Light and electron microscopic study of the bacterial adhesion to termite flagellates applying lectin cytochemistry

Summary Many of the flagellates inhabiting the hindgut of lower termites are associated with ectobiotic, rod-like bacteria or spirochetes. Different types of attachment sites are present. Electron dense material underlies, e.g., the plasma membrane ofJoenia annectens at the contact site, whereas other attachment sites do not show any visible specializations. The host cell's glycocalyx may, however, be reduced at the attachment sites as it is the case inDevescovina glabra. The thick glycocalyx ofStephanonympha nelumbium is not changed at the sites where bacterial rods attach, but spirochetes penetrate to a certain extent. Bacteria which colonize the extracellular surface structures ofMicrorhopalodina multinucleata express their own glycocalyx to mediate a contact. In this study we focussed on the examination of one common mode of interaction between bacteria and their host cells, i.e., adhesion via lectins and sugars. The sugar composition was analysed by light and electron microscopic labelling experiments using the lectins Con A, WGA and SBA. In general, only the posterior body surface ofJoenia which is colonized with bacteria is labelled. The demonstrated sugars are found in fibrous glycocalyx portions surrounding the attachment sites of the bacteria. Such glycocalyx fibres in combination with the electron dense material supporting the attachment sites seem to be the prerequisites for bacterial attachment. InD. glabra, however, a role for sugars in mediating the attachment could not be demonstrated. Removal of the ectobiotes using antibiotics revealed that the specialized contact sites ofJoenia are present in the absence of bacteria and thus possibly serve to attract bacteria. Nothing, however, remains of the former attachment sites in bacteria-freeDevescovina cells. Attachment sites in this case could be induced by bacterial contact. There is not one general mechanism for bacterial attachment to termite flagellates; rather, adhesion seems to follow different strategies.Abbreviations Con A concanavalin A - DAB 3,3-diaminobenzidine tetrahydrochloride - DAPI 4,6-diamidino-2-phenylindole - DIC differential interference contrast - FA formaldehyde - FITC fluorescein isothiocyanate - GA glutaraldehyde - PB Soerensen's phosphate buffer - PC phase contrast - pen/strep penicillin and streptomycin - SBA soybean agglutinin - SEM scanning electron microscope - TBS Tris buffer saline - TEM transmission electron microscope - WGA wheat germ agglutinin Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Bacterial adhesion to different termite flagellates: Ultrastructural and functional evidence for distinct molecular attachment modes

Summary The attachment modes of rodlike ectobiotic bacteria to the surface of two different termite flagellates were studied.Devescovina glabra was covered by laterally attached bacteria. Treatment with chemicals that disturb hydrophobic interactions and solubilize proteins removed the ectobionts. Freeze-fracture and freeze-etching electron microscopy revealed rows of intramembrane particles that occurred exclusively along the attachment sites. The adhering Gram-negative bacteria possessed an S-layer (surface layer) composed of globular protein particles. The S-layer could be removed by protein-solubilizing chemicals, e.g., urea, as shown by ultrathin-section electron microscopy. Therefore, it seems plausible that the attachment was mediated by hydrophobic interactions between the flagellate's plasma membrane and the S-layer of the bacteria. The bacteria of the second flagellate,Joenia annectens, adhered by their tips. The attachment was extremely strong. Chemicals disturbing ionic or hydrophobic bindings or solubilizing proteins did not detach the ectobionts. Globular intramembrane protein particles were preferentially found in a ringlike array at the external fracture face of the flagellate's contact sites.Abbreviations DIC differential interference contrast - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - TEM transmission electron microscope - Tween 20 polyoxyethylenesorbitan     

Colonization of termite hindgut walls by oxymonad flagellates and prokaryotes in Incisitermes tabogae,I. marginipennis and Reticulitermes flavipes

We studied the colonization of the paunch wall of three lower termites, Reticulitermes flavipes, Incisitermes tabogae, and Incisitermes marginipennis, by light and electron microscopy. In addition to various prokaryotes, oxymonad flagellates were attached to the wall of the paunch in all three species. The prokaryotic layer found in R. flavipes is relatively thin, since most organisms are attached laterally. Large members of the flagellate genus Pyrsonympha protrude into the gut lumen. The prokaryotes are very abundant on the gut wall in I. tabogae and I. marginipennis, forming a thick carpet of mostly vertically attached rods and wavy spirochetes. The adhering oxymonads are relatively small and almost hidden in the thick bacterial biofilm. Three small morphotypes were seen in I. tabogae; two possessing a short rostellum and one amoeboid. The only oxymonad found in I. tabogae so far, Oxymonas clevelandi, is not identical to any of the present oxymonads. I. marginipennis contains a mid-sized oxymonad with ectobiotic spirochetes, probably identical to Oxymonas hubbardi, and a tiny unknown morphotype. The spatial organization of the pro- and eukaryotic microorganisms on the gut wall of the three termites is described and discussed concerning oxygen stress.     

The hierarchy quorum sensing network in Pseudomonas aeruginosa

Pseudomonas aeruginosa causes severe and persistent infections in immune compromised individuals and cystic fibrosis sufferers. The infection is hard to eradicate as P. aeruginosa has developed strong resistance to most conventional antibiotics. The problem is further compounded by the ability of the pathogen to form biofilm matrix, which provides bacterial cells a protected environment withstanding various stresses including antibiotics. Quorum sensing (QS), a cell density-based intercellular communication system, which plays a key role in regulation of the bacterial virulence and biofilm formation, could be a promising target for developing new strategies against P. aeruginosa infection. The QS network of P. aeruginosa is organized in a multi-layered hierarchy consisting of at least four interconnected signaling mechanisms. Evidence is accumulating that the QS regulatory network not only responds to bacterial population changes but also could react to environmental stress cues. This plasticity should be taken into consideration during exploration and development of anti-QS therapeutics.     

A Process of Reproduction of Trypanosoma conorhini Different from Binary or Multiple Fission

SYNOPSIS. In cultures of Trypanosoma conorhini the authors have detected "cyst-like bodies" (CLBs) that, in the initial phase, have the appearance of 2 or more overgrown crithidiae fused together; after disorganization of the internal structure of the parent flagellates and division of their organelles, new crithidiae are formed within the CLB. The wall of the CLB consists of an irregularly thick layer of cytoplasm surrounded by the periplast of the parent flagellates and a true cystic membrane is not evident. The daughter crithidiae eventually escape from the CLB leaving the periplast and other residues of the parent flagellates. It is suggested that this represents a method of reproduction entirely different from binary or multiple fission, that is probably common to other trypanosomes of the Section STERCORARIA, and that it may provide for genetic exchange even if it is not a true sexual process.     

Development of quasi-multicellular bodies of Treponema denticola

The formation of quasi-multicellular bodies of Treponema denticola was analysed using different electron microscopical methods. These bacteria could develop four different conformations: (i) normal helical forms; (ii) twisted spirochetes, forming plaits; (iii) twisted spirochetes, forming club-like structures; (iv) spherical bodies in different size. Treponemes within spherical bodies, plaits, and clubs proved to be enclosed in a common outer sheath in which the normal arrangement of their axial flagella was lost. The development of the quasi-multicellular bodies starting from the monoforme spirochetes was elucidated and this morphogenetic process is illustrated by a schematic drawing. Factors which might be involved in the induction of the structures are discussed and their possible pathogenetic importance is considered.     

Primitive mitotic mechanisms.

Unorthodox mitotic mechanisms are reviewed and their contribution to the understanding of evolution of the orthodox mitotic apparatus is considered. Dinoflagellates and hypermastigote flagellates are of particular significance because the microtubular mitotic apparatus is entirely extranuclear with the nuclear membrane persisting through mitosis. Chromosomes are attached to the nuclear membrane. In hypermastigole flagellates early kinetochore separation is on the nuclear membrane without any contribution from microtubules. In dinoflagellates the chromosomes are also attached to the nuclear membrane, but at least in some species cytoplasmic microtubules connect to the attachment site. In Syndinium the attachment site resembles a typical kinetochore, but is inserted in the nuclear membrane. A similar kinetochore is found in certain Radiolaria, but with an intranuclear spindle apparatus the association with the nuclear membrane is no longer necessary and has been lost. Mitosis in the yeast Saccharomyces is essentially orthodox, though chromosomes do not condense. No kinetochores are seen, but a single microtubule makes direct contact with the 20 nm chromatin fiber of each chromosome and shortens during anaphase. About 5-10 microtubules are continuous between the spindle pole bodies and form the elongating central spindle.     

Possible formation and development of spirochaete attachment sites found on the surface of symbiotic polymastigote flagellates of the termite Reticulitermes flavipes.

We propose that spirochaete attachment sites arise from peripheral protrusions which appear on the surface of polymastigote flagellates. These protrusions develop into bracket-shaped structures which then form the mature attachment site. Next the site becomes detached from the surface of the cell; this latter process may be facilitated by the fusion of vesicles located in the region immediately beneath the spirochaete attachment site. This theory could explain the variability in the number and distribution of attachment sites on the surface of the flagellates.     

土壤原生动物对川滇高山栎恢复时间的响应及生长季动态

青藏高原东缘生态环境脆弱, 森林频繁遭到砍伐, 生物多样性受到严重威胁, 森林砍伐后的生态恢复成为研究热点。原生动物在生态恢复中作为指示生物起着重要作用。本文就未砍伐、砍伐后不同恢复时期(20年、10年和1年)生的川滇高山栎(Quercus aquifolioides)林的土壤理化性质和原生动物在生长季的变化进行比较研究, 以探讨生长季不同月份、不同恢复期的原生动物数量变化规律, 分析土壤理化性质与其相关性。结果表明: (1)恢复10年和1年的林地的鞭毛虫数量(193个/g干土, 164个/g干土)显著高于原始林地(22个/g干土), 肉足虫在恢复1年的林地中数量最多(600个/g干土), 纤毛虫数量则随次生演替进程逐渐增多。(2)在生长季不同月份原生动物的数量呈先增加后减少的趋势。鞭毛虫和肉足虫的峰值分别出现在7月和8月, 而纤毛虫的数量在7、8、9月明显高于6月。(3)原生动物数量与土壤理化性质密切相关。鞭毛虫数量与pH值呈显著正相关(P = 0.019), 纤毛虫数量与铵态氮(P = 0.002)和碳氮比呈显著正相关(P = 0.022), 肉足虫数量与硝态氮(P = 0.008)和碳氮比(P = 0.016)呈显著负相关。结果显示, 三种原生动物数量在生长季不同月份表现出较大的波动性, 其数量变化受土壤理化性质等多种因素调控。纤毛虫数量对川滇高山栎林砍伐恢复有正响应, 而鞭毛虫、肉足虫数量有负响应。     

On the trophic fate of Phaeocystis pouchetii (Hariot). II. Grazing rates of Calanus hyperboreus (Krøyer) on diatoms and different size categories of Phaeocystis pouchetii

Experiments were conducted with CIV and C V copepodites of Calanus hyperboreus (Krøyer) to determine if they would feed on the prymnesiophyte Phaeocystis pouchetii (Hariot). We used analysis of gut pigment to estimate ingestion and clearance rates. In applying this methodology we have demonstrated that pigments can be completely extracted from whole animals within 90 min, and that laborious procedures of tissue homogenization and centrifugation are not required. We conducted two experiments. In the first experiment Stage IV copepodites were exposed to ≈1 mg C·1⁻¹ of either P. pouchetii flagellates, small colonies (25–200 μm), large colonies (> 200 μm) or mixed diatoms > 25 μm (primarily Chaetoceros socialis Lauder and Nitzschia grunowii Hasle). Ingestion rates and daily rations were almost four times greater on both sizes of colonies than on either Phaeocystis pouchetii flagellates or mixed diatoms. Daily rations of copepodites feeding on colonies ranged from 8.1 to 12.4% · day⁻¹, well within the range previously reported for Calanus hyperboreus or sympatric copepods of similar size. From the second experiment we determined that Stage V copepodites obtained a daily ration of 6.2 to 10.8% · day⁻¹ when feeding on small colonies of Phaeocystis pouchetii. We conclude that a diet of P. pouchetii colonies should sustain the metabolic and growth requirements of Calanus hyperboreus copepodites.     

Studies on the rumen flagellate Sphaeromonas communis.

The rumen flagellate Sphaeromonas communis showed a significant increase in population density 1 to 2 h after the host sheep commenced feeding, followed by a reduction in numbers to the pre-feeding level after a further 2 to 3 h. The life-history of the organism was shown to consist of a motile flagellate which germinated to produce a vegetative stage comprising a limited rhizoidal system on which up to three reproductive bodies were borne together with (in vitro) other spherical bodies of unknown function; in vivo, the reproductive bodies were stimulated to liberate flagellates by a component of the diet of the host. The vegetative stage strongly resembled that of certain species of aquatic phycomycete fungi, and the flagellates may therefore by zoospores. Flagellates liberated in vivo lost their motility within 2 to 3 h and developed into the reproductive vegetative phase, producing a rapid decrease in numbers of flagellates. Conditions of maximum flagellate production (pH 6.5, 39 degrees C, presence of CO2, absnece of oxygen) approximated to those found in the rumen. The organism was cultured in vitro in an undefined medium in the absnece of bacteria and other flagellates.     

Surface morphology of Saccinobaculus (Oxymonadida): implications for character evolution and function in oxymonads

Examination of surface morphology of the oxymonad genus Saccinobaculus from the gut of the wood-feeding cockroach Cryptocercus punctulatus with scanning and transmission electron microscopy reveals several new characters not observable with light microscopy. These include small concavities covering the external surface, a glycocalyx, coated pinocytotic vesicles, and, in one species, unidentified, membrane-bounded organelles with a granular matrix that may represent peroxisomal or mitochondrial derivatives. Unlike representatives of some other oxymonad families, Saccinobaculus lacks extracellular surface structures, a holdfast, and, generally, ectobiotic bacteria. We examined the evolution of these and other characters in light of previously published phylogenies of oxymonads based on molecular data. The presence of concavities in Saccinobaculus and families Pyrsonymphidae and Oxymonadidae strengthens support for a clade comprising these three families. A glycocalyx appears to be a synapomorphy of all oxymonads, and the presence of ectobiotic bacteria also appears to be ancestral to oxymonads, but lost in Saccinobaculus. A holdfast appears to have arisen multiple times. We hypothesize that concavities may play a role in a two-step mechanism for the accumulation and internalization of specific solutes, and that the highly motile and morphologically plastic nature of Saccinobaculus cells limits the possibility of retaining a covering of ectobiotic bacteria.     

Phylogenetic position of Cryothecomonas inferred from nuclear-encoded small subunit ribosomal RNA

The systematic position of the genus Cryothecomonas has been determined from an analysis of the nuclear-encoded small subunit ribosomal RNA gene of Cryothecomonas longipes and two strains of Cryothecomonas aestivalis. Our phylogenetic trees inferred from maximum likelihood, distance and maximum parsimony methods robustly show that the genus Cryothecomonas clusters within the phylum Cercozoa, and is related to the sarcomonad flagellate Heteromita globosa. Morphological data supporting the taxonomic placement of Cryothecomonas near the sarcomonad flagellates has been compiled from the literature. The high number of nucleotide substitutions found between two morphologically indistinguishable strains of Cryothecomonas aestivalis suggests the possibility of cryptic species within Cryothecomonas aestivalis.     

Organization of the flagellar apparatus and associate cytoplasmic microtubules in the quadriflagellate alga Polytomella agilis

The organization of microtubular systems in the quadriflagellate unicell Polytomella agilis has been reconstructed by electron microscopy of serial sections, and the overall arrangement confirmed by immunofluorescent staining using antiserum directed against chick brain tubulin. The basal bodies of the four flagella are shown to be linked in two pairs of short fibers. Light microscopy of swimming cells indicates that the flagella beat in two synchronous pairs, with each pair exhibiting a breast-stroke-like motion. Two structurally distinct flagellar rootlets, one consisting of four microtubules in a 3 over 1 pattern and the other of a striated fiber over two microtubules, terminate between adjacent basal bodies. These rootlets diverge from the basal body region and extend toward the cell posterior, passing just beneath the plasma membrane. Near the anterior part of the cell, all eight rootlets serve as attachment sites for large numbers of cytoplasmic microtubules which occur in a single row around the circumference of the cell and closely parallel the cell shape. It is suggested that the flagellar rootless may function in controlling the patterning and the direction of cytoplasmic microtubule assembly. The occurrence of similar rootlet structures in other flagellates is briefly reviewed.     

Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists

Some species of protists inhabiting the hindgut of lower-termites have a large number of ectosymbiotic spirochetes on the cell surface. The phylogenetic positions of the ectosymbiotic spirochetes of three oxymonad protists, Dinenympha porteri in the gut of Reticulitermes speratus, and Pyrsonympha sp. and Dinenympha sp. in Hodotermopsis sjoestedti, were investigated without cultivation of these organisms. Protist fractions carefully collected with a micromanipulator were used as templates for the amplification of small subunit ribosomal RNA genes (SSU rDNA). The phylogenetic tree inferred from the nucleotide sequences of the SSU rDNA showed that they were affiliated with the Treponema cluster of spirochetes and they were divided into two clusters. One was grouped together with the spirochetal sequences reported previously from the gut of termites and the other was related to the Treponema bryantii subgroup of treponemes (denoted as termite Treponema clusters I and II, respectively). Whole-cell in situ hybridization using a fluorescent-labeled oligonucleotide probe specific for the group of sequences in cluster II identified most of the ectosymbiotic spirochetes of the oxymonad protists in the gut of R. speratus and H. sjoestedti. However, not all of the ectosymbiotic spirochetes could be detected by means of this cluster II group-specific probe and the population of ectosymbiotic spirochetes of cluster II was different among the oxymonad species. In the case of D. porteri, an oligonucleotide probe specific for one member of cluster II recognized a portion of the ectosymbiotic spirochetes of cluster II, and their population was also different depending on the cell-type of D. porteri in terms of the attachment of ectosymbiotic spirochetes. The results indicate that the spirochetes of cluster II and probably those of a part of cluster I can be assigned to ectosymbiotic species of oxymonad protists and that the population of ectosymbiotic spirochetes associated with a single protist consists of at least three species of phylogenetically distinct spirochetes.     

Sulfatide mediates attachment of Pseudomonas aeruginosa to human pharyngeal epithelial cells

Pseudomonas aeruginosa infections are particularly common in people with cystic fibrosis and despite regular treatment with antibiotics, lung damage due to chronic infection with P. aeruginosa remains the major cause of death in those patients. In order to initiate an infection, P. aeruginosa needs contact with the respiratory epithelial surface and by means of its adhesins i.e., fimbria, hemagglutinins,etc., it recognizes and adheres to the corresponding epithelial receptors. We treated P. aeruginosa strains isolated from sputum of cystic fibrosis patients with several glycolipids such as sulfatide, sulfated ganglioside mixture (GM1a, GD1b, GT1b), asialo-GM1 and galactocerebrosides to determine their effect on attachment with pharyngeal epithelial cells. Sulfated ganglioside mixture and sulfatide inhibited the attachment of P. aeruginosa significantly, whereas asialo-GM1, Gal-Cer and sodium sulfite had no effect on attachment inhibition. This finding suggests that sulfated glycoconjugates found in the extracellular matrix, in mucus and on the surface of epithelial cells of human trachea and lung mediates attachment of P. aeruginosa.     

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine

In this work the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine (HCQ) was studied. The minimal bactericidal concentration (MBC) of HCQ against the mobile spirochetes was >32 μg/ml at 37 °C, and >128 μg/ml at 30 °C. Incubation with HCQ significantly reduced the conversion of mobile spirochetes to cystic forms. When incubated at 37 °C, the MBC for young biologically active cysts (1-day old) was >8 μg/ml, but it was >32 μg/ml for old cysts (1-week old). Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that the contents of the cysts were partly degraded when the concentration of HCQ was ≥MBC. At high concentrations of HCQ (256 μg/ml) about 95% of the cysts were ruptured. When the concentration of HCQ was ≥MBC, core structures did not develop inside the cysts, and the amount of RNA in these cysts decreased significantly. Spirochetal structures inside the cysts dissolved in the presence of high concentrations of HCQ. When the concentration of HCQ was ≥MBC, the core structures inside the cysts were eliminated. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of HCQ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi. Electronic Publication     

Spirochete round bodies Syphilis, Lyme disease & AIDS: Resurgence of “the great imitator”?

We advocate investigation of spirochete cyclical symbioses (e.g.,Borrelia sp.,Leptospira sp., Treponema sp.) given the newly established verification of a developmental history in these gram-negative motile helical eubacteria, both in pure culture and in mammals. Symbiotic spirochetes can be compared to free-living relatives for their levels of integration (behavioral, metabolic, gene product or genetic levels), Detailed research that correlates life histories of symbiotic spirochetes to changes in the immune system of associated vertebrates is sorely needed. Genome analyses show that in necrotrophic symbioses (Borrelia andTreponema sp.) of humans and other primates, integration of the bionts occurs at the gene product and genetic level. Spirochete round bodies (also called cysts, L-forms and sphaeroplasts) can be induced by many types of unfavorable conditions (e.g., threats of starvation, desiccation, oxidation, penicillin and other antibiotics). Reversion to familiar helical, motile active swimmers by placement of pure cultures into favorable environments in some cases can be controlled. These observations are supported by a European literature, especially Russian, apparently unknown to American medicine and medical research.     

HPLC测定灵芝子实体水提物及相关产品中三萜的含量

灵芝药品大多以灵芝子实体水提物为原料,为快速准确测定灵芝子实体水提物及相关产品中三萜的含量,建立了具有较好分离效果的HPLC分析测定方法。通过优化色谱柱和洗脱条件,优选出Agilent Zorbax SB-Aq C18色谱柱(250 mm×4.6 mm,5μm),以乙腈-乙酸水溶液(0.01%)为流动相梯度洗脱,流速为1.0 mL/min,检测波长252 nm,柱温30℃,该条件下灵芝酸A、灵芝酸F等10种灵芝酸得到较好的分离。方法学考察显示该分析方法精密度、重复性、稳定性和加样回收率的RSD值均小于5%,可以用于灵芝酸C2、灵芝酸G、灵芝烯酸B、灵芝酸B等10种灵芝酸的定量检测。通过对灵芝子实体原料、水提物和市售灵芝产品中10种三萜类成分分析发现,灵芝子实体水提物中均含有这10种三萜,含量为2.52%–6.83%,较子实体原料大幅提高,市售的灵芝产品中的三萜含量为0.27%–0.84%。该方法的建立为灵芝水提物及其产品质量标准的建立奠定基础。