Anita A Koshy

The intracellular parasite Toxoplasma gondii has multiple strategies to alter host cell function, including the injection of rhoptry proteins into the cytosol of host cells as well as bystander populations, but the consequence of these events is unclear. Here, a reporter system using fluorescent parasite strains that inject Cre recombinase with their rhoptry proteins (Toxoplasma-Cre) was combined with Ai6 Cre reporter mice to identify cells that have been productively infected, that have been rhoptry injected but lack the parasite, or that have phagocytosed T. gondii. The ability to distinguish these host-parasite interactions was then utilized to dissect the events that lead to the production of interleukin-12 p40 (IL-12p40), which is required for resistance to T. gondii. In vivo, the use of invasion-competent or invasion-inhibited (phagocytosed) parasites with IL-12p40 (YET40) reporter mice revealed that dendritic cell (DC) and macrophage populations that phagocytose the parasite or are infected can express IL-12p40 but are not the major source, as larger numbers of uninfected cells secrete this cytokine. Similarly, the use of Toxoplasma-Cre parasite strains indicated that dendritic cells and inflammatory monocytes untouched by the parasite and not cells injected by the parasite are the primary source of IL-12p40. These results imply that a soluble host or parasite factor is responsible for the bulk of IL-12p40 production in vivo, rather than cellular interactions with T. gondii that result in infection, infection and clearance, injection of rhoptry proteins, or phagocytosis of the parasite.

The classic anti-viral cytokine interferon (IFN)-β can be induced during parasitic infection, but relatively little is know about the cell types and signaling pathways involved. Here we show that inflammatory monocytes (IMs), but not neutrophils, produce IFN-β in response to T. gondii infection. This difference correlated with the mode of parasite entry into host cells, with phagocytic uptake predominating in IMs and active invasion predominating in neutrophils. We also show that expression of IFN-β requires phagocytic uptake of the parasite by IMs, and signaling through Toll-like receptors (TLRs) and MyD88. Finally, we show that IMs are major producers of IFN-β in mesenteric lymph nodes following in vivo oral infection of mice, and mice lacking the receptor for type I IFN-1 show higher parasite loads and reduced survival. Our data reveal a TLR and internalization-dependent pathway in IMs for IFN-β induction to a non-viral pathogen.

The local production of gamma interferon (IFN-γ) is important to control Toxoplasma gondii in the brain, but the basis for these protective effects is not fully understood. The studies presented here reveal that the ability of IFN-γ to inhibit parasite replication in astrocytes in vitro is dependent on signal transducer and activator of transcription 1 (STAT1) and that mice that specifically lack STAT1 in astrocytes are unable to limit parasite replication in the central nervous system (CNS). This susceptibility is associated with a loss of antimicrobial pathways and increased cyst formation in astrocytes. These results identify a critical role for astrocytes in limiting the replication of an important opportunistic pathogen.

Toxoplasma gondii infection occurs through the oral route, but we lack important information about how the parasite interacts with the host immune system in the intestine. We used two-photon laser-scanning microscopy in conjunction with a mouse model of oral T. gondii infection to address this issue. T. gondii established discrete foci of infection in the small intestine, eliciting the recruitment and transepithelial migration of neutrophils and inflammatory monocytes. Neutrophils accounted for a high proportion of actively invaded cells, and we provide evidence for a role for transmigrating neutrophils and other immune cells in the spread of T. gondii infection through the lumen of the intestine. Our data identify neutrophils as motile reservoirs of T. gondii infection and suggest a surprising retrograde pathway for parasite spread in the intestine.

Toxoplasma gondii is an intracellular parasite that transitions from acute infection to a chronic infective state in its intermediate host via encystation, which enables the parasite to evade immune detection and clearance. It is widely accepted that the tissue cyst perimeter is highly and specifically decorated with glycan modifications; however, the role of these modifications in the establishment and persistence of chronic infection has not been investigated. Here we identify and biochemically and biologically characterize a Toxoplasma nucleotide-sugar transporter (TgNST1) that is required for cyst wall glycosylation. Toxoplasma strains deleted for the TgNST1 gene (Δnst1) form cyst-like structures in vitro but no longer interact with lectins, suggesting that Δnst1 strains are deficient in the transport and use of sugars for the biosynthesis of cyst-wall structures. In vivo infection experiments demonstrate that the lack of TgNST1 activity does not detectably impact the acute (tachyzoite) stages of an infection or tropism of the parasite for the brain but that Δnst1 parasites are severely defective in persistence during the chronic stages of the infection. These results demonstrate for the first time the critical role of parasite glycoconjugates in the persistence of Toxoplasma tissue cysts.