The cellular and molecular mechanisms that underlie species-specific membrane fusion between male and female gametes remain largely unknown. Here, by use of gene discovery methods in the green alga Chlamydomonas, gene disruption in the rodent malaria parasite Plasmodium berghei, and distinctive features of fertilization in both organisms, we report discovery of a mechanism that accounts for a conserved protein required for gamete fusion. A screen for fusion mutants in Chlamydomonas identified a homolog of HAP2, an Arabidopsis sterility gene. Moreover, HAP2 disruption in Plasmodium blocked fertilization and thereby mosquito transmission of malaria. HAP2 localizes at the fusion site of Chlamydomonas minus gametes, yet Chlamydomonas minus and Plasmodium hap2 male gametes retain the ability, using other, species-limited proteins, to form tight prefusion membrane attachments with their respective gamete partners. Membrane dye experiments show that HAP2 is essential for membrane merger. Thus, in two distantly related eukaryotes, species-limited proteins govern access to a conserved protein essential for membrane fusion.[Keywords: Gamete fusion; cell-cell fusion; malaria; HAP2; Chlamydomonas, Plasmodium] Supplemental material is available at http://www.genesdev.org. Received January 28, 2008; revised version accepted February 22, 2008. Fusion of gametes of opposite sex (or mating type) to form a zygote is the defining moment in the life of a eukaryote. In the first phase of gamete interactions, cell adhesion molecules displayed on the surfaces of the gametes bring the two cells together. In animals, the sperm plasma membrane binds to the extracellular matrix of the egg (the zona pellucida in mammals and the jelly coat in many invertebrates). The interacting gametes use this first-phase adhesion step not only to bind to each other, but also to initiate a signal transduction cascade that activates the sperm and exposes new, fusogenic regions of the sperm plasma membrane. In the second phase of fertilization, the membrane fusion reaction, the plasma membranes of the two gametes come into intimate contact and then fuse, bringing about cytoplasmic continuity (Primakoff and Myles 2002;Rubinstein et al. 2006). Although these two steps-prefusion attachment of the plasma membranes of gametes and merger of their lipid bilayers-have been experimentally separated using in vitro bioassays, gene disruption studies to date have failed to distinguish the two, and no genes have been identified whose disruption allows prefusion attachment and disallows membrane merger. In mice, several proteins involved in gamete membrane interactions have been described, including ADAMS family members and CRISP proteins on sperm and integrins and tetraspanin family members CD9 and CD81 on eggs (for review, see Ellerman et al. 2006;Inoue et al. 2007;Primakoff and Myles 2007). Izumo, an immunoglobulin superfamily sperm protein that appears to be limited to mammals, is gamete-specific and shown by gene disruption to be essential at a late step in ferti...

Yersinia pseudotuberculosis employs a type III secretion system for targeting of several virulence factors directly to the cytosol of eukaryotic cells. This protein translocation mechanism mediates the ability of Yersinia to resist phagocytosis and is required for sustained extracellular bacterial replication. In the present study, the Yersinia outer protein E (YopE) was used as a carrier molecule for type III-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. In comparison to wild-type Yersinia, an attenuated Y. pseudotuberculosis yopK-null mutant strain hypertranslocates chimeric YopE/LLO into the cytosol of macrophages, resulting in enhanced major histocompatibility complex (MHC) class I-restricted antigen presentation of an LLO-derived CD8 T-cell epitope. Remarkably, T-cell activation assays also revealed a superior ability of translocated over secreted LLO to induce MHC class II-restricted antigen presentation. These in vitro observations were confirmed after immunization of mice with a single dose of the yopK-null mutant strain. Animals orally inoculated with recombinant Yersinia expressing translocated chimeric YopE/LLO revealed high numbers of gamma interferon-producing LLO-specific CD4 and CD8 T cells. For the first time, it is shown that cytosolic antigen display mediated by an extracellular bacterial carrier vaccine results in simultaneous CD4 and CD8 T-cell priming, conferring protection against an intracellular pathogen.

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The sequencing of bacterial genomes has opened new perspectives for identification of targets for treatment of infectious diseases. We have identified a set of novel virulence-associated genes (vag genes) by comparing the genome sequences of six human pathogens that are known to cause persistent or chronic infections in humans: Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi, Streptococcus pneumoniae, and Treponema pallidum. This comparison was limited to genes annotated as hypothetical in the T. pallidum genome project. Seventeen genes with unknown functions were found to be conserved among these pathogens. Insertional inactivation of 14 of these genes generated nine mutants that were attenuated for virulence in a mouse infection model. Out of these nine genes, five were found to be specifically associated with virulence in mice as demonstrated by infection with Yersinia pseudotuberculosis in-frame deletion mutants. In addition, these five vag genes were essential only in vivo, since all the mutants were able to grow in vitro. These genes are broadly conserved among bacteria. Therefore, we propose that the corresponding vag gene products may constitute novel targets for antimicrobial therapy and that some vag mutants could serve as carrier strains for live vaccines.Antibacterial therapy is becoming less effective in the treatment of infections due to the spread of multiple-antibioticresistant bacterial strains (59). This has lead to a concerted effort to develop mechanistically novel antimicrobial agents. This search is being facilitated by the wealth of information accumulating in databases via different large-scale genome projects. To date, about 100 bacterial genomes, including several important pathogens have been sequenced, revealing around 25% of the open reading frames (ORFs) to be conserved hypothetical genes without known function (17). Presumably, among this group of ORFs, protein classes that constitute suitable molecular targets for novel therapeutics will be identified. In fact, a number of genome-based studies have already identified homologous gene classes from different bacteria that encode products of unknown or hypothetical function (4,7,20,55). By combining the bioinformatic approaches with mutagenesis, genes essential for in vitro viability have been identified (4,20,55). Indeed, these proteins constitute potential targets for the development of novel antibacterial agents that would serve to inhibit bacterial growth.An alternative approach would be to target genes required only for in vivo growth and not for growth outside the animal host. Finding these nontraditional targets by using a bioinformatic approach is a hitherto-unproven strategy (29). The approach presented here makes use of the fact that several obligatory pathogenic bacteria have genome sizes that are considerably smaller than those of free-living eubacteria. During evolution, these pathogenic bacteria have condensed their genomes by eliminating genes not required for growth in the animal host ...

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Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins [Yops]) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) fromListeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and major histocompatibility complex (MHC) class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the Yersinia T3SS. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen-processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it did not require the function either of the proteasome or of transporters associated with antigen processing. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T-cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen.Protein antigens are recognized by T cells as short peptide fragments bound either to major histocompatibility class (MHC) I or to MHC class II molecules on the surface of antigen-presenting cells (APCs). The location of antigens in distinct intracellular compartments of APCs influences their proteolytic processing as well as access to MHC molecules (16, 39). Classically, peptides generated in the cytosol (e.g., derived from viral proteins) by proteasomal degradation are bound to MHC class I molecules after transport across the endoplasmic reticulum membrane by the transporters associated with antigen processing (TAP) (90). Subsequently, MHC class I molecules present these endogenous antigenic peptides to CD8 T cells. In contrast, exogenous antigens (e.g., antigens derived from engulfed bacteria and soluble antigens) are directed into the endosomal/lysosomal pathway for degradation (63). In late endosomal compartments, degraded protein fragments interact with MHC class II molecules and are further trimmed into peptides for presentation to CD4 T cells.However, the synchrony of this system has been challenged by biochemical and functional studies of professional and nonprofessional APCs. Epitopes derived from a diverse ...

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Experimental oral infections of rabbits with a wild-type Yersinia pseudotuberculosis strain (pIB102), and two null-mutants (yopK and ypkA) were carried out with the aim to explore the possibility to use mutant strains of Y. pseudotuberculosis as live carrier vaccine strains. The infectious process of the three strains proceed with passing hyperthermia, leucocytosis with granulocytosis, moderate monocytosis and a transient lymphopenia, better demonstrated at mutant strain infections. Short-term bacterial dissemination into the brain and viscera was observed at yopK infection. An augmented resistance to bactericidal activity of leucocytes at the initial phase of infection was followed by an increased sensitivity discovered earlier in case of yopK strain accompanied by at least 70- and 20-fold, respectively, for ypkA lower virulence for mice. The level of attenuation of yopK was accompanied by significant Yersinia specific IgG and IgM antibody response. Inflammatory foci were found by morphological examination in brain, lung and small intestines after infection with the wild-type strain, while such foci were only observed in brain and mesenterial lymph nodes after infection with the yopK mutant. After infection with the ypkA mutant foci were found in brain and spleen of the infected animals. Morphological changes in the lymphatic tissue of rabbits infected with mutant strains were consistent with induction of immunogenesis. The data suggest that genetically constructed yopK null-mutant exhibits characteristics that makes the strain suitable to be used as a live carrier vaccine to deliver heterologous antigens.