Toxoplasma MIC2 Is a Major Determinant of Invasion and Virulence

Citation: Huynh MH, Carruthers VB ( Toxoplasma MIC2 Is a Major Determinant of Invasion and Virulence My-Hang Huynh 0 1 2 Vern B. Carruthers 0 1 2 0 Current address: Department of Microbiology and Immunology, University of Michigan Medical School , Ann Arbor, Michigan , United States of America 1 Editor: John C. Boothroyd, Stanford University , United States of America 2 W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland , United States of America 3 www.plospathogens.org Like its apicomplexan kin, the obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and uses a unique form of gliding motility. The recent identification of several transmembrane adhesive complexes, potentially capable of gripping external receptors and the sub-membrane actinomyosin motor, suggests that the parasite has multiple options for host-cell recognition and invasion. To test whether the transmembrane adhesin MIC2, together with its partner protein M2AP, participates in a major invasion pathway, we utilized a conditional expression system to introduce an anhydrotetracycline-responsive mic2 construct, allowing us to then knockout the endogenous mic2 gene. Conditional suppression of MIC2 provided the first opportunity to directly determine the role of this protein in infection. Reduced MIC2 expression resulted in mistrafficking of M2AP, markedly defective host-cell attachment and invasion, the loss of helical gliding motility, and the inability to support lethal infection in a murine model of acute toxoplasmosis. Survival of mice infected with MIC2-deficient parasites correlated with lower parasite burden in infected tissues, an attenuated inflammatory immune response, and induction of long-term protective immunity. Our findings demonstrate that the MIC2 protein complex is a major virulence determinant for Toxoplasma infection and that MIC2-deficient parasites constitute an effective live-attenuated vaccine for experimental toxoplasmosis. - Apicomplexan parasites cause significant human and animal diseases such as toxoplasmosis (Toxoplasma gondii), malaria (Plasmodium spp.), cryptosporidiosis (Cryptosporidium spp.), and coccidiosis (Eimeria spp.). Among the invasionrelated apical structures shared by these parasites are the secretory micronemes, which harbor adhesive proteins involved in gliding motility and cell invasion [13]. Recent knockout studies have shown that several Toxoplasma micronemal proteins (MICs) have significant roles in invasion and virulence; however, no single gene disruption completely abolished infection in vitro or in vivo [46]. The transmembrane adhesin MIC2 has long been proposed to play a central role in gliding motility and cell invasion, yet its precise contribution has not been fully established due to lack of strong genetic evidence. Although MIC2 contains several conserved extracellular adhesive domains for receptor-binding and a short cytosolic domain that connects via aldolase to the actinomyosin glideosome [7], it remains unclear whether it functionally overlaps with the numerous other transmembrane adhesins that have emerged from the recent sequencing of the Toxoplasma genome. Moreover, the recent discovery of other proteins that are more intimately associated with the moving junction raises questions regarding the precise contribution of MIC2 to active entry [8]. Since MIC2 is a member of the conserved thrombospondin-related anonymous protein (TRAP) family of adhesins, it may serve as a valuable model for understanding the function of this family in active cell invasion by other apicomplexan parasites. To address the role of MIC2 in T. gondii, we generated a conditional knockout mutant using a tetracycline-regulatable system [9]. We show that MIC2-deficient parasites are severely defected in their ability to attach to and invade host cells and are transformed to a non-lethal strain in the mouse model of infection. Reduced MIC2 Expression Severely Compromises Attachment and Invasion Since previous attempts to directly disrupt the mic2 gene have been unsuccessful, a conditional knockout system was employed. This scheme utilizes a transcriptional transactivator (tTA) protein that regulates expression of genes containing tetracycline operator cassettes upstream of the transcription start site. Genes are constitutively transcribed until the addition of tetracycline releases the tTA from the operator cassettes, preventing further transcription. The parental strain was generated by introducing the tTA into the RH type I strain [9]. The reference strain in this study, tTA-dhfr, expresses tTA and a dihydrofolate reductasethymidylate synthase (dhfr-ts) to control for expression of this selectable marker. Strains generated in this study are shown schematically in Figure 1A. The tetracycline-responsive mic2, henceforth referred to as mic2i, was transfected into the tTA strain using the dhfr-ts marker and an individual clone, named mic2e/mic2i to indicate the presence of both the Toxoplasma gondii is a protozoan parasite that infects a broad range of hosts including humans. In people with weakened immunity resulting from HIV/AIDS or immune-suppressive treatment following organ transplantation, reactivation of a chronic T. gondii infection represents a serious threat, potentially leading to lethal disease within the brain, heart, or lungs. As an intracellular parasite, invasion into a host cell is a critical first step in ensuring parasite survival during infection. By using a regulatable expression system, this study shows that an adhesive protein called MIC2 is a limiting component of the parasites invasion system and that it is required for the corkscrew-like movement of the parasite. Moreover, infection of mice with parasites lacking MIC2 no longer resulted in an acute infection leading to death. Not only do mice survive infection, they are protected from infection with a lethal dose of wild-type parasites, indicating an induction of protective immunity. In addition to having implications for the development of live-attenuated vaccines, this work suggests that novel treatment strategies directed at MIC2 may limit the severity of Toxoplasma infections. endogenous and inducible mic2 genes, was isolated by pyrimethamine selection. A knockout plasmid containing the 59 and 39 mic2 genomic flanking regions and the fulllength chloramphenicol acetyl transferase (CAT) selectable marker was then used to replace the endogenous mic2 by homologous recombination in the mic2e/mic2i clone; a resulting knockout clone, Dmic2e/mic2i, was isolated by chloramphenicol selection. The presence of the inducible mic2 or absence of the endogenous mic2 was confirmed by PCR and Southern blotting (Figure S1). To determine the localization of mic2e and mic2i in all strains, immunofluorescence staining of intracellular parasites was performed. Figure 1B illustrates the spatia (...truncated)