A new microsporidium Percutemincola moriokae gen. nov., sp. nov. from Oscheius tipulae: A novel model of microsporidia-nematode associations.

Here, we describe a new microsporidium Percutemincola moriokae gen. nov., sp. nov., which was discovered in the intestinal and hypodermal cells of a wild strain of the nematode Oscheius tipulae that inhabits in the soil of Morioka, Iwate Prefecture, Japan. The spores of Pe. moriokae had an average size of 1.0 × 3.8 µm and 1.3 × 3.2 µm in the intestine and hypodermis, respectively, and electron microscopy revealed that they exhibited distinguishing features with morphological diversity in the hypodermis. Isolated spores were able to infect a reference strain of O. tipulae (CEW1) through horizontal transmission but not the nematode Caenorhabditis elegans. Upon infection, the spores were first observed in the hypodermis and then in the intestine the following day, suggesting a unique infectious route among nematode-infective microsporidia. Molecular phylogenetic analysis grouped this new species with the recently identified nematode-infective parasites Enteropsectra and Pancytospora forming a monophyletic sister clade to Orthosomella in clade IV, which also includes human pathogens such as Enterocytozoon and Vittaforma. We believe that this newly discovered species and its host could have application as a new model in microsporidia-nematode association studies.

Molecular dissection of Caenorhabditis elegans ATP-binding cassette transporter protein HAF-4 to investigate its subcellular localization and dimerization.

Caenorhabditis elegans HAF-4 and HAF-9 are half-type ATP-binding cassette (ABC) transporter proteins, which are highly homologous to the human peptide transporter protein, transporter associated with antigen processing-like (TAPL, ABCB9). TAPL forms homodimers and localizes to lysosomes, whereas HAF-4 and HAF-9 form heterodimers and localize to intestine-specific non-acidified organelles. Both TAPL and HAF-4/HAF-9 are predicted to have four amino-terminal transmembrane helices [transmembrane domain 0 (TMD0)] additional to the six transmembrane helices that form the canonical core domain of ABC transporters with a cytosolic ABC region. TAPL requires its amino-terminal domain for localization to lysosomes; however, molecular mechanisms underlying HAF-4 and HAF-9 localization to their target organelles had not been elucidated. Here, we demonstrate that the mechanisms underlying HAF-4 localization differ from those underlying TAPL localization. Using transgenic C. elegans expressing mutant HAF-4 proteins labeled with green fluorescent protein, we reveal that the TMD0 of HAF-4 was not sufficient for proper localization of the protein. The mutant HAF-4, which lacked TMD0, localized to intracellular organelles similarly to the wild-type protein and functioned normally in the biogenesis of its localizing organelles, indicating that the TMD0 of HAF-4 is dispensable for both its localization and function.

Characterization of HAF-4- and HAF-9-localizing organelles as distinct organelles in Caenorhabditis elegans intestinal cells.

BACKGROUND: The intestinal cells of Caenorhabditis elegans are filled with heterogeneous granular organelles that are associated with specific organ functions. The best studied of these organelles are lipid droplets and acidified gut granules associated with GLO-1, a homolog of the small GTPase Rab38. In this study, we characterized a subset of the intestinal granules in which HAF-4 and HAF-9 localize on the membrane. HAF-4 and HAF-9 are ATP-binding cassette (ABC) transporter proteins that are homologous to the mammalian lysosomal peptide transporter TAPL (transporter associated with antigen processing-like, ABCB9). RESULTS: Using transgenic worms expressing fluorescent protein-tagged marker proteins, we demonstrated that the HAF-4- and HAF-9-localizing organelles are not lipid droplets and do not participate in yolk protein transport. They were also ruled out as GLO-1-positive acidified gut granules. Furthermore, we clarified that the late endosomal protein RAB-7 localizes to the HAF-4- and HAF-9-localizing organelles and is required for their biogenesis. CONCLUSIONS: Our results indicate that the HAF-4- and HAF-9-localizing organelles are distinct intestinal organelles associated with the endocytic pathway.

Co-operative function and mutual stabilization of the half ATP-binding cassette transporters HAF-4 and HAF-9 in Caenorhabditis elegans.

Caenorhabditis elegans HAF-4 and HAF-9 are half ABC (ATP-binding-cassette) transporters that are highly homologous to the human lysosomal peptide transporter TAPL [TAP (transporter associated with antigen processing)-like; ABCB9]. We reported previously that both HAF-4 and HAF-9 localize to the membrane of a subset of intestinal organelles, and are required for the formation of these organelles and other physiological aspects. In the present paper, we report the genetic and physical interactions between HAF-4 and HAF-9. Overexpression of HAF-4 and HAF-9 did not rescue the intestinal organelle defect of the haf-9 and haf-4 deletion mutants respectively, indicating that they cannot substitute for each other. Double haf-4 and haf-9 mutants do not exhibit more severe phenotypes than the single mutants, suggesting their co-operative function. Immunoprecipitation experiments demonstrated their physical interaction. The results of the present study suggest that HAF-4 and HAF-9 form a heterodimer. Furthermore, Western blot analysis of the deletion mutants and RNAi (RNA interference) knockdown experiments in GFP (green fluorescent protein)-tagged HAF-4 or HAF-9 transgenic worms suggest that HAF-4-HAF-9 heterodimer formation is required for their stabilization. The findings provide a clue as to how ABC transporters adopt a stable functional form.

Functional analysis of the honeybee (Apis mellifera L.) salivary system using proteomics.

In insects, specific proteins and physiologically active molecules whose functions are related to their lifestyles are secreted from the salivary system. To investigate proteins/molecules related to the sociality of the European honeybee (Apis mellifera L.), we performed a proteomic analysis of the honeybee salivary system. The honeybee salivary system comprises two secretory glands: the postcerebral gland (PcG) and the thoracic gland (TG), both of which are connected to a common duct that opens in the mouthpart. Although most (31 out of 35) of the major proteins identified from the PcG and TG were housekeeping proteins, the spot intensities for aldolase and acetyl-CoA acyltransferase 2 were stronger in the PcG than in the TG in the 2-dimensional gel electrophoresis. Immunoblotting confirmed that the expression of these proteins was stronger in the PcG than in the TG, whereas expression was almost not detectable in the hypopharyngeal gland (HpG), suggesting that carbohydrate metabolism is enhanced in the honeybee PcG. In addition, imaginal disc growth factor 4 (IDGF4) was synthesized in the honeybee salivary system. Immunoblotting indicated IDGF4 expression was very strong in the PcG, moderate in the TG, and very weak in the HpG. A considerable amount of IDGF4 was detected in the royal jelly, while less was detected in honey, strongly suggesting that the honeybee salivary system secretes IDGF4 into the royal jelly and honey. The secreted IDGF4 might therefore affect growth and physiology of the other colony members.

Morph-dependent expression and subcellular localization of host serine carboxypeptidase in bacteriocytes of the pea aphid associated with degradation of the endosymbiotic bacterium Buchnera.

Pea aphids form a mutualistic association with the endosymbiotic bacterium Buchnera, which is harbored in specialized host cells called bacteriocytes. The adult aphids display dimorphism In which there are winged and wingless morphs. We previously reported that the Buchnera density in bacteriocytes of the winged morph (alate) decreases around final ecdysis, whereas that in the wingless morph (aptera) does not decrease; the decrease in density in alatae is accompanied by activation of the host lysosomal system and by Buchnera degradation. In the present study, we performed a proteomic analysis to clarify the molecular mechanisms underlying the decrease in Buchnera density. By comparing the protein expression profiles of bacteriocytes in alatae and apterae Just after final ecdysis, we identified three and one protein spots that were preferentially expressed in alatae and apterae, respectively. Among the three alate-preferential spots, two were an identical aphid protein, carboxypeptidase vitellogenic-like (CPVL), whereas the other was a mixture of four proteins: gamma-glutamyl hydrolase, acyl-CoA dehydrogenase, aphid short chain acyl-CoA dehydrogenase, and Buchnera S-adenosylmethionine synthetase. The aptera-preferential spot was Buchnera outer membrane protein A. Immunoblot and immunohistochemical analyses using aphid bacteriocytes Just after final ecdysis revealed that expression of aphid CPVL was preferentially upregulated in alatae and was localized around Buchnera cells in the bacterlocytes, suggesting the involvement of CPVL in Buchnera degradation in alatae.

Age- and morph-dependent activation of the lysosomal system and Buchnera degradation in aphid endosymbiosis.

Endosymbiosis in aphids is maintained through a mutualistic association between the host and a symbiotic bacterium, Buchnera, which is harbored in specialized host cells called bacteriocytes. Here, we examined the changes in the Buchnera density in bacteriocytes in relation to the development and polyphenism of the host aphid. Buchnera density in the winged morph aphids, alatae, decreased drastically around the final ecdysis, whereas in the wingless morph aphids, apterae, Buchnera density decreased after the final ecdysis. Thereafter, in both apterae and alatae, Buchnera density was maintained at a constant level until 10 days and then again decreased gradually until 18 days after the final ecdysis. Cytochemical analysis with LysoTracker reagent and quantitative RT-PCR analysis revealed that the number of lysosome-like acidic organelles and the amount of lysosome-related gene (lysozyme and cathepsin L) transcripts increased drastically in the bacteriocytes of alatae around the final ecdysis. Electron microscopy of alatae bacteriocytes around the final ecdysis revealed many Buchnera with irregular electron-dense areas in their cytoplasm that were enclosed by a distended symbiosome membrane. These findings indicated that age- and morph-dependent decreases in Buchnera density coincided with activation of the host lysosomal system and the increased degradation of Buchnera.

Properties of an intracellular poly(3-hydroxybutyrate) depolymerase (PhaZ1) from Rhodobacter spheroides.

The gene of an intracellular poly(3-hydroxybutyrate) (iPHB) depolymerase from Rhodobacter sphaeroides was cloned and sequenced. The nucleotide sequence of the cloned gene was homologous to that of the iPHB depolymerase gene from Ralstonia eutropha H16 (phaZ1(Reu)) and the gene was designated phaZ1(Rsh). PhaZ1(Rsh) was purified from E. coli harboring an expression vector containing phaZ1(Rsh) and its properties were examined. PhaZ1(Rsh) degraded amorphous PHB granules, and the 3-hydroxybutyrate tetramer and pentamer, but not crystalline PHB granules. The enzyme activity was inhibited by p-chloromercuribenzoate and Triton X-100. Diisopropylfluorophosphate, phenylmethylsulfonylfluoride, and dithiothreitol had no effect on the activity. A mutant having alanine instead of cysteine at 178 lost the activity. These results show that PhaZ1(Rsh) is a quite similar enzyme to PhaZ1(Reu).