The Toxoplasma Kinase ROP18: An Active Member of a Degenerate Family

I ntracellular parasitism defines the most intimate of interactions between a pathogen and host. Inherent in this equation are the needs of the pathogen to enter the cell and establish a replication-permissive niche while neutralizing and subverting cellular defenses. The protozoan parasite Toxoplasma gondii is particularly adept at every stage of intracellular parasitism, which has contributed to its being among the most cosmopolitan parasites on the planet [1]. Toxoplasma is a member of the Apicomplexa, so noted by the presence of specialized cytoskeletal and secretory organelles (the apical complex) that play a critical role in invasion [2]. Most prominent among these secretory organelles are the club-shaped rhoptries [3,4]. Recent work, including a detailed cataloging of the rhoptry proteome [5], coupled with the availability of the T. gondii genome and gene expression data (http://www.ToxoDB.org; [6]), have resulted in new insights into rhoptry functions. Among the activities inferred from the analysis of the proteomic and genomic data was a family of predicted serine threonine kinases related to the rhoptry protein ROP2 [7]. While ROP2 and several other members of the ROP2 family retain ‘‘molecular fossils,’’ reflecting their ancestry as kinases, they do not possess kinase activity on account of degeneracy and deletions at key catalytic sites [7]. However, bioinfomatic and structural prediction studies reveal that two members, ROP16 and ROP18, do retain all the elements needed to classify them as true kinases [7]. The focus of this Opinion is the rhoptry-derived kinase ROP18, the initial biochemical analysis of which appears in this issue of PLoS Pathogens from the laboratory of Jean François Dubremetz [8]. This characterization is particularly timely given a pair of recent papers from the Boothroyd [9] and Sibley [10] laboratories identifying ROP18 as a critical contributor to virulence, a finding that represents the first virulence factor identified in Toxoplasma using classical forward genetic approaches. I hope in this article to integrate the results in these seminal papers and present them in the context of Toxoplasma biology, virulence, and microbial pathogenesis in general. Rhoptries are unique secretory/excretory organelles found exclusively in the Apicomplexa that are discharged at the time of invasion and are critical in the establishment of a productive infection [11,12]. Functional studies emanating from the analysis of the rhoptry proteome [5] identified factors critical in establishing the machinery for both parasite invasion and the establishment of the nascent parasitophorous vacuole [13,14]. The parasitophorous vacuole, which defines the replication-permissive niche, is delimited from the host cytoplasm by the parasitophorous vacuole membrane (PVM), a remarkable and enigmatic organelle in its own right [15]. To date, all proteins secreted from the rhoptry either associate with the PVM or are transported across it into the host cell cytoplasm [5,16]. Among these members of the ROP2 family are several particularly interesting proteins. ROP2, the founding member of this family, is secreted to the PVM at the time of parasite invasion, where it is believed to establish itself as an integral membrane protein with its N-terminus exposed to the host cell cytoplasm [17]. The mechanism for membrane association, however, is controversial in light of recent molecular modeling analyses [7]. The presence of an Nterminal signal on the secreted protein that is reminiscent of a canonical mitochondrial import signal has implicated ROP2 as the mediator of the high affinity binding of mitochondria to the PVM [18,19]. The availability of the T. gondii genome led the Dubremetz group to extend the ROP2 family to 16 members based upon both bioinformatic criteria and functional proteomic data [7]. A somewhat surprising revelation regarding the provenance of ROP2 family suggests they have all evolved from a progenitor serine threonine kinase on the basis of the presence of key signature motifs [7]. These motifs, however, have degenerated, particularly at the N-terminus, where critical structural and sequence components for kinase activity are either missing of significantly mutated in most of the members [7]. Most of these degenerate ROP2 family members are predicted [7] or have been demonstrated [20] to lack detectable kinase activity. Interestingly, in most of these proteins lacking kinase activity, the key structural element, a kinase fold at the Cterminus, remains unchanged, which suggests a strong selective pressure to retain it [7]. This fold in ROP2, and the other closely related proteins, appear to be transmembrane domains [17], which may suggest that a divergence of function has accompanied the evolution of this family. What makes ROP18 and ROP16 stand out in this family is that they retain all of the bioinformatic and structural elements required to designate them as putative functional kinases [7]. There is a significant difference in the in silico prediction of an enzymatic activity and its biochemical demonstration. The studies described in this issue of the journal provide the first biochemical and functional data that define ROP18 as a