The Interactions of Parasite Calreticulin With Initial Complement Components: Consequences in Immunity and Virulence.

Because of its capacity to increase a physiologic inflammatory response, to stimulate phagocytosis, to promote cell lysis and to enhance pathogen immunogenicity, the complement system is a crucial component of both the innate and adaptive immune responses. However, many infectious agents resist the activation of this system by expressing or secreting proteins with a role as complement regulatory, mainly inhibitory, proteins. Trypanosoma cruzi, the causal agent of Chagas disease, a reemerging microbial ailment, possesses several virulence factors with capacity to inhibit complement at different stages of activation. T. cruzi calreticulin (TcCalr) is a highly-conserved, endoplasmic reticulum-resident chaperone that the parasite translocates to the extracellular environment, where it exerts a variety of functions. Among these functions, TcCalr binds C1, MBL and ficolins, thus inhibiting the classical and lectin pathways of complement at their earliest stages of activation. Moreover, the TcCalr/C1 interaction also mediates infectivity by mimicking a strategy used by apoptotic cells for their removal. More recently, it has been determined that these Calr strategies are also used by a variety of other parasites. In addition, as reviewed elsewhere, TcCalr inhibits angiogenesis, promotes wound healing and reduces tumor growth. Complement C1 is also involved in some of these properties. Knowledge on the role of virulence factors, such as TcCalr, and their interactions with complement components in host-parasite interactions, may lead toward the description of new anti-parasite therapies and prophylaxis.

Triatoma infestans Calreticulin: Gene Cloning and Expression of a Main Domain That Interacts with the Host Complement System.

Triatoma infestans is an important hematophagous vector of Chagas disease, a neglected chronic illness affecting approximately 6 million people in Latin America. Hematophagous insects possess several molecules in their saliva that counteract host defensive responses. Calreticulin (CRT), a multifunctional protein secreted in saliva, contributes to the feeding process in some insects. Human CRT (HuCRT) and Trypanosoma cruzi CRT (TcCRT) inhibit the classical pathway of complement activation, mainly by interacting through their central S domain with complement component C1. In previous studies, we have detected CRT in salivary gland extracts from T. infestans We have called this molecule TiCRT. Given that the S domain is responsible for C1 binding, we have tested its role in the classical pathway of complement activation in vertebrate blood. We have cloned and characterized the complete nucleotide sequence of CRT from T. infestans, and expressed its S domain. As expected, this S domain binds to human C1 and, as a consequence, it inhibits the classical pathway of complement, at its earliest stage of activation, namely the generation of C4b. Possibly, the presence of TiCRT in the salivary gland represents an evolutionary adaptation in hematophagous insects to control a potential activation of complement proteins, present in the massive blood meal that they ingest, with deleterious consequences at least on the anterior digestive tract of these insects.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Trypanosoma cruzi calreticulin: a novel virulence factor that binds complement C1 on the parasite surface and promotes infectivity.

In Trypanosoma cruzi, calreticulin (TcCRT) translocates from the endoplasmic reticulum (ER) to the area of flagellum emergence. We propose herein that the parasite uses this molecule to capture complement C1, in an infective apoptotic mimicry strategy. Thus, TcCRT/C1 interactions, besides inhibiting the classical pathway of complement activation as previously shown in our laboratories, will also promote infectivity. This fact correlates with significant increases in TcCRT mRNA levels during early infection stages of a VERO cell line. In vitro, the collagenous and globular C1q domains simultaneously bind TcCRT and antigen aggregated Igs, respectively. Accordingly, mouse immunizations with TcCRT induced humoral responses that, after challenge, correlated with increased parasitemia. Thus, on the parasite surface, whole Igs anti-TcCRT promote C1 deposits on trypomastigotes while, as expected, F(ab')2 fragments decrease it. Likewise, pretreatment of the parasites with whole anti-TcCRT antibodies augmented parasitemia and mortality in mice. In contrast, pretreatment with F(ab')2 fragments anti-TcCRT, devoid of their capacity to provide additional C1q binding sites, was protective. Most important, while pretreatment of trypomastigotes with C1q increased infectivity in the RAW murine cell line, as well as mice mortality and parasitemia, the F(ab')2 fragments significantly interfered with the C1q-dependent infectivity. Differently from other surface molecules involved in infectivity, TcCRT uses C1 as an adaptor molecule to recognize host cells. As expected, since TcCRT is one of several cell surface parasite molecules participating in infectivity, attempts to interfere with the C1/TcCRT interactions with F(ab')2 fragments, were moderately but significantly effective, both in vitro and in vivo.

Inhibitory effect of bilirubin on complement-mediated hemolysis.

We investigated the in vitro action of the bile pigments, unconjugated bilirubin (UB) and bilirubin monoglucuronide (BMG) on complement (C) cascade reaction. Both UB and BMG inhibited hemolysis in the classical pathway (CP) in a dose-dependent manner at low micromolar concentrations, UB showing a stronger effect than BMG. The analysis of the action of UB on the hemolytic activity of the C1, C4, C2 and C-EDTA components of the C cascade revealed that the C1 step was the most inhibited. An enzyme immunoassay was developed to evaluate the effect of UB on the binding of C1q, one of the subcomponents of C1, to human IgM and IgG. The study demonstrated that the unconjugated pigment interferes both the C1q-IgM and -IgG interactions, thus tentatively explaining the inhibitory action of UB on hemolytic activity of C1. We conclude that the anti-complement effect of UB is mainly exerted on the C1 component, the recognition unit of CP. The potential clinical implication of the reported effects in hyperbilirubinemia is discussed.

Complement fixation by Brazilian Pemphigus foliaceus autoantibodies.

This study was undertaken to determine if Brazilian Pemphigus foliaceus (BPF) autoantibodies will fix complement as do P. vulgaris (PV) autoantibodies. When sections of human skin were examined following indirect immunofluorescence (IF) staining, BPF autoantibodies reacted with the upper layers of epidermis, while PV autoantibodies were reactive with the lower layers. When tissue culture epidermal cells were used for indirect IF, BPF autoantibodies were not detected after plating until 24 h, while PV autoantibodies reacted within 18 h of plating. Using complement IF staining methods, BPF autoantibodies were found to fix C1q, C4 and C3 to whole skin sections in an intercellular pattern and to the surface of cultured keratinocytes. Although reactive with different epidermal cell surface antigens, autoantibodies in BPF will fix complement in a fashion similar to autoantibodies in PV.