A Bare-bones Review of the Complement System
The complement system consists of ~30 seemingly innocuous proteins floating in our tissue fluids, with the highest concentrations being found in our bloodstream. Upon activation, they morph sequentially into several protease complexes (see figure below). Each protease cleaves specific complement components, generating the subunits for the next protease complex in the cascade. The cascade ends with assembly of the membrane attack complex, a pore that kills the microbe. It is important to keep in mind that the membrane attack complex is not the only weapon that the complement system uses to kill invading microbes. Some of the complement fragments generated by the proteases also help ignite inflammation, in part by attracting phagocytes that engulf the target microbe.
The complement system is triggered when a complex of complement proteins bearing a recognition subunit, either C1q, a ficolin, or mannose-binding lectin (MBL), bind to certain microbial surface molecules. C1q can also attach to the Fc region of antibodies bound to the microbe. Binding of the recognition subunit to the microbe activates the protease component of the complex. The protease (C1r/C1s and MASP in the figure below) cleaves the complement components C4 and C2, leading to the formation of another protease complex on the surface of the microbe, a C3 convertase with the composition C4b-C2a. Although the classical pathway is triggered by C1q and the lectin pathway by MBL and the ficolins, note that both pathways lead to formation of the C3 convertase.
There is also a third pathway. The alternative pathway is triggered by covalent binding of exposed hydroxyl groups on membrane surfaces to C3b, which is generated by the slow, spontaneous cleavage of C3. Of course all cell surfaces, whether pathogen or host, bear hydroxyl groups. To prevent tissue damage, complement regulators quickly inactivate any C3b molecules that end up binding host cells. C3b molecules that end up bound to the microbe capture factor B, which is subsequently cleaved by the protease factor D, resulting in the formation of another type of C3 convertase, one that comprises the C3b and Bb subunits.
Regardless of its composition, C3 convertase cleaves C3, generating even more C3b, which end up sticking covalently to the microbial surface. C3b is a key player in the complement system. C3b flags the unlucky microbe for destruction by phagocytes, which are attracted to the site of infection by soluble protein fragments generated by the complement proteases. In addition, C3b can also join with either type of C3 convertase, resulting in yet another protease complex called the C5 convertase. Cleavage of C5 by C5 convertase releases C5b, which assembles the complement proteins C6 through C9 into the lethal membrane attack complex.
|A "simplified" view of complement activation. Ficolins are not shown. Source|
What They Did
In an earlier study, Schuijt and colleagues identified the Ixodes scapularis tick protein TSLPI as a target for antibodies generated by rabbits that were immunized with tick saliva. Since TSLPI also possessed anti-complement activity, its properties were examined further by the investigators.
Since TSLPI is actually a glycoprotein, the investigators mass produced TSLPI in an insect cell line so that the recombinant protein, like the native one, would be decorated with sugar molecules. Following production of TSLPI, the glycoprotein was purified for use in the test-tube experiments described below.
Serum contains all the components necessary to execute the complement cascade, including assembly of the membrane attack complex (MAC). An easy way to test whether a bacterial strain is susceptible to complement-mediated killing by MAC is to see how well it survives in serum. When a strain of Borrelia garinii, a European Lyme disease species, was mixed with human serum, the MAC assembled on the surface of the spirochete, and the spirochetes perished. Addition of TSLPI allowed the spirochetes to survive in serum.
As I mentioned earlier, the complement system also has other methods of killing microbes. Some of the complement components that end up attached to the microbe are recognized by phagocytes attracted to the infection site by the complement cleavage products C3a and C5a. This activity, known as opsonophagocytosis, is not reflected in the serum killing assay. To see whether activation of complement produced products that promoted phagocytosis of B. garinii, the investigators added human neutrophils to the mixtures of B. garinii and serum. The neutrophils engulfed the spirochetes within minutes. Fewer spirochetes were engulfed when TSLPI was also present, indicating that TSLPI protected B. garinii from opsonophagocytosis.
A U.S. strain of B. burgdorferi was also tested. Most B. burgdorferi strains survive in human serum unless antibodies against the spirochete are also present. Therefore, TSLPI was tested with serum pooled from seropositive Lyme disease patients. TSLPI promoted survival of B. burgdorferi in immune serum. TSLPI also limited the number of spirochetes that ended up engulfed when neutrophils were added to the immune serum.
The investigators next tried to figure out which of the three complement pathways was being blocked by TSLPI. Fortunately simple assays are available to recreate the classical pathway with the CH50 assay and the alternative pathway with the AP50 assay. Both assays are set up so that activation of the complement pathway leads to the assembly of the membrane attack complex in the plasma membrane of red blood cells, causing them to lyse and release their hemoglobin. The extent of lysis is determined simply by measuring the absorbance of the freed hemoglobin in a spectrophotometer.
In the CH50 assay, human serum is mixed with sheep red blood cells that are coated with anti-sheep red blood cell antibodies. The classical pathway is triggered when C1q, the recognition subunit of the C1 protease complex, binds the antibody. In the figure below, TSLPI or BSA (control) was mixed with different dilutions of human serum prior to addition of the antibody-sensitized red blood cells. Based on the serum killing assay with B. burgdorferi, one may expect the classical pathway to be hindered by TSLPI. Surprisingly, the results indicated that TSLPI had no effect whatsoever ("control" vs. rTSLPI in graph below). The anti-C1q and anti-C3 antibodies, additional controls, blocked the classical pathway, as expected.
|Fig. 3C from Schuijt et al., 2011. Effect of TSLPI on the classical complement pathway determined with a CH50 assay.|
The AP50 assay is identical to the CH50 assay except for a couple of modifications. The red blood cells come from rabbits because sheep red blood cells are not sensitive targets for the human alternative complement pathway. The other critical difference is that the chelator EGTA is added to prevent activation of the classical pathway, which requires calcium for activity. The results (below) show that TSLPI failed to block the alternative pathway as well.
|Fig. 3D from Schuijt et al., 2011. Effect of TSLPI on the alternative complement pathway determined with an AP50 assay.|
No comparable assay is available to examine the lectin pathway. Instead the investigators added dilutions of human serum to mannan-coated ELISA plates to look at deposition of the C4b protein, a component of C3 convertase. Mannan is a polysaccharide made up of the simple sugar mannose and is the molecule recognized by MBL. C4b will be generated only if the lectin pathway is triggered by binding of MBL to mannan. C4b binds to the mannan coat. After the incubation, unbound proteins are washed away, and adherence of C4b is detected by doing a standard ELISA with anti-C4 antibody. The results indicate that TSLPI blocked the deposition of C4b, and thus the lectin pathway, in a dose-dependent manner (see graph below).
|Fig. 3E from Schuijt et al., 2011. Effect of TSLPI on the mannose arm of the lectin-binding pathway determined by ELISA measurement of C4b deposition onto mannan-coated wells.|
Additional experiments showed that TSLPI blocked the very first step in the lectin pathway, binding of MBL to mannan. TSLPI also interfered with the binding of L-ficolin to its target.
To confirm that the lectin pathway killed Borrelia, the authors looked at the killing activity of serum pooled from individuals deficient in MBL. Such individuals were not hard to find. According to the authors, about 25% of the population is deficient in MBL!
A lower percentage of B. garinii ended up being killed in MBL-deficient serum than in normal serum. TSLPI further reduced the killing activity of MBL-deficient sera, suggesting that recognition of B. garinii by ficolin also triggered the lectin pathway. The balance of the lethal activity of human serum was accounted for by the classical pathway; C1 esterase inhibitor, which blocks both the classical and lectin pathways, completely eliminated the killing activity of human serum.
The investigators next determined whether TSLPI aided B. burgdorferi infection of laboratory mice. They knocked down TSLPI expression in Ixodes scapularis by RNA interference and used the altered ticks to transmit B. burgdorferi into mice. As a control, they also had another group of mice that were inoculated with B. burgdorferi with unmodified ticks, which were producing normal levels of TSLPI. When the altered ticks were used to inoculate B. burgdorferi, the bacterial loads in the heart, joint, and skin (distant from the site of tick feeding) turned out to be much less than when the unaltered ticks were used. These results demonstrate that TSLPI protected B. burgdorferi from being killed inside the host.
Even though the complement system encounters pathogens early in infection, all of the in vitro B. burgdorferi experiments were done with serum containing anti-B. burdgorferi antibodies, which are not produced until later during infection. For this reason, the in vitro experiments fail to shed any light on how complement killed B. burgdorferi in the mouse experiment when TSLPI was not around to provide protection. The authors surmised that B. burgdorferi is susceptible to killing by complement-mediated opsonophagocytosis, which should not require antibody since complement components such as C3b can serve as opsonins. Unfortunately, the neutrophil phagocytosis experiment with B. burgdorferi was conducted only with immune serum.
The commentary written by Marconi and McDowell is also worth a look. Although they concede that the evidence for the lectin pathway playing a crucial role in killing Borrelia is compelling, they point out that other studies have implicated the classical and alternative pathways in controlling Lyme Borrelia infections. They also wonder how exactly TSLPI, which most likely acts at the site of the tick bite, influenced the bacterial load in tissues distant from the tick bite. If you're wondering why the mouse experiment wasn't done with B. garinii, Marconi and McDowell explain that tick-animal models for B. garinii infections are not as developed as they are for B. burgdorferi infections.
The bottom line is that the tick salivary glycoprotein TSLPI helps B. burgdorferi and possibly B. garinii establish an infection. The in vitro experiments suggest that TSLPI facilitates infection by protecting Borrelia from being killed by the lectin complement pathway, although it's not clear how the lectin pathway would kill B. burgdorferi if TSLPI was not around to provide protection.
Schuijt, T.J., Coumou, J., Narasimhan, S., Dai, J., DePonte, K., Wouters, D., Brouwer, M., Oei, A., Roelofs, J.J.T.H., van Dam, A.P., van der Poll, T., van't Veer, C., Hovius, J.W., & Fikrig, E. (2011). A tick mannose-binding lectin inhibitor interferes with the vertebrate complement cascade to enhance transmission of the Lyme disease agent Cell Host & Microbe, 10 (2), 136-146 DOI: 10.1016/j.chom.2011.06.010
Marconi, R.T., & McDowell, J.V. (2011). Tick salivary proteins offer the Lyme disease spirochetes an easy ride and another way to hide Cell Host & Microbe, 10 (2), 95-96 DOI: 10.1016/j.chom.2011.08.003 (Commentary)
Schuijt, T.J., Narasimhan, S., Daffre, S., DePonte, K., Hovius, J.W.R., Veer, C., van der Poll, T., Bakhtiari, K., Meijers, J.C.M., Boder, E.T., van Dam, A.P., & Fikrig, E. (2011). Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display PLoS ONE, 6 (1) DOI: 10.1371/journal.pone.0015926
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