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.

Differential gene expression in the mandibular glands of queen and worker honeybees, Apis mellifera L.: implications for caste-selective aldehyde and fatty acid metabolism.

In honeybees, queens synthesize the "queen pheromone," whereas workers synthesize fatty acid components of "royal jelly" in their mandibular glands (MGs). To identify candidate proteins involved in the caste-selective MG function, we performed a proteomic analysis and identified three proteins that were expressed selectively in queen MGs (aldehyde dehydrogenase 1 [ALDH1], medium-chain acyl-CoA dehydrogenase [MCAD], and electron transfer flavoprotein alpha [ETFalpha)]), and a protein that was expressed selectively in worker MGs (fatty acid synthase [FAS)]). The quantitative reversed transcription-polymerase chain reaction demonstrated that the level of aldh1 transcription in MGs was significantly higher, whereas that of fas transcription was lower in queens than in workers. Among the eight genes encoding proteins similar to ALDH1 that are registered in the honeybee genome database, aldh6, aldh7, and aldh1 were expressed at significantly higher levels in queen MGs than in worker MGs. In situ hybridization showed that in the queen head, aldh1 was expressed in MG cells, whereas aldh6 and aldh7 were expressed in fat cells attached to the MGs. These results suggest caste- and cell type-selective aldehyde/fatty acid metabolism in honeybee MGs.

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.

In vivo gene transfer into the honeybee using a nucleopolyhedrovirus vector.

The honeybee Apis mellifera L. is a social insect and one of the most industrially important insects. We examined whether a baculovirus-mediated retrotransposon is applicable to in vivo transfer of exogenous genes to the honeybees. Honeybee larvae and pupae were injected with two types of recombinant Autographa californica nucleopolyhedrovirus (AcNPV) vectors, one that includes the enhanced green fluorescent protein gene (egfp) as a reporter to be inserted into the honeybee genome, and another that includes the reverse transcriptase gene responsible for the insertion. Fluorescence was observed in most of the viral-injected larvae and pupae. Reverse transcription-polymerase chain reaction and immunoblotting confirmed egfp mRNA and eGFP expression in these honeybees, although egfp insertion into the honeybee genome was not confirmed. These results indicate that AcNPV vectors can be used for the transfer and transient expression of an exogenous gene in the larval and pupal honeybees.

Identification of proteins whose expression is up- or down-regulated in the mushroom bodies in the honeybee brain using proteomics.

To identify protein(s) with different expression patterns in the mushroom bodies (MBs) in the honeybee brain, we compared the protein profiles of MBs and optic lobes (OLs) using proteomics. Two-dimensional gel electrophoresis revealed that five and three spots were selectively expressed in the MBs or OLs, respectively. Liquid chromatography tandem mass spectrometry analysis identified juvenile hormone diol kinase and glyceraldehyde-3-phosphate dehydrogenase as MB- and OL-selective proteins, respectively. In situ hybridization revealed that jhdk expression was upregulated in MB neuron subsets, whereas gapdh expression was downregulated, indicating that MBs have a distinct gene and protein expression profile in the honeybee brain.

Hundreds of flagellar basal bodies cover the cell surface of the endosymbiotic bacterium Buchnera aphidicola sp. strain APS.

Buchnera aphidicola is the endosymbiotic bacterium of the pea aphid. Due to its small genome size, Buchnera lacks many essential genes for autogenous life but obtains nutrients from the host. Although the Buchnera cell is nonmotile, it retains clusters of flagellar genes that lack the late genes necessary for motility, including the flagellin gene. In this study, we show that the flagellar genes are actually transcribed and translated and that the Buchnera cell surface is covered with hundreds of hook-basal-body (HBB) complexes. The abundance of HBB complexes suggests a role other than motility. We discuss the possibility that the HBB complex may serve as a protein transporter not only for the flagellar proteins but also for other proteins to maintain the symbiotic system.

Identification of soldier caste-specific protein in the frontal gland of nasute termite Nasutitermes takasagoensis (Isoptera: Termitidae).

The termite soldier is unique because of its defensive task in a colony. In Nasutitermitinae (family Termitidae), soldiers use in their defense frontal glands, which contain various chemical substances. To isolate the gene products related to the chemical defense, we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles of soldier heads with those of workers of the nasute termite Nasutitermes takasagoensis. We identified a 26-kDa soldier-specific protein (Ntsp1) that exists most abundantly in the dorsal head including the frontal gland. We determined the N-terminal amino acid sequence of Ntsp1, and then cloned the Ntsp1 cDNA by rapid amplification of the cDNA ends-polymerase chain reaction (RACE-PCR). A putative signal peptide was detected upstream of the N-terminus and the Ntsp1 protein showed sequence homologies with known insect secretory carrier proteins, which bind to hydrophobic ligands such as juvenile hormone, suggesting that Ntsp1 belongs to this class of proteins. Northern blot analysis confirmed that the expression level of Ntsp1 was high only in the soldier head. In addition, the localization of Ntsp1 expression was limited in epithelial cells of the frontal gland reservoir, suggesting that this protein binds to some terpenoid(s) preserved in the frontal gland reservoir.

Venomous protease of aphid soldier for colony defense.

In social aphids, morphological, behavioral, and physiological differences between soldiers and normal insects are attributed to differences in gene expression between them, because they are clonal offspring parthenogenetically produced by the same mothers. By using cDNA subtraction, we identified a soldier-specific cysteine protease of the family cathepsin B in a social aphid, Tuberaphis styraci, with a second-instar soldier caste. The cathepsin B gene was specifically expressed in soldiers and first-instar nymphs destined to be soldiers. The cathepsin B protein was preferentially produced in soldiers and showed a protease activity typical of cathepsin B. The cathepsin B mRNA and protein were localized in the midgut of soldiers. For colony defense, soldiers attack enemies with their stylet, which causes paralysis and death of the victims. Notably, after soldiers attacked moth larvae, the cathepsin B protein was detected from the paralyzed larvae. Injection of purified recombinant cathepsin B protein certainly killed the recipient moth larvae. From these results, we concluded that the cathepsin B protein is a major component of the aphid venom produced by soldiers of T. styraci. Soldier-specific expression of the cathepsin B gene was found in other social aphids of the genus Tuberaphis. The soldier-specific cathepsin B gene showed an accelerated molecular evolution probably caused by the action of positive selection, which had been also known from venomous proteins of other animals.

Identification of honeybee antennal proteins/genes expressed in a sex- and/or caste selective manner.

We identified three candidate proteins/genes involved in caste and/or sex-specific olfactory processing in the honeybee Apis mellifera L., that are differentially expressed between the antennae of the worker, queen, and drone honeybees using SDS-polyacrylamide gel electrophoresis or the differential display method. A protein was identified, termed D-AP1, that was expressed preferentially in drone antennae when compared to those of workers. cDNA cloning revealed that D-AP1 is homologous to carboxylesterases. Enzymatic carboxylesterase activity in the drone antennae was higher than in the workers, suggesting its dominant function in the drone antennae. In contrast, two proteins encoded by genes termed W-AP1 and Amwat were expressed preferentially in worker antennae when compared to those of queens. W-AP1 is homologous to insect chemosensory protein, and Amwat encodes a novel secretory protein. W-AP1 is expressed selectively in worker antennae, while Amwat is expressed both in the antennae and legs of the workers. These findings suggest that these proteins are involved in the antennal function characteristic to drone or worker honeybees.

A new heat shock gene, AgsA, which encodes a small chaperone involved in suppressing protein aggregation in Salmonella enterica serovar typhimurium.

We discovered a novel small heat shock protein (sHsp) named AgsA (aggregation-suppressing protein) in the thermally aggregated fraction from a Salmonella enterica serovar Typhimurium dnaK-null strain. The -10 and -35 regions upstream of the transcriptional start site of the agsA gene are characteristic of sigma(32)- and sigma(72)-dependent promoters. AgsA was strongly induced by high temperatures. The similarity between AgsA and the other two sHsps of Salmonella serovar Typhimurium, IbpA and IbpB, is rather low (around 30% amino acid sequence identity). Phylogenetic analysis suggested that AgsA arose from an ancient gene duplication or amplification at an early evolutionary stage of gram-negative bacteria. Here we show that overproduction of AgsA partially complements the DeltadnaK52 thermosensitive phenotype and reduces the amount of heat-aggregated proteins in both DeltadnaK52 and DeltarpoH mutants of Escherichia coli. These data suggest that AgsA is an effective chaperone capable of preventing aggregation of nonnative proteins and maintaining them in a state competent for refolding in Salmonella serovar Typhimurium at high temperatures.

Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones.

BACKGROUND: It is well known that expression of certain bacterial genes responds rapidly to such stimuli as exposure to toxic chemicals and physical agents. It is generally believed that the proteins encoded in these genes are important for successful survival of the organism under the hostile conditions. Analogously, the proteins induced in bacterial cells exposed to antibiotics are believed to affect the organisms' susceptibility to these agents. RESULTS: We demonstrated that Escherichia coli cells exposed to levofloxacin (LVFX), a fluoroquinolone (FQ), induce the syntheses of heat shock proteins and RecA. To examine whether the heat shock proteins affect the bactericidal action of FQs, we constructed E. coli strains with mutations in various heat shock genes and tested their susceptibility to FQs. Mutations in dnaK, groEL, and lon increased this susceptibility; the lon mutant exhibited the greatest effects. The increased susceptibility of the lon mutant was corroborated by experiments in which the gene encoding the cell division inhibitor, SulA, was subsequently disrupted. SulA is induced by the SOS response and degraded by the Lon protease. The findings suggest that the hypersusceptibility of the lon mutant to FQs could be due to abnormally high levels of SulA protein resulting from the depletion of Lon and the continuous induction of the SOS response in the presence of FQs. CONCLUSION: The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease. FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility. Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.

Increased expression of Hsp70 for resistance to deuterium oxide in a yeast mutant cell line.

Labeling with stable isotopes, typically deuterium (D), is powerful tool for studying the functional structure of biomolecules by NMR. Biosynthesis of certain deuterated proteins in microorganisms cultured in deuterium oxide (D(2)O) is an attractive strategy. However, the growth of almost all microorganisms is inhibited at high concentrations of D(2)O. We isolated a mutant of yeast that grows well in D(2)O. The expression of Hsp70 was enhanced in the mutant. The increased expression also endowed the yeast with cold-resistance. The mutant might be useful for biosynthesis of D-labeled biomolecules.

An experimental validation of orphan genes of Buchnera, a symbiont of aphids.

Although Buchnera sp. APS, an intracellular symbiont of pea aphids, is a close relative of Escherichia coli, its genome has been extensively modified because of its prolonged intracellular life. In our previous studies on the Buchnera genome, computer analysis predicted three "orphan" genes, yba2, yba3, and yba4, which are open reading frames (ORFs) with no homologs in the database. In this paper, we successfully validated all these orphan genes by RT-PCR and Northern hybridization. The present study also revealed that yba3 and yba4 formed an operon, suggesting that they function in concert. Sequences around transcriptional start sites suggests that these genes are under the control of sigma 70. In view of codon usage and AT bias observed in these genes, it is likely that Buchnera have maintained them for an evolutionarily long time.

The ATP-dependent lon protease of Salmonella enterica serovar Typhimurium regulates invasion and expression of genes carried on Salmonella pathogenicity island 1.

An early step in the pathogenesis of Salmonella enterica serovar Typhimurium infection is bacterial penetration of the intestinal epithelium. Penetration requires the expression of invasion genes found in Salmonella pathogenicity island 1 (SPI1). These genes are controlled in a complex manner by regulators in SPI1, including HilA and InvF, and those outside SPI1, such as two-component regulatory systems and small DNA-binding proteins. We report here that the expression of invasion genes and the invasive phenotype of S. enterica serovar Typhimurium are negatively regulated by the ATP-dependent Lon protease, which is known to be a major contributor to proteolysis in Escherichia coli. A disrupted mutant of lon was able to efficiently invade cultured epithelial cells and showed increased production and secretion of three identified SPI1 proteins, SipA, SipC, and SipD. The lon mutant also showed a dramatic enhancement in transcription of the SPI1 genes hilA, invF, sipA, and sipC. The increases ranged from 10-fold to almost 40-fold. It is well known that the expression of SPI1 genes is also regulated in response to several environmental conditions. We found that the disruption of lon does not abolish the repression of hilA and sipC expression by high-oxygen or low-osmolarity conditions, suggesting that Lon represses SPI1 gene expression by a regulatory pathway independent of these environmental signals. Since HilA is thought to function as a central regulator of SPI1 gene expression, it is speculated that Lon may regulate SPI1 gene expression by proteolysis of putative factors required for activation of hilA expression.

DNA Replication in Permeabilized Cells of Sea Urchin Embryos: (DNA replication/permeabilized embryonic cells/sea urchin).

For study of the regulation of DNA replication in sea urchin embryos during the early stages of development, an embryonic cell system that was permeable to exogenously supplied nucleotides was established. Embryos were permeabilized by incubating them in hypotonic buffer containing 0.3 M glucose. The permeabilized embryonic cells maintained their morphological integrity, and synthesized DNA when supplied with exogenous dNTPs. DNA synthesis in these permeabilized embryonic cells required the presence of ATP and three other deoxyribonucleoside triphosphates in addition to labeled dTTP. DNA synthesis was almost completely inhibited by N-ethylmaleimide, and proceeded in a discontinuous fashion. Only cells permeabilized during the S phase could incorporate nucleoside triphosphates into DNA: cells permeabilized during other phases did not synthesize DNA. During a 60 min-incubation period, over 10% of the genomic DNA was replicated under the experimental conditions used.