Hiroyuki Yamaguchi

Effect of egg yolk phospholipids plasma elimination and tissue distribution of coenzyme Q10 administered in an emulsion to rats

When coenzyme Q10 (CoQ10) in fat emulsion emulsified with egg yolk phospholipids (PL) was administered intravenously to rats, CoQ10 was eliminated more rapidly than when it was solubilized with a polyoxyethylene derivative of hydrogenated castor oil. Although CoQ10 in emulsion form is distributed mainly to liver, increase of PL concentration in the emulsion increased the distribution of CoQ10 to heart which is a target organ for CoQ10.

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.

Ciliates rapidly enhance the frequency of conjugation between Escherichia coli strains through bacterial accumulation in vesicles.

The mechanism underlying bacterial conjugation through protozoa was investigated. Kanamycin-resistant Escherichia coli SM10λ+ carrying pRT733 with TnphoA was used as donor bacteria and introduced by conjugation into ciprofloxacin-resistant E. coli clinical isolate recipient bacteria. Equal amounts of donor and recipient bacteria were mixed together in the presence or absence of protozoa (ciliates, free-living amoebae, myxamoebae) in Pages amoeba saline for 24 h. Transconjugants were selected with Luria broth agar containing kanamycin and ciprofloxacin. The frequency of conjugation was estimated as the number of transconjugants for each recipient. Conjugation frequency in the presence of ciliates was estimated to be approximately 10⁻⁶, but in the absence of ciliates, or in the presence of other protozoa, it was approximately 10⁻⁸. Conjugation also occurred in culture of ciliates at least 2 h after incubation. Successful conjugation was confirmed by the polymerase chain reaction. Addition of cycloheximide or latrunculin B resulted in suppression of conjugation. Heat killing the ciliates or bacteria had no effect on conjugation frequency. Co-localization of green fluorescent protein-expressing E. coli and PKH-67-vital-stained E. coli was observed in the same ciliate vesicles, suggesting that both donor and recipient bacteria had accumulated in the same vesicle. In this study, the conjugation frequency of bacteria was found to be significantly higher in vesicles purified from ciliates than those in culture suspension. We conclude that ciliates rapidly enhance the conjugation of E. coli strains through bacterial accumulation in vesicles.

Factors influencing the tissue distribution of coenzyme Q10 intravenously administered in an emulsion to rats: emulsifying agents and lipoprotein lipase activity

The tissue distribution of coenzyme Q10 (CoQ10) administered intravenously in an emulsion prepared with egg yolk phosphatidylcholine (PC), egg yolk sphingomyelin (SPM) or a combination of PC and a polyoxyethylene derivative of hydrogenated castor oil (HCO‐60) (PC + HCO‐60) was investigated. The disappearance from the plasma of CoQ10 administered in three different emulsions of lipid particle size less than 0.5 μm varied with the particular emulsifer. Its disappearance occurred most rapidly from the PC emulsion; with the addition of HCO‐60, its disappearance was much slower. In the reticuloendothelial system, the concentration of CoQ10 was higher in the spleen, for both the SPM and PC + HCO‐60 emulsions than for the PC emulsion. HCO‐60 reduced the CoQ10 distribution in the liver from the PC emulsion. Differences in disappearance rates from the plasma are thus considered to be due to the extent of CoQ10 distribution in the liver. CoQ10 concentration in the heart, a target organ, was greatest with the PC emulsion. Its distribution was related to lipoprotein lipase (LPL) activity in this organ. The effects caused by HCO‐60, however, could not be explained by LPL activity alone. CoQ10 distribution in the adrenal gland and kidney can be explained partly by LPL activity but in the presence of HCO‐60, the distribution mechanism apparently involves other factors.

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.

Frequency of Chlamydia trachomatis in Ureaplasma-positive healthy women attending their first prenatal visit in a community hospital in Sapporo, Japan

BackgroundAlthough Chlamydia trachomatis is the most commonly reported pathogen that causes urogenital infection such as urethritis or cervicitis, Ureaplasma parvum and Ureaplasma urealyticum, which are commensals in the genital tract, have also now been recognized as contributors to urogenital infection. However, whether the presence of either U. parvum or U. urealyticum is related to that of C. trachomatis in the urogenital tract remains unknown. We therefore attempted to estimate by PCR the prevalence of C. trachomatis, U. parvum and U. urealyticum in endocervical samples obtained from healthy women attending their first prenatal visit in Sapporo, Japan.MethodsThe samples were taken from 303 apparently healthy women, and the extracted DNAs (n = 280) were used for PCR detection targeting C. trachomatis, U. parvum and U. urealyticum. Statistical analysis of the data was performed by Fishers exact test.ResultsPCR detection revealed that the prevalence of C. trachomatis, U. parvum and U. urealyticum was 14.3% (40/280), 41.7% (117/280) and 8.9% (25/280), respectively. C. trachomatis ompA genotype D was most frequently identified. Surprisingly, either C. trachomatis or Ureaplasma spp. was detected in almost half of the healthy women. Mixed infection of C. trachomatis with either U. parvum or U. urealyticum was also observed in 9.2% (26/280) of the women. There was a significant association between C. trachomatis and either U. parvum (p = 0.023) or Ureaplasma total (p = 0.013), but not U. urealyticum (p = 0.275).ConclusionThis study demonstrated that the presence of Ureaplasma had a significant effect on the presence of C. trachomatis in the genital tract of healthy women, suggesting that mixed infection is an important factor in bacterial pathogenesis in the genital tract.

Prevalence of Helicobacter and Acanthamoeba in natural environment

Aims:u2002 We examined whether the presence of Helicobacter is related to that of Acanthamoeba in river and soil environments.

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.

Ciliates promote the transfer of the gene encoding the extended-spectrum β-lactamase CTX-M-27 between Escherichia coli strains

OBJECTIVESnThe mechanism by which Escherichia coli acquires multidrug resistance genes from other bacteria in the natural environment or livestock is still unclear. The ability of ciliates to promote the transfer of genes encoding extended-spectrum β-lactamases (ESBLs) between the CTX-M-27 donor and clinically isolated recipient E. coli strains was investigated.nnnMETHODSnEqual amounts (∼10(9) cfu) of donor cefotaxime-resistant E. coli and recipient ciprofloxacin-resistant E. coli strains were mixed together in the presence or absence of 10(5) ciliates in Pages amoeba saline for 24 h, in the presence or absence of certain drugs (cytochalasin D, cycloheximide and latrunculin B).nnnRESULTSnGene transfer frequency in the presence of ciliates was estimated at ∼10(-6); in the absence of ciliates it was ∼10(-10). Protein synthesis (cycloheximide) or phagocytosis (cytochalasin D or latrunculin B) inhibitors significantly reduced the frequency of gene transfer.nnnCONCLUSIONSnCiliates promote the transfer of genes encoding ESBLs between E. coli strains, implying that the presence of ciliates may provide a significant impact on emerging multidrug-resistant bacteria.