Apicomplexa are unicellular eukaryotes and obligate intracellular parasites, including Plasmodium, the causative agent of malaria and Toxoplasma, one of the most widespread zoonotic pathogens. Rhoptries, one of their specialized secretory organelles, undergo regulated exocytosis during invasion 1 . Rhoptry proteins are injected directly into the host cell to support invasion and subversion of host immune function 2 . The mechanism by which they are discharged is unclear and appears distinct from those in bacteria, yeast, animals or plants.Here we show that rhoptry secretion in Apicomplexa shares structural and genetic elements with the exocytic machinery of ciliates, their free-living relatives. Rhoptry exocytosis depends on intramembranous particles in the shape of a rosette embedded into the plasma membrane of the parasite apex. Formation of this rosette requires multiple Non-discharge (Nd) proteins conserved and restricted to Ciliata, Dinoflagellata, and Apicomplexa, that together constitute the superphylum Alveolata. We identified Nd6 at the site of exocytosis in association with an apical vesicle. Sandwiched between the rosette and the tip of the rhoptry, this vesicle appears as a central element of the rhoptry secretion machine. Our results describe a conserved secretion system that was adapted to provide defense for free-living unicellular eukaryotes and host cell injection in intracellular parasites.Apicomplexan parasites are invasive and defined by the presence of an apical complex used to recognize and gain entry into host cells. It includes two secretory organelles: micronemes and rhoptries 3 . Microneme proteins are secreted to the parasite surface and mediate motility, host cell recognition and invasion 4 . Rhoptry proteins are injected directly into the host cell 2 , where they anchor the machinery propelling the parasite into the host cell 5 , facilitate nutrient

Members of the Apicomplexa phylum, including Plasmodium and Toxoplasma , have two types of secretory organelles (micronemes and rhoptries) whose sequential release is essential for invasion and the intracellular lifestyle of these eukaryotes. During invasion, rhoptries inject an array of invasion and virulence factors into the cytoplasm of the host cell, but the molecular mechanism mediating rhoptry exocytosis is unknown. Here we identify a set of parasite specific proteins, termed rhoptry apical surface proteins (RASP) that cap the extremity of the rhoptry. Depletion of RASP2 results in loss of rhoptry secretion and completely blocks parasite invasion and therefore parasite proliferation in both Toxoplasma and Plasmodium . Recombinant RASP2 binds charged lipids and likely contributes to assembling the machinery that docks/primes the rhoptry to the plasma membrane prior to fusion. This study provides important mechanistic insight into a parasite specific exocytic pathway, essential for the establishment of infection.

Plesiomonas shigelloides is the unique member of the Enterobacteriaceae family able to produce polar flagella when grow in liquid medium and lateral flagella when grown in solid or semisolid media. In this study on P. shigelloides 302-73 strain, we found two different gene clusters, one exclusively for the lateral flagella biosynthesis and the other one containing the biosynthetic polar flagella genes with additional putative glycosylation genes. P. shigelloides is the first Enterobacteriaceae were a complete lateral flagella cluster leading to a lateral flagella production is described. We also show that both flagella in P. shigelloides 302-73 strain are glycosylated by a derivative of legionaminic acid (Leg), which explains the presence of Leg pathway genes between the two polar flagella regions in their biosynthetic gene cluster. It is the first bacterium reported with O-glycosylated Leg in both polar and lateral flagella. The flagella O-glycosylation is essential for bacterial flagella formation, either polar or lateral, because gene mutants on the biosynthesis of Leg are non-flagellated. Furthermore, the presence of the lateral flagella cluster and Leg O-flagella glycosylation genes are widely spread features among the P. shigelloides strains tested.

In this study, we report the identification of genes required for the biosynthesis of the core lipopolysaccharides (LPSs) of two strains of Proteus mirabilis. Since P. mirabilis and Klebsiella pneumoniae share a core LPS carbohydrate backbone extending up to the second outer-core residue, the functions of the common P. mirabilis genes was elucidated by genetic complementation studies using well-defined mutants of K. pneumoniae. The functions of strain-specific outer-core genes were identified by using as surrogate acceptors LPSs from two well-defined K. pneumoniae core LPS mutants. This approach allowed the identification of two new heptosyltransferases (WamA and WamC), a galactosyltransferase (WamB), and an N-acetylglucosaminyltransferase (WamD). In both strains, most of these genes were found in the so-called waa gene cluster, although one common core biosynthetic gene (wabO) was found outside this cluster.Gram-negative motile and frequently swarming bacteria of the genus Proteus and the family Enterobacteriaceae are opportunistic human pathogens (33). Currently, the genus consists of five species (Proteus mirabilis, P. penneri, P. vulgaris, P. myxofaciens, and P. hauseri) and three genomospecies (4, 5, and 6) (33, 35). P. mirabilis is a common uropathogen that causes urinary tract infections especially in individuals with functional or anatomical abnormalities of the urinary tract (52) and elderly persons undergoing long-term catheterization (53) but less frequently in normal hosts (43). Potentially serious complications arising from P. mirabilis infections include bladder and kidney stone formation, catheter obstruction due to the formation of encrusting biofilms, and bacteremia (reviewed in reference 2). This bacterium is found more frequently than Escherichia coli in kidney infections (14) and may be associated with rheumatoid arthritis (38). Studies aimed at the identification of P. mirabilis virulence factors showed that flagella and fimbriae (MR/P and PMF) are required for entry into and colonization of the bladder, respectively (reviewed in reference 12). Other important virulence factors are urease, hemolysin, and iron acquisition (12). More recently, an extracellular metalloprotease (37) and several putative DNA binding regulatory, cell-envelope related, and plasmid-encoded proteins have been identified by signature-tagged mutagenesis (8, 21).The lipopolysaccharide (LPS), as in other members of the family Enterobacteriaceae, consists of three domains, an endotoxic glycolipid (lipid A), an O-polysaccharide (O-PS) chain or O-antigen, and an intervening core oligosaccharide (OS) region. The O-antigen is the major surface antigen, and its serological O specificity, in contrast to that of other Gram-negative bacteria (31), is defined by the structure of the O-PS chain and that of the core OS (51). On the basis of immunospecificity, 60 O serogroups (28, 36) have been recognized in P. mirabilis and P. vulgaris, and several new Proteus O serogroups have been proposed for P. penneri (27,55). The LPS is a pote...

Plesiomonas shigelloides, the only species of the genus, is an emergent pathogenic bacterium associated with human diarrheal and extraintestinal disease. We present the whole-genome sequence analysis of the representative strain for the O1 serotype (strain 302-73), providing a tool for studying bacterial outbreaks, virulence factors, and accurate diagnostic methods.

Thrombospondin type I repeat (TSR) domains are commonly O -fucosylated by protein O -fucosyltransferase 2 (PoFUT2), and this modification is required for optimal folding and secretion of TSR-containing proteins. The human malaria parasite Plasmodium falciparum expresses proteins containing TSR domains, such as the thrombospondin-related anonymous protein (TRAP) and circumsporozoite surface protein (CSP), which are O -fucosylated. TRAP and CSP are present on the surface of sporozoites and play essential roles in mosquito and human host invasion processes during the transmission stages. Here, we have generated PoFUT2 null-mutant P. falciparum and Plasmodium berghei (rodent) malaria parasites and, by phenotyping them throughout their complete life cycle, we show that PoFUT2 disruption does not affect the growth through the mosquito stages for both species. However, contrary to what has been described previously by others, P. berghei PoFUT2 null mutant sporozoites showed no deleterious motility phenotypes and successfully established blood stage infection in mice. This unexpected result indicates that the importance of O -fucosylation of TSR domains may differ between human and RODENT malaria parasites; complicating our understanding of glycosylation modifications in malaria biology.

bWe report here the identification of waa clusters with the genes required for the biosynthesis of the core lipopolysaccharides (LPS) of two Plesiomonas shigelloides strains. Both P. shigelloides waa clusters shared all of the genes besides the ones flanking waaL. In both strains, all of the genes were found in the waa gene cluster, although one common core biosynthetic gene (wapG) was found in a different chromosome location outside the cluster. Since P. shigelloides and Klebsiella pneumoniae share a core LPS carbohydrate backbone extending up at least to the second outer-core residue, the functions of the common P. shigelloides genes were elucidated by genetic complementation studies using well-defined K. pneumoniae mutants. The function of strainspecific inner-or outer-core genes was identified by using as a surrogate acceptor LPS from three well-defined K. pneumoniae core LPS mutants. Using this strategy, we were able to assign a proteomic function to all of the P. shigelloides waa genes identified in the two strains encoding six new glycosyltransferases (WapA, -B, -C, -D, -F, and -G). P. shigelloides demonstrated an important variety of core LPS structures, despite being a single species of the genus, as well as high homologous recombination in housekeeping genes.

Thank you for your assistance. HighlightsThe Plesiomonas shigelloides 302-73 strain (serotype O1) wb gene cluster. The cluster encodes 15 proteins in agreement with chemical structure. The mutants lacking this O1-antigen LPS unable to survive in serum or adhere. First report on genetics from a P. shigelloides O-antigen LPS not shared by Shigella. The Plesiomonas shigelloides 302-73 strain (serotype O1) wb gene cluster encodes 15 proteins which are consistent with the chemical structure of the O1-antigen lypopolysaccharide (LPS) previously described for this strain. The P. shigelloides O1-antigen LPS export uses the Wzy-dependent pathway as correspond to heteropolysaccharides structures. By the isolation of two mutants lacking this O1-antigen LPS, we could establish that the presence of the O1-antigen LPS is crucial for to survive in serum mainly to become resistant to complement. Also, it is an important factor in the bacterial adhesion and invasion to some eukaryotic cells, and in the ability to form biofilms. This is the first report on the genetics from a P. shigelloides O-antigen LPS cluster (wb) not shared by Shigella like P. shigelloides O17, the only one reported until now.