BB0562 is a nutritional virulence determinant with lipase activity important for Borrelia burgdorferi infection and survival in fatty acid deficient environments.

The Lyme disease spirochete Borrelia burgdorferi relies on uptake of essential nutrients from its host environments for survival and infection. Therefore, nutrient acquisition mechanisms constitute key virulence properties of the pathogen, yet these mechanisms remain largely unknown. In vivo expression technology applied to B. burgdorferi (BbIVET) during mammalian infection identified gene bb0562, which encodes a hypothetical protein comprised of a conserved domain of unknown function, DUF3996. DUF3996 is also found across adjacent encoded hypothetical proteins BB0563 and BB0564, suggesting the possibility that the three proteins could be functionally related. Deletion of bb0562, bb0563 and bb0564 individually and together demonstrated that bb0562 alone was important for optimal disseminated infection in immunocompetent and immunocompromised mice by needle inoculation and tick bite transmission. Moreover, bb0562 promoted spirochete survival during the blood dissemination phase of infection. Gene bb0562 was also found to be important for spirochete growth in low serum media and the growth defect of Δbb0562 B. burgdorferi was rescued with the addition of various long chain fatty acids, particularly oleic acid. In mammals, fatty acids are primarily stored in fat droplets in the form of triglycerides. Strikingly, addition of glyceryl trioleate, the triglyceride form of oleic acid, to the low serum media did not rescue the growth defect of the mutant, suggesting bb0562 may be important for the release of fatty acids from triglycerides. Therefore, we searched for and identified two canonical GXSXG lipase motifs within BB0562, despite the lack of homology to known bacterial lipases. Purified BB0562 demonstrated lipolytic activity dependent on the catalytic serine residues within the two motifs. In sum, we have established that bb0562 is a novel nutritional virulence determinant, encoding a lipase that contributes to fatty acid scavenge for spirochete survival in environments deficient in free fatty acids including the mammalian host.

A Minimal Replicon Enables Efficacious, Species-Specific Gene Deletion in Chlamydia and Extension of Gene Knockout Studies to the Animal Model of Infection Using Chlamydia muridarum.

The genus Chlamydia consists of diverse, obligate intracellular bacteria that infect various animals, including humans. Although chlamydial species share many aspects of the typical intracellular lifestyle, such as the biphasic developmental cycle and the preference for invasion of epithelial cells, each chlamydial strain also employs sophisticated species-specific strategies that contribute to an extraordinary diversity in organ and/or tissue tropism and disease manifestation. In order to discover and understand the mechanisms underlying how these pathogens infect particular hosts and cause specific diseases, it is imperative to develop a mutagenesis approach that would be applicable to every chlamydial species. We present functional evidence that the region between Chlamydia trachomatis and Chlamydia muridarum pgp6 and pgp7, containing four 22-bp tandem repeats that are present in all chlamydial endogenous plasmids, represents the plasmid origin of replication. Furthermore, by introducing species-specific ori regions into an engineered 5.45-kb pUC19-based plasmid, we generated vectors that can be successfully transformed into and propagated under selective pressure by C. trachomatis serovars L2 and D, as well as C. muridarum. Conversely, these vectors were rapidly lost upon removal of the selective antibiotic. This conditionally replicating system was used to generate a tarP deletion mutant by fluorescence-reported allelic exchange mutagenesis in both C. trachomatis serovar D and C. muridarum. The strains were analyzed using in vitro invasion and fitness assays. The virulence of the C. muridarum strains was then assessed in a murine infection model. Our approach represents a novel and efficient strategy for targeted genetic manipulation in Chlamydia beyond C. trachomatis L2. This advance will support comparative studies of species-specific infection biology and enable studies in a well-established murine model of chlamydial pathogenesis.

The Chlamydia trachomatis Tarp effector targets the Hippo pathway.

Chlamydia trachomatis injects bacterial effector proteins into human epithelial cells to facilitate the establishment of new infections. The chlamydial type III secreted effector translocated actin recruiting phosphoprotein (Tarp) has been shown to nucleate and bundle actin filaments. It is also believed to initiate new signaling pathways via an N-terminal phosphorylation domain. A comprehensive understanding of the host pathways that are controlled by Tarp to aid in the establishment of a successful infection remains incomplete. To gain further insight into the cell signaling regulated by Tarp, we generated transgenic fruit flies engineered to express the N-terminal domain of Tarp. As many signaling pathways are conserved between flies and mammals, we hypothesized that expression of the Tarp N-domain in the fruit fly might disrupt key pathways, resulting in developmental defects. Tarp N-domain expression in the fruit fly resulted in a mechanosensory bristle duplication phenotype similar to a previously characterized fly phenotype found to be a consequence of defects in the Hippo pathway. Tarp-dependent disruption of the Hippo pathway was confirmed in a C. trachomatis tissue culture infection model. The capability of Tarp to alter Hippo pathway signaling in infected epithelial cells is a previously unrecognized pathway commandeered by chlamydia and likely contributes to the establishment of chlamydia's intracellular niche.

Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion.

The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia's ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

Chlamydia trachomatis Tarp harbors distinct G and F actin binding domains that bundle actin filaments.

All species of Chlamydia undergo a unique developmental cycle that transitions between extracellular and intracellular environments and requires the capacity to invade new cells for dissemination. A chlamydial protein called Tarp has been shown to nucleate actin in vitro and is implicated in bacterial entry into human cells. Colocalization studies of ectopically expressed enhanced green fluorescent protein (EGFP)-Tarp indicate that actin filament recruitment is restricted to the C-terminal half of the effector protein. Actin filaments are presumably associated with Tarp via an actin binding alpha helix that is also required for actin nucleation in vitro, but this has not been investigated. Tarp orthologs from C. pneumoniae, C. muridarum, and C. caviae harbor between 1 and 4 actin binding domains located in the C-terminal half of the protein, but C. trachomatis serovar L2 has only one characterized domain. In this work, we examined the effects of domain-specific mutations on actin filament colocalization with EGFP-Tarp. We now demonstrate that actin filament colocalization with Tarp is dependent on two novel F-actin binding domains that endow the Tarp effector with actin-bundling activity. Furthermore, Tarp-mediated actin bundling did not require actin nucleation, as the ability to bundle actin filaments was observed in mutant Tarp proteins deficient in actin nucleation. These data shed molecular insight on the complex cytoskeletal rearrangements required for C. trachomatis entry into host cells.

Chlamydia trachomatis TmeB antagonizes actin polymerization via direct interference with Arp2/3 activity.

Chlamydia trachomatis is an obligate intracellular pathogen that actively promotes invasion of epithelial cells. A virulence-associated type III secretion system contributes to chlamydial entry and at least four effectors have been described that are deployed during this time. Two of these invasion-related effectors, the translocated membrane-associated effectors A and B (TmeA and TmeB), are encoded in a bi-cistronic operon. TmeA directly activates host N-WASP to stimulate Arp2/3-dependent actin polymerization. According to current working models, TmeA-mediated N-WASP activation contributes to invasion. TmeB has not been functionally characterized. Unlike a tmeA null strain, loss of tmeB does not impact invasion efficiency of C. trachomatis. Using strains deficient for multiple genes, we provide evidence that TmeA is dispensable for invasion in the absence of TmeB. Our data indicate that overabundance of TmeB interferes with invasion and that this activity requires active Arp2/3 complex. We further show that TmeB is capable of interfering with Arp2/3-mediated actin polymerization. In aggregate, these data point to opposing functions for TmeA and TmeB that manifest during the invasion process. These studies raise intriguing questions regarding the dynamic interplay between TmeA, TmeB, and branched actin polymerization during chlamydial entry.

Borrelia burgdorferi-mediated induction of miR146a-5p fine tunes the inflammatory response in human dermal fibroblasts.

Colonization of a localized area of human skin by Borrelia burgdorferi after a bite from an infected tick is the first step in the development of Lyme disease. The initial interaction between the pathogen and the human host cells is suggested to impact later outcomes of the infection. MicroRNAs (miRNAs) are well known to be important regulators of host inflammatory and immune responses. While miRNAs have been shown to play a role in the inflammatory response to B. burgdorferi at late stages of infection in the joints, the contributions of miRNAs to early B. burgdorferi infection have yet to be explored. To address this knowledge gap, we used the published host transcriptional responses to B. burgdorferi in erythema migrans skin lesions of early Lyme disease patients and a human dermal fibroblasts (HDFs)/B. burgdorferi co-culture model to predict putative upstream regulator miRNAs. This analysis predicted a role for miR146a-5p in both, B. burgdorferi-infected skin and -stimulated HDFs. miR146a-5p was confirmed to be significantly upregulated in HDF stimulated with B. burgdorferi for 24 hours compared to uninfected control cells. Furthermore, manipulation of miR146a-5p expression (overexpression or inhibition) altered the B. burgdorferi driven inflammatory profile of HDF cells. Our results suggest that miR146a-5p is an important upstream regulator of the transcriptional and immune early response to early B. burgdorferi infection.

LMAN1 is a receptor for house dust mite allergens.

Development of therapies with the potential to change the allergic asthmatic disease course will require the discovery of targets that play a central role during the initiation of an allergic response, such as those involved in the process of allergen recognition. We use a receptor glycocapture technique to screen for house dust mite (HDM) receptors and identify LMAN1 as a candidate. We verify the ability of LMAN1 to directly bind HDM allergens and demonstrate that LMAN1 is expressed on the surface of dendritic cells (DCs) and airway epithelial cells (AECs) in vivo. Overexpression of LMAN1 downregulates NF-κB signaling in response to inflammatory cytokines or HDM. HDM promotes binding of LMAN1 to the FcRγ and recruitment of SHP1. Last, peripheral DCs of asthmatic individuals show a significant reduction in the expression of LMAN1 compared with healthy controls. These findings have potential implications for the development of therapeutic interventions for atopic disease.

Chlamydia trachomatis Tarp cooperates with the Arp2/3 complex to increase the rate of actin polymerization.

Actin polymerization is required for Chlamydia trachomatis entry into nonphagocytic host cells. Host and chlamydial actin nucleators are essential for internalization of chlamydiae by eukaryotic cells. The host cell Arp2/3 complex and the chlamydial translocated actin recruiting phosphoprotein (Tarp) are both required for entry. Tarp and the Arp2/3 complex exhibit unique actin polymerization kinetics individually, but the molecular details of how these two actin nucleators cooperate to promote bacterial entry is not understood. In this study we provide biochemical evidence that the two actin nucleators act synergistically by co-opting the unique attributes of each to enhance the dynamics of actin filament formation. This process is independent of Tarp phosphorylation. We further demonstrate that Tarp colocalization with actin filaments is independent of the Tarp phosphorylation domain. The results are consistent with a model in which chlamydial and host cell actin nucleators cooperate to increase the rate of actin filament formation.

The conserved Tarp actin binding domain is important for chlamydial invasion.

The translocated actin recruiting phosphoprotein (Tarp) is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.

The Chlamydia trachomatis Early Effector Tarp Outcompetes Fascin in Forming F-Actin Bundles In Vivo.

The intracellular pathogen Chlamydia trachomatis secretes multiple early effectors into the host cell to promote invasion. A key early effector during host cell entry, Tarp (translocated actin-recruiting phosphoprotein) is comprised of multiple protein domains known to have roles in cell signaling, G-actin nucleation and F-actin bundle formation. In vitro, the actin bundles generated by Tarp are uncharacteristically flexible, however, in vivo, the biological significance of Tarp-mediated actin bundles remains unknown. We hypothesize that Tarp's ability to generate unique actin bundles, in part, facilitates chlamydial entry into epithelial cells. To study the in vivo interaction between Tarp and F-actin, we transgenically expressed Tarp in Drosophila melanogaster tissues. Tarp expressed in Drosophila is phosphorylated and forms F-actin-enriched aggregates in tissues. To gain insight into the significance of Tarp actin bundles in vivo, we utilized the well-characterized model system of mechanosensory bristle development in Drosophila melanogaster. Tarp expression in wild type flies produced curved bristles, indicating a perturbation in F-actin dynamics during bristle development. Two F-actin bundlers, Singed/Fascin and Forked/Espin, are important for normal bristle shape. Surprisingly, Tarp expression in the bristles displaced Singed/Fascin away from F-actin bundles. Tarp's competitive behavior against Fascin during F-actin bundling was confirmed in vitro. Loss of either singed or forked in flies leads to highly deformed bristles. Strikingly, Tarp partially rescued the loss of singed, reducing the severity of the bristle morphology defect. This work provides in vivo confirmation of Tarp's F-actin bundling activity and further uncovers a competitive behavior against the host bundler Singed/Fascin during bundle assembly. Also, we demonstrate the utility of Drosophila melanogaster as an in vivo cell biological platform to study bacterial effector function.

Chlamydia trachomatis TmeA Directly Activates N-WASP To Promote Actin Polymerization and Functions Synergistically with TarP during Invasion.

Chlamydia trachomatis is a medically significant human pathogen and is an epithelial-tropic obligate intracellular parasite. Invasion of nonprofessional phagocytes represents a crucial step in the infection process and has likely promoted the evolution of a redundant mechanism and routes of entry. Like many other viral and invasive bacterial pathogens, manipulation of the host cell cytoskeleton represents a focal point in Chlamydia entry. The advent of genetic techniques in C. trachomatis, such as creation of complete gene deletions via fluorescence-reported allelic exchange mutagenesis (FRAEM), is providing important tools to unravel the contributions of bacterial factors in these complex pathways. The type III secretion chaperone Slc1 directs delivery of at least four effectors during the invasion process. Two of these, TarP and TmeA, have been associated with manipulation of actin networks and are essential for normal levels of invasion. The functions of TarP are well established, whereas TmeA is less well characterized. We leverage chlamydial genetics and proximity labeling here to provide evidence that TmeA directly targets host N-WASP to promote Arp2/3-dependent actin polymerization. Our work also shows that TmeA and TarP influence separate, yet synergistic pathways to accomplish chlamydial entry. These data further support an appreciation that a pathogen, confined by a reductionist genome, retains the ability to commit considerable resources to accomplish bottle-neck steps during the infection process.IMPORTANCE The increasing genetic tractability of Chlamydia trachomatis is accelerating the ability to characterize the unique infection biology of this obligate intracellular parasite. These efforts are leading to a greater understanding of the molecular events associated with key virulence requirements. Manipulation of the host actin cytoskeleton plays a pivotal role throughout Chlamydia infection, yet a thorough understanding of the molecular mechanisms initiating and orchestrating actin rearrangements has lagged. Our work highlights the application of genetic manipulation to address open questions regarding chlamydial invasion, a process essential to survival. We provide definitive insight regarding the role of the type III secreted effector TmeA and how that activity relates to another prominent effector, TarP. In addition, our data implicate at least one source that contributes to the functional divergence of entry mechanisms among chlamydial species.

Phylogenetic analysis of Chlamydia trachomatis Tarp and correlation with clinical phenotype.

Chlamydia trachomatis is the leading cause of infectious blindness worldwide and is the most commonly reported pathogen causing sexually transmitted infections. Tarp (translocated actin recruiting phosphoprotein), a type III secreted effector that mediates actin nucleation, is central to C. trachomatis infection. The phylogenetic analysis of tarP from reference strains as well as ocular, genital, and lymphogranuloma venereum (LGV) clinical isolates demonstrated an evolutionary relationship with disease phenotype, with LGV and ocular isolates branched into clades that were separate from the urogenital isolates. The sequence analysis of Tarp indicated a high degree of variability and identified trends within clinical groupings. Tarps from LGV strains contained the highest number of tyrosine-rich repeat regions (up to nine) and the fewest (two) predicted actin binding domains. The converse was noted for Tarp proteins from ocular isolates that contained up to four actin binding domains and as few as one tyrosine-rich repeat region. The results suggest that Tarp is among the few known genes to play a role in C. trachomatis adaptations to specific niches within the host.

Identification of the in vivo role of a viral bcl-2.

Many gamma-herpesviruses encode candidate oncogenes including homologues of host bcl-2 and cyclin proteins (v-bcl-2, v-cyclin), but the physiologic roles of these genes during infection are not known. We show for the first time in any virus system the physiologic role of v-bcl-2. A gamma-herpesvirus v-bcl-2 was essential for efficient ex vivo reactivation from latent infection, and for both persistent replication and virulence during chronic infection of immunocompromised (interferon [IFN]-gamma(-/-)) mice. The v-cyclin was also critical for the same stages in pathogenesis. Strikingly, while the v-bcl-2 and v-cyclin were important for chronic infection, these genes were not essential for viral replication in cell culture, viral replication during acute infection in vivo, establishment of latent infection, or virulence during acute infection. We conclude that v-bcl-2 and v-cyclin have important roles during latent and persistent gamma-herpesvirus infection and that herpesviruses encode genes with specific roles during chronic infection and disease, but not acute infection and disease. As gamma-herpesviruses primarily cause human disease during chronic infection, these chronic disease genes may be important targets for therapeutic intervention.

Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases.

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.

Targeted Disruption of Chlamydia trachomatis Invasion by in Trans Expression of Dominant Negative Tarp Effectors.

Chlamydia trachomatis invasion of eukaryotic host cells is facilitated, in part, by the type III secreted effector protein, Tarp. The role of Tarp in chlamydiae entry of host cells is supported by molecular approaches that examined recombinant Tarp or Tarp effectors expressed within heterologous systems. A major limitation in the ability to study the contribution of Tarp to chlamydial invasion of host cells was the prior absence of genetic tools for chlamydiae. Based on our knowledge of Tarp domain structure and function along with the introduction of genetic approaches in C. trachomatis, we hypothesized that Tarp function could be disrupted in vivo by the introduction of dominant negative mutant alleles. We provide evidence that transformed C. trachomatis produced epitope tagged Tarp, which was secreted into the host cell during invasion. We examined the effects of domain specific Tarp mutations on chlamydial invasion and growth and demonstrate that C. trachomatis clones harboring engineered Tarp mutants lacking either the actin binding domain or the phosphorylation domain had reduced levels of invasion into host cells. These data provide the first in vivo evidence for the critical role of Tarp in C. trachomatis pathogenesis and indicate that chlamydial invasion of host cells can be attenuated via the introduction of engineered dominant negative type three effectors.

Chlamydial TARP is a bacterial nucleator of actin.

Chlamydia trachomatis entry into host cells results from a parasite-directed remodeling of the actin cytoskeleton. A type III secreted effector, TARP (translocated actin recruiting phosphoprotein), has been implicated in the recruitment of actin to the site of internalization. To elucidate the role of TARP in actin recruitment, we identified host cell proteins that associated with recombinant GST-TARP fusions. TARP directly associated with actin, and this interaction promoted actin nucleation as determined by in vitro polymerization assays. Domain analysis of TARP identified an actin-binding domain that bears structural and primary amino acid sequence similarity to WH2 domain family proteins. In addition, a proline-rich domain was found to promote TARP oligomerization and was required for TARP-dependent nucleation of new actin filaments. Our findings reveal a mechanism by which chlamydiae induce localized cytoskeletal changes by the translocated effector TARP during entry into host cells.

Two separate, conserved acidic amino acid domains within the Toxoplasma gondii MIC2 cytoplasmic tail are required for parasite survival.

Apicomplexan parasites rely on actin-based motility to drive host cell invasion. Motility and invasion also require thrombospondin-related anonymous protein (TRAP) adhesins, which are secreted apically and translocated to the posterior end of the parasite before they are shed by the activity of a rhomboid protease. TRAP orthologs, including Toxoplasma gondii MIC2 (microneme protein 2), possess a short cytoplasmic tail, which is essential for motility. Previous studies have shown that aldolase forms a critical bridge between actin filaments and the cytoplasmic domains of MIC2 and TRAP. The cytoplasmic tails of TRAP family members harbor a conserved penultimate tryptophan, which is essential for aldolase binding, and clustered acidic residues. Herein, we determined the role of the conserved acidic residues by using alanine point mutants to investigate aldolase binding in vitro and to test functionality in the parasite. Our studies revealed two separate acidic residue clusters in the cytoplasmic domain of MIC2 that are essential for parasite survival. One region, located at the extreme C terminus, is required for the direct interaction with aldolase, whereas the second upstream acidic region is not necessary for aldolase binding but is nonetheless essential to parasite survival. Both acidic domains are conserved throughout TRAP orthologs, implicating a central role for these motifs in apicomplexan motility.

A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma.

Apicomplexan parasites cause serious human and animal diseases, the treatment of which requires identification of new therapeutic targets. Host-cell invasion culminates in the essential cleavage of parasite adhesins, and although the cleavage site for several adhesins maps within their transmembrane domains, the protease responsible for this processing has not been discovered. We have identified, cloned, and characterized the five nonmitochondrial rhomboid intramembrane proteases encoded in the recently completed genome of Toxoplasma gondii. Four T. gondii rhomboids (TgROMs) were active proteases with similar substrate specificity. TgROM1, TgROM4, and TgROM5 were expressed in the tachyzoite stage responsible for the disease, whereas TgROM2 and TgROM3 were expressed in the oocyst stage involved in transmission. Although both TgROM5 and TgROM4 localized to the cell surface in tachyzoites, TgROM5 was primarily at the posterior of the parasite, whereas adhesins were sequestered in internal micronemes. Upon microneme secretion, as occurs during invasion, the MIC2 adhesin was secreted to the apical end and translocated to the posterior, the site of cleavage, where it colocalized only with TgROM5. Moreover, only TgROM5 was able to cleave MIC adhesins in a cell-based assay, indicating that it likely provides the key protease activity necessary for invasion. T. gondii rhomboids have clear homologues in other apicomplexans including malaria; thus, our findings provide a model for studying invasion by this deadly pathogen and offer a target for therapeutic intervention.

The toxoplasma proteins MIC2 and M2AP form a hexameric complex necessary for intracellular survival.

Toxoplasma gondii parasites gain entry into host cells through a process that depends on apically stored adhesins that are strategically released during invasion. One of these adhesins, microneme protein 2 (MIC2), is a type one transmembrane protein that binds to an accessory protein known as MIC2-associated protein (M2AP). Together the MIC2 x M2AP complex participates in host cell attachment and invasion. The short cytoplasmic C-domain of MIC2 is implicated in protein trafficking and mediating an association with the parasite cytoskeleton. To define the role of the cytoplasmic domain of MIC2, proteins lacking the C-domain were expressed in transgenic T. gondii. Surprisingly, protein trafficking and secretion were not affected. We hypothesized that mutant mic2 lacking the C-domain might be escorted to the micronemes by association with endogenous wild-type MIC2 possessing functional transmembrane and cytoplasmic domains. To investigate this interaction, native blue gels and gel filtration were employed to identify a stable macromolecular MIC2 x M2AP complex of approximately 450 kDa. Our findings reveal that MIC2 and M2AP proteins form stable hexamers consisting of three alphabeta dimers. Resolution of this complex has implications for how MIC2 x M2AP associates with host cell receptors and the cytoskeleton to facilitate parasite motility and invasion.