displays an unusual population structure in that 3 clonal lineages predominate in Europe and North America 
. Type I strains are highly virulent in mice, whereas strain types II and III are less virulent and can establish latent infection. Importantly, there is evidence that Type I strains also cause more serious disease in humans 
. Studies in mice suggest that the immune response is an important determinant of virulence, although its exact role is unclear. For example, infection with Type I strains is associated with overproduction of proinflammatory cytokines, whereas the response during Type II infection is more restrained 
. Paradoxically, Type II tachyzoites stimulate higher levels of MØ IL-12 production compared to Type I parasites during in vitro infection 
strain type is an important determinant of activation of STAT signaling pathways during intracellular infection. Forward genetic analysis identified ROP16 as a polymorphic parasite kinase controlling strain-specific activation of STAT3 and STAT6, as well as modulating production of IL-12p40 
. Here, we employed a reverse genetic approach to generate ROP16 deletion mutants as well as control complementation mutants to gain insight into the biological role of this rhoptry molecule during infection.
Previously, it was found that Type I strain parasites induces rapid and sustained STAT3 activation associated with low-level IL-12 production in mouse MØ 
. In contrast, Type II Toxoplasma
fails to sustain STAT3 activation, and this was genetically linked to high level IL-12 synthesis 
. We found Type I parasites lacking ROP16 were severely defective in STAT3 tyrosine phosphorylation. Nevertheless, ΔROP16 parasites maintained an ability to trigger early STAT3 activation. This result is essentially identical to previous data from genetic crosses indicating that Type I ROP16 is required for sustained STAT3 activation rather than the initial response 
. Nevertheless, recent in vitro kinase studies have shown that both STAT3 and STAT6 serve as direct substrates for ROP16 tyrosine kinase activity 
. Therefore, we propose that there are two STAT3 activation phases. The first occurs rapidly and independently of ROP16, and possibly involves activation of JAK molecules. The second wave is necessary for sustained STAT3 activation in Toxoplasma
-infected cells and is likely dependent upon direct tyrosine kinase activity of Type I ROP16. Interestingly, STAT6 tyrosine phosphorylation differed from STAT3 tyrosine phosphorylation insofar as deletion of ROP16 completely eliminated the parasite's ability to activate this signal transducing molecule. Therefore, STAT6 activation during infection is entirely dependent upon the parasite kinase.
Deletion of ROP16 converts Type I parasites from low to high inducers of IL-12. Here, we show that this response, like that induced by the Type II Toxoplasma
ME49 strain, is highly dependent upon the common adaptor MyD88. This molecule is involved in signal transduction through most TLR, as well as signaling through receptors for IL-1β and IL-18 
. We assessed whether TLR2, 4, 9 and 11, MyD88-dependent TLR implicated in the response to Toxoplasma 
, were responsible for high level IL-12 production induced by the ΔROP16 strain. However, using TLR knockout MØ, we found no evidence for involvement of these TLR, and we formally ruled out autocrine IL-1β and IL-18 activity using MØ from caspase-1 knockout mice. The ROP16-dependent and MyD88-dependent IL-12 production that we observe may result from redundant functions of multiple TLR, or it is possible that Toxoplasma
itself may be capable of bypassing TLR and use a novel mechanism to directly trigger MyD88-dependent signaling.
is known to inhibit signaling through TLR ligands such as LPS, and we previously found that this activity was dependent upon STAT3 
. Here, we show that ROP16 controls the ability to suppress TLR4-triggered IL-12p40 and TNF-α production. We reported recently that T. gondii
interferes with LPS-induced chromatin remodeling at the TNF promoter by blocking phosphorylation and acetylation of histone H3, suggesting one mechanism for the suppressive effects of the parasite 
. However, ROP16 does not appear to be involved in this activity, because ΔROP16 parasites maintain the ability to inhibit TLR4-mediated histone H3 modification (data not shown). The biological significance of the down-regulatory effects of Toxoplasma
on TLR signaling is not yet clear. Since Toxoplasma
infection is naturally acquired via oral ingestion of parasites, one possibility is that inhibition is a way to evade the activating effects of bacterial TLR ligands that the host is exposed to during T. gondii
infection in the intestine 
. Alternatively, it is possible that down-regulating TLR signaling is a way for the parasite to avoid the activating effects of its own TLR ligands.
also interferes with signaling mediated by IFN-γ 
. In order to assess this response, we first examined bone marrow-derived and thioglycollate-elicited MØ NO production. In our hands these cells produced undetectable amounts of NO in response to IFN-γ (data not shown). However, both astrocytes and microglial cells are known to produce NO 
, and indeed we found that IFN-γ stimulation of these cells resulted in NO release. While wild-type parasites were able to suppress the response, the ΔROP16 strain was defective in inhibitory activity. Insight into the functional significance of this response may come from the observation that ability to express inducible NO, in particular by microglial cells, is involved in controlling chronic infection in the mouse brain, whereas animals survive acute infection without the iNOS enzyme 
. Therefore, expression of ROP16 may be a parasite mechanism to increase transmission potential by escaping the microbicidal effects of NO in the central nervous system.
Despite the finding that absence of ROP16 results in enhanced IL-12 production and defective ability to inhibit production of proinflammatory mediators, replication and dissemination of tachyzoites was enhanced by deletion of ROP16. We obtained evidence that the reduced replication and dissemination of parental RH parasites compared to ΔROP16 parasites was due to arginine starvation resulting from ROP16-dependent STAT6-mediated induction of arginase-1. The dependence of Toxoplasma
-induced arginase-1 expression on STAT6 activation we observed is in contrast to previously published results by El Kasmi et al. 
. In that study, the authors observed STAT6 activation by Type II ROP16, a result not seen by us or others 
. Likewise, the authors reported STAT6-independent arginase-1 induction during infection with Type II ROP16-expressing parasites, a result that also stands in contrast to our findings. At present we do not understand the reason for these discrepant results, but because El Kasmi et al. used ME49 without manipulation of ROP16 a certain degree of caution regarding STAT6-dependent arginase-1 induction may be warranted.
Regardless, our data are consistent with a view that ROP16-mediated induction of arginase-1 functions to limit parasite replication, and that this is a strategy to facilitate host survival and establishment of latent infection to increase transmission potential. An alternative view comes from the consideration that arginase-1 and iNOS compete for the same substrate - namely, arginine. Thus, ROP16-mediated arginase-1 induction, and consequent arginine depletion in infected cells, may represent a mechanism used by Type I strain parasites to evade the potentially lethal effects of high-level NO production. Consistent with this concept, we previously reported that Type II ME49 infection, in contrast to Type I RH infection, resulted in NO production during in vivo infection in the spleen even though overall cytokine synthesis was greater during Type I infection 
More evidence for an in vivo role of arginase-1 during infection comes from recent findings with Leishmania major
and Schistosoma mansoni 
. In those studies, arginase-1 induction in myeloid cells promoted infection by localized depletion of arginine, in turn leading to suppressed T cell responses. This could possibly have relevance to in vivo responses during Toxoplasma
infection, insofar as several older studies suggested that acute infection is associated with nonspecific T cell suppression 
. We are currently re-examining this issue.
Our study demonstrates ROP16 manipulates host cell signaling pathways that determine availability of arginine for parasite replication and dissemination, and host production of NO. By manipulating arginase-1 levels and consequently arginine availability to both host and parasite, ROP16 may act as a central regulator of parasite replication and transmission potential. In addition to ROP16-mediated control of host arginase-1, it seems possible, and even likely, that ROP16 has additional functions during intracellular infection. The ROP16 molecule is involved in activation of STAT3 and STAT6, transcription factors that each possess their own unique targets. Microarray analysis of host cell responses also suggests that ROP16 has multiple downstream targets 
. Thus, ROP16 is emerging as a molecule at the nexus of the host-parasite interaction, and as such may function as one of the key determinants of strain-specific virulence and transmissibility.
While this manuscript was under review similar findings were reported by an independent group (Jensen et al. Cell Host and Microbe 2011. 9:472).