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

Patricia M Sikorski,Alessandra G Commodaro,Michael E Grigg

doi:10.3389/fimmu.2019.03105

Abstract

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.

Highlights

At early stages of infection, intracellular protozoan parasites are vulnerable to humoral attack in the extracellular environment
Radiolabeled C3 was added to non-immune human serum (NHS) to detect levels of C3b that bound the parasite surface upon activation [7]
20 and 40% NHS rapidly saturated within minutes, and 5 and 2.5% were less sensitive in the absolute level of C3b detected, we found that 10% NHS was the optimal concentration, it did not saturate in minutes and was still sensitive enough to detect variable levels of C3b deposited depending on the conditions used (Figure 1A)

Summary

Introduction

At early stages of infection, intracellular protozoan parasites are vulnerable to humoral attack in the extracellular environment. The complement system, a humoral effector system that activates on pathogen surfaces, is considered the first line of defense. Complement activation is initiated by three pathways, known as the classical, lectin, and alternative pathways. Recognition of immune complexes and microbial carbohydrates activate the classical (CP) and lectin (LP) pathways, respectively. The alternative pathway (AP) is activated by the spontaneous hydrolysis of C3 and can further activate on non-self surfaces that lack host regulator proteins. Recognition triggers a sequential cascade of proteolytic events that results in the assembly of complexes known as convertases, that generate active proteins C3a and C3b, and culminates in the formation a pore complex to mediates direct lysis of pathogens. Can result in damage and inflammation in nearby tissues, it must be tightly regulated by the host [2]

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Results

Conclusion