Gonococcal lipooligosaccharide sialylation: virulence factor and target ...

Pathogens and Disease, 75, 2017, ftx049 doi: 10.1093/femspd/ftx049 Advance Access Publication Date: 27 April 2017 Minireview

MINIREVIEW

Gonococcal lipooligosaccharide sialylation: virulence factor and target for novel immunotherapeutics Sanjay Ram∗ , Jutamas Shaughnessy, Rosane B. de Oliveira, Lisa A. Lewis, Sunita Gulati and Peter A. Rice Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605, USA ∗

Corresponding author: Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Lazare Research Building, Room 322, 364 Plantation Street, Worcester, MA 01605, USA. Tel: +1-508-856-6269; Fax: +1-508-856-8447; E-mail: [email protected] One sentence summary: Lipooligosaccharide sialic acid, a key virulence factor for gonococci, can be targeted by novel immunotherapeutics to overcome multidrug-resistant gonorrhea. Editor: Alison Criss

ABSTRACT Gonorrhea has become resistant to most conventional antimicrobials used in clinical practice. The global spread of multidrug-resistant isolates of Neisseria gonorrhoeae could lead to an era of untreatable gonorrhea. New therapeutic modalities with novel mechanisms of action that do not lend themselves to the development of resistance are urgently needed. Gonococcal lipooligosaccharide (LOS) sialylation is critical for complement resistance and for establishing infection in humans and experimental mouse models. Here we describe two immunotherapeutic approaches that target LOS sialic acid: (i) a fusion protein that comprises the region in the complement inhibitor factor H (FH) that binds to sialylated gonococci and IgG Fc (FH/Fc fusion protein) and (ii) analogs of sialic acid that are incorporated into LOS but fail to protect the bacterium against killing. Both molecules showed efficacy in the mouse vaginal colonization model of gonorrhea and may represent promising immunotherapeutic approaches to target multidrug-resistant isolates. Disabling key gonococcal virulence mechanisms is an effective therapeutic strategy because the reduction of virulence is likely to be accompanied by a loss of fitness, rapid elimination by host immunity and consequently, decreased transmission. Keywords: gonorrhea; lipooligosaccharide; sialic acid; complement; factor H; immunotherapeutic

ABBREVIATIONS LOS: lipooligosaccharide LNnT: lacto-N-neotetraose CMP-Neu5Ac: cytidine-5 -monophospho-N-acetylneuraminic acid FH: factor H aHUS: atypical hemolytic uremic syndrome STase: sialyltransferase PorB: Porin B Opa: opacity protein SCR: short consensus repeat

CCP: complement control protein C4BP: C4b-binding protein MBL: mannan binding lectin MASP: mannan binding lectin-associated serine protease NulO: nonulosonate Leg: legionaminic acid

INTRODUCTION Gonorrhea is a major international public health problem. About 78 million new cases of gonococcal infection are estimated to occur worldwide, annually (Newman et al. 2015); almost 400 000

Received: 28 January 2017; Accepted: 26 April 2017 C FEMS 2017. All rights reserved. For permissions, please e-mail: [email protected]

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of these are reported in the USA. Gonorrhea can result in serious sequelae in women, including infertility, ectopic pregnancy and chronic pelvic pain. Furthermore, concomitant infection with HIV and gonorrhea can enhance the rate of transmission of HIV 5-fold (Laga et al. 1993). Neisseria gonorrhoeae has developed resistance to every antimicrobial it has encountered (Unemo and Shafer 2011). The emergence of multidrug-resistant isolates in Asia, Europe and Australia (Ohnishi et al. 2011; Camara et al. 2012; Lahra, Ryder and Whiley 2014) has led to N. gonorrhoeae being called a ‘superbug’ (Unemo and Nicholas 2012) and has ushered in an era of potentially untreatable gonococcal infection (Bolan, Sparling and Wasserheit 2012). The US Centers for Disease Control and Prevention (CDC), for the first time, has prioritized drugresistant N. gonorrhoeae as ‘urgent’ (one of three bacterial organisms) in a list of microbes that are otherwise considered serious (12) or concerning (3) (CDC 2013). The importance of addressing this problem using novel approaches cannot be overemphasized. Interfering with bacterial virulence mechanisms offers an attractive option for therapeutics. In this review, we discuss the importance of a 9-carbon sugar called sialic acid in gonococcal virulence and how gonococcal sialic acid can be targeted by two novel immunotherapeutics.

LIPOOLIGOSACCHARIDE AND GONOCOCCAL PATHOGENESIS The discovery of lipooligosaccharide sialylation as the cause of ‘unstable’ serum resistance In 1970, Ward and colleagues showed that gonococci recovered from male urethral secretions and directly examined (i.e. without subpassage onto routine culture media) were fully resistant to killing by homologous normal serum obtained from the infected individual within 3 days of onset of symptoms (Ward, Watt and Glynn 1970). However, several strains lost the ability to resist killing by homologous complement following even a single culture (passage) on routine gonococcal media. The ability of gonococcal strains to resist complement only when tested directly ex vivo, but not following passage onto gonococcal media was termed ‘unstable serum resistance’ and data suggested that the gonococcal surface likely was modified in vivo, which enabled it to resist killing by complement. Almost two decades later, a series of elegant and detailed studies carried out by Harry Smith and his colleagues in Birmingham, UK, identified a low molecular mass factor present in genital secretions (Martin et al. 1982), serum and extracts of red or white blood cells (Nairn et al. 1988; Smith, Cole and Parsons 1992) that was associated with unstable serum resistance. This compound was identified as cytidine-5 -monophospho-Nacetylneuraminic acid (CMP-Neu5Ac) (Nairn et al. 1988; Smith, Parsons and Cole 1995). Lipooligosaccharide (LOS) was the only gonococcal molecule that incorporated radiolabel following incubation of bacteria in media containing 14 C-labeled CMPNeu5Ac. Subsequently, several groups have confirmed that CMPNeu5Ac added to growth media confers serum resistance to gonococci (Mandrell et al. 1990; Wetzler et al. 1992; Ram et al. 1998), even at nanomolar concentrations (Emond, Dublanchet and Goldner 1995). Mandrell et al. (1990) showed that mammalian α(2,3)- and α(2,6)-sialyltransferases (STases) could each transfer Neu5Ac from CMP-Neu5Ac on to gonococcal LOS; α(2,3)- and α(2,6)-STase added Neu5Ac onto distinct higher (∼4.5 kDa) and lower (∼4 kDa) glycoforms found in strain F62, suggesting two distinct LOS glycan species served as acceptors for Neu5Ac.

Mass spectroscopic analyses of LOS revealed that gonococci elaborated glycans from heptose I (HepI) that mimicked host glycans that also could be sialylated—for example, the lacto-Nneotetraose (LNnT; Galβ1-4GlcNAcβ1-3Galβ1-4Glc (a schematic is shown in Fig. 2)) is identical to the terminal tetrasaccharide of paragloboside, a precursor of the major human blood group antigen; globotriose is identical to the terminal trisaccharide of the PK -like blood group antigen (Galα1-4Galβ1-4Glc) (Mandrell, Griffiss and Macher 1988; Mandrell 1992). It is worth noting that the LOS of N. meningitidis, Haemophilus influenzae and Campylobacter jejuni also mimic host glycans and can be sialylated (Mandrell et al. 1992; Mandrell and Apicella 1993; Tsai 2001; Houliston et al. 2011).

Gonococcal sialyltransferase (STase) All gonococcal isolates examined possess the LOS sialyltransferase (lst) gene that translates into LOS STase activity, the latter evidenced by transfer of radiolabeled Neu5Ac from CMP-Neu5Ac to 4.5 kD LNnT-expressing LOS (Mandrell et al. 1993; Packiam et al. 2006). Although gonococcal STase can transfer Neu5Ac from CMP-Neu5Ac onto lactose (Gal-Glc) present in LOS in bacterial Triton extracts (Mandrell et al. 1993), to our knowledge the addition of Neu5Ac to LOS lactose residues on intact gonococci has not been demonstrated. Using Tn 1545 delta-3 transposon mutagenesis, Bramley et al. (1995) isolated an STase-deficient mutant in strain F62. Shell et al. (2002) showed that gonococcal LOS STase is located in the outer membrane of the bacterium. Pyruvate and lactate in growth media increase sialylation when compared to sialylation in the presence of glucose (McGee and Rest 1996; Parsons et al. 1996a,b; Regan et al. 1999). A gonococcal lactate permease deletion mutant that was incapable of taking up lactate showed decreased LOS sialylation when grown in CMP-Neu5Ac and was less virulent in the mouse model compared to the wild-type strain (Exley et al. 2007). Packiam et al. (2006) showed considerable variation in the level of LOS STase activity across 16 gonococcal strains tested. Interestingly, when considered as a group, the average STase activity of these gonococcal strains was 2.2-fold more than the average LOS STase activity of 16 meningococcal strains. Lower LOS STase activity in meningococci is the result of a 105-bp transposon-like Correia element 5 to the lst open-reading frame, which is absent in N. gonorrhoeae (Packiam et al. 2006). LOS sialylation is increased when gonococci are grown anaerobically (Frangipane and Rest 1993). About 16-fold more CMP-Neu5Ac is required to confer the same level of serum resistance to bacteria grown aerobically on a plate compared to bacteria grown under anaerobic conditions (Frangipane and Rest 1993). Gonococci grown anaerobically in media containing CMP-Neu5Ac become serum resistant two to three times faster than aerobic gonococci and incorporated almost six times as much sialic acid into their LOS, attributable to 4-fold increased STase activity as well as increased expression of sialylatable LNnT LOS (Frangipane and Rest 1993). These findings are relevant to humans, where gonococci encounter anaerobic conditions (Chow, Patten and Marshall 1979; Burnakis and Hildebrandt 1986), in part because of competition between neutrophils and bacteria for oxygen (Britigan and Cohen 1986).

LOS sialylation and complement resistance Complement activation on microbial surfaces occurs through three pathways, namely the classical, lectin and alternative

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Figure 1. Schematic representing the activation of the complement cascade. The fragments released into solution are indicated in blue font. The key fluid-phase regulators are indicated in green font. CRP, C-reactive protein; SAP, serum amyloid P component; PTX3, pentraxin 3; C1 inh, C1 inhibitor; α2-M, α2-macroglobulin; C4BP, C4b-binding protein; FHL-1, factor H like protein-1. From Ram et al. (2010).

(Fig. 1). The classical pathway is triggered by the binding of antibodies (IgM and IgG (mainly the IgG3 and IgG1 subclasses in humans)) to an antigen, which engages the C1 complex that then activates C4, which leads to C4b deposition. The lectin pathway leads to C4b deposition on surfaces following binding of complexes of mannan binding lectin (MBL), ficolins or collectins and the MBL-associated serine proteases (MASPs). The alternative pathway is characterized by a positive feedback loop, where C3b deposited on the bacterial surface is amplified by factors B and D that lead to the formation of C3 convertases. All three complement pathways converge at the level of C3 deposition. Downstream activation of complement results in the formation of C5 convertases, which lead to generation of the C5b-9 complex, also called the membrane attack complex that can insert into the membrane of Gram-negative bacteria and kill the organism. In physiological conditions, the complement cascade is kept under control by inhibitors such as C4b-binding protein (C4BP), factor H (FH) and vitronectin. A detailed description of the complement pathways and its inhibition, while beyond the scope of this review, can be found elsewhere (Walport 2001; Ricklin et al. 2010). LOS sialic acid inhibits all three pathways of complement as described below.

The classical pathway and LOS sialic acid An intact classical pathway is essential for complementdependent killing of wild-type N. gonorrhoeae (Ingwer, Petersen and Brooks 1978). Killing of serum-sensitive (unsiaylated) gonococci by non-immune normal human sera is initiated at least in part by IgM directed against LOS (Ward and Glynn 1972; Schneider et al. 1982; Rice 1989; Griffiss et al. 1991; Jarvis 1995); killing by non-immune sera is reduced by LOS sialylation (Parsons et al. 1989; de la Paz, Cooke and Heckels 1995). LOS sialylation reduced binding of some (Elkins et al. 1992) but not all (Wetzler et al. 1992; de la Paz, Cooke and Heckels et al. 1995) antibodies directed against PorB. In contrast, binding of mAbs directed against opacity protein (Opa) was not diminished by LOS sialylation. In accordance with these findings, we showed that LOS sialylation decreased binding of IgG present in pooled normal human serum to the bacterial surface (Gulati et al. 2015). How LOS sialylation blocks binding of Abs directed against specific non-LOS structures remains unclear. Zaleski and Densen (1996) have suggested that LOS sialic acid may also interfere with the ability of Ab to engage C1q.

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The MBL pathway and LOS sialylation MBL preferentially recognizes glycans that terminate in mannose, glucose, fucose or N-acetylglucosamine (GlcNAc) (Weis, Drickamer and Hendrickson 1992). MBL binds to several strains of N. gonorrhoeae in varying amounts (Gulati et al. 2002). Binding of MBL to N. gonorrhoeae strains MS11 (Devyatyarova-Johnson et al. 2000) and 24-1 (Gulati et al. 2002) decreases following LOS sialylation. Surface-bound MBL-MASP complexes activate complement on N. gonorrhoeae only when purified MBL-MASP is incubated with bacteria prior to the addition of serum. However, the MBL pathway does not appear to contribute to C4b deposition in the context of ‘whole’ serum or when MBL-deficient serum is first reconstituted with purified MBL-MASP and then added to bacteria (Gulati et al. 2002). This MASP function is blocked by C1 inhibitor and α 2 -macroglobulin in serum (Gulati et al. 2002). Although not understood, a possible explanation for why these inhibitors do not block complement activation when purified MBLMASP is pre-incubated with bacteria is that they cannot associate with MASP once the MBL-MASP complex has associated

with gonococci. Overall, the role of the lectin pathway in host defenses against N. gonorrhoeae remains controversial. The alternative pathway: interactions with factor H (FH) The role of sialic acid on host cell surfaces in regulating the alternative pathway has been studied extensively. Desialylation of human erythrocytes with neuraminidase renders the cells susceptible to hemolysis by homologous serum (Fearon 1978). Jarvis (1994) showed that sialylation of the LOS of strain F62 did not affect IgM binding, but reduced C3b deposition, providing evidence for alternative pathway inhibition on sialylated gonococci. Erythrocyte surface-associated sialic acid enhances the affinity of FH for C3b (Kazatchkine, Fearon and Austen 1979). FH plays a key role in ‘self-nonself’ discrimination (Pangburn 2000; Pangburn, Ferreira and Cortes 2008). FH comprises 20 domains, sometimes called short consensus repeat (SCR) or complement control protein (CCP) domains that are each arranged in a linear head-to-tail fashion (Ripoche et al. 1988) (Fig. 2). Only the first four N-terminal domains are necessary and sufficient for

Figure 2. FH structure and the proposed mechanisms of FH/Fc activity. (A) Schematic representation of the domain structure of human FH. The short consensus repeat (SCR, also called complement control protein or CCP) domains that are required for complement inhibition, binding to C3b, glycosaminoglycans (GAGs) and most pathogens are shown. (B) Proposed mechanism of action of the FH/Fc fusion protein. FH domains 18–20 bind to sialylated gonococci; Neu5Ac α(2,3)-linked to LNnT LOS and gonococcal PorB are both required for FH–gonococcal interactions. FH domains 18–20 are fused to the Fc region of IgG. Upon binding to gonococci, adjacent Fc fragments engage the C1 complex, which activates C4 and deposits C4b on the microbe (not shown). Subsequent complement activation deposits C3b and could result in membrane attack complex (C5b-9) insertion into the membrane. Conversion of C3b to iC3b can enhance uptake of the microbe by professional phagocytes through complement receptors such as CR3, in conjunction with engagement of Fc receptors (Fcγ Rs) by the Fc portion of FH/Fc. Blocking binding of host FH to the microbial surface constitutes a third possible mechanism of action of FH/Fc (left side).

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complement inhibition (Sharma and Pangburn 1996), while some of the other domains—specifically domains 6 and 7, and domains 19 and 20—interact with host cells (Prosser et al. 2007b; Kajander et al. 2011). The two C-terminal domains of FH play a major role in restricting unwanted complement activation on host tissue. The currently proposed model suggests that domains 19 and 20 bind to cell-surface-bound C3 fragments and glycosaminoglycans, respectively (Kajander et al. 2011). Blaum et al. (2015) showed that the interaction between sialic acid and FH domain 20 is restricted only to α(2,3) linked Neu5Ac; α(2,6), α(2,8) or α(2,9) linked Neu5Ac do not interact with FH. Mutations in FH that affect its interactions with glycosaminoglycans and/or C3 fragments, most of which are located in domains 19 and 20 (de Cordoba et al. 2012), result in excessive complement activation and are often associated with a condition called atypical hemolytic uremic syndrome (aHUS) (Pickering and Cook 2008; Kavanagh and Goodship 2010). Sialic acid linkage and glycan backbone specificity of FH binding to sialoglycans is relevant to FH interactions with N. gonorrhoeae as discussed below. Domains 6 and 7 also interact with certain host polyanions (Blackmore et al. 1998; Prosser et al. 2007a). A polymorphism in FH domain 7 (402H) decreases the binding of FH to malondialdehydes that accumulates in drusen (Weismann et al. 2011), the retinal lesions that characterize the dry form of age-related macular degeneration, thereby permitting relatively uninhibited alternative pathway activation and accelerated disease progression. Several microbes, including N. gonorrhoeae and N. meningitidis, mimic their human hosts and bind to FH to escape killing by complement (Blom, Hallstrom and Riesbeck 2009; Ram et al. 2016). Not surprisingly, most microbes also interact with FH through the same domains that interact with host cells—domains 6 and 7, and/or domains 19 and 20. Elegant cocrystallographic studies by Schneider et al. (2009) showed that meningococcal factor H-binding protein (FHbp) bound FH domains 6 and 7 in a manner that simulated polyanion interactions with this region in FH. Sialylation of gonococcal LNnT LOS enhances FH binding (Ram et al. 1998). Akin to human cells, FH binds to siaylated gonococci through its C-terminal domains (Ram et al. 1998; Ngampasutadol et al. 2008). Sialylation of gonococcal LNnT LOS, but not sialylation of the PK -like LOS, enhances FH binding (Gulati et al. 2005). Similar to the observations made by Wakarchuk et al with sialylated meningococcal PK -like LOS (also called the L1 LOS immunotype; Scholten et al. 1994), mass spectroscopic analysis of sialylated gonococcal PK -like LOS revealed α(2,6)-linked Neu5Ac to the terminal Gal. These data are consistent with the linkage specificity of Neu5Ac determining interactions with FH domain 20 (Blaum et al. 2015). The requirement for gonococcal PorB expression in permitting FH to bind to sialylated gonococci is another important consideration. Replacing gonococcal PorB with meningococcal PorB abrogated FH binding (measured by flow cytometry) to sialylated gonococci. Although not proven experimentally, we speculate that the C-terminus of FH interacts with sialylated gonococci through a tripartite interaction— PorB and LOS α(2,3)-linked LOS Neu5Ac together interact with FH domains 19 and 20 (depicted in Fig. 2B) in a manner analogous with FH interactions between C3 fragments and Neu5Ac together with other (unidentified) components on host cells (Kajander et al. 2011; Blaum et al. 2015). The requirement for gonococcal PorB may also explain why sialylation of only gonococcal but not meningococcal LNnT LOS enhances direct interactions with the C-terminus of FH (Madico et al. 2007). Direct binding of FH to meningococci occurs exclusively through FH domains 6 and 7 through meningococcal FHbp, Neisserial surface

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protein A (NspA), PorB2 and certain variants of PorB3 (Madico et al. 2006; Lewis et al. 2010, 2013b; Giuntini et al. 2015). In conclusion, studies to date suggest that LOS sialic acid inhibits all three pathways of complement through several independent mechanisms: the classical pathway is inhibited by reducing antibody binding and possibly by reducing in C1q engagement by antibody; the lectin pathway is inhibited by reducing MBL binding; and increased FH binding downregulates the alternative pathway. Furthermore, Neisserial LOS also serves as a target for C4b and C3b (Lewis et al. 2008); although not proven experimentally, sialic acid may obscure sites on LOS that are targets for deposition of C3 and/or C4 fragments.

LOS sialylation and opsonophagocytosis Gonococci may be taken up and killed by neutrophils either through opsonic (i.e. antibody and complement dependent) or non-opsonic (independent of Fc and complement receptors) means (Rest et al. 1982). Sialylation of LOS decreases opsonic killing of gonococci (Kim et al. 1992; Rest and Frangipane 1992; Gill et al. 1996), which may be in part because of decreased complement activation and C3 fragment deposition on the surface of sialylated bacteria (Jarvis 1994; Ram et al. 1998). Lactate present in PMNs may further facilitate LOS sialylation (Parsons et al. 1996a,b) and promote bacterial survival. A well-characterized mechanism of non-opsonic uptake of gonococci by human PMNs involves Opa-CEACAM3 interactions (Schmitter et al. 2004; Sarantis and Gray-Owen 2007). Rest and Frangipane showed that LOS sialylation, in a dose-dependent manner, significantly inhibited the ability of Opa-positive gonococci to adhere to neutrophils and stimulate neutrophil oxidative burst. However, killing of Opa-positive bacteria by human PMNs was not significantly affected by sialyation (Rest and Frangipane 1992). The interactions between gonococci and neutrophils has been reviewed in detail by Criss and Seifert (2012).

Inhibition of invasion into epithelial cells by LOS sialylation Sialylation of gonococcal LOS dramatically reduces Opamediated invasion of N. gonorrhoeae into human epithelial cell lines (van Putten 1993; Rest and Mandrell 1995; Smith, Parsons and Cole 1995). The degree of inhibition of invasion is proportional to the extent of LOS sialylation (van Putten 1993). It is worth noting that LOS sialylation has no effect on adherence of bacteria to epithelial cells (van Putten 1993), in contrast to decreased adherence of gonococci to PMNs (Rest and Frangipane 1992), LOS sialylation can also impede colonization of the male urethra as discussed next.

A critical role for LOS sialylation in vivo Sialylation of LNnT LOS was shown in male urethral secretions by Apicella et al. (1990) by electron microscopy. The importance of LNnT sialylation for virulence in humans was demonstrated in male volunteers who were challenged with a variant of N. gonorrhoeae MS11 that in vitro expressed predominantly lactose (Galβ1-4Glc) from HepI; however, bacteria recovered by culture from infected men expressed the sialylatable ∼4.5 kDa LNnT LOS species (Schneider et al. 1991, 1995). Intraurethral inoculation of human male volunteers with ∼40 000 CFU of MS11 variants that expressed either LNnT or lactose from HepI resulted in infection of 5/5 or only 2/5 subjects, respectively (Schneider et al. 1995). Subsequently, Wu and Jerse (2006) showed that a

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gonococcal mutant of strain MS11 that lacked sialyltransferase was less virulent than its wild-type counterpart in the mouse vaginal colonization model of gonococcal infection. These findings in mice were also confirmed with strain F62 (Lewis et al. 2015). It is likely that the amount of sialylation in vivo needs to be ‘fine-tuned’ for optimal colonization in humans; while excessive sialylation may block invasion of bacteria into epithelial cells, the complete absence of sialic acid may render the organisms susceptible to eradication by host immunity. Consistent with this hypothesis, intra-urethral inoculation of male volunteers with 5000 CFU ‘pre’-sialylated gonococci (strain MS11 mkC) infected only one of five (20%) of subjects, while inoculation of the same number of unsialylated bacteria infected five of six (86%) of individuals (Schneider et al. 1996). Ketterer et al. (2016) showed that cervical secretions obtained from women infected with gonorrhea contain sialidase in quantities sufficient to desialylate LOS, which may facilitate transmission of infection from women to men. Collectively, these data all support a key role for LOS sialylation in pathogenesis, thereby making it an attractive target for immunotherapeutic strategies to combat multidrug resistance.

NOVEL IMMUNOTHERAPEUTICS AGAINST NEISSERIAGONORRHOEAE THAT TARGET LOS SIALYLATION Gonococci have become resistant to almost every conventional antibiotic currently in clinical use and we are entering an era of untreatable gonorrhea (Unemo and Shafer 2014). There is an urgent need to develop novel therapies against gonorrhea. We reasoned that LOS sialylation constituted a good target for novel immunotherapies given its importance in gonococcal pathogenesis.

FH/Fc fusion proteins In the first approach, we have fused the region in FH that binds to sialylated gonococci (domains 18–20) to the Fc region of IgG (Shaughnessy et al. 2016). Because the complement inhibiting activity of FH resides in the first four N-terminal domains (Sharma and Pangburn 1996), this FH/Fc fusion molecule lacks any alternative pathway inhibitory activity. FH/Fc has three possible modes of action: (i) activation of the classical pathway of complement by the Fc region; (ii) enhancement of phagocytosis through Fcγ R and C3 fragments; and (iii) blocking binding of host FH to bacteria, which will permit unimpeded activation of the alternative pathway (Fig. 2). As discussed above, the C-terminus of FH is critical for blocking unwanted complement activation on host cells (Kajander et al. 2011). Therefore, if left unmodified, FH domains 18, 19 and 20 in FH/Fc will bind to and activate complement on host cells and cause unwanted tissue damage. Thus, it was necessary to introduce a mutation(s) in the FH region of FH/Fc such that the molecule did not bind to host cells but retained binding to sialylated N. gonorrhoeae. A condition called atypical hemolytic uremic syndrome is characterized by excessive alternative pathway activation, caused either by loss-of-function mutations in complement inhibitors including FH, factor I (FI) and CD46 or by gain-of-function mutations in molecules involved in complement activation such as C3 and FB (Holers 2008; Kavanagh and Goodship 2010; Loirat and Fremeaux-Bacchi 2011; Rodriguez de Cordoba et al. 2014; Nester et al. 2015). As expected, the majority of aHUS-associated mutations in FH occur in its two

C-terminal domains (de Cordoba et al. 2012). Based on the work by Ferreira et al. (2009), who characterized the effects of several of the aHUS FH mutations on host cell and C3b binding, we constructed four FH/Fc molecules that each contained one of the following amino-acid point mutations that were predicted not to bind to human RBCs and interfere with the function of endogenous serum FH: D→G at position 1119 (domain 19), 1182 R→S, 1183 W→R or 1215 R→G (all in domain 20) (Shaughnessy et al. 2016). Of these four mutations, the D1119G mutant showed maximal complement-dependent bactericidal activity against sialylated gonococcal strains F62 (PorB.1B) and 252 (PorB1.A) that was comparable to activity of the ‘wild-type’ FH/Fc. Importantly, the D1119G mutant did not cause hemolysis of anti-CD59-treated human RBCs (Shaughnessy et al. 2016), which led to selection of this molecule as the lead immunotherapeutic candidate. FH domains 18–20 with the D1119G mutation fused to Fc will henceforth be referred to as FH∗ /Fc. FH∗ /Fc bound to each of 15 sialylated gonococcal strains tested and was able to kill 10 of these 15 strains in a serum bactericidal assay (Fig. 3A) (Shaughnessy et al. 2016). We noted that FH∗ /Fc enhanced C3 deposition (measured as fluorescence in a flow cytometry assay) at least 8-fold compared to complement alone on each of the five strains that were not killed in serum bactericidal assays (data with strain FA1090 are shown in Fig. 3B). Increased C3 deposition was sufficient to support opsonophagocytic killing by freshly isolated human PMNs (Shaughnessy et al. 2016); an Opa-negative mutant of FA1090 was used to eliminate disposal of bacteria through non-opsonic Opa-CEACAM3 interactions (Sarantis and Gray-Owen 2007) (Fig. 3C). The efficacy of FH∗ /Fc when administered topically was shown in vivo using the mouse vaginal colonization model of gonorrhea with strain F62 (Shaughnessy et al. 2016) (Fig. 4). We have confirmed the activity of topically (intravaginally) administered FH∗ /Fc against two additional strains, FA1090 and ceftriaxone-resistant isolate H041 (Ohnishi et al. 2011) (our unpublished observations). These data suggest that FH∗ /Fc may be a useful agent against gonorrhea, either as an immunoprophylactic when administered topically to women at a high risk of acquiring gonorrhea or as an adjunctive therapeutic when administered systemically to individuals with established infection.

Potential limitations of FH∗ /Fc We acknowledge that further studies are needed to ensure that FH∗ /Fc does not cross react with host tissue and result in complement-mediated damage. Furthermore, the use of FH∗ /Fc could result in development of antibodies against neoepitopes created by fragmenting FH, or by the introduction of the 1119G mutation. Such antibodies could decrease efficacy of the drug or could interfere with the function of endogenous host FH if it were to react with its C-terminus. Antibodies that block binding of FH to host cell surfaces could result in dysregulation of complement, perhaps resulting in aHUS. Sialidases elaborated by microbes (fe.g. Gardnerella vaginalis; von Nicolai et al. 1984; Briselden et al. 1992) that often are concomitantly present in the genital tract of gonorrhea-infected women can desialylate LNnT LOS. This may reduce FH∗ /Fc binding and subsequent complement activation, thereby compromising efficacy of the molecule. However, sialidase levels vary by as much as 89-fold across gonorrhea–infected women (Ketterer et al. 2016); therefore, the extent of desialylation of N. gonorrhoeae can also be expected to vary widely. Sialic acid levels also vary in female genital secretions during different phases of the menstrual cycle (Iacobelli, Garcea and Angeloni 1971).

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Figure 3. Activity of FH∗ /Fc (FH domains 18–20 that contains the D→G mutation at position 1119 in domain 19 fused to IgG Fc) against N. gonorrhoeae in vitro. (A) Complement-dependent bactericidal activity of FH∗ /Fc against N. gonorrhoeae. Sialylated N. gonorrhoeae strains (n = 16) were incubated with FH∗ /Fc (33.3 μg/ml; gray bars) or buffer alone (controls; black bars), followed by the addition of 20% (v/v) human complement (normal human serum depleted of IgG and IgM) for 30 min at 37◦ C. Percent survival (±SEM) of bacterial counts at 30 min relative to counts at the beginning of the assay (t0 min) is shown on the Y-axis. Strains that resisted killing by FH∗ /Fc plus complement (>50% survival of CFU at 30 min) are indicated (‘#’). (B) C3 fragment deposition on a representative sialylated strain (FA1090) that resisted direct complement-dependent killing. Sialylated FA1090 was incubated with FH∗ /Fc (33.3 μg/ml) and 10% (v/v) human complement. C3 fragments (C3b/iC3b) deposited on bacteria in the presence of FH∗ /Fc were detected by flow cytometry (histogram shown with a solid black line). C3 fragment deposition on bacteria incubated with complement alone shown by the gray shaded histogram and antibody conjugate controls (bacteria plus anti-C3c FITC) by the dashed histogram. The number next to each histogram represents median fluorescence. Similarly, each of the remaining four resistant isolates showed >10-fold increases in C3 deposition in the presence of complement plus FH∗ /Fc compared to C3 deposited by complement alone. (C) Opsonophagocytic killing of the sialylated Opa-negative mutant of N. gonorrhoeae FA1090 by FH∗ /Fc and complement. Opa-negative FA1090 grown in media containing CMP-Neu5Ac to sialylate LOS (107 CFU) was incubated with FH∗ /Fc (16.7 μg/ml) and 10% (v/v) human complement, followed by the addition of 106 freshly isolated human PMNs for 60 min at 37◦ C (MOI 10:1). Bacterial survival at 60 min relative to t0 is shown on the Y-axis (mean (±SD) of four independently performed experiments). Controls included reactions where complement was heat inactivated (indicated by ‘–’ in the ‘Active complement’ row), and/or where FHD1119G/Fc was omitted. ∗ , P < 0.05; ∗ ∗ , P < 0.01 (ANOVA). Reproduced from Shaughnessy et al. (2016). Copyright 2016. The American Association of Immunologists, Inc.

R Figure 4. FH∗ /Fc reduces the duration and burden of gonococcal infection in the murine vaginal model of gonococcal colonization. Two groups of Premarin -treated wild-type BALB/c mice were infected with 1.5 × 106 CFU of N. gonorrhoeae strain F62 and given either 12 μg FH∗ /Fc (n = 14) or a corresponding volume of PBS (n = 12) as a vehicle control, daily for the duration of the experiment. Vaginal swabs were obtained daily to quantify N. gonorrhoeae CFUs. (A) Kaplan-Meier analysis of time to clearance. (B) Colonization of bacteria (log10 CFU) measured daily. (C) Bacterial burdens consolidated over time (area under the curve [log10 CFU] analysis) for the two groups. Reproduced from Shaughnessy et al. (2016). Copyright 2016. The American Association of Immunologists, Inc.

While the importance of LOS sialylation has been demonstrated in men (Apicella et al. 1990), the extent of LOS sialylation in gonococci, particularly when sialidase-producing organisms are present, and its role in the female genital tract remains to be fully elucidated. Differences in how gonococci interact with male and female genital epithelial cells, and modulation of these interactions by LOS sialic acid, have been reviewed by Edwards and Apicella (2004). ‘Pros and cons’ of the mouse model also need to be considered. Findings in male human volunteers have been replicated in mice; as an example, an lptA mutant that was unable to add phosphoethanolamine to lipid A and rendered bacteria susceptible to cationic peptides and complement (Lewis et al. 2009, 2013a) was attenuated both in mice and men (Hobbs et al. 2013). However, mice lack several receptors that gonococci use to adhere to and invade epithelial cells. Furthermore, scavenging

of iron and complement evasion by gonococci are also host restricted (Jerse et al. 2011). As discussed above, sialidases that are often present in the genital tracts of women may not be present in mouse vaginal secretions. Further studies to evaluate the efficacy of FH∗ /Fc against N. gonorrhoeae in transgenic mice that better simulate the human genital tracts should include expression of the relevant human receptors that facilitate epithelial invasion; human complement inhibitors to provide the barrier that FH∗ /Fc must surmount in humans and the inclusion of sialidaseproducing organisms such as G. vaginalis.

Analogs of sialic acid A second approach currently being studied to counteract the pathogenic role of LOS Neu5Ac is to identify nonulosonate (NulO) analogs of CMP-Neu5Ac that are (i) used as substrates by

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Figure 5. Schematic of the structures of CMP-N-acetylneuraminic acid (CMPNeu5Ac) and CMP-diacetyl legionaminic acid (CMP-Leg5Ac7Ac). Chair chemical structures of the CMP-NulOs are shown. For reference, the 9 carbon atoms of the NulOs are numbered.

N. gonorrhoeae LOS sialyltransferase and incorporated into LOS and (ii) do not shield bacteria against killing by complement (and possibly other arms of the immune system subverted by Neu5Ac). Two analogs were identified that fulfilled both criteria: a 9-azido derivative of CMP-Neu5Ac called CMP-Neu5Ac9Az and a di-acetyl legionaminic acid (Leg) derivate called CMPLeg5Ac7Ac (Fig. 5 shows a schematic of CMP-Neu5Ac and the analog CMP-Leg5Ac7Ac; Fig 6A shows full serum sensitivity of F62 that expressed either Leg5Ac7Ac or Neu5Ac9Az on its’ LOS) (Gulati et al. 2015). Two additional analogs, a 9-acetyl derivative (CMP-Neu5Ac9Ac) and a 5-glycolyl 8-methyl derivative (CMPNeu5Gc8Me), were also incorporated into LOS, but conferred only partial protection against complement-dependent killing (i.e. resistance to 3%, but not 10% normal human serum) (Gulati et al. 2015); therefore, these compounds were not consider further for drug development. In accordance with their serum-sensitive phenotype, gonococci ‘coated’ with Neu5Ac9Az or Leg5Ac7Ac activated complement in quantities comparable to that of the unsialylated strain (Fig. 6B–F). Activation of both, the classical and the alternative pathways of complement proceeded in an unimpeded manner on bacteria with NulO LOS substitutions, evidenced by high IgG binding (Fig. 6B) and C4 deposition (Fig. 6C; indicators of classical pathway activation), factor Bb deposition (Fig. 6D;

Figure 6. Incorporation of Leg5Ac7Ac and Neu5Ac9Az into LNnT LOS permits uninhibited complement activation and killing of N. gonorrhoeae. (A) Serum-sensitive N. gonorrhoeae F62 was incubated with ∼30 μM of the CMP salts of each of the indicated NulOs, and serum bactericidal assays were performed using 3.3%, 6.7% or 10% NHS. The percent survival of bacteria at 30 min relative to bacterial counts at the beginning of the assay (t0 min) is shown on the Y-axis. Only Neu5Ac incorporation into LOS rendered the bacteria resistant to killing by 10% NHS; Neu5Ac9Az and Leg5Ac7Ac incorporation did not confer resistance even to 3.3% serum. Adapted from Gulati et al. (2015) with permission. (B-F) Incorporation of Neu5Ac9Az or Leg5Ac7Ac does not inhibit complement activation on N. gonorrhoeae. Bacteria (strain F62) were grown in media with or without 20 μg/ml of each of the indicated CMP-NulOs, then incubated with 10% NHS; IgG (B), C4 (C), C3 (D), FB (E) deposited on the bacteria were measured by ELISA, and FH binding to bacteria (F) was measured by flow cytometry following incubation of bacteria with purified FH (20 μg/ml). Adapted from Gulati et al. (2015).

Ram et al.

Figure 7. CMP-Leg5Ac7Ac prevents serum resistance mediated by CMP-Neu5Ac. Neisseria gonorrhoeae strain H041 (ceftriaxone-resistant (CRO-R)) was grown in media alone, or media containing 20 μg/ml CMP-Neu5Ac. After 15 min, the indicated concentrations of CMP-Leg5Ac7Ac were added to media and bacteria were grown for an additional 2 h. Resistance to killing by NHS was measured by serum bactericidal assay. The percent survival of bacteria at 30 min relative to bacterial counts at the beginning of the assay (t0 min) is indicated on the Y-axis. ∗ ∗ ∗ ∗ , P < 0.0001 by one-way ANOVA compared to all other groups. CMP-Neu5Ac9Az showed similar results as CMP-Leg5Ac7Ac (data not shown). Adapted from Gulati et al. (2015).

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sensitive (∼90% killing) even in the presence of 100-fold less CMP-Leg5Ac7Ac compared to CMP-Neu5Ac and despite mass spectrometric analysis showing a preponderance of Neu5Acsubstituted LOS on bacteria under these growth conditions (Gulati et al. 2015). An analysis of complement activation on bacteria whose LOS was substituted with both NeuAc and Leg5Ac7Ac showed that the complement inhibition seen in the presence of CMP-Neu5Ac alone (Fig. 6B–F) did not occur when CMP-Leg5Ac7Ac was concomitantly present (Gulati et al. 2015). The reason for the ‘dominant suppressive’ effect of uninhibited complement activation in the presence of LOS Leg5Ac7Ac over complement inhibition in the presence of Neu5Ac alone is not understood. Encouraged by the observation that CMP-Leg5Ac7Ac permitted relatively uninhibited complement activation even in the presence of CMP-Neu5Ac, we tested the efficacy of topically administered CMP-Leg5Ac7Ac on the duration and burden of H041 vaginal colonization in the BALB/c model. As shown in Fig. 8, the duration of infection and overall bacterial burden were significantly reduced in the CMP-Leg5Ac7Ac-treated animals. The data presented above show that CMP-NulO analogs could serve as effective topical immunoprophylatics when administered locally, for example, through vaginal rings (Baum et al. 2012).

Potential limitations of CMP-NulOs alternative pathway activation) and C3 deposition (Fig. 6E; convergence of all complement pathways) (Gulati et al. 2015). Consistent with the importance of the exocyclic glycerol chain of Neu5Ac in binding FH as reported by Blaum et al. (2015), and alternative pathway inhibition reported by Fearon (1978) and Michalek, Mold and Bremer (1988), neither Neu5Ac9Az nor Leg5Ac7Ac coated bacteria showed enhanced FH binding (Fig. 6F). In order to be effective in the treatment of gonorrhea, the bacteria-associated NulOs must activate complement even in the presence of LOS substituted with Neu5Ac. The addition of CMP-NulO concomitantly with or even 15 min after adding CMPNeu5Ac to bacterial cultures resulted in the bacteria remaining relatively serum sensitive (Fig. 7; only results with CMPLeg5Ac7Ac are shown) (Gulati et al. 2015). Of note, serum sensitivity was seen even when the competing CMP-NulO was present at a 10-fold lower concentration than CMP-Neu5Ac on strain H041, as shown in Fig. 7. Strain F62 remained fully serum

As with FH∗ /Fc, further studies are needed to establish the stability and safety of CMP-NulOs. It is important to ensure that the NulOs are not displayed on host cell surfaces, which could be viewed as a foreign antigen by the host immune system and elicit anti-NulO Ab. The presence of anti-Neu5Gc antibodies in some humans is one such example. This is because humans (but not lower primates or other mammals) lack CMPhydroxylase activity and therefore cannot convert Neu5Ac to Neu5Gc (Chou et al. 1998). Consumption of animal proteins that contain Neu5Gc-substituted glycans by humans results in Neu5Gc incorporation on cell surfaces, which may elicit antiNeu5Gc antibodies that could bind to ‘self’ tissue and incite inflammation (Tangvoranuntakul et al. 2003). CMP-substituted sugars are not transported across mammalian membranes; thus, the use of CMP-NulOs may minimize decoration of host cells by NulO analogs. However, enzymes such as ST6GalI that may be present in genital secretions could transfer NulO from the activated (CMP) NulO to host glycans (Watson et al. 2015a,b).

Figure 8. Treatment with CMP-Leg5Ac7Ac decreases the duration and burden of infection with multidrug-resistant N. gonorrhoeae strain H041 in the mouse model of gonorrhea. Wild-type BALB/c mice were infected intravaginally with 9 × 105 CFU of strain H041. One group (n = 10) received CMP-Leg5Ac7Ac 10 μg/ml intravaginally daily; the other group (n = 10 received saline (vehicle control). Vaginal swabs were obtained daily to quantify N. gonorrhoeae CFUs. (A) Kaplan Meier analysis of time to clearance. (B) Bacterial burdens consolidated over time (area under the curve [log 10 CFU] analysis) for the two groups. Adapted from Gulati et al. (2015).

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The acidic pH of the healthy vagina would render the CMP-NulOs susceptible to hydrolysis. Therefore, it is important to select and formulate a therapeutic CMP-NulO such that sustained release without significant loss of activity in cervico-vaginal secretions is achieved. Sialidases elaborated by the microbial flora that coexist with N. gonorrhoeae may cleave the NulO from LOS (Ketterer et al. 2016), rendering the CMP-NulO treatment ineffective. Thus, resistance to sialidases is another desirable property of a therapeutic CMP-NulO. In this regard, Leg5Ac7Ac is far less susceptible to cleavage than Neu5Ac (Watson et al. 2011).

CONCLUDING REMARKS Although advances in sequencing technologies and protein methods have greatly advanced our knowledge of the gonococcal transcriptome and proteome, our understanding of the gonococcal glycome has lagged. The role of sialic acid substitution of LOS in gonococcal pathogenesis has been characterized by several groups. While the role of sialic acid in downregulating complement and enhancing serum resistance has been studied extensively, it is likely that Neu5Ac modulates several other aspects of gonococcal pathogenesis and merits further study. On a molar basis, LOS is the most abundant molecule on the gonococcal surface, is essential for survival of the bacterium and plays a key role in pathogenesis. LOS is therefore an attractive target for vaccines and immunotherapeutics. We acknowledge that LOS glycan extensions are under the control of phase variable genes and thus could be risky target for vaccines and therapies (Tan et al. 2016). However, recognizing and understanding the critical importance of LOS sugars, such as sialic acid, for establishing infection makes them viable targets for therapeutics. By targeting a N. gonorrhoeae LOS epitope important for virulence and therefore expressed by almost every clinical N. gonorrhoeae isolate, two major objectives will be accomplished: (i) should LOS phase variation occur to eliminate FH∗ /Fc binding or CMP-NulO incorporation, the bacteria would be less fit and rendered susceptible to host innate immunity and (ii) attainment of broad strain coverage, which has been a challenge for N. gonorrhoeae vaccine or therapeutic antibody development because its surface structures display extensive antigenic and phase variation. The immunotherapeutics discussed here represent two examples of novel approaches against drug-resistant gonorrhea and further studies to establish the safety and feasibility of these approaches are merited.

ACKNOWLEDGMENT We acknowledge the invaluable contributions of our collaborators including Dr Michael K. Pangburn (University of Texas, Tyler), Dr Christopher Elkins (University of North Carolina, Chapel Hill), Dr Sakari Jokiranta and Dr Arnab Bhattacharjee (Haartman Institute, Helsinki), Dr Ian C. Schoenhofen, Dr Dennis Whitfield and Dr Andrew D. Cox (National Research Council, Ottawa), Dr Brian Akerley and Dr Sandy M. Wong (University of Mississippi, Jackson), Dr Dan M. Granoff (Childrens Hospital Oakland Research Institute, Oakland), Dr Viviana Ferreira (University of Toledo College of Medicine and Life Sciences, Toledo), Dr Ann E. Jerse (Uniformed Services University of Health Sci¨ ences, Bethesda, MD), Dr Magnus Unemo (Orebro University ¨ Hospital, Orebro, Sweden), Dr Xiao-Hong Su (Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, P. R. China), Dr Ajit Varki (University of California, San Diego) and current and previous colleagues at University of Massachusetts

(Nancy Nowak, Samuel Fountain, Dr Sarika Agarwal, Dr Bo Zheng, Srinjoy Chakraborti, Dr Tathagat Dutta Ray, Dr Douglas T. Golenbock, Dr Alberto Visintin and Brian G. Monks).

FUNDING This work was funded by National Institutes of Health/National Institute of Allergy and Infectious Diseases grants AI114710, AI119327, AI114790, AI118161 and AI111728. Conflict of interest. None declared.

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