More than just immune evasion: Hijacking complement by Plasmodium falciparum.

G Model

ARTICLE IN PRESS

MIMM-4627; No. of Pages 14

Molecular Immunology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Molecular Immunology journal homepage: www.elsevier.com/locate/molimm

Review

More than just immune evasion: Hijacking complement by Plasmodium falciparum Christoph Q. Schmidt a,∗ , Alexander T. Kennedy b , Wai-Hong Tham b,∗∗ a

Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Helmholtzstraße 20, Ulm, Germany Department of Medical Biology, University of Melbourne and Division of Infection and Immunity, The Walter and Eliza Hall Institute, Parkville, Victoria 3052, Australia b

a r t i c l e

i n f o

Article history: Received 18 January 2015 Received in revised form 4 March 2015 Accepted 4 March 2015 Available online xxx Keywords: Plasmodium falciparum Complement Parasite invasion Severe malaria pathogenesis Complement receptor 1 Evasion strategies

a b s t r a c t Malaria remains one of the world’s deadliest diseases. Plasmodium falciparum is responsible for the most severe and lethal form of human malaria. P. falciparum’s life cycle involves two obligate hosts: human and mosquito. From initial entry into these hosts, malaria parasites face the onslaught of the first line of host defence, the complement system. In this review, we discuss the complex interaction between complement and malaria infection in terms of hosts immune responses, parasite survival and pathogenesis of severe forms of malaria. We will focus on the role of complement receptor 1 and its associated polymorphisms in malaria immune complex clearance, as a mediator of parasite rosetting and as an entry receptor for P. falciparum invasion. Complement evasion strategies of P. falciparum parasites will also be highlighted. The sexual forms of the malaria parasites recruit the soluble human complement regulator Factor H to evade complement-mediated killing within the mosquito host. A novel evasion strategy is the deployment of parasite organelles to divert complement attack from infective blood stage parasites. Finally we outline the future challenge to understand the implications of these exploitation mechanisms in the interplay between successful infection of the host and pathogenesis observed in severe malaria. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Malaria remains a major global health threat accounting for a yearly death toll of about 700,000 people on a background of about 200 million infections worldwide (WHO Malaria Report 2014). There are five species of Plasmodium that infect humans: P. falciparum, P. knowlesi, P. vivax, P. ovale and P. malariae. P. falciparum is responsible for the majority of human fatalities worldwide. Severe P. falciparum malaria remains an important cause of maternal and childhood morbidity and mortality. An emerging view is that the innate immune response to malaria infection driven by the complement system may be a determinant in the severity of infections (reviewed in Silver et al. (2010)). This review will cover the complex interaction between the human complement system and malaria infection in terms of hosts’ immune responses, parasites survival strategies and implications for the pathogenesis of severe complications of malaria.

∗ Corresponding author. Tel.: +49 73150065615. ∗∗ Corresponding author. E-mail addresses: [email protected] (C.Q. Schmidt), [email protected] (W.-H. Tham).

Malaria parasites have a complex life cycle involving an insect vector, the female Anopheles mosquito, and a vertebrate host, the human (Fig. 1). Infection in humans is initiated through the bite of an infected female mosquito. The mosquito bite injects Plasmodium parasites, in the form of sporozoites, into the human bloodstream. Sporozoites travel to the liver to invade hepatocytes beginning the liver stage infection. Within 10 days, a single sporozoite multiplies asexually into thousands of merozoites which are released into the blood stream when the infected liver cell ruptures. These merozoites are capable of invading healthy red blood cells initiating the blood stage cycle of human infection. Within 48 h of invading a red blood cell, a single merozoite progresses through the ring and trophozoite forms to replicate and divide via schizogony into 16–32 new daughter merozoites. The infected red blood cell subsequently ruptures to release newly formed merozoites which are capable of re-infecting other healthy red blood cells. The blood stage cycle is responsible for all the clinical symptoms such as fever and chills. Some of the infected red blood cells will leave the asexual cycle to develop into the sexual forms of the parasites, called gametocytes, which freely circulate in the blood stream. When a female Anopheles mosquito bites an infected human, gametocytes are ingested into the mosquito midgut. The rest of the sexual stages occur within the mosquito host, where gametocytes are activated to mature into

http://dx.doi.org/10.1016/j.molimm.2015.03.006 0161-5890/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Schmidt, C.Q., et al., More than just immune evasion: Hijacking complement by Plasmodium falciparum. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.03.006

G Model MIMM-4627; No. of Pages 14

ARTICLE IN PRESS C.Q. Schmidt et al. / Molecular Immunology xxx (2015) xxx–xxx

2

Fig. 1. Plasmodium falciparum life cycle. The bite of an infected female mosquito injects sporozoites into the bloodstream to initiate infection in humans (mosquito on the right). Sporozoites migrate to the liver to invade hepatocytes and develop into merozoites which are released into the blood stream. Merozoites invade red blood cells and grow into ring, trophozoite and schizont forms. The infected red blood cell subsequently ruptures to release newly formed merozoites which are capable of re-infecting other healthy red blood cells. Some infected red blood cells develop into the sexual forms of the parasites, called gametocytes, which freely circulate in the blood stream. When a mosquito bites an infected human, gametocytes are ingested. The sexual stages of the malaria parasites occur within the mosquito midgut, where gametocytes are activated to mature into male and female gametes that undergo fertilisation to produce a diploid zygote. The diploid zygote develops into a mobile ookinete to an oocyst and finally into the sporozoite form that migrate to the mosquito salivary glands, ready to be transmitted to humans upon the next mosquito bite.

male and female gametes that undergo fertilisation to produce a diploid zygote. The diploid zygote develops into a mobile ookinete that crosses the midgut wall to embed itself as an oocyst on the exterior of the midgut. Thousands of sporozoites develop within the oocyst, which are subsequently released to migrate to the mosquito salivary glands and ready to be transmitted to the next human via a mosquito bite. Within both the human and mosquito host, the malaria parasite in its various sexual and asexual forms is exposed to the hosts’ immune system. 2. The complement system 2.1. Activation and regulation As an important part of the “first line of defence”, complement proteins act rapidly upon entry of pathogens into the human host to facilitate opsonisation of pathogens for phagocytosis, lyse pathogens directly and stimulate the adaptive immune response. This is exemplified by the correlation of deficiencies in certain complement components with susceptibility to recurrent infections caused by e.g. Streptococcus pneumoniae, Haemophilus influenza, Neisseria meningitidis (reviewed in Skattum et al. (2011)). Triggering one or all three of complement’s initiation pathways (classical, lectin or alternative pathway) leads to the proteolytic activation of C3 into the split products C3a and C3b (Fig. 2). In absence of complement regulation on the pathogen surface any initial number of tethered C3b opsonins becomes amplified by C3 convertases. Continuous C3b deposition leads to assembly of C5 convertases that cleave C5 into the potent, chemotactic anaphylatoxin C5a and into C5b, which initiates the terminal and lytic membrane attack complex (MAC) (Dunkelberger and Song, 2010). While the classical pathway (CP) and lectin pathway (LP) are initiated in response to danger associated or pathogen associated molecular patterns (e.g. immune complexes and microbial sugars), the alternative pathway (AP) is continuously auto-activated at low level (so called tick-over activation) (Kemper and Atkinson, 2007). Several soluble and membrane-bound host regulators are in place to restrict complement activation and amplification on self surfaces (Dunkelberger and Song, 2010; Ricklin et al., 2010; Rodriguez de Cordoba et al., 1985). While membrane-bound

complement regulators protect the cell surfaces that they are linked to, soluble regulators control complement in the fluid phase and also on host cells, which they specifically recognise through certain host cell markers (Blaum et al., 2015; Clark et al., 2010). Especially, the opsonin C3b with its auto-amplification capacity and the C3 and C5 convertases are tightly regulated (Table 1). One important regulatory mechanism is to accelerate the natural decay of the bi- and tri-molecular C3 and C5 convertase complexes. The enzymatic converting function of the convertases is lost as soon as the complexes are disassembled into their singular constituents (Ricklin et al., 2010). Another crucial regulatory function is the ‘cofactor activity’ for the regulatory protease Factor I. Factor I proteolytically inactivates C4b and C3b molecules both of which can be covalently attached to surfaces via their thioester and act as platforms for convertase assembly. However, Factor I can perform this task only in presence of a cofactor. Five homologous proteins, which belong to the “regulators of complement activation” (RCA) family (Kirkitadze and Barlow, 2001), perform the essential tasks of decay-acceleration and/or cofactor activity: Complement receptor 1 (CR1 or CD35), Membrane cofactor protein (MCP or CD46), Decay-accelerating factor (DAF or CD55), C4bbinding protein (C4BP) and Factor H (FH) (including its alternative splice product Factor H-like 1 (FHL-1)) (Table 1). These RCA proteins comprise mainly of ‘complement control protein’ (CCP) structural modules which typically contain about 60 amino acid residues, including four conserved cysteines arranged in two disulfide bonds and a conserved tryptophan buried in a beta-sandwich structure. Three of the five regulators are membrane proteins anchored either by a transmembrane domain (CR1, MCP) or tethered to the cell surface via a GPI-anchor (DAF). C4BP, FH and FHL-1 are soluble proteins. FH and FHL-1 are unique in being composed entirely from CCP domains and exclusively regulates the AP of complement activation via decay-accelerating and cofactor activities (Schmidt et al., 2008a). FH has the ability to specifically recognise and target to polyanionic host cell surface markers such as sialic acids or glycosaminoglycans via specialised CCP domains (Schmidt et al., 2008a,b; Zipfel and Skerka, 2009). This mechanism aims to reinforce protection from complement on host surfaces that are void of or do not bear sufficient membrane bound regulators (Ferreira et al., 2006; Meri and Pangburn, 1990; Schmidt et al., 2008a,b)


G Model

ARTICLE IN PRESS


C.Q. Schmidt et al. / Molecular Immunology xxx (2015) xxx–xxx

Classical (CP) & Lectin Pathway (LP) PAMPs & DAMPs

CP & LP pattern recognition

Alternative Pathway (AP) low-level autoactivation of C3

C3 activation by enzymatic cleavage

CP C3-convertase (C4b2a)

3

early AP C3-convertase

C3a C3b

AP AP C3-convertase amplification (C3bBb) loop

iC3b C3b

C3dg

anaphylatoxins: inflammatory mediators opsonins: mediators of phagocytosis

AP-factors D&B

CP C3-convertase (C4b2a)

AP C3-convertase (C3bBb)

CP C5-convertase (C4b2aC3b)

AP C5-convertase (C3bBbC3b)

C5

C5a MAC

C5b

Fig. 2. Scheme of the complement cascade. The complement cascade is initiated by either CP, LP or AP. Within the CP and LP recognition of Pathogen-Associated or Danger Associated-Molecular-Patterns (PAMPS or DAMPS, respectively) lead to formation of the CP C3 convertase. At low level, the conformational autoactivation of C3 initiates the AP leading to the formation of the early AP C3 convertase. Failure to inactivate and regulate the C3 convertases through complement regulators results in the cascade proceeding to its central step: the proteolytic activation of C3 into C3a and C3b. Nascent C3b molecules can covalently attach to cell surfaces. Complement regulators with cofactor activity aid the serum protease Factor I in inactivating the opsonin C3b into iC3b and C3dg. The latter two opsonins are not able to form new convertases. Lack of C3b inactivation leads to procession into the AP amplification loop: C3b molecules can form new C3 convertases, thus activating further C3 molecules and autoamplifing itself. With more C3b molecules being produced, the bimolecular C3-convertases recruit freshly produced C3b molecules to form tri-molecular C5-convertases thus initiating the terminal (lytic) pathway via proteolytic activation of C5 into C5a and C5b. The latter binds several other complement components to form the membrane attack complex. On host surfaces several complement regulators keep the cascade in check.

and also helps the complement system to distinguish ‘self’ from ‘foreign’.

2.2. Complement evasion and exploitation by pathogens For millions of years the phylogenetically old complement system has co-evolved with pathogens (Dunkelberger and Song, 2010). Therefore it is not surprising that bacteria, viruses, fungi and parasites have evolved several mechanisms to circumvent destruction by complement (Lambris et al., 2008). Strategies that pathogens deploy to evade complement include (i) recruitment of host regulators, (ii) secretion of pathogen-encoded regulators which mimic host regulators, (iii) inactivation of complement effectors by secreted proteases, (iv) interference with convertase formation and stability, (v) inhibition of MAC formation and (vi) interference with anaphylatoxin signalling. Not surprisingly, some pathogens utilise simultaneously several complement evasion strategies to ensure efficient complement evasion (Lambris et al., 2008). In this regard

Staphylococcus and Streptococcus species appear to be especially skilful. The recruitment of host regulators is often achieved by means of expressing surface proteins that specifically bind to the soluble complement regulators FH or C4BP. In some cases bacterial proteins such as OspE of B. burgdorferi, fhbA of B. hermsii and Tuf of P. aeruginosa, can form a tri-molecular complex with the soluble host regulator FH and the complement protein C3b. Formation of such tripartite complexes was shown to enhance the regulator’s functional activity (Meri et al., 2013). For some pathogens the loss of one complement evasion strategy diminishes survival. FH-binding protein (FHBP) of N. meningitidis is a definite virulence factor and essential for the survival within the human host making FHBP an excellent vaccine target (McNeil et al., 2013; Scarselli et al., 2011). Several viruses exploit surface-bound complement proteins as means of entering host cells. Examples are Epstein–Barr virus which interacts with Complement receptor 2 (CR2) (Nemerow et al., 1987), measles virus and human herpesvirus 6 which bind to MCP (Naniche et al., 1993; Santoro et al., 2003), and certain


G Model

ARTICLE IN PRESS


C.Q. Schmidt et al. / Molecular Immunology xxx (2015) xxx–xxx

4

Table 1 Regulation of C3 and C5 activation (by regulators of RCA gene cluster).

Cofactors for Factor I mediated cleavage of C4b or C3b

Decay of C3 & C5 Convertases CP

CP & AP

AP

pathway specificity

CP (C4b only)

CP & AP

AP (C3b only)

C4BP

DAF (CD55) & CR1 (CD35)

FH & FHL-1

regulator name

C4BP

MCP (CD46) & CR1 (CD35)

FH & FHL-1

C4BP: C4-binding protein; CR1: complement receptor 1; DAF: decay accelerating factor; MCP: membrane cofactor protein; FH: Factor H; FHL-1: Factor H like 1; RCA: regulators of complement activation; CP: classical pathway; AP: alternative pathway. Membrane anchored regulatory proteins are indicated by an underline.

enteroviruses, which bind to DAF (Bernet et al., 2003). Remarkably, some intracellular pathogens also exploit the complement system by ‘voluntarily’ allowing their surfaces to be opsonised; the covalently fixed C3 activation products on the pathogen subsequently interact with complement receptors CR1 and Complement receptor 3 (CR3) to facilitate entry into the host cell. HIV, Mycobacterium tuberculosis, Legionella pneumophila and Leishmania spp. were reported to benefit from a ‘voluntary opsonisation strategy’ for host cell entry (Datta and Rappaport, 2006; Payne and Horwitz, 1987; Robledo et al., 1994; Schlesinger et al., 1990; Schorey et al., 1997; Da Silva et al., 1989).

3. Complement receptor 1 and malaria CR1 is a type one integral membrane glycoprotein composed of an N-terminal ectodomain of varying polypeptide size, a transmembrane region and a C-terminal cytoplasmic domain (Fig. 3) (Krych-Goldberg and Atkinson, 2001). In humans, this glycoprotein is expressed on all peripheral blood cells with the exception of platelets, natural killer cells and most T cells (Fearon, 1980; Tedder et al., 1983). Apart from peripheral blood, CR1 has also been identified on follicular dendritic cells in the germinal centres of the lymph nodes, where the glycoprotein captures and retains antigens for B-cell stimulation (Reynes et al., 1985). In contrast to leucocytes, which display about 10,000–30,000 CR1 copies per cell, the number of CR1 molecules on erythrocytes is approximately 300–800 (Moulds, 2010). However, due to the high proportion of erythrocytes in blood, erythrocyte-bound CR1 represent almost 90% of CR1 molecules in circulation (Krych-Goldberg and Atkinson, 2001).

CR1 is characterised by an unusual size polymorphism (Dykman et al., 1984; Holers et al., 1987). Four polypeptide sizes are known (Table 2) and have arisen through duplication or deletion of highly homologous repeating units (Moulds et al., 1991). The frequencies of these CR1 size variants are similar among Caucasians and Africans (Dykman et al., 1984). The ectodomain of the most common CR1 allelic variant CR1*1 is composed of 30 CCPs. According to internal homology the first 28 CCPs can be subdivided into four long homologous repeats (LHR) of seven CCPs each: LHR A, LHR B, LHR C and LHR D. The complement regulatory functions of CR1 map to the three most amino-terminal CCPs within LHRs A, B and C (Klickstein et al., 1988; Krych et al., 1991). CCPs 1–3 in LHR A represent site 1, which binds reasonably to C4b and weakly to C3b. Site 1 harbours the decay accelerating activity for the C3 and C5 convertases of the CP and AP (Krych-Goldberg et al., 1999). The two copies of Site 2 (i.e. CCPs 8–10 and 15–17) bind C3b and C4b efficiently (Tetteh-Quarcoo et al., 2012) and act as cofactors for Factor I cleavage of C3b and C4b (Ross et al., 1982). In the full length ectodomain (which corresponds to soluble CR1 (sCR1)) these sites cooperate to bind the principle ligands C3b and C4b thus leading to higher affinities than observed for each single site (TettehQuarcoo et al., 2012; Tham et al., 2011). In addition to complement regulation the shuttling of C3b- and C4b-opsonized immune complexes (immune adherence) is another major function of CR1 on red blood cells (Birmingham and Hebert, 2001). A second, ‘quantitative’ CR1 polymorphism locates to intron 27 and determines the number of CR1 molecules expressed on erythrocytes (Wilson et al., 1986). Subsequently, this intronic variation has been linked to several extronic substitutions in CR1 (Xiang et al., 1999). In earlier studies, comparing a limited set of geographic regions, the frequencies of the high (H) and low (L) copy number

Fig. 3. Schematic diagram of the most common size variant of CR1: CR*1. CR1*1 comprises an ectodomain of 30 extracellular complement control protein domains (CCP) (circles), a transmembrane (TM) and a cytoplasmatic domain (CYT). The first 28 CCP domains are, according to homology, organised in four long homolgous repeats (LHR) with 7 CCPs per LHR. CCPs 1–3 of LHR A constitute the functional “site 1” which harbours the decay accelerating function and exhibits moderate affinities for C4b and C3b. CCPs 8–10 and CCPs 15–17 in LHR B and C, respectively, constitute “site 2” responsible for cofactor activity. “Site 2” exhibits higher affinity for the principle ligands C3b and C4b than “site 1”. sCR1 comprises the ectodomain of CR1 (CCPs 1–30) and shows the tightest binding behaviour to C3b and C4b due to avidity among the three active sites (“site1” and twice “site 2”). Affinity values are derived from Tetteh-Quarcoo et al. and Tham et al., 2010. CCPs 8–10 and 15–17 are nearly identical and differ only by three amino acids (same shading indicates very high sequence identity). Also CCPs 3, 10 and 17 are highly similar and differ in sequence by up to three amino acids. CCP 25 in LHR D harbours most of the Knops blood group antigens (see Table 2).




5

Table 2 Genetic polymorphic variants of CR1.

types

genomic change

phenotype

CR1*1 (4 LHR:220 kDa)#

Polypeptide size variants

deletion or duplication of genomic DNA

CR1*2 (5 LHR: 250 kDa) CR1*3 (3 LHR: 190 kDa) CR1*4 (6 LHR: 280 kDa)

quantitative variants

CR1 genetic poly-

(number of CR1 molecules on erythrocytes vary)

~100 CR1 molecules per cell (homozygous for low expression: LL)

intronic & extronic SNPs (linkage disequilibrium)

intermediate expression (heterozygous: LH)

~1000 CR1 molecules per cell (homozygous for high expression; HH)

morphic variants

comment

CR1*1 allotype is most frequent (>0.82); combined frequency of CR1*1 and CR1*2 is almost ~1.0. H and L alleles affect number of CR1 molecules only on erythrocytes (not on other cell types);

association of HH, HL, LL genotype with CR1 erythrocyte expression phenotype is not true for Africans

Kna & Knb Knops antigens: KN1 / KN2 (in CCP25: V1561M )

McCa & McCb Knops antigens: KN3 / KN6 (in CCP25: K1590E)

Sl1 & Sl2

Knops blood group system*

extronic SNPs

Knops antigens: KN4 / KN7 (in CCP25: R1601G) (alternative nomenclature: a c d Sl & Vil , or McC & McC )

single antigen: Yka Knops antigens: KN5 (=T1408) (in linker between CCPs 22/23: a/aYk corresponds to T1408M)

single antigen: KCAM+

Sl2 (G1601) phenotype reduces rosetting, is common in Africans, but rare in Caucasoids;

Together Sl1 (R1601) and S1610 comprise the conformational epitope Sl3 (= Knops antigens: KN8)

Knops antigens: KN9 (=I1615) (in CCPs 25: CKAM+/corresponds to I1615V)

* #

Kn, Knops; McC, McCoy; Sl, Swain–Langley; Yk, York. MW estimated on SDS-PAGE under reducing conditions.

alleles were found to be about 0.75 and 0.25, respectively (Herrera et al., 1998; Wilson et al., 1986; Xiang et al., 1999). Homozygotes for the L allele typically express fewer than 200 copies of CR1, with homozygotes for the H allele exceeding several times this number (typically 3- to 4-fold, but ratios up to about 10-fold are described (Wilson et al., 1986)), and heterozygotes being intermediate. Erythrocytes with very low CR1 copy number (typically fewer than 100 CR1 copy per red cell) have the “null” serologic phenotype, which is also known as Helgeson phenotype (Moulds et al., 1992). Intriguingly, in Africans the L allele does not correlate with CR1 copy number on red cells (Herrera et al., 1998; Rowe et al., 2002). A later

study extended the mapping of the number of H/L alleles to a wider geographic range and found that the L allele frequency was higher in malaria endemic regions of Asia but not in Africa (Thomas et al., 2005). Another CR1 polymorphism of interest relates to the Knops blood group system. This system consists of nine antigens (KN1–KN9) comprising the three antigenic pairs V1561M, K1590E and R1601G and one conformational epitope (R1601 with S1610) in CCP25, and the single antigens T1408 located in the linker between CCPs 22–23 and I1615 in CCP25 (Table 2) (Covas et al., 2007; Moulds et al., 1991, 2001, 2002, 2005; Veldhuisen et al., 2011).


G Model MIMM-4627; No. of Pages 14 6


3.1. CR1 polymorphism in malaria endemic regions: implications for rosetting and immune adherence

associate with malaria pathology in this region (Bellamy et al., 1998).

Quantitative blood group polymorphisms impact on CR1’s immune adherence function and its ability to form rosettes with P. falciparum infected erythrocytes. Rosetting is additionally influenced by the Knops blood group polymorphisms. Since immune adherence takes place when erythroid CR1 binds C3b- and C4bopsonised substrates, it is comprehensible that the number of CR1 molecules on red cells influences this function. Opsonised immune complexes bind CR1 on red cells, which act as an inert shuttle to deliver their cargo to the liver and spleen. There, the opsonised substrates transfer to macrophages to be ingested and eliminated, while the erythrocytes return to circulation. This mechanism ensures safe removal of immune complexes (IC) to maintaining tissue homeostasis. Failure to do so exposes vulnerable tissue to non-cleared IC and associates with pathologies including severe forms of malaria. Rosetting is a phenomenon where P. falciparum-infected erythrocytes bind to uninfected red cells to form clusters (Rowe et al., 2009) (Fig. 4). These clusters of red cells are thought to obstruct small blood vessels thus contributing to severe, life-threatening forms of malaria (Kaul et al., 1991). Interactions between infected and uninfected erythrocytes are established by the parasite protein PfEMP1 which is expressed on the surface of infected erythrocytes in structures called knobs (Chen et al., 2000). Rosetting is mediated either by PfEMP1 binding to ABO blood groups antigens (Rowe et al., 2007) and/or to CR1 (Rowe et al., 1997). The interaction site of PfEMP1 on CR1 has been mapped to the functional site 2 (Rowe et al., 2000).

3.1.2. CR1 Knops blood group polymorphism Intriguingly, in the African population the frequency of the Knops antigens E1590 (McCb ) and G1601 (Sl2) are unusually high (Moulds et al., 2001; Thathy et al., 2005). This led to the hypothesis that these antigens were under selective pressure in order to provide protection against severe malaria in malariaendemic regions. Laboratory experiments with the E1590/G1601 phenotype showed reduction of rosette formation (Rowe et al., 1997). More recently the underlying Knops antigens were examined for their ability to influence CR1 functional activities, but no difference was found (Tetteh-Quarcoo et al., 2012). In addition, the Knops antigen variants did not exhibit any inhibition of either parasites invasion or rosette formation (Tetteh-Quarcoo et al., 2012). It has also been investigated whether the Sl2/McCb alleles associate with reduced susceptibility to severe malaria, but several studies failed to produce concordant results (Gandhi et al., 2009; Jallow et al., 2009; Thathy et al., 2005; Zimmerman et al., 2003). Again, regional population or pathogen variations may account for these observed discrepancies. It is important to highlight that the selective pressure on the Knops polymorphism may have arisen from the interaction of CR1 with another infective agent like Mycobacterium tuberculosis (Noumsi et al., 2011). Collectively, these contradictory reports underline the complexity of CR1 in malaria pathogenesis and call for further studies.

3.1.1. CR1 expression polymorphism In regards to protecting the host from adverse effects during P. falciparum infections, the requirements in terms of erythroid CR1 copy number are contrary when immune adherence and rosetting are concerned: while many CR1 molecules may facilitate efficient clearance of IC observed during infection (Mibei et al., 2005), more CR1 copies may support rosetting and thus promote more severe forms of the disease. In people of Caucasian and Asian origin the erythroid CR1 copy number correlates with high (H) and low (L) expression alleles in the CR1 gene (Table 2). In Papua New Guinea (PNG), a malaria endemic region, an erythroid CR1 density lower than 200 molecules was very common (∼80%). In PNG, low CR1 numbers were found to protect against severe forms of malaria (Cockburn et al., 2004; Thomas et al., 2005). Intriguingly, only the HL genotype associated with protection whereas homozygotes for LL exhibiting only a non-significant trend towards protection. In disagreement with this finding, in Thailand two reports showed that low CR1 densities (homozygotes for LL) are not associated with protection from severe disease forms (Nagayasu et al., 2001; Teeranaipong et al., 2008). Several other studies further contribute to this discrepancy between reports (Fowkes et al., 2008; Kosoy et al., 2011; Lin et al., 2010; Panda et al., 2012; Rout et al., 2011; Sinha et al., 2009). Overall, no general rules can be proposed whether or not CR1 density on erythrocytes protects from malaria pathologies. Regional differences within parasite and human populations may account for these observed discrepancies. Also, in some specific conditions it may be more beneficial to strengthen the ability to remove immune complexes (high CR1 density), while in others reduction of rosettes (low CR1 density) may lead to a more promising outcome. Since in Africans the expression polymorphism is not in effect (Herrera et al., 1998; Rowe et al., 2002; Thomas et al., 2005; Xiang et al., 1999), it is hardly surprising that the low expression allele L does not

3.2. CR1 as entry receptor for P. falciparum merozoites Being an obligate intracellular organism, malaria parasites must invade red blood cells to grow and replicate (Fig. 1). The invasive forms of the malaria parasites, the merozoites, invade red blood cells through a multi-step process that involves initial contact with the red blood cell, reorientation of the apical tip of the merozoite to allow close juxtaposition with the red blood cell surface and the formation of a tight junction which moves progressively towards the posterior end of the parasite until host cell membrane fusion is completed (reviewed in (Tham et al., 2012)). These steps in invasion are dependent on specific interactions between multiple parasite adhesins and their respective host red blood cell receptors (Butcher et al., 1973; Gratzer and Dluzewski, 1993). To date, two P. falciparum gene families have been identified to be involved in recognition and binding to red blood cell surface receptors; the P. falciparum erythrocyte binding antigens (PfEBAs: EBA-181, EBA-175, EBA-165, EBA-140 and EBL-1) (Adams et al., 1992; Gilberger et al., 2003; Lobo et al., 2003; Maier et al., 2003; Mayer et al., 2001; Sim et al., 1994; Thompson et al., 2001) and P. falciparum reticulocyte binding homolog proteins (PfRhs: PfRh1, PfRh2a, PfRh2b, PfRh3, PfRh4 and PfRh5) (Baum et al., 2009; Duraisingh et al., 2003; Hayton et al., 2008; Kaneko et al., 2002; Rayner et al., 2000, 2001; Stubbs et al., 2005; Triglia et al., 2001). All members of these families are expressed and functional except EBA-165 and PfRh3, which appear to encode pseudo genes (Taylor et al., 2001; Triglia et al., 2001). Several red blood cell receptors have been identified as entry points for P. falciparum. The glycophorins were the first red blood cells proteins identified to be involved in P. falciparum invasion and exclusively bound members of the PfEBA family of proteins (glycophorin A to EBA-175, glycophorin B to EBL-1 and glycophorin C to EBA-140) (Lobo et al., 2003; Maier et al., 2003; Mayer et al., 2002; Sim et al., 1994). More recently, it was identified that PfRh5 bound to basigin to mediate an essential role in parasite invasion (Crosnier et al., 2011). From a complement perspective, P. falciparum also uses CR1 to enter red blood cells (Spadafora et al., 2010; Tham et al., 2010) (Fig. 4). The parasite adhesin, PfRh4 is responsible for




7

Fig. 4. Complement and Malaria. A. Complement evasion of malaria parasites in the mosquito. Emerging sexual stages of malaria parasites bind to FH to protect themselves from complement-mediated lysis (shown in yellow). FH is recruited to the parasite surface by GAP50. B. Complement effects and malaria parasites in human blood stage. (i) P. falciparum invasion into red blood cells uses the parasite ligand PfRh4 to recognises CR1 on the surface of red blood cells for parasite entry. This interaction allows P. falciparum parasites to invade red blood cells via an independent pathway to glycophorin-mediated entry. (ii) During the trophozoite stages, P. falciparum parasites express PfEMP-1 which is trafficked to the surface of the infected red blood cells where it interacts with CR1 to mediate rosetting. Rosetting has been implicated in causing vascular obstruction and pathogenesis. iii) During schizont rupture, merozoites and digestive vacuoles (in green) are released into the blood stream. Digestive vacuoles can activate and deposit complement on its surface (shown in yellow) which leads to phagocytosis by macrophages. Complement-deposited digestive vacuoles can lead to functional exhaustion of macrophages and increased sepsis seen in severe malaria and create a bystander effect that promotes lysis of adjacent red blood cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

binding directly to CR1 to mediate successful invasion into red blood cells in an alternate pathway to glycophorins (Reiling et al., 2012; Stubbs et al., 2005; Tham et al., 2009). PfRh4 binding was directly correlated with the levels of CR1 on the surface. In addition, PfRh4-mediated P. falciparum invasion is reduced in the presence of low CR1 versus high CR1 levels on the red blood cell surface (Tham et al., 2010). These results will have important implications for P. falciparum field isolates as malarious regions such as PNG have reported a higher prevalence of low CR1 phenotypes (Cockburn et al., 2004). Furthermore, P falciparum clinical isolates obtained from Kenyan children with acute malaria infection are able to invade via the alternate CR1 pathway, indicating that the PfRh4-CR1 invasion pathway is used in the field (Awandare et al., 2011). Through mapping studies it has been possible to demarcate the region within CR1 that PfRh4 binds to and to ascertain the effect it has on CR1 function (Fig. 3). The use of multiple recombinant CR1 fragments narrowed the binding site to reside with CCP1-3 of CR1 (Tham et al., 2011). The addition of soluble CCP1-3 to parasite cultures prevents PfRh4 from interacting with membrane-bound CR1

on red blood cells and represents the most effective inhibitor of the CR1-PfRh4 invasion pathway. Binding of PfRh4 did not affect CR1’s ability to simultaneously bind to its normal complement components C3b and C4b providing potential avenues for investigating the effect of complement deposition on the PfRh4-CR1 invasion pathway (Tham et al., 2011). While PfRh4 binding also did not affect co-factor activity (which resides in site 2 of CR1), it resulted in a dramatic reduction of CR1’s decay-accelerating activity. It should be noted that parasite invasion of red blood cells is a rapid process, often taking only a couple of minutes from initial attachment to resealing of the host membrane after internalisation. Therefore, the inhibition of CR1’s activity is extremely transient. We propose that PfRh4 binding to CR1 has minor effects on global complement regulation of the host and that malaria parasites have taken advantage of binding to an essential functional site of CR1 that maybe less mutatable. The exact amino acid residues within CR1 required for its interaction with PfRh4 were identified using extensive mutagenesis analyses (Park et al., 2014). Truncation and deletion constructs of




Fig. 5. PfRh4 binding site on CCP1 of CR1. Six amino acids comprise the set of crucial interaction residues for PfRh4 binding. The four negatively charged side-chains of E6, D18, E19 and E21 (orange atom representation with oxygen and nitrogen atoms being coloured red and blue, respectively) surround the two hydrophobic amino acids W7 and F20 (green atom representation with oxygen and nitrogen atoms coloured as described above). Of all six amino acids indicated in PfRh4 binding, only residue W7 was found to crucially participate in CR1 decay accelerating regulatory function. Other amino acids identified to bear important roles for CR1 site 1 functionality (i.e. decay acceleration of C3 and C5 convertases, C4b- and C3bbinding) locate to the interface between CCP1 and 2 and to CCP2. Coordinates are derived from pdb entry 2MCZ (numbering of amino acids according to CR1 amino acid sequence without considering the signal peptide). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

LHR A implicated the major binding site for PfRh4 to be within CCP1. CCP8, which shares 60% amino acid identity with CCP1, has no capability to bind PfRh4. By replacing small clusters of adjacent amino acids within CCP1 with the corresponding amino acids in CCP8, it was identified that residues 6–9, 18–19 and 20–21 were required for PfRh4 binding (Park et al., 2014). Further systematic single amino acid mutation of these clusters showed that D18N and F20S completely abolished binding of PfRh4 to CR1 (Fig. 5). Collectively, the mutagenesis approach identified six amino acids with important contributions to PfRh4 binding. Illustrating these six amino acids on the available protein structure identifies the proposed binding patch for PfRh4 adherence: two hydrophobic amino acids surrounded by four negatively residues mapped to one site of CCP1 (Fig. 5). Intriguingly an engineered binding site that was created by substitution of putative PfRh4-interacting residues from CCP1 into their homologous positions within CCP8 resulted in a 30fold higher affinity for PfRh4 than the native one in CCP 1 (Park et al., 2014). Further investigations using parasite growth and erythrocyte binding assays will determine if this engineered site will serve as a more potent inhibitor of the PfRh4-CR1 invasion pathway than CCP1-3.

4. Complement evasion of malaria parasites 4.1. Complement evasion strategy in the sexual stages of malaria parasites Gametocytes, the sexual forms of the malaria parasites, also develop within human red blood cells (Fig. 1). When a female mosquito takes a blood meal, gametocytes are ingested. Upon arrival at the midgut, external stimuli trigger the release of gametocytes from their red blood cells to fully mature into female and male gametes. Within 1 h post-blood meal, fertilisation of these gametes produces a zygote that develops into a diploid ookinete. The motile ookinete is now able to exit the midgut lumen and embed itself on the exterior wall to develop as an oocyst. Sporozoites develops within the oocyst and when released, they travel to the salivary glands. When the mosquito bites a human, it will inject sporozoites into the blood stream to begin infection in the human host.

The emerging macrogametes and fertilised zygotes recruit FH (from the human blood ingested during the blood meal) to their surface to evade human complement attack within the mosquito midgut (Simon et al., 2013) (Fig. 4). Human complement, in particular the AP, present in the blood meal was found to be active within 1–6 h post-feeding. In the presence of active serum, the numbers of exflagellation centres, macrogametes and zygotes are reduced compared to heat-inactivated serum. However, when utilising an anti-FH monoclonal to inhibit FH function, further reduction in extracellular stage parasites was observed in active serum (Simon et al., 2013). To prevent complete killing by residual human complement in the blood meal, the surface of macrogametes and zygotes recruit FH. While bound to the surface of macrogametes and zygotes, FH still retains functional co-factor activity thus providing protection from complement attack. While most of the studies were performed using in vitro cultured gametocytes, complement’s devastating effects on the sexual parasite forms was also evident in the transmission of parasites to the mosquito host. When mosquitoes were fed on mature gametocytes cultures in the presence of anti-FH monoclonal, a complete inhibition of parasite transmission was observed (Simon et al., 2013). This is thought to be due the destruction of gametocytes in the presence of active complement. FH and FHL-1 are recruited to the surface of the extracellular sexual stages through its interaction with parasite protein GAP50 (Simon et al., 2013). GAP50 is part of a multi-protein complex known as the glideosome, which is required for parasite invasion and substrate gliding motility empowered by an actin-myosin motor. Its identification as the interacting partner is surprising, as GAP50 has been previously localised to the inner membrane complex and therefore thought to not be accessible to the extracellular milieu (Baum et al., 2006). Within FH, the CCP modules of 5–7 were solely responsible for binding to the surface of activated gametocytes. The addition of anti-GAP50 antibodies showed also a reduction in parasite transmission albeit not as striking as the use of anti-FH monoclonal antibody. Further investigation on how anti-GAP50 regulates complement activation on the surface of sexual stages will shed some understanding on the molecular mechanisms at play. 4.2. Complement activation by malaria parasites: implications for pathogen clearance Within 48 h inside a red blood cell, a single malaria parasite grows and replicates to form 16–32 new daughter merozoites (Fig. 1). To support its maturation, the parasite uses a specialised organelle called the digestive vacuole to breakdown haemoglobin for a source of amino acids. A product of haemoglobin digestion is toxic heme which is subsequently converted to hemozoin, a safe crystalline form, within the digestive vacuole. Upon completion of this growth phase, the infected red blood cell ruptures to release newly formed merozoites, the digestive vacuole and other contents within the red blood cell cytosol. The released digestive vacuole and its effect on complement activation, phagocytosis and severe malaria-associated bacterial sepsis has been the focus of several studies (Fig. 4). Intact digestive vacuoles activate the alternative pathway and the intrinsic pathway of coagulation (Dasari et al., 2011, 2012). Furthermore, it has been observed that C3 and C5b-9 are present on the membrane of intact digestive vacuoles. Complement activation on the surface results in the rapid phagocytosis of the organelle by polymorphonuclear granulocytes (PMNs) (Dasari et al., 2011, 2012). Phagocytosis of digestive vacuoles removes it from circulation and induces the generation of reactive oxygen species (ROS) surrounding the intact vacuole upon uptake. However, it also appears the overloading of PMNs with phagocytosed digestive vacuoles have direct effects on




the control of bacterial infections in severe malaria cases (Dasari et al., 2011). Children with severe malaria sometimes succumb to septicaemia by bacteria infection that normally would not result in fatality. Dasari et al. provide a potential link between the functional exhaustion of PMNs with phagocytosed digestive vacuoles and reduced microbicidal activity. They observed that PMNs with ingested digestive vacuoles have a lower ROS generation and reduced microbicidal killing upon a subsequent challenge with S. aureus. Reduced microbicidal killing was also observed using S. typhimurium (Dasari et al., 2011). From these studies it is clear that phagocytosis of digestive vacuoles is important in modulating malaria infections, but in the presence of high parasite loads, as seen in severe malaria cases, it may promote associated pathogenesis of severe malaria such as increased susceptibility to bacterial sepsis. Complement-deposition on digestive vacuoles is thought to also contribute to malaria anaemia (Fig. 4). The interaction of complement-deposited digestive vacuoles with healthy red blood cells results in the deposition of C3 on the adjacent red blood cells and as a consequence, increased erythrophagocytosis. Increased bystander deposition and erythrophagocytosis were enhanced in the presence of erythrocytes lacking DAF and CD59 (Dasari et al., 2014). A previous study also described hematin promoting opsonisation and clearance of red blood cells with the highest levels of CR1 as potentially leading to increased red blood cell destruction (Pawluczkowycz et al., 2007). To determine if the bystander effect occurs within human tissues, the same study performed immunohistochemical staining on sections obtained from brain autopsies of cerebral malaria patients, which showed activated C3 and C5b-9 on erythrocytes adjacent to digestive vacuoles but not on erythrocytes away from a digestive vacuoles (Dasari et al., 2014). It is thought that these bystander effects may contribute to the development of malaria anaemia in severe malaria patients through increased lysis or erythrophagocytosis. An intriguing observation from these studies is the protection of merozoites from complement deposition. Upon rupture of the spent erythrocyte, released merozoites are completely exposed to the human immune system. However in the presence of non-immune active serum, merozoites show no evidence of C3 deposition or being phagocytosed by PMNs (Dasari et al., 2012). While most of these studies were done using immunofluorescence assays, it remains an unanswered question of how merozoites are protected from complement attack on a molecular level. The merozoite surface is covered with numerous merozoite surface coat proteins that may provide a potential protective role towards complement attack, though this suggestion will require further work within the field. 5. Complement and severe malaria 5.1. Complement and cerebral malaria Cerebral malaria represents one of the most severe complications of human malaria infections. It is characterised by unarousable coma with associated parasitemia. It can affect both adults and children with survivors often suffering long-term cognitive and neurological damage. In 1981 Adam et al. investigated the role of immune complexes and complement in cerebral malaria. They found that hypocomplementaemia and elevated levels of the complement activation product C3d were associated with patients who had cerebral malaria compared to those with benign infections (Adam et al., 1981). Since then, the activation of the complement system has been repeatedly implicated in the severest forms of malarial disease. To understand the mechanisms by which complement activation can lead to cerebral malaria a common mouse model of

9

cerebral malaria using P. berghei ANKA has been employed. Mice defective in either C5 synthesis or function are resistant to cerebral malaria, while mice with normal C5 function are susceptible (Patel et al., 2008). C5a has been implicated as the key cytokine driving this reaction as it could potentiate the levels of TNF and IL6 secreted by peripheral blood mononuclear cells (PBMCs) when they are activated by P. falciparum GPI (PfGPI), an abundant surface glycolipid normally used by the parasite to anchor surface proteins, can activate inflammation via the Toll-like receptor signalling (Patel et al., 2008). It has been noted that this property is similar to what occurs during bacterial infection where LPS, another Tolllike receptor pathway activator, potentiates inflammation leading to sepsis (Mäck et al., 2001; Patel et al., 2008; Riedemann et al., 2004; Schaeffer et al., 2007). A role for C5b and the terminal complex has also been suggested since C5aR deletion mice still remain susceptible after a slight delay to cerebral malaria, yet purported blockade of soluble terminal complex formation using an anti-C9 antibody leads to enhanced survival (Ramos et al., 2011). This is an area of some contention as another study has reported C5aR deletion, but not deletion of the related receptor C5L2, leads to improved survival and reduced levels of inflammatory molecules leading to lower levels of sequestered parasitised erythrocytes and mononuclear cells, easing the cerebral malaria burden (Kim et al., 2014). C5aR deletion mice also displayed enhanced angiogenic stability and enhanced blood brain barrier integrity, loss of which are hallmarks of cerebral malaria, indicating that C5a-C5aR contribute to the loss (Kim et al., 2014). A similar process is seen in experimental lupus, a disease that can cause neurological symptoms (Jacob et al., 2010, 2011; Kim et al., 2014). A fuller mechanistic understanding of the role C5 products plays in cerebral malaria will only be reached by studying deletions in other components of the pathway, such as a C6 deletion, to establish the relative effects of each pathway. Deletion of carboxypeptidase M (CPM), which traditionally processes C3a and C5a to less active C3aDesArg and C5aDesArg forms shed little light on which C5 product is primarily responsible in mouse cerebral malaria given the overall disruption to the inflammatory system caused by CPM deletion (Darley et al., 2012). What is known is that regardless of C5a or C5b, the canonical pathways of C5 activation are not required, although may contribute, since C5 activates in mice defective in C3 and C4 pathways. It is likely this activation instead occurs by cleavage by thrombin and other serine proteases (Amara et al., 2010; Ramos et al., 2012). Furthermore, caution is required interpreting these findings as they are all based on observations in mouse models, although they do fit with models of sepsis which is believed to be similar to cerebral malaria (Patel et al., 2008). In humans, C5a levels were significantly elevated in cerebral malaria patients compared to uncomplicated patients supporting the murine data of a role for C5 in cerebral malaria pathogenesis (Kim et al., 2014). Thus, overactive complement in the form of C5 fragment production may contribute to severe malarial disease and efforts to regulate this could lead to better patient outcomes. 5.2. Placental malaria and complement Malaria infection during pregnancy can lead to a severe form of the disease known as placental malaria which is a risk factor for miscarriage, low birth weight and premature delivery. The disease is mediated by the up to 60 members of the PfEMP-1 family of proteins encoded by var genes. PfEMP-1 proteins are exported to the erythrocyte surface where they can mediate adherence and attachment. The members of the family are antigenically diverse facilitating immune evasion. A particular variant called Var2CSA mediates placental malaria by recognising erythrocytes within




the blood vessels of the placenta, along with mononuclear cell infiltrates, occluding blood vessels and leading to inflammation. Placental malaria is a particular risk factor for primiparous woman (those in their first pregnancy) because they have not had the opportunity to develop Var2CSA neutralising antibodies as the placenta is a novel environment (Conroy et al., 2011). In vitro work has revealed that infected erythrocytes can activate complement leading to production of C5a, although the precise source of activation is unclear and it is hypothesised that production of C5a may drive placental malaria (Conroy et al., 2009). The receptor for C5a, C5aR, is upregulated on PBMC surfaces in response to PfGPI in vitro. While PfGPI causes secretion of cytokines such as TNF, IL-6 and IL-1B on its own, C5a acts in synergy to enhance the levels of cytokines produced, perhaps creating a dysregulated response (Conroy et al., 2009). In support of the in vitro observation for C5a production, cohort studies have also found elevated levels of C5a in woman with placental malaria compared to those without placental malaria (Conroy et al., 2013; Khattab et al., 2013). A cohort study of 146 Gabonese woman identified elevated levels of terminal complement complex TCC in the placental and cord blood of pregnant woman with placental malaria versus uninfected woman. In addition, they found participants with elevated TCC were significantly more likely to birth babies in the bottom 25th percentile of birth weight, suggesting a role for complement activation in placental malaria pathogenesis (Khattab et al., 2013). Parity was also examined as a risk factor for low birth weight and regression modelling identified it as a greater risk factor than placental malaria. However, preeclampasia, which may also induce elevated TCC, was not a criteria for patient exclusion from the study (Khattab et al., 2013). However, this study still suggests a role for complement in placental malaria. This conclusion is supported by another study of Malawian woman which found elevated C5a levels were associated with low birth weight after accounting for gravidity (a risk factor for small for gestational age rather than pre-term delivery), gestational age and placental malaria status (Conroy et al., 2013). One of the most remarkable findings with complement in respect to placental malaria is its interplay with angiogenic factors. Early on it was found that C5a and PfGPI led to increased expression of soluble fms-like tyrosine kinase 1 (sFlt1) by PBMCs (Conroy et al., 2009). sFlt1 is an important anti-angiogenic factor also known as soluble VEGF receptor 1. It functions as a decoy receptor for VEGF receptor signalling to prevent angiogenesis (CharnockJones and Burton, 2000). Two other angiogenic factors important in blood vessel remodelling and formation during pregnancy are Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2). Ang-1 and Ang-2 are involved in trophoblast migration and stimulating DNA synthesis for blood vessel development of the placenta, respectively (Dunk et al., 2000; Geva et al., 2002). A study of Malawian woman found that patients with placental malaria had alterations in their angiogenic factors with decreased Ang-1 and elevated sFlt1 and Ang-2 compared to the control group, which was free of placental malaria (Conroy et al., 2013). Structural equation modelling led to a model where parasitised erythrocyte sequestration leads to mononuclear infiltration. The infiltration is inversely proportional to maternal haemoglobin levels, itself a predictor of small for gestational age and foetal growth restriction. This also contributes to C5a production which dysregulates angiogenesis also driving foetal growth restriction. Both foetal growth restriction and “small for gestatinal age” combine to create a low birth weight outcome (Conroy et al., 2013). The mechanistic relationship between placental malaria and angiogenesis has been studied using a mouse model of placental malaria. Deletion of C5aR leads to improved birth weights implicating C5a in foetal growth restriction within the mouse model

(Conroy et al., 2013). Furthermore, in mice with placental malaria Ang-1 levels decreases and Ang-2 and VEGF levels increases regardless of C5aR status, indicating these changes are likely in response to placental malaria to alleviate the increased arterial resistance by increasing angiogenesis. In wildtype mice, sFlt1 expression also increased, leading to an unchanged VEFG/sFlt1 ratio, which would lead to no changes in angiogenesis, whereas in C5aR knockout mice the sFlt1 did not increase, preventing sFLt1 mediated inhibition of VEGF signalling and angiogenesis (Conroy et al., 2013). By imaging the placenta of infected mice they found that infection leads to an increase in the amount of blood vessels. However, C5a-C5aR causes sFlt1 production inhibiting this process, as indicated by a lower level of blood vessels in wild-type PM compared to C5aR deletion PM. This meant that increased arterial resistance was only resolved in C5aR deletion mice. Without resolution arterial resistance can lead to foetal growth restrictions and low birth weights. Despite this, an effect of C5a inflammation on growth hormones cannot be excluded (Conroy et al., 2013). The role C5a plays in development of placental malaria may explain the abundance of a naturally occurring MASP2 inactivating allele in the lectin pathway in African populations (Holmberg et al., 2012). The allele in question, MASP2 rs12085877, inactivates the MBL-MASP2 complex (Fig. 2) due to a substitution of arginine 439 for histidine (Thiel et al., 2009). This could prevent over activation of the complement cascade leading to lower levels of C5a generation conferring protection from placental malaria (Khattab et al., 2013).

6. Mosquito immunity to malaria To complete its life cycle, malaria parasites have to evade the human immune system and to outsmart the innate defence mechanisms of the mosquito host. Several studies have looked at how Anopheles mosquitoes respond to Plasmodium infection. The discovery of thioester protein 1 (TEP1) revealed that there was a complement-like system present in Anopheles driven by thioester dependent deposition of the TEP1 protein (Levashina et al., 2001). Subsequently Leucine rich repeat (LRR) proteins (i.e. Leucine-rich repeat immune protein 1 (LRIM1) and Anopheles Plasmodiumresponsive leucine-rich repeat 1 (APL1) protein family were found to be involved in activating this complement-like defence response in the mosquito against malaria parasites (Fraiture et al., 2009; Riehle et al., 2006, 2008). The APL1 family consists of three proteins APL1A, APL1B and APL1C, which all contain an N-terminal signal peptide, leucine rich repeat motif and a C-terminal coiled coil domain (Riehle et al., 2008). LRIM1 is a member of the LRIM group of Anopheles immune genes and showed promising anti-malarial effects including being required for melanisation of malarial parasites (Osta et al., 2004). TEP1 has demonstrated an ability to control infection of Plasmodium berghei, the murine malaria, in Anopheles gambiae mosquitoes. This is due to TEP1 deposition targeting parasites for either lysis or melanisation as they cross the midgut (Blandin et al., 2004). TEP-1 function is regulated by the heterodimer complex APL1:LRIM1, which has been hypothesised to act as a convertase for conversion of other TEP1 molecules, or as a targeting or protection mechanism for non-self surfaces (Baxter et al., 2010; Fraiture et al., 2009). The complement-like system apparently has capacity for fine pathogen distinction (Mitri et al., 2009) since different APL1 genes are implicated in protection against specific pathogens: the pair of APL1C and LRIM1 has been implicated in protecting the mosquito from P. berghei (Mitri et al., 2009; Riehle et al., 2008), whereas APL1A has been implicated in protection of Anopheles mosquitoes from P. falciparum. In addition to the complement-like defence




system two intracellular immune signalling pathways (Toll/Rel1 and Imd/Rel2) have been identified (Frolet et al., 2006). Plasmodium actively seeks to evade this complement-like system with particular strains, such as NF54, able to survive even in refractory mosquito strains. This ability to survive appears to correlate with geographic location, with strains that are from similar regions to the mosquito strain being better adapted to survival (Molina-Cruz et al., 2012). One mechanism that allows survival is expression of the correct isoform of an oocyst surface protein, Pfs47. Expression of certain Pfs47 isoforms on the parasite surface leads to lower nitration activation, a key activating step in the TEP1 pathway. When Pfs47 is knocked out parasites are susceptible to melanisation and lysis (Molina-Cruz et al., 2013). The skirmish between Plasmodium and the Anopheles immune complex is an exciting area of future research with transmission blocking potential. 7. Conclusions and future perspectives Upon completion of its life cycle, P. falciparum has successfully evaded the immune defence of its two hosts. The erythroid receptor and complement regulator CR1 is implicated in malaria pathologies at various levels. High CR1 densities aid the efficient clearance of IC, which occur at high number during infection, thus protecting vulnerable host tissues and organs such as kidney and brain from inflammation and damage. High expression levels of CR1 may also support the red cells to better control bystander complement activation induced by digestive vacuoles. Keeping complement activation on erythrocyte surfaces to a minimum reduces the risk for anaphylatoxins release, opsono-phagocytosis and direct complement mediated lysis, essentially preventing inflammation and anaemia. However, high CR1 density also has direct antagonising effects, since it promotes rosetting of infected red blood cells and efficient parasite invasion into erythrocytes. Considering these multi-faceted but opposing effects of CR1, it is hardly surprising that several studies have produced conflicting data on how CR1 polymorphisms correlate with malaria disease and severity. Possible explanations for these discrepancies are differences in regional environment together with differences in parasite and human population under study. At first glance it seems counterproductive to consider therapeutic dampening of the human immune system during an infection. However, recent findings clearly correlated severe forms of malaria with increased levels of complement activation products: C3b on opsonised erythrocytes, C5a and MAC. These findings hint at the vague prospect to alleviate the more severe forms of malaria with the help of complement modifying agents. It will be critical to define when and where in the complement cascade and for which condition complement intervention may be beneficial. Interfering at the level of C3 will prevent opsonisation of erythrocytes and subsequent clearance by macrophages, but it expectedly will also tamper with the efficient removal of IC and haemozoin. C5 inhibition may seem the most promising approach given the link between C5a, MAC and complicated malaria. Eculizumab, a therapeutic monoclonal antibody directed against C5, which is approved for therapeutic use in two complement mediated conditions, could theoretically be tested in clinical trials, but the high cost of the drug will likely be an insurmountable obstacle in malaria endemic regions. Increased susceptibility to infection (N. meningitidis) observed under eculizumab therapy poses another obstacle, especially since some severe forms of malaria are per se characterised by a higher incidence of bacterial infections. Alternatively, strategies that target specifically C5a signalling in severe malaria (e.g. C5a neutralising agents or C5aR antagonists) may represent a promising way forward, given the documented correlation of C5a and disease severity. Considering that many pathogens use

11

several simultaneous strategies to evade complement, we expect to see novel interactions between Plasmodium parasites and the complement cascade in the future. A particular interest will be to understand how merozoites avoid destruction by complement once they have emerged from the ruptured erythrocyte after being completely exposed to the onslaught of the human immune system.

Acknowledgements WHT is supported by the Australian Research Council Future Fellowship. CQS is supported by the Deutsche Forschungsgemeinschaft, Germany.

References Adam, C., Géniteau, M., Gougerot-Pocidalo, M., Verroust, P., Lebras, J., Gibert, C., Morel-Maroger, L., 1981. Cryoglobulins, circulating immune complexes, and complement activation in cerebral malaria. Infect. Immun. 31, 530–535. Adams, J.H., Sim, B.K., Dolan, S.A., Fang, X., Kaslow, D.C., Miller, L.H., 1992. A family of erythrocyte binding proteins of malaria parasites. Proc. Natl. Acad. Sci. U. S. A. 89, 7085–7089. Amara, U., Flierl, M.A., Rittirsch, D., Klos, A., Chen, H., Acker, B., Bruckner, U.B., Nilsson, B., Gebhard, F., Lambris, J.D., et al., 2010. Molecular intercommunication between the complement and coagulation systems. J. Immunol. 185, 5628–5636. Awandare, G.A., Spadafora, C., Moch, J.K., Dutta, S., Haynes, J.D., Stoute, J.A., 2011. Plasmodium falciparum field isolates use complement receptor 1 (CR1) as a receptor for invasion of erythrocytes. Mol. Biochem. Parasitol. 177, 57–60. Baum, J., Richard, D., Healer, J., Rug, M., Krnajski, Z., Gilberger, T.-W., Green, J.L., Holder, A.A., Cowman, A.F., 2006. A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites. J. Biol. Chem. 281, 5197–5208. Baum, J., Chen, L., Healer, J., Lopaticki, S., Boyle, M., Triglia, T., Ehlgen, F., Ralph, S.A., Beeson, J.G., Cowman, A.F., 2009. Reticulocyte-binding protein homologue 5 – an essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum. Int. J. Parasitol. 39, 371–380. Baxter, R.H.G., Steinert, S., Chelliah, Y., Volohonsky, G., Levashina, E.A., Deisenhofer, J., 2010. A heterodimeric complex of the LRR proteins LRIM1 and APL1C regulates complement-like immunity in Anopheles gambiae. Proc. Natl. Acad. Sci. U. S. A. 107, 16817–16822. Bellamy, R., Kwiatkowski, D., Hill, A.V., 1998. Absence of an association between intercellular adhesion molecule 1, complement receptor 1 and interleukin 1 receptor antagonist gene polymorphisms and severe malaria in a West African population. Trans. R. Soc. Trop. Med. Hyg. 92, 312–316. Bernet, J., Mullick, J., Singh, A.K., Sahu, A., 2003. Viral mimicry of the complement system. J. Biosci. 28, 249–264. Birmingham, D.J., Hebert, L.A., 2001. CR1 and CR1-like: the primate immune adherence receptors. Immunol. Rev. 180, 100–111. Blandin, S., Shiao, S.-H., Moita, L.F., Janse, C.J., Waters, A.P., Kafatos, F.C., Levashina, E.A., 2004. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116, 661–670. Blaum, B.S., Hannan, J.P., Herbert, A.P., Kavanagh, D., Uhrín, D., Stehle, T., 2015. Structural basis for sialic acid-mediated self-recognition by complement factor H. Nat. Chem. Biol. 11, 77–82. Butcher, G.A., Mitchell, G.H., Cohen, S., 1973. Letter: mechanism of host specificity in malarial infection. Nature 244, 40–41. Charnock-Jones, D.S., Burton, G.J., 2000. Placental vascular morphogenesis. Baillièreaposs Best Pract. Amp Res. Clin. Obstet. Amp. Gynaecol. 14, 953–968. Chen, Q., Heddini, A., Barragan, A., Fernandez, V., Pearce, S.F., Wahlgren, M., 2000. The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors. J. Exp. Med. 192, 1–10. Clark, S.J., Perveen, R., Hakobyan, S., Morgan, B.P., Sim, R.B., Bishop, P.N., Day, A.J., 2010. Impaired binding of the age-related macular degeneration-associated complement factor H 402H allotype to Bruch’s membrane in human retina. J. Biol. Chem. 285, 30192–30202. Cockburn, I.A., Mackinnon, M.J., O’Donnell, A., Allen, S.J., Moulds, J.M., Baisor, M., Bockarie, M., Reeder, J.C., Rowe, J.A., 2004. A human complement receptor 1 polymorphism that reduces Plasmodium falciparum rosetting confers protection against severe malaria. Proc. Natl. Acad. Sci. U. S. A. 101, 272–277. Conroy, A., Serghides, L., Finney, C., Owino, S.O., Kumar, S., Gowda, D.C., Liles, W.C., Moore, J.M., Kain, K.C., 2009. C5a enhances dysregulated inflammatory and angiogenic responses to malaria in vitro: potential implications for placental malaria. PLoS ONE 4, e4953. Conroy, A.L., McDonald, C.R., Silver, K.L., Liles, W.C., Kain, K.C., 2011. Complement activation: a critical mediator of adverse fetal outcomes in placental malaria? Trends Parasitol. 27, 294–299. Conroy, A.L., Silver, K.L., Zhong, K., Rennie, M., Ward, P., Sarma, J.V., Molyneux, M.E., Sled, J., Fletcher, J.F., Rogerson, S., et al., 2013. Complement activation and the resulting placental vascular insufficiency drives fetal growth restriction associated with placental malaria. Cell Host Amp. Microbe 13, 215–226.




Covas, D.T., de Oliveira, F.S., Rodrigues, E.S., Abe-Sandes, K., Silva, W.A., Fontes, A.M., 2007. Knops blood group haplotypes among distinct Brazilian populations. Transfusion (Paris) 47, 147–153. Crosnier, C., Bustamante, L.Y., Bartholdson, S.J., Bei, A.K., Theron, M., Uchikawa, M., Mboup, S., Ndir, O., Kwiatkowski, D.P., Duraisingh, M.T., et al., 2011. Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature 480, 534–537. Darley, M.M., Ramos, T.N., Wetsel, R.A., Barnum, S.R., 2012. Deletion of carboxypeptidase N delays onset of experimental cerebral malaria. Parasite Immunol. 34, 444–447. Dasari, P., Reiss, K., Lingelbach, K., Baumeister, S., Lucius, R., Udomsangpetch, R., Bhakdi, S.C., Bhakdi, S., 2011. Digestive vacuoles of Plasmodium falciparum are selectively phagocytosed by and impair killing function of polymorphonuclear leukocytes. Blood 118, 4946–4956. Dasari, P., Heber, S.D., Beisele, M., Torzewski, M., Reifenberg, K., Orning, C., Fries, A., Zapf, A.-L., Baumeister, S., Lingelbach, K., et al., 2012. Digestive vacuole of Plasmodium falciparum released during erythrocyte rupture dually activates complement and coagulation. Blood 119, 4301–4310. Dasari, P., Fries, A., Heber, S.D., Salama, A., Blau, I.-W., Lingelbach, K., Bhakdi, S.C., Udomsangpetch, R., Torzewski, M., Reiss, K., et al., 2014. Malarial anemia: digestive vacuole of Plasmodium falciparum mediates complement deposition on bystander cells to provoke hemophagocytosis. Med. Microbiol. Immunol. (Berl.) 203, 383–393. Datta, P.K., Rappaport, J., 2006. HIV and complement: hijacking an immune defense. Biomed. Pharmacother. Biomed. Pharmacother. 60, 561–568. Dunk, C., Shams, M., Nijjar, S., Rhaman, M., Qiu, Y., Bussolati, B., Ahmed, A., 2000. Angiopoietin-1 and angiopoietin-2 activate trophoblast Tie-2 to promote growth and migration during placental development. Am. J. Pathol. 156, 2185–2199. Dunkelberger, J.R., Song, W.-C., 2010. Complement and its role in innate and adaptive immune responses. Cell Res. 20, 34–50. Duraisingh, M.T., Triglia, T., Ralph, S.A., Rayner, J.C., Barnwell, J.W., McFadden, G.I., Cowman, A.F., 2003. Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes. EMBO J. 22, 1047–1057. Dykman, T.R., Hatch, J.A., Atkinson, J.P., 1984. Polymorphism of the human C3b/C4b receptor. Identification of a third allele and analysis of receptor phenotypes in families and patients with systemic lupus erythematosus. J. Exp. Med. 159, 691–703. Fearon, D.T., 1980. Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. J. Exp. Med. 152, 20–30. Ferreira, V.P., Herbert, A.P., Hocking, H.G., Barlow, P.N., Pangburn, M.K., 2006. Critical role of the C-terminal domains of factor H in regulating complement activation at cell surfaces. J. Immunol. 177, 6308–6316. Fowkes, F.J.I., Michon, P., Pilling, L., Ripley, R.M., Tavul, L., Imrie, H.J., Woods, C.M., Mgone, C.S., Luty, A.J.F., Day, K.P., 2008. Host erythrocyte polymorphisms and exposure to Plasmodium falciparum in Papua New Guinea. Malar. J. 7, 1. Fraiture, M., Baxter, R.H.G., Steinert, S., Chelliah, Y., Frolet, C., Quispe-Tintaya, W., Hoffmann, J.A., Blandin, S.A., Levashina, E.A., 2009. Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of Plasmodium. Cell Host Amp. Microbe 5, 273–284. Frolet, C., Thoma, M., Blandin, S., Hoffmann, J.A., Levashina, E.A., 2006. Boosting NFkappaB-dependent basal immunity of Anopheles gambiae aborts development of Plasmodium berghei. Immunity 25, 677–685. Gandhi, M., Singh, A., Dev, V., Adak, T., Dashd, A.P., Joshi, H., 2009. Role of CR1 Knops polymorphism in the pathophysiology of malaria: Indian scenario. J. Vector Borne Dis. 46, 288–294. Geva, E., Ginzinger, D.G., Zaloudek, C.J., Moore, D.H., Byrne, A., Jaffe, R.B., 2002. Human placental vascular development: vasculogenic and angiogenic (branching and nonbranching) transformation is regulated by vascular endothelial growth factor-A, angiopoietin-1, and angiopoietin-2. J. Clin. Endocrinol. Metab. 87, 4213–4224. Gilberger, T.-W., Thompson, J.K., Triglia, T., Good, R.T., Duraisingh, M.T., Cowman, A.F., 2003. A novel erythrocyte binding antigen-175 paralogue from Plasmodium falciparum defines a new trypsin-resistant receptor on human erythrocytes. J. Biol. Chem. 278, 14480–14486. Gratzer, W.B., Dluzewski, A.R., 1993. The red blood cell and malaria parasite invasion. Semin. Hematol. 30, 232–247. Hayton, K., Gaur, D., Liu, A., Takahashi, J., Henschen, B., Singh, S., Lambert, L., Furuya, T., Bouttenot, R., Doll, M., et al., 2008. Erythrocyte binding protein PfRH5 polymorphisms determine species-specific pathways of Plasmodium falciparum invasion. Cell Host Microbe 4, 40–51. Herrera, A.H., Xiang, L., Martin, S.G., Lewis, J., Wilson, J.G., 1998. Analysis of complement receptor type 1 (CR1) expression on erythrocytes and of CR1 allelic markers in Caucasian and African American populations. Clin. Immunol. Immunopathol. 87, 176–183. Holers, V.M., Chaplin, D.D., Leykam, J.F., Gruner, B.A., Kumar, V., Atkinson, J.P., 1987. Human complement C3b/C4b receptor (CR1) mRNA polymorphism that correlates with the CR1 allelic molecular weight polymorphism. Proc. Natl. Acad. Sci. U. S. A. 84, 2459–2463. Holmberg, V., Onkamo, P., Lahtela, E., Lahermo, P., Bedu-Addo, G., Mockenhaupt, F.P., Meri, S., 2012. Mutations of complement lectin pathway genes MBL2 and MASP2 associated with placental malaria. Malar. J. 11, 61.

Jacob, A., Hack, B., Chiang, E., Garcia, J.G.N., Quigg, R.J., Alexander, J.J., 2010. C5a alters blood-brain barrier integrity in experimental lupus. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 24, 1682–1688. Jacob, A., Hack, B., Chen, P., Quigg, R.J., Alexander, J.J., 2011. C5a/CD88 signaling alters blood–brain barrier integrity in lupus through nuclear factor-␬B. J. Neurochem. 119, 1041–1051. Jallow, M., Teo, Y.Y., Small, K.S., Rockett, K.A., Deloukas, P., Clark, T.G., Kivinen, K., Bojang, K.A., Conway, D.J., Pinder, M., et al., 2009. Genome-wide and fine-resolution association analysis of malaria in West Africa. Nat. Genet. 41, 657–665. Kaneko, O., Mu, J., Tsuboi, T., Su, X., Torii, M., 2002. Gene structure and expression of a Plasmodium falciparum 220-kDa protein homologous to the Plasmodium vivax reticulocyte binding proteins. Mol. Biochem. Parasitol. 121, 275–278. Kaul, D.K., Roth, E.F., Nagel, R.L., Howard, R.J., Handunnetti, S.M., 1991. Rosetting of Plasmodium falciparum-infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions. Blood 78, 812–819. Kemper, C., Atkinson, J.P., 2007. T-cell regulation: with complements from innate immunity. Nat. Rev. Immunol. 7, 9–18. Khattab, A., Kremsner, P.G., Meri, S., 2013. Complement activation in primiparous women from a malaria endemic area is associated with reduced birthweight. Placenta 34, 162–167. Kim, H., Erdman, L.K., Lu, Z., Serghides, L., Zhong, K., Dhabangi, A., Musoke, C., Gerard, C., Cserti-Gazdewich, C., Liles, W.C., et al., 2014. Functional roles for C5a and C5aR but not C5L2 in the pathogenesis of human and experimental cerebral malaria. Infect. Immun. 82, 371–379. Kirkitadze, M.D., Barlow, P.N., 2001. Structure and flexibility of the multiple domain proteins that regulate complement activation. Immunol. Rev. 180, 146–161. Klickstein, L.B., Bartow, T.J., Miletic, V., Rabson, L.D., Smith, J.A., Fearon, D.T., 1988. Identification of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis. J. Exp. Med. 168, 1699–1717. Kosoy, R., Ransom, M., Chen, H., Marconi, M., Macciardi, F., Glorioso, N., Gregersen, P.K., Cusi, D., Seldin, M.F., 2011. Evidence for malaria selection of a CR1 haplotype in Sardinia. Genes Immun. 12, 582–588. Krych, M., Hourcade, D., Atkinson, J.P., 1991. Sites within the complement C3b/C4b receptor important for the specificity of ligand binding. Proc. Natl. Acad. Sci. U. S. A. 88, 4353–4357. Krych-Goldberg, M., Atkinson, J.P., 2001. Structure-function relationships of complement receptor type 1. Immunol. Rev. 180, 112–122. Krych-Goldberg, M., Hauhart, R.E., Subramanian, V.B., Yurcisin, B.M., Crimmins, D.L., Hourcade, D.E., Atkinson, J.P., 1999. Decay accelerating activity of complement receptor type 1 (CD35). Two active sites are required for dissociating C5 convertases. J. Biol. Chem. 274, 31160–31168. Lambris, J.D., Ricklin, D., Geisbrecht, B.V., 2008. Complement evasion by human pathogens. Nat. Rev. Microbiol. 6, 132–142. Levashina, E.A., Moita, L.F., Blandin, S., Vriend, G., Lagueux, M., Kafatos, F.C., 2001. Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae. Cell 104, 709–718. Lin, E., Tavul, L., Michon, P., Richards, J.S., Dabod, E., Beeson, J.G., King, C.L., Zimmerman, P.A., Mueller, I., 2010. Minimal association of common red blood cell polymorphisms with Plasmodium falciparum infection and uncomplicated malaria in papua new Guinean school children. Am. J. Trop. Med. Hyg. 83, 828–833. Lobo, C.-A., Rodriguez, M., Reid, M., Lustigman, S., 2003. Glycophorin C is the receptor for the Plasmodium falciparum erythrocyte binding ligand PfEBP-2 (baebl). Blood 101, 4628–4631. Mäck, C., Jungermann, K., Götze, O., Schieferdecker, H.L., 2001. Anaphylatoxin C5a actions in rat liver: synergistic enhancement by C5a of lipopolysaccharidedependent alpha(2)-macroglobulin gene expression in hepatocytes via IL-6 release from Kupffer cells. J. Immunol. 167, 3972–3979. Maier, A.G., Duraisingh, M.T., Reeder, J.C., Patel, S.S., Kazura, J.W., Zimmerman, P.A., Cowman, A.F., 2003. Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations. Nat. Med. 9, 87–92. Mayer, D.C., Kaneko, O., Hudson-Taylor, D.E., Reid, M.E., Miller, L.H., 2001. Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175. Proc. Natl. Acad. Sci. U. S. A. 98, 5222–5227. Mayer, D.C.G., Mu, J.-B., Feng, X., Su, X., Miller, L.H., 2002. Polymorphism in a Plasmodium falciparum erythrocyte-binding ligand changes its receptor specificity. J. Exp. Med. 196, 1523–1528. McNeil, L.K., Zagursky, R.J., Lin, S.L., Murphy, E., Zlotnick, G.W., Hoiseth, S.K., Jansen, K.U., Anderson, A.S., 2013. Role of factor H binding protein in Neisseria meningitidis virulence and its potential as a vaccine candidate to broadly protect against meningococcal disease. Microbiol. Mol. Biol. Rev. 77, 234–252. Meri, S., Pangburn, M.K., 1990. Discrimination between activators and nonactivators of the alternative pathway of complement: regulation via a sialic acid/polyanion binding site on factor H. Proc. Natl. Acad. Sci. U. S. A. 87, 3982–3986. Meri, T., Amdahl, H., Lehtinen, M.J., Hyvärinen, S., McDowell, J.V., Bhattacharjee, A., Meri, S., Marconi, R., Goldman, A., Jokiranta, T.S., 2013. Microbes bind complement inhibitor factor H via a common site. PLoS Pathog. 9, e1003308. Mibei, E.K., Orago, A.S.S., Stoute, J.A., 2005. Immune complex levels in children with severe Plasmodium falciparum malaria. Am. J. Trop. Med. Hyg. 72, 593–599. Mitri, C., Jacques, J.-C., Thiery, I., Riehle, M.M., Xu, J., Bischoff, E., Morlais, I., Nsango, S.E., Vernick, K.D., Bourgouin, C., 2009. Fine pathogen discrimination within




the APL1 gene family protects Anopheles gambiae against human and rodent malaria species. PLoS Pathog. 5, e1000576. Molina-Cruz, A., DeJong, R.J., Ortega, C., Haile, A., Abban, E., Rodrigues, J., JaramilloGutierrez, G., Barillas-Mury, C., 2012. Some strains of Plasmodium falciparum, a human malaria parasite, evade the complement-like system of Anopheles gambiae mosquitoes. Proc. Natl. Acad. Sci. U. S. A. 109, E1957–E1962. Molina-Cruz, A., Garver, L.S., Alabaster, A., Bangiolo, L., Haile, A., Winikor, J., Ortega, C., van Schaijk, B.C.L., Sauerwein, R.W., Taylor-Salmon, E., et al., 2013. The human malaria parasite Pfs47 gene mediates evasion of the mosquito immune system. Science 340, 984–987. Moulds, J.M., 2010. The Knops blood-group system: a review. Immunohematol. Am. Red Cross 26, 2–7. Moulds, J.M., Nickells, M.W., Moulds, J.J., Brown, M.C., Atkinson, J.P., 1991. The C3b/C4b receptor is recognized by the Knops, McCoy, Swain-langley, and York blood group antisera. J. Exp. Med. 173, 1159–1163. Moulds, J.M., Moulds, J.J., Brown, M., Atkinson, J.P., 1992. Antiglobulin testing for CR1related (Knops/McCoy/Swain-Langley/York) blood group antigens: negative and weak reactions are caused by variable expression of CR1. Vox Sang 62, 230–235. Moulds, J.M., Zimmerman, P.A., Doumbo, O.K., Kassambara, L., Sagara, I., Diallo, D.A., Atkinson, J.P., Krych-Goldberg, M., Hauhart, R.E., Hourcade, D.E., et al., 2001. Molecular identification of Knops blood group polymorphisms found in long homologous region D of complement receptor 1. Blood 97, 2879–2885. Moulds, J.M., Zimmerman, P.A., Doumbo, O.K., Diallo, D.A., Atkinson, J.P., KrychGoldberg, M., Hourcade, D.E., Moulds, J.J., 2002. Expansion of the Knops blood group system and subdivision of Sl(a). Transfusion (Paris) 42, 251–256. Moulds, J.M., Pierce, S., Peck, K.B., Tulley, M.L., Doumbo, O., Moulds, J.J., 2005. KAM: a new allele in the Knops blood group system. Transfusion 45 (Suppl. 27A), 45. Nagayasu, E., Ito, M., Akaki, M., Nakano, Y., Kimura, M., Looareesuwan, S., Aikawa, M., 2001. CR1 density polymorphism on erythrocytes of P. falciparum malaria patients in Thailand. Am. J. Trop. Med. Hyg. 64, 1–5. Naniche, D., Varior-Krishnan, G., Cervoni, F., Wild, T.F., Rossi, B., Rabourdin-Combe, C., Gerlier, D., 1993. Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. J. Virol. 67, 6025–6032. Nemerow, G.R., Mold, C., Schwend, V.K., Tollefson, V., Cooper, N.R., 1987. Identification of gp350 as the viral glycoprotein mediating attachment of Epstein–Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d. J. Virol. 61, 1416–1420. Noumsi, G.T., Tounkara, A., Diallo, H., Billingsley, K., Moulds, J.J., Moulds, J.M., 2011. Knops blood group polymorphism and susceptibility to Mycobacterium tuberculosis infection: knops polymorphism and M. tuberculosis infection. Transfusion (Paris) 51, 2462–2469. Osta, M.A., Christophides, G.K., Kafatos, F.C., 2004. Effects of mosquito genes on Plasmodium development. Science 303, 2030–2032. Panda, A.K., Panda, M., Tripathy, R., Pattanaik, S.S., Ravindran, B., Das, B.K., 2012. Complement receptor 1 variants confer protection from severe malaria in Odisha, India. PLOS ONE 7, e49420. Park, H.J., Guariento, M., Maciejewski, M., Hauhart, R., Tham, W.-H., Cowman, A.F., Schmidt, C.Q., Mertens, H.D.T., Liszewski, M.K., Hourcade, D.E., et al., 2014. Using mutagenesis and structural biology to map the binding site for the Plasmodium falciparum merozoite protein PfRh4 on the human immune adherence receptor. J. Biol. Chem. 289, 450–463. Patel, S.N., Berghout, J., Lovegrove, F.E., Ayi, K., Conroy, A., Serghides, L., Min-oo, G., Gowda, D.C., Sarma, J.V., Rittirsch, D., et al., 2008. C5 deficiency and C5a or C5aR blockade protects against cerebral malaria. J. Exp. Med. 205, 1133–1143. Pawluczkowycz, A.W., Lindorfer, M.A., Waitumbi, J.N., Taylor, R.P., 2007. Hematin promotes complement alternative pathway-mediated deposition of C3 activation fragments on human erythrocytes: potential implications for the pathogenesis of anemia in malaria. J. Immunol. 179, 5543–5552. Payne, N.R., Horwitz, M.A., 1987. Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors. J. Exp. Med. 166, 1377–1389. Ramos, T.N., Darley, M.M., Hu, X., Billker, O., Rayner, J.C., Ahras, M., Wohler, J.E., Barnum, S.R., 2011. Cutting edge: the membrane attack complex of complement is required for the development of murine experimental cerebral malaria. J. Immunol. 186, 6657–6660. Ramos, T.N., Darley, M.M., Weckbach, S., Stahel, P.F., Tomlinson, S., Barnum, S.R., 2012. The C5 convertase is not required for activation of the terminal complement pathway in murine experimental cerebral malaria. J. Biol. Chem. 287, 24734–24738. Rayner, J.C., Galinski, M.R., Ingravallo, P., Barnwell, J.W., 2000. Two Plasmodium falciparum genes express merozoite proteins that are related to Plasmodium vivax and Plasmodium yoelii adhesive proteins involved in host cell selection and invasion. Proc. Natl. Acad. Sci. U. S. A. 97, 9648–9653. Rayner, J.C., Vargas-Serrato, E., Huber, C.S., Galinski, M.R., Barnwell, J.W., 2001. A Plasmodium falciparum homologue of Plasmodium vivax reticulocyte binding protein (PvRBP1) defines a trypsin-resistant erythrocyte invasion pathway. J. Exp. Med. 194, 1571–1581. Reiling, L., Richards, J.S., Fowkes, F.J.I., Wilson, D.W., Chokejindachai, W., Barry, A.E., Tham, W.-H., Stubbs, J., Langer, C., Donelson, J., et al., 2012. The Plasmodium falciparum erythrocyte invasion ligand Pfrh4 as a target of functional and protective human antibodies against malaria. PLOS ONE 7, e45253. Reynes, M., Aubert, J.P., Cohen, J.H., Audouin, J., Tricottet, V., Diebold, J., Kazatchkine, M.D., 1985. Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens. J. Immunol. 135, 2687–2694. Ricklin, D., Hajishengallis, G., Yang, K., Lambris, J.D., 2010. Complement: a key system for immune surveillance and homeostasis. Nat. Immunol. 11, 785–797.

13

Riedemann, N.C., Guo, R.-F., Hollmann, T.J., Gao, H., Neff, T.A., Reuben, J.S., Speyer, C.L., Sarma, J.V., Wetsel, R.A., Zetoune, F.S., et al., 2004. Regulatory role of C5a in LPS-induced IL-6 production by neutrophils during sepsis. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 18, 370–372. Riehle, M.M., Markianos, K., Niare, O., Xu, J., Li, J., Touré, A.M., Podiougou, B., Oduol, F., Diawara, S., Diallo, M., et al., 2006. Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region. Science 312, 577–579. Riehle, M.M., Xu, J., Lazzaro, B.P., Rottschaefer, S.M., Coulibaly, B., Sacko, M., Niare, O., Morlais, I., Traore, S.F., Vernick, K.D., 2008. Anopheles gambiae APL1 is a family of variable LRR proteins required for Rel1-mediated protection from the malaria parasite, Plasmodium berghei. PLoS ONE 3, e3672. Robledo, S., Wozencraft, A., Valencia, A.Z., Saravia, N., 1994. Human monocyte infection by Leishmania (Viannia) panamensis. Role of complement receptors and correlation of susceptibility in vitro with clinical phenotype. J. Immunol. 152, 1265–1276. Rodriguez de Cordoba, S., Lublin, D.M., Rubinstein, P., Atkinson, J.P., 1985. Human genes for three complement components that regulate the activation of C3 are tightly linked. J. Exp. Med. 161, 1189–1195. Ross, G.D., Lambris, J.D., Cain, J.A., Newman, S.L., 1982. Generation of three different fragments of bound C3 with purified factor I or serum. I. Requirements for factor H vs CR1 cofactor activity. J. Immunol. 129, 2051–2060. Rout, R., Dhangadamajhi, G., Mohapatra, B.N., Kar, S.K., Ranjit, M., 2011. High CR1 level and related polymorphic variants are associated with cerebral malaria in eastern-India. Infect. Genet. Evol. 11, 139–144. Rowe, J.A., Moulds, J.M., Newbold, C.I., Miller, L.H., 1997. P. falciparum rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature 388, 292–295. Rowe, J.A., Rogerson, S.J., Raza, A., Moulds, J.M., Kazatchkine, M.D., Marsh, K., Newbold, C.I., Atkinson, J.P., Miller, L.H., 2000. Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates. J. Immunol. 165, 6341–6346. Rowe, J.A., Raza, A., Diallo, D.A., Baby, M., Poudiougo, B., Coulibaly, D., Cockburn, I.A., Middleton, J., Lyke, K.E., Plowe, C.V., et al., 2002. Erythrocyte CR1 expression level does not correlate with a HindIII restriction fragment length polymorphism in Africans; implications for studies on malaria susceptibility. Genes Immun. 3, 497–500. Rowe, J.A., Handel, I.G., Thera, M.A., Deans, A.-M., Lyke, K.E., Koné, A., Diallo, D.A., Raza, A., Kai, O., Marsh, K., et al., 2007. Blood group O protects against severe Plasmodium falciparum malaria through the mechanism of reduced rosetting. Proc. Natl. Acad. Sci. U. S. A. 104, 17471–17476. Rowe, J.A., Opi, D.H., Williams, T.N., 2009. Blood groups and malaria: fresh insights into pathogenesis and identification of targets for intervention. Curr. Opin. Hematol. 16, 480–487. Santoro, F., Greenstone, H.L., Insinga, A., Liszewski, M.K., Atkinson, J.P., Lusso, P., Berger, E.A., 2003. Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46. J. Biol. Chem. 278, 25964–25969. Scarselli, M., Aricò, B., Brunelli, B., Savino, S., Di Marcello, F., Palumbo, E., Veggi, D., Ciucchi, L., Cartocci, E., Bottomley, M.J., et al., 2011. Rational design of a meningococcal antigen inducing broad protective immunity. Sci. Transl. Med. 3, 91ra62. Schaeffer, V., Cuschieri, J., Garcia, I., Knoll, M., Billgren, J., Jelacic, S., Bulger, E., Maier, R., 2007. The priming effect of C5a on monocytes is predominantly mediated by the p38 MAPK pathway. Shock Augusta Ga 27, 623–630. Schlesinger, L.S., Bellinger-Kawahara, C.G., Payne, N.R., Horwitz, M.A., 1990. Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. J. Immunol. 144, 2771–2780. Schmidt, C.Q., Herbert, A.P., Hocking, H.G., Uhrín, D., Barlow, P.N., 2008a. Translational mini-review series on complement factor H: structural and functional correlations for factor H. Clin. Exp. Immunol. 151, 14–24. Schmidt, C.Q., Herbert, A.P., Kavanagh, D., Gandy, C., Fenton, C.J., Blaum, B.S., Lyon, M., Uhrín, D., Barlow, P.N., 2008b. A new map of glycosaminoglycan and C3b binding sites on factor H. J. Immunol. 181, 2610–2619. Schorey, J.S., Carroll, M.C., Brown, E.J., 1997. A macrophage invasion mechanism of pathogenic mycobacteria. Science 277, 1091–1093. Da Silva, R.P., Hall, B.F., Joiner, K.A., Sacks, D.L., 1989. CR1, the C3b receptor, mediates binding of infective Leishmania major metacyclic promastigotes to human macrophages. J. Immunol. 143, 617–622. Silver, K.L., Higgins, S.J., McDonald, C.R., Kain, K.C., 2010. Complement driven innate immune response to malaria: fuelling severe malarial diseases. Cell. Microbiol. 12, 1036–1045. Sim, B.K., Chitnis, C.E., Wasniowska, K., Hadley, T.J., Miller, L.H., 1994. Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum. Science 264, 1941–1944. Simon, N., Lasonder, E., Scheuermayer, M., Kuehn, A., Tews, S., Fischer, R., Zipfel, P.F., Skerka, C., Pradel, G., 2013. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13, 29–41. Sinha, S., Jha, G.N., Anand, P., Qidwai, T., Pati, S.S., Mohanty, S., Mishra, S.K., Tyagi, P.K., Sharma, S.K., Venkatesh, V., et al., 2009. CR1 levels and gene polymorphisms exhibit differential association with falciparum malaria in regions of varying disease endemicity. Hum. Immunol. 70, 244–250. Skattum, L., van Deuren, M., van der Poll, T., Truedsson, L., 2011. Complement deficiency states and associated infections. Mol. Immunol. 48, 1643–1655.




Spadafora, C., Awandare, G.A., Kopydlowski, K.M., Czege, J., Moch, J.K., Finberg, R.W., Tsokos, G.C., Stoute, J.A., 2010. Complement receptor 1 is a sialic acidindependent erythrocyte receptor of Plasmodium falciparum. PLoS Pathog. 6, e1000968. Stubbs, J., Simpson, K.M., Triglia, T., Plouffe, D., Tonkin, C.J., Duraisingh, M.T., Maier, A.G., Winzeler, E.A., Cowman, A.F., 2005. Molecular mechanism for switching of P. falciparum invasion pathways into human erythrocytes. Science 309, 1384–1387. Taylor, H.M., Triglia, T., Thompson, J., Sajid, M., Fowler, R., Wickham, M.E., Cowman, A.F., Holder, A.A., 2001. Plasmodium falciparum homologue of the genes for Plasmodium vivax and Plasmodium yoelii adhesive proteins, which is transcribed but not translated. Infect. Immun. 69, 3635–3645. Tedder, T.F., Fearon, D.T., Gartland, G.L., Cooper, M.D., 1983. Expression of C3b receptors on human be cells and myelomonocytic cells but not natural killer cells. J. Immunol. 130, 1668–1673. Teeranaipong, P., Ohashi, J., Patarapotikul, J., Kimura, R., Nuchnoi, P., Hananantachai, H., Naka, I., Putaporntip, C., Jongwutiwes, S., Tokunaga, K., 2008. A functional single-nucleotide polymorphism in the CR1 promoter region contributes to protection against cerebral malaria. J. Infect. Dis. 198, 1880–1891. Tetteh-Quarcoo, P.B., Schmidt, C.Q., Tham, W.-H., Hauhart, R., Mertens, H.D.T., Rowe, A., Atkinson, J.P., Cowman, A.F., Rowe, J.A., Barlow, P.N., 2012. Lack of evidence from studies of soluble protein fragments that Knops blood group polymorphisms in complement receptor-type 1 are driven by malaria. PLOS ONE 7, e34820. Tham, W.-H., Wilson, D.W., Reiling, L., Chen, L., Beeson, J.G., Cowman, A.F., 2009. Antibodies to reticulocyte binding protein-like homologue 4 inhibit invasion of Plasmodium falciparum into human erythrocytes. Infect. Immun. 77, 2427–2435. Tham, W.-H., Wilson, D.W., Lopaticki, S., Schmidt, C.Q., Tetteh-Quarcoo, P.B., Barlow, P.N., Richard, D., Corbin, J.E., Beeson, J.G., Cowman, A.F., 2010. Complement receptor 1 is the host erythrocyte receptor for Plasmodium falciparum PfRh4 invasion ligand. Proc. Natl. Acad. Sci. U. S. A. 107, 17327–17332. Tham, W.-H., Schmidt, C.Q., Hauhart, R.E., Guariento, M., Tetteh-Quarcoo, P.B., Lopaticki, S., Atkinson, J.P., Barlow, P.N., Cowman, A.F., 2011. Plasmodium falciparum

uses a key functional site in complement receptor type-1 for invasion of human erythrocytes. Blood 118, 1923–1933. Tham, W.-H., Healer, J., Cowman, A.F., 2012. Erythrocyte and reticulocyte bindinglike proteins of Plasmodium falciparum. Trends Parasitol. 28, 23–30. Thathy, V., Moulds, J.M., Guyah, B., Otieno, W., Stoute, J.A., 2005. Complement receptor 1 polymorphisms associated with resistance to severe malaria in Kenya. Malar. J. 4, 54. Thiel, S., Kolev, M., Degn, S., Steffensen, R., Hansen, A.G., Ruseva, M., Jensenius, J.C., 2009. Polymorphisms in mannan-binding lectin (MBL)-associated serine protease 2 affect stability, binding to MBL, and enzymatic activity. J. Immunol. 182, 2939–2947. Thomas, B.N., Donvito, B., Cockburn, I., Fandeur, T., Rowe, J.A., Cohen, J.H.M., Moulds, J.M., 2005. A complement receptor-1 polymorphism with high frequency in malaria endemic regions of Asia but not Africa. Genes Immun. 6, 31–36. Thompson, J.K., Triglia, T., Reed, M.B., Cowman, A.F., 2001. A novel ligand from Plasmodium falciparum that binds to a sialic acid-containing receptor on the surface of human erythrocytes. Mol. Microbiol. 41, 47–58. Triglia, T., Thompson, J.K., Cowman, A.F., 2001. An EBA175 homologue which is transcribed but not translated in erythrocytic stages of Plasmodium falciparum. Mol. Biochem. Parasitol. 116, 55–63. Veldhuisen, B., Ligthart, P.C., Vidarsson, G., Roels, I., Folman, C.C., van der Schoot, C.E., de Haas, M., 2011. Molecular analysis of the York antigen of the Knops blood group system. Transfusion (Paris) 51, 1389–1396. Wilson, J.G., Murphy, E.E., Wong, W.W., Klickstein, L.B., Weis, J.H., Fearon, D.T., 1986. Identification of a restriction fragment length polymorphism by a CR1 cDNA that correlates with the number of CR1 on erythrocytes. J. Exp. Med. 164, 50–59. Xiang, L., Rundles, J.R., Hamilton, D.R., Wilson, J.G., 1999. Quantitative alleles of CR1: coding sequence analysis and comparison of haplotypes in two ethnic groups. J. Immunol. 163, 4939–4945. Zimmerman, P.A., Fitness, J., Moulds, J.M., McNamara, D.T., Kasehagen, L.J., Rowe, J.A., Hill, A.V.S., 2003. CR1 Knops blood group alleles are not associated with severe malaria in the Gambia. Genes Immun. 4, 368–373. Zipfel, P.F., Skerka, C., 2009. Complement regulators and inhibitory proteins. Nat. Rev. Immunol. 9, 729–740.


Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

Plasmodium falciparum erythrocyte invasion: combining function with immune evasion.

Plasmodium vivax populations are more genetically diverse and less structured than sympatric Plasmodium falciparum populations.

Biomarkers: more than just markers!

More than just a job.

Complement activation by merozoite antigens of Plasmodium falciparum.

More than just multiple sclerosis.

More than just lip service.

A Unique Virulence Gene Occupies a Principal Position in Immune Evasion by the Malaria Parasite Plasmodium falciparum.

Complement Evasion by Pathogenic Leptospira.

It is more than just beds.

Safety in more than just numbers.

Tumour microenvironment: more than just a mutagen.

Epileptogenesis: More Than Just the Latent Period.

More than just a daily ritual.

Emergency preparedness: more than just a drill.

Glycosylphosphatidylinositols: More than just an anchor?

More than just optic disc swelling.

More than just "histone-like" proteins.

One Health: more than just collaboration.

Alkaptonuria, more than just a mere disease.

Protein prenylation: more than just glue?

More than just reassurance on tiotropium safety.

Metachondromatosis: more than just multiple osteochondromas.