines IL-1b, IL-6, and TNF-a is partially affected by cyto-chalasin D treatment of GBS-infected DCs. As reported forStreptococcus pyogenes-infected macrophages (von Delwiget al. 2002), surface expression of co-stimulatory moleculesis not affected by cytochalasin D. These results suggest thatmultiple mechanisms are involved in GBS modulation of DCactivation (Lemire et al.2). Chemokines are also important inregulating innate and adaptive immune responses. GBS-exposed DCs secrete high levels of the neutrophil chemoat-tractant CXCL1 and the monocyte chemoattractant CCL2, asalready reported with mouse macrophages (Draper et al.2006; Fan et al. 2007). Results with cytochalasin D indicatethat both intracellular and extracellular signals lead toCXCL1 and CCL2 production by DCs. High levels of secre-tion of these chemokines might contribute to host innate de-fense against GBS but might also result in increasedpathology (Lemire et al.2). On the other hand, DC stimulatedwith GBS fails to produce significant levels of CXCL9 andproduces low and delayed levels of the CXCL10 chemokine.CXCL10 shares with CXCL9 the ability to signal throughCXCR3, which is present on T cells and NK cells. Although

upregulation of Cxcl10 gene expression was observed byDNA microarray analyses of mouse peritoneal macrophages(Draper et al. 2006), GBS was reported to be unable to in-duce CXCL10 secretion by these cells (Fan et al. 2007). Acritical role of type I IFN-dependent CXCL10 has been iden-tified in host defense during microbial sepsis by increasingneutrophil recruitment and function (Kelly-Scumpia et al.2010). Mancuso et al. (2009) reported that GBS inducestype I IFN by DCs via a lysosomal TLR7-dependent path-way. This novel bacterial recognition system operates in con-ventional DCs but not in macrophages. Thus, GBSmodulation of this pathway in DCs may have important con-sequences for both innate and adaptive immune responsesagainst this pathogen. Mancuso et al. (2009) suggested thatby disrupting phagosomal maturation and (or) integrity, bac-terial pathogens may avoid not only direct killing but alsoimmune recognition in lysosomal compartments and the sub-sequent establishment of host-protective responses dependenton interferon-regulatory transcription factor-1. In agreementwith these results, CXCL10 production was almost com-pletely abrogated by cytochalasin D treatment of GBS-infected cells, confirming that intracellular bacteria is re-quired for activation of this pathway (Lemire et al.2). BesidesTLR7, receptors involved in GBS recognition and modula-tion of DC functions have not been characterized.Encapsulated S. suis also triggers mouse bmDC production

of high levels of pro-inflammatory, Th1-driving and regula-tory cytokines. In addition, high levels of the chemokinesCXCL1, CXCL10, and CCL2, and low, albeit significant,levels of CXCL9 were observed in S. suis-infected DCs(Lecours et al. 2011a). In comparison to GBS, the effect ofS. suis CPS on cytokine production is more marked. Indeed,CPS severely interferes with the release of most of the cyto-kines produced by S. suis-infected DCs. This is in agreementwith the strong antiphagocytic effect of S. suis CPS. Simi-larly, S. suis activation of cytokine release by mouse or hu-man phagocytes has been reported as a phagocytosis-independent event (Segura et al. 1999). The S. suis CPS alsohighly interferes with the release of IL-6, IL-8, IL-12p40,and TNF-a by swine bmDCs (Lecours et al. 2011b). Theability of S. suis to induce the maturation of DCs was alsoinvestigated by evaluating the surface expression of the co-stimulatory molecules CD80/CD86 and MHC-II on swine-or mouse-origin bmDCs. Encapsulated S. suis fail to inducethe expression of either CD80/CD86 or MHC-II on swinebmDCs (Lecours et al. 2011b). The CPS has been shown tobe responsible for the impaired expression of CD80/CD86 onDCs and also seems to interfere, at least in part, with MHC-II expression. This differs with results obtained with mousebmDCs, where encapsulated S. suis induced maturation lev-els similar to those observed with the nonencapsulated mu-tant (Lecours et al. 2011a). These differences could berelated to the cell origin (swine versus mouse) and also tothe fact that mouse DC are derived from inbred mouse lines,while swine DCs are originated from outbred animals. Albeitthese differences, increased exposure of cell wall componentsdue to the absence of a capsule may account for the highercapacity of the nonencapsulated S. suis mutant to induce DCactivation, and may confirm the role of cell wall componentsas major immunomodulators (Segura et al. 1999, 2006;Graveline et al. 2007; Domínguez-Punaro et al. 2010). Pre-

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vious studies using monocytes, macrophages, or total periph-eral blood mononuclear cells of either mouse or human ori-gin demonstrate an important contribution of TLR2, TLR6,TLR9, CD14 but not of TLR4 or TLR1 in cytokine produc-

tion by these cells (Segura et al. 2002; Graveline et al. 2007;Domínguez-Punaro et al. 2010; Zheng et al. 2011). The con-tribution of these receptors in DC activation by S. suis is lesscharacterized. It has been shown that both encapsulated

Fig. 1. Proposed model of Streptococcus suis type 2 interactions with dendritic cells (DCs). (1) Encapsulated S. suis interacts with severalmembrane receptor(s) (mRc) and induces DC maturation (CD40, CD86, and MHC-II expression) as well as the production of several cyto-kines (IL-1b, TNF-a, IL-6, IL-10, IL-12p70, IL-23, CXCL1, CXCL9, CXCL10, and CCL2) via an endocytosis-independent mechanism. Thisinitial DC activation upon S. suis contact is thought to be mediated by exposed cell wall component interaction mainly with TLR2/6. OtherTLRs as well as CD14 might also be involved. MYD88 and MAPK are part of the signaling platform involved in S. suis-activated events.(2) The presence of capsular polysaccharide (CPS) markedly impairs encapsulated S. suis phagocytosis through destabilization of lipidmicrodomains, preventing accumulation of lactosylceramide (LacCer) at the interaction points of S. suis with the membrane and consequentinhibition of key signaling pathways, including nitric oxide production (NO). (3) Blockade of other endocytosis mechanisms cannot be ruledout and remains to be elucidated. (4) If S. suis fails to avoid internalization, it will rapidly and efficiently killed by DCs upon ingestion.Finally, results with the nonencapsulated mutant showed that CPS dramatically impairs the production of most cytokines by infected DCs (asindicated by ↓). The surface expression of co-stimulatory molecules by DCs also seems to be affected by the presence of CPS (as indicated bya dotted ↓).

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Fig. 2. Proposed model of Group B Streptococcus (GBS) type III interactions with dendritic cells (DCs). (1) Encapsulated GBS interacts withunknown membrane receptor(s) (mRc) and induces DC maturation (CD40, CD86, and MHC-II expression) as well as the production of sev-eral cytokines (IL-1b, TNF-a, IL-6, CXCL1, and CCL2) in part via an endocytosis-independent mechanism. This initial DC activation uponGBS contact is followed by bacterial endocytosis via two major pathways. (2) The presence of capsular polysaccharide (CPS) directs GBSthrough lipid raft-dependent (but caveolin-1-independent) endocytosis. CPS-recognizing receptor(s) within lipid rafts (lrRc) are probably in-volved in this step. (3) Other exposed cell wall components might lead to bacterial internalization via clathrin-mediated endocytosis uponreceptor ligation (cRc). (4) As total inhibition of GBS internalization could not be observed with inhibitors of either pathway, other compen-satory endocytosis mechanisms are probably involved. (5) Once internalized, encapsulated GBS resides in LAMP-1+ phagosomes but sur-vives for at least 6 h. These phagosomes might eventually undergo maturation and fusion with lysosomes (5a). It is hypothesized that CPS-mediated interactions of GBS with lipid rafts might promote GBS survival (5b). (6) GBS internalization is required for DC production of IL-12p70, IL-23, IL-10, and CXCL10. As reported by Mancuso et al. (2009), GBS recognition by TLR7 in the phagolysosomes of DCs resultsin IFN-a/b production. Type I IFN autocrine–paracrine loop might be involved in cytokine production, particularly the chemokine CXCL10.Finally, results with the nonencapsulated mutant showed that CPS impairs, at least in part, GBS-induced production of several cytokines byDCs (as indicated by ↓).

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S. suis and its nonencapsulated mutant strain increase the ex-pression of TLR2 and TLR6 mRNA in infected swinebmDCs. No differences were noticeable between the twostrains for the expression of TLR2. However, the expressionof TLR6 was increased more rapidly after swine bmDC in-fection with the nonencapsulated strain than with the wild-type strain (Lecours et al. 2011b). Preliminary data usingmouse bmDCs derived from C57BL/6, TLR2–/–, TLR4–/–,MyD88–/–, or NOD2–/– mice suggest that a multimodal recog-nition, involving a combination of these different receptors, isessential for DC effective response to S. suis (M. Segura, un-published observations).

Concluding remarksGBS type III and S. suis type 2 share the common charac-

teristic of being the sole Gram-positive bacteria harbouring asialylated CPS, yet their interactions with DCs seems to thor-oughly differ. Streptococcus suis extracellular localizationconfers to this pathogen a survival advantage, and the CPSis essential to it. In fact, S. suis CPS was shown to modulatemost interactions with DCs by protecting bacteria againstphagocytosis mainly through lipid raft destabilization. Over-all, CPS-impaired S. suis interactions with DCs would resultin low bacterial uptake as well as low DC activation and ma-turation, which might translate in reduced antigen processingand T cell activation. The S. suis CPS could therefore be con-sidered as an escape mechanism for S. suis (summarized inFig. 1). On the other hand, encapsulated GBS mobilizes dif-ferent components of the DC endocytic machinery, whichmight result in differential modulation of DC functions. In-deed, the route of bacterial uptake by APCs can influencethe repertoire of epitopes presented to T cells (von Delwig etal. 2002; Burgdorf et al. 2007). Lipid rafts appear to be thepreferred site for encapsulated GBS entry. A possible advant-age of invasion through lipid rafts could be the avoidance ofintracellular degrading mechanisms (Duncan et al. 2002;Wang and Hajishengallis 2008). As such, GBS CPS mighttarget lipid rafts as a safe door for intracellular survival withconsequent modulation of key cytokine pathways involve-ment in protective immune responses (summarized in Fig. 2).

AcknowledgementsThis work was supported by Natural Sciences and Engi-

neering Research Council of Canada (NSERC) through agrant to M.S. (342150-07). M.S. is the recipient of a Fondsde recherche en santé du Québec (FRSQ) Career Award. Iwould like to thank Dr. Giuseppe Teti (University of Mes-sina, Italy) for helpful discussions.

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