Fueling the fire of B cell activation.

Antigen activated naïve B cells undergoing germinal center responses have distinct metabolic requirements.

A Flow Cytometry-Based Assay to Measure C3b Deposition on Protozoan Parasites Such as Toxoplasma gondii.

Flow cytometry is a powerful tool that can be used to study protozoan parasite interactions with the complement system. We developed a flow cytometric assay to measure the deposition of complement activation product C3b and to assess resistance to complement-mediated lysis. This assay involves exposing cultured parasites to human serum (the source of human complement) and staining parasites with antibodies against complement proteins to detect and quantify complement components on the parasite surface by flow cytometry. The assay can be used to compare complement activation across a variety of different species of protozoan parasites. As a proof of concept, we describe protocols to study C3 deposition on the single-cell protist Toxoplasma gondii. This parasite actively regulates C3 deposition and proteolytic inactivation to eliminate the formation of lytic pores targeted to the parasite surface coat, which is the end-product of the complement cascade. The antibodies used in this assay recognize both active and inactive forms of C3 deposited on parasite surfaces. Hence, the assay facilitates the identification and characterization of parasite resistance factors that regulate complement deposition and catabolic inactivation. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Culturing human foreskin fibroblasts and Toxoplasma gondii strains Basic Protocol 2: In vitro complement activation assay Support Protocol: Screening of normal human serum Basic Protocol 3: Flow cytometric analysis of C3b deposition.

A Protective and Pathogenic Role for Complement During Acute Toxoplasma gondii Infection.

The infection competence of the protozoan pathogen Toxoplasma gondii is critically dependent on the parasite's ability to inactivate the host complement system. Toxoplasma actively resists complement-mediated killing in non-immune serum by recruiting host-derived complement regulatory proteins C4BP and Factor H (FH) to the parasite surface to inactivate surface-bound C3 and limit formation of the C5b-9 membrane attack complex (MAC). While decreased complement activation on the parasite surface certainly protects Toxoplasma from immediate lysis, the biological effector functions of C3 split products C3b and C3a are maintained, which includes opsonization of the parasite for phagocytosis and potent immunomodulatory effects that promote pro-inflammatory responses and alters mucosal defenses during infection, respectively. In this review, we discuss how complement regulation by Toxoplasma controls parasite burden systemically but drives exacerbated immune responses locally in the gut of genetically susceptible C57BL/6J mice. In effect, Toxoplasma has evolved to strike a balance with the complement system, by inactivating complement to protect the parasite from immediate serum killing, it generates sufficient C3 catabolites that signal through their cognate receptors to stimulate protective immunity. This regulation ultimately controls tachyzoite proliferation and promotes host survival, parasite persistence, and transmissibility to new hosts.

Toxoplasma gondii Recruits Factor H and C4b-Binding Protein to Mediate Resistance to Serum Killing and Promote Parasite Persistence in vivo.

Regulating complement is an important step in the establishment of infection by microbial pathogens. Toxoplasma gondii actively resists complement-mediated killing in non-immune human serum (NHS) by inactivating C3b, however the precise molecular basis is unknown. Here, a flow cytometry-based C3b binding assay demonstrated that Type II strains had significantly higher levels of surface-bound C3b than Type I strains. However, both strains efficiently inactivated C3b and were equally resistant to serum killing, suggesting that resistance is not strain-dependent. Toxoplasma activated both the lectin (LP) and alternative (AP) pathways, and the deposition of C3b was both strain and lectin-dependent. A flow cytometry-based lectin binding assay identified strain-specific differences in the level and heterogeneity of surface glycans detected. Specifically, increased lectin-binding by Type II strains correlated with higher levels of the LP recognition receptor mannose binding lectin (MBL). Western blot analyses demonstrated that Toxoplasma recruits both classical pathway (CP) and LP regulator C4b-binding proteins (C4BP) and AP regulator Factor H (FH) to the parasite surface to inactivate bound C3b-iC3b and C3dg and limit formation of the C5b-9 attack complex. Blocking FH and C4BP contributed to increased C5b-9 formation in vitro. However, parasite susceptibility in vitro was only impacted when FH was blocked, indicating that down regulation of the alternative pathway by FH may be more critical for parasite resistance. Infection of C3 deficient mice led to uncontrolled parasite growth, acute mortality, and reduced antibody production, indicating that both the presence of C3, and the ability of the parasite to inactivate C3, was protective. Taken together, our results establish that Toxoplasma regulation of the complement system renders mice resistant to acute infection by limiting parasite proliferation in vivo, but susceptible to chronic infection, with all mice developing transmissible cysts to maintain its life cycle.