Kaori Takahashi

LIL3, a light-harvesting-like protein, plays an essential role in chlorophyll and tocopherol biosynthesis

The light-harvesting chlorophyll-binding (LHC) proteins are major constituents of eukaryotic photosynthetic machinery. In plants, six different groups of proteins, LHC-like proteins, share a conserved motif with LHC. Although the evolution of LHC and LHC-like proteins is proposed to be a key for the diversification of modern photosynthetic eukaryotes, our knowledge of the evolution and functions of LHC-like proteins is still limited. In this study, we aimed to understand specifically the function of one type of LHC-like proteins, LIL3 proteins, by analyzing Arabidopsis mutants lacking them. The Arabidopsis genome contains two gene copies for LIL3, LIL3:1 and LIL3:2. In the lil3:1/lil3:2 double mutant, the majority of chlorophyll molecules are conjugated with an unsaturated geranylgeraniol side chain. This mutant is also deficient in α-tocopherol. These results indicate that reduction of both the geranylgeraniol side chain of chlorophyll and geranylgeranyl pyrophosphate, which is also an essential intermediate of tocopherol biosynthesis, is compromised in the lil3 mutants. We found that the content of geranylgeranyl reductase responsible for these reactions was severely reduced in the lil3 double mutant, whereas the mRNA level for this enzyme was not significantly changed. We demonstrated an interaction of geranylgeranyl reductase with both LIL3 isoforms by using a split ubiquitin assay, bimolecular fluorescence complementation, and combined blue-native and SDS polyacrylamide gel electrophoresis. We propose that LIL3 is functionally involved in chlorophyll and tocopherol biosynthesis by stabilizing geranylgeranyl reductase.

Amoebal endosymbiont Neochlamydia genome sequence illuminates the bacterial role in the defense of the host amoebae against Legionella pneumophila.

Previous work has shown that the obligate intracellular amoebal endosymbiont Neochlamydia S13, an environmental chlamydia strain, has an amoebal infection rate of 100%, but does not cause amoebal lysis and lacks transferability to other host amoebae. The underlying mechanism for these observations remains unknown. In this study, we found that the host amoeba could completely evade Legionella infection. The draft genome sequence of Neochlamydia S13 revealed several defects in essential metabolic pathways, as well as unique molecules with leucine-rich repeats (LRRs) and ankyrin domains, responsible for protein-protein interaction. Neochlamydia S13 lacked an intact tricarboxylic acid cycle and had an incomplete respiratory chain. ADP/ATP translocases, ATP-binding cassette transporters, and secretion systems (types II and III) were well conserved, but no type IV secretion system was found. The number of outer membrane proteins (OmcB, PomS, 76-kDa protein, and OmpW) was limited. Interestingly, genes predicting unique proteins with LRRs (30 genes) or ankyrin domains (one gene) were identified. Furthermore, 33 transposases were found, possibly explaining the drastic genome modification. Taken together, the genomic features of Neochlamydia S13 explain the intimate interaction with the host amoeba to compensate for bacterial metabolic defects, and illuminate the role of the endosymbiont in the defense of the host amoebae against Legionella infection.

Functional Analysis of Light-harvesting-like Protein 3 (LIL3) and Its Light-harvesting Chlorophyll-binding Motif in Arabidopsis

Background: The light-harvesting complex (LHC) motif is an amino acid consensus sequence found in various thylakoid proteins. Results: Modification and replacement of the transmembrane domain encompassing the LHC motif of a plant protein retains its membrane-anchoring function but impairs its protein-protein interaction. Conclusion: This domain functions in membrane anchoring and complex formation. Significance: This study provides new insights regarding the function of the LHC motif. The light-harvesting complex (LHC) constitutes the major light-harvesting antenna of photosynthetic eukaryotes. LHC contains a characteristic sequence motif, termed LHC motif, consisting of 25–30 mostly hydrophobic amino acids. This motif is shared by a number of transmembrane proteins from oxygenic photoautotrophs that are termed light-harvesting-like (LIL) proteins. To gain insights into the functions of LIL proteins and their LHC motifs, we functionally characterized a plant LIL protein, LIL3. This protein has been shown previously to stabilize geranylgeranyl reductase (GGR), a key enzyme in phytol biosynthesis. It is hypothesized that LIL3 functions to anchor GGR to membranes. First, we conjugated the transmembrane domain of LIL3 or that of ascorbate peroxidase to GGR and expressed these chimeric proteins in an Arabidopsis mutant lacking LIL3 protein. As a result, the transgenic plants restored phytol-synthesizing activity. These results indicate that GGR is active as long as it is anchored to membranes, even in the absence of LIL3. Subsequently, we addressed the question why the LHC motif is conserved in the LIL3 sequences. We modified the transmembrane domain of LIL3, which contains the LHC motif, by substituting its conserved amino acids (Glu-171, Asn-174, and Asp-189) with alanine. As a result, the Arabidopsis transgenic plants partly recovered the phytol-biosynthesizing activity. However, in these transgenic plants, the LIL3-GGR complexes were partially dissociated. Collectively, these results indicate that the LHC motif of LIL3 is involved in the complex formation of LIL3 and GGR, which might contribute to the GGR reaction.

Survival and transfer ability of phylogenetically diverse bacterial endosymbionts in environmental Acanthamoeba isolates

Obligate intracellular bacteria are commonly found as endosymbionts of acanthamoebae; however, their survival in and ability to transfer to amoebae are currently uncharacterized. In this study, six bacterial endosymbionts, found in five environmental Acanthamoeba isolates (S13, R18, S23, S31, S40) from different locations of Sapporo city, Japan, were characterized. Phylogenetic analysis revealed that three bacterial endosymbionts (eS23, eS31, eS40a) belonged to α- and β-Proteobacteria phyla and the remaining endosymbionts (eS13, eR18, eS40b) belonged to the order Chlamydiales. The Acanthamoeba isolate (S40) contained two phylogenetically different bacterial endosymbionts (eS40a, eS40b). Fluorescent in situ hybridization analysis showed that all bacterial endosymbionts were diffusely localized within amoebae. Transmission electron microscopy also showed that the endosymbionts were rod-shaped (eS23, eS31, eS40a) or sphere- or crescent-shaped (eS13, eR18, eS40b). No successful culture of these bacteria was achieved using conventional culture methods, but the viability of endosymbionts was confirmed by live/dead staining and RT-PCR methods. However, endosymbionts (except eR18) derived from original host cells lost the ability to be transferred to another Acanthamoebae strains [ATCC strain (C3), environmental strains (S14, R23, S24)]. Thus, our data demonstrate that phylogenetically diverse bacterial endosymbionts found in amoebae maintain a stable interaction with amoebae, but the transferability is limited.

Mechanisms of concealed ventricular bigeminy: The concept of concealed conduction in the reentrant pathway

To clarify the presence of concealed conduction in the reentrant pathway of extrasystoles, 20 patients with ventricular extrasystoles were studied in whom two forms of interectopic periods were found in the same recording. One form is the XS1S2X period, in which two sinus QRS complexes (S1 and S2) intervene between an interpolated extrasystole (the first X) and the next extrasystole (the second X). The other is the XS2X or XS1X period, in which one sinus QRS complex intervenes between two extrasystoles. In all patients except one, the XX interval in the XS1S2X period was longer than that in the XS2X or XS1X period though shorter than twice the latter XX interval. This strongly suggests the presence of two-level block in the reentrant pathway of the extrasystoles. It appears that the sinus impulse S1 in the XS1S2X period invaded a large portion of the reentrant pathway and then was blocked at a distal site of the pathway; namely, that concealed conduction of the impulse S1 occurred in the pathway. It is suggested that such concealed conduction prolonged the conduction time of the following sinus impulse, S2, in the reentrant pathway, resulting in lengthening of the XX interval. The presence of three- or four-level block is also suggested. By the use of such multilevel block, mechanisms of concealed ventricular bigeminy are explained.

Environmental Chlamydiae Alter the Growth Speed and Motility of Host Acanthamoebae

Symbiosis between living beings is an important driver of evolutionary novelty and ecological diversity; however, understanding the mechanisms underlying obligate mutualism remains a significant challenge. Regarding this, we have previously isolated two different Acanthamoeba strains harboring endosymbiotic bacteria, Protochlamydia (R18 symbiotic amoebae: R18WT) or Neochlamydia (S13 symbiotic amoebae; S13WT). In this study, we treated the symbiotic amoebae R18WT and S13WT with doxycycline (DOX) and rifampicin (RFP), respectively, to establish the aposymbiotic amoebae R18DOX and S13RFP, respectively. Subsequently, we compared the growth speed, motility, phagocytosis, pinocytosis, and morphology of the symbiotic and aposymbiotic amoebae. The growth speed of R18DOX was decreased, although that of S13RFP was increased. A marked change in motility was observed only for R18DOX amoebae. There was no difference in phagocytic and pinocytic activities between the symbiotic and aposymbiotic amoebae. Meanwhile, we observed a significant change in the phalloidin staining pattern and morphological changes in R18DOX (but not S13RFP) aposymbiotic amoebae, indicating a change in actin accumulation upon removal of the Protochlamydia. Infection of C3 (a reference strain) or S13RFP amoebae with Protochlamydia had a harmful effect on the host amoebae, but R18DOX amoebae re-infected with Protochlamydia showed recovery in both growth speed and motility. Taken together, we conclude that endosymbiont environmental chlamydiae alter the growth speed and/or motility of their host Acanthamoeba, possibly implying an close mutual relationship between amoebae and environmental chlamydiae.

Host range of obligate intracellular bacterium Parachlamydia acanthamoebae

The obligate intracellular bacterium Parachlamydia acanthamoebae is a potential human pathogen, but the host range of the bacteria remains unknown. Hence, the growth of P. acanthamoebae Bn9 in protozoa (Tetrahymena, Acanthamoeba, Dictyostelium) and mammalian cells (HEp‐2, Vero, THP‐1, PMA‐stimulated THP‐1, Jurkat) was assessed using an AIU assay which had been previously established by the current authors. P. acanthamoebae grew in Acanthamoeba but not in the other cell types. The growth was also confirmed using DAPI staining, FISH and TEM. These results indicate that the host range of P. acanthamoebae is limited.

Protochlamydia Induces Apoptosis of Human HEp-2 Cells through Mitochondrial Dysfunction Mediated by Chlamydial Protease-Like Activity Factor

Obligate amoebal endosymbiotic bacterium Protochlamydia with ancestral pathogenic chlamydial features evolved to survive within protist hosts, such as Acanthamoba, 0.7–1.4 billion years ago, but not within vertebrates including humans. This observation raises the possibility that interactions between Protochlamydia and human cells may result in a novel cytopathic effect, leading to new insights into host-parasite relationships. Previously, we reported that Protochlamydia induces apoptosis of the immortalized human cell line, HEp-2. In this study, we attempted to elucidate the molecular mechanism underlying this apoptosis. We first confirmed that, upon stimulation with the bacteria, poly (ADP-ribose) polymerase (PARP) was cleaved at an early stage in HEp-2 cells, which was dependent on the amount of bacteria. A pan-caspase inhibitor and both caspase-3 and -9 inhibitors similarly inhibited the apoptosis of HEp-2 cells. A decrease of the mitochondrial membrane potential was also confirmed. Furthermore, lactacystin, an inhibitor of chlamydial protease-like activity factor (CPAF), blocked the apoptosis. Cytochalasin D also inhibited the apoptosis, which was dependent on the drug concentration, indicating that bacterial entry into cells was required to induce apoptosis. Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis. We also confirmed that the Protochlamydia used in this study possessed a homologue of the cpaf gene and that two critical residues, histidine-101 and serine-499 of C. trachomatis CPAF in the active center, were conserved. Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis. More interestingly, because C. trachomatis infection can block the apoptosis, our finding implies unique features of CPAF between pathogenic and primitive chlamydiae.

Endosymbiotic bacterium Protochlamydia can survive in acanthamoebae following encystation.

Obligate intracellular bacteria are commonly seen as endosymbionts of acanthamoebae. However, whether endosymbionts can survive amoebal encystations remains a significant challenge in cellular biology. The survival of the endosymbiotic bacteria Protochlamydia belonging to environmental chlamydiae found in an amoebal isolate that we have previously reported (Environmental Microbiology Reports, DOI: 10.1111/j.1758-2229.2009.00094.x, 2009) following encystation was therefore assessed. The bacteria were observed in cysts and trophozoites reverted from cysts by analysis with transmission electron microscope, and the bacterial 16S rRNA transcripts were detected in amoeba cultures following encystations by reverse transcription polymerase chain reaction method. Furthermore, the bacterial growth was also confirmed, by fluorescent in situ hybridization analysis and the AIU assay that we have previously established (Applied Environmental Microbiology, 74: 6397-6404, 2008), in trophozoites reverted from cysts stored at 4°C for up to a month after encystation. Thus, these results demonstrated that Protochlamydia could survive in acanthamoebae following encystation. Our findings suggest that amoeba cysts might be further studied in order to understand their role in the environmental survival of endosymbionts.

Amoebal Endosymbiont Protochlamydia Induces Apoptosis to Human Immortal HEp-2 Cells

Protochlamydia, an environmental chlamydia and obligate amoebal endosymbiotic bacterium, evolved to survive within protist hosts, such as Acanthamobae, 700 million years ago. However, these bacteria do not live in vertebrates, including humans. This raises the possibility that interactions between Protochlamydia and human cells could induce a novel cytopathic effect, leading to new insights into host-parasite relationships. Therefore, we studied the effect of Protochlamydia on the survival of human immortal cell line, HEp-2 cells and primary peripheral blood mononuclear cells (PBMC). Using mainly 4′,6-diamidino-2-phenylindole staining, fluorescent in situ hybridization, transmission electron microscopy, and also TUNEL and Transwell assays, we demonstrated that the Protochlamydia induced apoptosis in HEp-2 cells. The attachment of viable bacterial cells, but not an increase of bacterial infectious progenies within the cells, was required for the apoptosis. Other chlamydiae [Parachlamydia acanthamoebae and Chlamydia trachomatis (serovars D and L2)] did not induce the same phenomena, indicating that the observed apoptosis may be specific to the Protochlamydia. Furthermore, the bacteria had no effect on the survival of primary PBMCs collected from five volunteers, regardless of activation. We concluded that Protochlamydia induces apoptosis in human-immortal HEp-2 cells and that this endosymbiont could potentially be used as a biological tool for the elucidation of novel host-parasite relationships.