Eimeria spp. are a highly successful group of intracellular protozoan parasites that develop within intestinal epithelial cells of poultry, causing coccidiosis. As a result of resistance against anticoccidial drugs and the expense of manufacturing live vaccines, it is necessary to understand the relationship between Eimeria and its host more deeply, with a view to developing recombinant vaccines. Eimeria possesses a family of microneme lectins (MICs) that contain microneme adhesive repeat regions (MARR). We show that the major MARR protein from Eimeria tenella, EtMIC3, is deployed at the parasite-host interface during the early stages of invasion. EtMIC3 consists of seven tandem MAR1-type domains, which possess a high specificity for sialylated glycans as shown by cell-based assays and carbohydrate microarray analyses. The restricted tissue staining pattern observed for EtMIC3 in the chicken caecal epithelium indicates that EtMIC3 contributes to guiding the parasite to the site of invasion in the chicken gut. The microarray analyses also reveal a lack of recognition of glycan sequences terminating in the N-glycolyl form of sialic acid by EtMIC3. Thus the parasite is well adapted to the avian host which lacks N-glycolyl neuraminic acid. We provide new structural insight into the MAR1 family of domains and reveal the atomic resolution basis for the sialic acid-based carbohydrate recognition. Finally, a preliminary chicken immunization trial provides evidence that recombinant EtMIC3 protein and EtMIC3 DNA are effective vaccine candidates.

P335 lactococcal phages infect the Gram؉ bacterium Lactococcus lactis using a large multiprotein complex located at the distal part of the tail and termed baseplate (BP). The BP harbors the receptor-binding proteins (RBPs), which allow the specific recognition of saccharidic receptors localized on the host cell surface. We report here the electron microscopic structure of the phage TP901-1 wild-type BP as well as those of two mutants bppL ؊ and bppU ؊ , lacking BppL (the RBPs) or both peripheral BP components (BppL and BppU), respectively. We also achieved an electron microscopic reconstruction of a partial BP complex, formed by BppU and BppL. This complex exhibits a tripod shape and is composed of nine BppLs and three BppUs. These structures, combined with lightscattering measurements, led us to propose that the TP901-1 BP harbors six tripods at its periphery, located around the central tube formed by ORF46 (Dit) hexamers, at its proximal end, and a ORF47 (Tal) trimer at its distal extremity. A total of 54 BppLs (18 RBPs) are thus available to mediate host anchoring with a large apparent avidity. TP901-1 BP exhibits an infection-ready conformation and differs strikingly from the lactococcal phage p2 BP, bearing only 6 RBPs, and which needs a conformational change to reach its activated state. The comparison of several Siphoviridae structures uncovers a close organization of their central BP core whereas striking differences occur at the periphery, leading to diverse mechanisms of host recognition.The first steps of phage infection require interactions between the phage receptor-binding proteins (RBPs) 2 (1, 2) and the receptors at the host cell surface. Although some RBPs are located at the tip of fibers (3), others belong to an elongated structure, the tail spike (4, 5). In bacteriophages infecting the Gram ϩ bacterium Lactococcus lactis such as p2 (936 group), TP901-1, and Tuc2009 (P335 group), RBPs are part of a large organelle (1-2 MDa) termed the baseplate (BP). We previously solved RBP structures of phages p2 (6, 7) and TP901-1 (8) as well as the RBP C-terminal domain ("head domain") of phage bIL170 (936 group) (9). It appeared that the RBP of phage TP901-1 (termed BppL, lower baseplate protein) was cleaved during crystallization, and the polypeptidic chain in the crystal structure starts either at residue 16 or at residue 32 (10). Proteolytic cleavage was also observed for the homologous RBP from Tuc2009 (11) as well as in the structure of a chimeric RBP comprising the N-terminal and linker domains of phage TP901-1 RBP fused to the C-terminal domain of phage p2 RBP (12). We demonstrated that individually expressed Tuc2009 BppU (upper baseplate protein) and BppL did not interact when mixed, and we attributed this to the proteolytic cleavage of the BppL N terminus that should normally plug into BppU (13). In contrast, co-expression of BppU and BppL yielded a well defined 3:9 complex (13).The BP architecture of the P335 phages TP901-1 and Tuc2009 has been investigated thoroughly using mutagenesis and immuno...

c Tailed phages are genome delivery machines exhibiting unequaled efficiency acquired over more than 3 billion years of evolution. Siphophages from the P335 and 936 families infect the Gram-positive bacterium Lactococcus lactis using receptor-binding proteins anchored to the host adsorption apparatus (baseplate). Crystallographic and electron microscopy (EM) studies have shed light on the distinct adsorption strategies used by phages of these two families, suggesting that they might also rely on different infection mechanisms. Here, we report electron microscopy reconstructions of the whole phage TP901-1 (P335 species) and propose a composite EM model of this gigantic molecular machine. Our results suggest conservation of structural proteins among tailed phages and add to the growing body of evidence pointing to a common evolutionary origin for these virions. Finally, we propose that host adsorption apparatus architectures have evolved in correlation with the nature of the receptors used during infection.

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

Background: TgMIC4 is an important microneme effector protein from Toxoplasma gondii. Results: The structure of TgMIC4 together with carbohydrate microarray analyses reveal a broad specificity for galactoseterminating sequences. Conclusion: Lectin activity within the fifth apple domain of TgMIC4 is reminiscent of the mammalian galectin family. Significance: TgMIC4 may contribute to parasite dissemination within the host or down-regulation of the immune response.

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Shortly preceding the submission of this manuscript, a paper by Müller et al. (Müller, J. J., Weiss, M. S., and Heinemann U. (2011) Acta Crystallogr. D 67, 936 -944) revealed the crystal structure of the Sarcocystis muris TgMIC4 homolog SML-2 in complex with 1-thio-␤-Dgalactose. This structure demonstrates that SML-2 binds to galactose via the same mode as demonstrated for TgMIC4-A5 in this manuscript, thereby superseding our prediction of a similar binding mode here. However, it should be noted that Müller et al. suggest that a H57Y substitution in TgMIC4-A5 decreases specificity for galactose and enables incorporation of a wider range of receptors, including N-acetylglucosamine. However, we have demonstrated here that TgMIC4-A5 binds with high specificity to galactose and is incapable of adhering to glucose or its derivatives.

Eimeria tenella is the causative agent of coccidiosis in domestic chickens. We report the complete backbone and side chain NMR assignments for the second microneme adhesive repeat of the microneme protein 3 of E. tenella.