The evasion of host immune defense is critical for pathogens to invade, establish infection and perpetuate in the host. The complement system is one of the first lines of innate immune defense in humans that destroys pathogens in the blood circulation. Activation of the complement system through direct encounter with pathogens or some other agents leads to osmolysis of pathogens, clearance of soluble immune complexes and recruitment of lymphocytes at the site of activation. Although malaria parasites are not exposed to the complement system owing to their intracellular development for most part of their life cycle in the human host, the extracellular stages must face the complement system of human or mosquito or both. In a recent issue of the Biochemical Journal, Sharma et al. reported that Plasmodiumfalciparum LCCL domain-containing protein 1 (PfCCp1) inhibited activation of the classical complement pathway and down-regulated effector responses of dendritic cells, which implicate PfCCp1 and related proteins in immunomodulation of the host that likely benefits the parasite. PfCCp1 belongs to a multi-domain protein family that exists as multimeric protein complexes. It needs to be investigated whether PfCCp1 or its multimeric protein complexes have an immunomodulatory effect in vivo and on the mosquito complement system

Pathogens must defend against immune attack of the host to establish infection and perpetuate. The complement system is one of the first lines of innate immune defense in humans, which inactivates pathogens in the blood circulation and links innate and adaptive immune responses to pathogens. As malaria parasites remain intracellular during most part of their life cycle in the human host, they are not in direct contact of the complement system. However, sporozoites, the parasite stage injected by mosquito into the human host, and merozoites, the stage that infects erythrocytes, are extracellular stages and are at risk of elimination by the complement system. Mature gametocytes and gametes in the mosquito midgut are exposed to the complement systems of both human and mosquito. Ookinete, oocyst and sporozoite stages must counter the complement system of mosquito as well. Only two malaria parasite proteins have been reported to have complement regulatory activities [15]. Sharma et al. [6], in a recent issue of the Biochemical Journal, reported that Plasmodium falciparum LCCL domain-containing protein 1 (PfCCp1) inhibited activation of the classical complement pathway and down-regulated effector responses of dendritic cells (DCs), which implicate PfCCp1 and related proteins in immunomodulation.

PfCCp1 belongs to a multi-domain protein family called the LCCL (named after the proteins it was first identified in: Limulus clotting factor C, Cochlear protein 5b2 and Lung gestation protein 1) domain-containing proteins (CCp) or LCCL/lectin adhesive-like proteins (LAPs) [710]. CCps/LAPs also contain one or more modules of other domains, including LH2 (lipoxygenase homology 2), SR (scavenger receptor cysteine-rich), PTX/LamG (pentraxin/laminin-G), RICIN (ricin B lectin-related carbohydrate-binding), COLFI (a conserved feature of fibrillary collagens), FN2 (fibronectin type 2), SCOPE1 (a galactose-binding-like domain fold) and Zn-f (zinc finger-like binding). CCp/LAP homologs are also present in other Apicomplexans, including Toxoplasma and Cryptosporidium. Malaria parasites have six CCp/LAP proteins (CCp1/LAP2, CCp2/LAP4, CCp3/LAP1, CCp4/LAP6, CCp5/LAP3 and FNPA/LAP5), of which FNPA/LAP5 lacks LCCL domain. PfCCps are predominantly expressed in gametocytes and form multimeric protein complexes (MPCs) on the surface of mature gametocytes, which are also secreted into extracellular milieu as gametes emerge from erythrocytes [8,1113]. Interestingly, the formation of MPCs appears to be critical for correct localization and/or stability of PfCCps, as gene knockout of any of the six PfCCp genes caused significant loss of all the six proteins without any apparent change at the transcript level [12,13]. PfCCp3 and its Plasmodium berghei homolog PbSR (P. berghei scavenger receptor protein) have been shown to be expressed in sporozoites [7]. However, other independent studies have reported the absence or negligible expression of PfCCps in ookinete, oocyst and sporozoite stages [8]. Knockouts of CCp/LAP genes did not affect asexual and sexual stage development, but caused developmental arrest at the oocyst stage, resulting in the failure to form salivary gland sporozoites [7,8,10,14]. Genetic crosses of LAP knockout P. berghei gametocytes with wild-type gametocytes indicated that LAP1, 2, 4 and 6 proteins produced in the female gamete are essential for oocyst maturation and subsequent formation of salivary gland sporozoites [14]. It is intriguing that the loss of function of CCps/LAPs did not affect the stages they are predominantly expressed in, rather CCps/LAPs carried over to the oocyst from the female gamete appear to be sufficient for subsequent development. Furthermore, specific functions of CCp/LAP family in parasite biology and disease pathogenesis remain to be identified.

Human complement system consists of ∼35 serum proteins and membrane-associated proteins, which are produced by the liver in inactive forms [15]. Encounter with pathogens or some other agents results in activation of the complement system through a series of sequential reactions. The activation can occur by (i) antibodies bound to an antigen/pathogen (classical pathway or CP), (ii) complexes of mannose-binding lectin/ficolin with carbohydrates on the pathogen surface (lectin pathway) and (iii) spontaneous production of C3 convertase (alternative pathway). All the three pathways merge at the formation of C3 convertase, which ultimately leads to the formation of a membrane attack complex on the pathogen surface that results in the osmolysis of pathogen, clearance of soluble immune complexes by phagocytes and recruitment of lymphocytes at the site of complement activation by anaphylatoxins. Several soluble and membrane-bound regulators of complement activation maintain complement proteins/their products in inactive state to protect from injury to self-cells [16].

Sharma et al. showed expression of PfCCp1 in asexual erythrocytic stages, and the same group has previously shown that it is secreted into the culture medium during asexual erythrocytic stage development [17]. Sharma et al. used multiple complementary approaches to investigate immunomodulatory activities of PfCCp1, which contains an N-terminal signal sequence followed by RICIN, SCOP1, COLF1, LCCL and Zn-f domains. The signal sequence has been proposed to mediate transport/secretion of the protein to the target location; RICIN is a ricin B lectin domain present in carbohydrate-binding proteins, SCOPE1 is a galactose-binding-like domain fold, COLF1 is a conserved feature of fibrillary collagens, LCCL domain is shared by diverse extracellular multi-domain proteins and it may bind to lipopolysaccharides, and Zn-f is a zinc finger domain that binds to zinc or other metal ions. The authors evaluated recombinant proteins containing the RICIN + SCOPE1 or LCCL domains of PfCCp1 for interaction with human complement proteins C3b and C4b and their effect on activation of the classical CP. The LCCL, but not the RICIN + SCOPE1, domain-containing protein interacted with C3b and C4b with sub- to low micromolar affinity. Recombinant LCCL domain inhibited production of C4d, a degradation product of C4b cleavage that is routinely used as a marker for complement activation. Hence, the authors propose that PfCCp1 negatively regulates complement pathway activation. Full-length PfCCp1 may have stronger inhibition of complement activation than that by individual domains, as the domains together may contribute to cooperative binding with the substrates. Since CCps/LAPs have been shown to exist as MPCs, similar assays with purified CCp/LAP MPCs should be done to evaluate the complement modulatory activity of this family. As PfCCp1 is secreted into the medium during asexual erythrocytic stage development and as PfCCps exist as MPCs on the surface of gametocytes [1113,17], further investigation is required whether PfCCp1 or other CCps are available on the surface of infected erythrocytes/merozoites and/or secreted into the extracellular milieu to interact with the host complement proteins. The culmination of complement activation against the parasite would be lysis of the parasite. It is necessary to test whether CCps/LAPs protect asexual and sexual stages from complement-mediated lysis. This could be tested by incubating these stages with immune/normal human serum in the presence of recombinant or, preferably, MPCs of CCps/LAPs.

The authors also report that compared with the control mice, initial parasite growth was enhanced in mice immunized with recombinant LCCL domain and the immunized mice had slightly increased mortality. The difference of parasite growth and mortality between the immunized and control groups is unlikely to be solely due to neutralization of PfCCp1 by antibodies, leading to its unavailability to inhibit complement activation. If that was the case, parasite growth in the immunized mice group would be less than that in the control mice group, as antibodies would neutralize PfCCp1, thereby relieving the inhibitory effect of PfCCp1 on complement proteins. One could address this by evaluating the effect of complement on erythrocytic parasites in the presence of anti-CCp antibodies and CCps. A similar approach has been used previously, which suggested a role for PfCCps in protection of egressing microgametes from complement-mediated lysis [18].

Mosquitoes have strong innate immunity, including a potent complement system. The key mosquito complement protein is the thioester-containing protein 1 (TEP1), a major antiparasitic factor present in the midgut epithelial cells and the hemolymph [19]. TEP1 is a homolog of human C3. Hence, it would be interesting to test if PfCCp1 or any other CCps/LAPs interact with TEP1 and protect the mosquito stages from the mosquito complement system.

Sharma et al. also show that recombinant RICIN + SCOP1 region of PfCCp1 bound to TLR2 on mouse DCs and modulated several processes, including decreased ROS and proinflammatory cytokine levels. Since PfCCp1 is secreted into the medium during asexual erythrocytic stage development and contains multiple-adhesive domains [17], its interaction with the host cells is plausible. However, it remains to be validated if the interaction of PfCCp1 domains with DCs has any immunomodulatory effect in vivo. One could compare immune responses of mice infected with CCp/LAP knockout and wild-type parasites to address the proposed immunomodulatory function of CCps/LAPs.

Immunomodulatory activities of PfCCp1 could benefit malaria parasites both in the vertebrate and mosquito hosts through targeting specific complement proteins. Since CCps/LAPs exist as MPCs, it needs to be investigated whether MPCs possess the activities observed with the recombinant domains of PfCCp1 and exert immunomodulatory effect in vivo. Given that CCp/LAP knockout parasites develop like wild-type parasites during asexual and sexual stage development, the primary function of these proteins is unlikely to protect the parasite from the complement system. Nonetheless, an accessory immunomodulatory function, as reported by the authors, cannot be ruled out and needs to be further investigated.

Abbreviations

     
  • COLFI

    a conserved feature of fibrillary collagens

  •  
  • CP

    classical pathway

  •  
  • DCs

    dendritic cells

  •  
  • FN2

    fibronectin type 2

  •  
  • LAP

    lectin adhesive-like proteins

  •  
  • MPCs

    multimeric protein complexes

  •  
  • PfCCp1

    Plasmodiumfalciparum LCCL domain-containing protein 1

  •  
  • RICIN

    ricin B lectin-related carbohydrate-binding

  •  
  • SR

    scavenger receptor cysteine-rich

  •  
  • TEP1

    thioester-containing protein 1

  •  
  • Zn-f

    zinc finger-like binding

Funding

P.S.S.'s laboratory is supported with funds from the Department of Biotechnology, India [grants SR/SO/BB/-0124/2012 and BT/COE/34/SP15138/2015] and the Council of Scientific & Industrial Research, India.

Competing Interests

The Author declares that there are no competing interests associated with this manuscript.

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