Mucus glycoproteins selectively secreted from bacteriocytes in gill filaments of the deep-sea clam Calyptogena okutanii

The deep-sea clam Calyptogena okutanii possesses a large gill containing vertically transmitted symbiotic sul-fur-oxidizing bacteria. It produces large amounts of highly viscoelastic mucus from the gill, which is thought to be a physical and chemical barrier. The mucus collected from the gill was shown to be composed of glycoproteins having the following sugar composition: Man (17.4%), GlcNAc (16.6%), GalNAc (15%), Glc (1.1%), Gal (29.9%), Xyl (3.0%), Fuc (14.4%), and unknown (2.6%), indicating that it contained mucin-like glycoproteins. In a monoclonal antibody li-brary against the gill tissue, we found a monoclonal antibody (mAb), CokG-B3C10, reacting to the mucus. Western blot analysis using the mAb showed that it reacted to several glycoproteins in the mucus from the gill tissue, but not with those of other tissues such as the mantle, foot, and ovary, where mucus production has been reported in bivalves. Fur-ther, immunohistochemical analysis showed the CokG-B3C10 mAb reacting to glycoproteins was detected in the inner area of the gill, which was occupied by many bacteriocytes in the row of gill filaments. Strong mAb signals were found on the outer surface of the bacteriocytes facing the interfilamental space, and in the interfilamental spaces between filaments. Weaker signals were also observed in the bacteriocyte cells. These results indicate that the CokG-B3C10 mAb-binding mucus glycoproteins were produced from cells including bacteriocytes and nonbacteriocyte cells in the inner area of the gill filaments.http://www.godac.jamstec.go.jp/darwin/cruise/natsushima/nt09-06_leg1/ehttp://www.godac.jamstec.go.jp/darwin/cruise/natsushima/nt10-01/ehttp://www.godac.jamstec.go.jp/darwin/cruise/natsushima/nt10-08/

Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia

Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires’ disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity. Because of the high mobility and high cell division rate of Paramecium spp., living with Paramecium spp. would be beneficial for L. pneumophila to expand its habitat. To control Legionaries’ disease, understanding the ecology of L. pneumophila in the environment is essential

A 63-kDa Periplasmic Protein of the Endonuclear Symbiotic Bacterium <i>Holospora obtusa</i> Secreted to the Outside of the Bacterium during the Early Infection Process Binds Weakly to the Macronuclear DNA of the Host <i>Paramecium caudatum</i>

The Gram-negative bacterium Holospora obtusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum. It is known that an infection of this bacterium induces high level expressions of the host hsp60 and hsp70 genes, and the host cell acquires both heat-shock and high salt resistances. In addition, an infectious form of H. obtusa-specific 63-kDa periplasmic protein with a DNA-binding domain in its amino acid sequence is secreted into the host macronucleus after invasion into the macronucleus and remain within the nucleus. These facts suggest that binding of the 63-kDa protein to the host macronuclear DNA causes changes in the host gene expressions and enhances an environmental adaptability of the host cells. This 63-kDa protein was renamed as periplasmic region protein 1 (PRP1) to distinguish it from other proteins with similar molecular weights. To confirm whether PRP1 indeed binds to the host DNA, SDS-DNA PAGE and DNA affinity chromatography with calf thymus DNA and P. caudatum DNA were conducted and confirmed that PRP1 binds weakly to the P. caudatum DNA with a monoclonal antibody raised for the 63-kDa protein

High degree of specificity in the association between symbiotic betaproteobacteria and the host Euplotes (Ciliophora,Euplotia)

TheBetaproteobacteria-Euplotesassociationisanobligatorysymbioticsysteminvolvingamonophyleticgroupofciliatespeciesandtwobetaproteobacteriaspecieswhichcanbealternativelypresent.Recentdatashowedthatthisrelationshiphasbeenestablishedmorethanonceandthatseveralsymbiont-substitutioneventstookplace,revealingacomplexandintriguingevolutionarypath.Duetothedifferentevolutionarypathwaysfollowedbythedifferentsymbionts,eachbacterialstraincouldhavedifferentiallyevolvedand/orlostfunctionaltraits.Therefore,weperformedre-infectionexperiments,bothbyphagocytosisandbymicroinjection,totestthepossiblefunctionalroleofthedifferentbacteriatowardstheciliates.Ourresultsconfirmthatthegrowthcapacityofthehostisindissolublylinkedtothepresenceofitsoriginalsymbionts.Resultsoftheattemptsofre-infectionbyphagocytosisshowedthatnoneofthebacteriaisabletosuccessfullycolonizethehostcytoplasminthisway,evenifregularlyingested.Re-infectionbymicroinjectionsucceedonlyinonecase.SuchresultspointtoahighdegreeofspecificityintheinteractionsbetweenbacteriaandEuplotesevenaftertheinvasionstep.Duetoaco-evolutivepathwayofreciprocaladaptation,differentdegreeofre-colonizationabilitycouldhavebeenconservedbythedifferentspeciesandstrainsofthesymbionts