The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans enlarges by distal growth and is rearranged during budding

The capsule of Cryptococcus neoformans can undergo dramatic enlargement, a phenomenon associated with virulence. A prior study that used Ab to the capsule as a marker for older capsular material concluded that capsule growth involved the intermixing of new and old capsular material with displacement of older capsular polysaccharide towards the surface. Here we have revisited that question using complement (C), which binds to capsular polysaccharide covalently, and cannot redistribute by dissociation and binding at different sites. The experimental approach involved binding of C to cells with small capsules, inducing capsule growth, and following the location of C relative to the cell wall as the capsule enlarged. C remained close to the cell wall during capsule growth, indicating that capsule enlargement occurred by addition of new polysaccharide near the capsule edge. This conclusion was confirmed by an independent method that employed radioactive metabolic labelling of newly synthesized capsule with 3H-mannose followed by gradual capsular stripping with gamma-radiation. Capsule growth proceeded to a certain size, which was a function of cell size, and was not degraded when the cells were transferred to a non-inducing medium. During budding, an opening appeared in the capsule of the mother cell that permitted the nascent bud to separate. Scanning EM suggested that a physical separation formed between the capsules of the mother and daughter cells during budding, which may avoid mixture between both capsules. Our results indicate that C. neoformans capsular enlargement also occurs by apical growth and that budding results in capsular rearrangements.     

A physico-chemical investigation of the jelly capsules surrounding eggs of the Common frog (Rana temporaria temporaria)

The mucopolysaccharide capsules surrounding eggs of the Common frog, Rana temporaria temporaria L. were investigated. Frogs were obtained from ponds in northern England ranging in altitude from 46 to 838 m. Egg capsules acted as insulators, keeping the centre of an egg mass warmer on average than the surrounding water. The size of an egg capsule and its insulating efficiency varied in different pond waters. Egg capsules in water from highland ponds were larger and more efficient insulators than egg capsules in water from lowland ponds. In a series of laboratory experiments the factors controlling capsular swelling were investigated). The ionic concentration of the fluid surrounding the eggs was found to be the principal factor involved, but the temperature and pH of the fluid and the valency and identity of the ions in solution were also important. The water chemistry of ponds varied with altitude. This accounted for the differential swelling of egg jelly in highland and lowland ponds. The fact that egg capsules are larger in highland pond water may increase egg survival at low temperatures. This is probably fortuitous as there is no evidence to suggest that frogs select their breeding ponds because the water in them will promote capsular swelling.     

Nodulation of soybean byRhizobium japonicum mutants with altered capsule synthesis

Spontaneous mutants with altered capsule synthesis were isolated from a marked strain of the symbiont,Rhizobium japonicum. Differential centrifugation was used to enrich serially for mutants incapable of forming capsules. The desired mutants were detected by altered colony morphology and altered ability to bind host plant lectin. Three mutants failed to form detectable capsules at any growth phase when cultured in vitro or in association with the host (soybean,Glycine max (L.) Merr.) roots. These mutants were all capable of nodulating and attaching to soybean roots, indicating that the presence of a capsule physically surrounding the bacterium is not required for attachment or for infection and nodulation. Nodulation by several of the mutants was linearly proportional to the amount of acidic exopolysaccharide that they released into the culture medium during the exponential growth phase, indicating that such polysaccharide synthesis is important and perhaps required for nodulation. Two of the mutants appeared to synthesize normal lectin-binding capsules when cultured in association with host roots, but not when cultured in vitro. Nodulation by these mutants appeared to depend on how rapidly after inoculation they synthesized capsular polysaccharide.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - FITC fluorescein isothiocyanate Contribution No. 719 of the C.F. Kettering Research Laboratory     

The surface hydrophobicity and avirulent character of an encapsulated strain of Klebsiella pneumoniae.

In order to elucidate how virulence is controlled in encapsulated bacteria, some surface properties of an encapsulated but avirulent strain of Klebsiella pneumoniae, strain 277, were examined. Although strain 277 was heavily fimbriated, the fimbriae did not demonstrate an avirulent character and were not responsible for the surface hydrophobicity of this strain. The surface hydrophobicity was well correlated with the capacity of the bacteria to associate with polymorphonuclear cells. More bacteria with hydrophobic surfaces associated with the PMN than nonhydrophobic bacteria. The hydrophobic surface character of this strain was not affected by either trypsin treatment or extraction with salt solution. We assume that the capsule of strain 277 has more hydrophobic polysaccharides than that of the virulent strain. Some chemical modifications might therefore exist in the capsular polysaccharides of the avirulent strain.     

Comparison of the capsular response to the Biocell RTV and Mentor 1600 Siltex breast implant surface texturing: a scanning electron microscopic study.

The utility of mammary prosthesis texturing in the prevention of capsular contracture was established some 20 years ago. Various models of implant texturing are currently on the market. We decided to study two of the most popular implants with two different surface texturings: the Biocell RTV and the Mentor 1600 Siltex. An observation at the electron microscopic level of the implants' surfaces was achieved. At the time of a prospective survey on 10 patients, the capsule fragments corresponding to these two prostheses have been analyzed at the electron microscopic level. All prostheses were removed from the patients because of asymmetry or bad positioning. The aim of our study was to establish a correlation between these two frequent texturing surfaces and their corresponding capsules. Our results showed that only the Biocell's capsules present a mirror image with correspondence of the depressions on the prosthesis and contacts on the capsule. This phenomenon seems linked to the existence of a critical size of the pores constituting the implant surface. This observation leads us to the hypothesis of an adhesive effect between the prosthesis and its capsule. If this last is not directly linked to the prevention of capsular contracture, it can have an effect on implant stabilization in the primary mammary reconstruction and in the secondary corrections of asymmetry or bad position.     

Encapsulation of R. planticola Rs-2 from alginate-starch-bentonite and its controlled release and swelling behavior under simulated soil conditions

The plant growth-promoting bacteria (PGPR) Raoultella planticola Rs-2 was encapsulated with the various blends of alginate, starch, and bentonite for development of controlled-release formulations. The stability and release characteristics of these different capsule formulations were evaluated. The entrapment efficiency of Rs-2 in the beads (capsules) was more than 99%. The diameter of dry beads ranged from 0.98 to 1.41 mm. The bacteria release efficiency, swelling ratio, and biodegradability of the different bead formulations were enhanced by increasing the starch or alginate contents, but were impeded by higher bentonite content. The release kinetics of viable cells from capsules and the swelling ratio of capsules were studied in simulated soil media of varying temperature, moisture, pH, and salt content. The release of loaded Rs-2 cells and swelling of capsules are greatly affected by moisture, temperature, pH and salt content of the release medium. The release of viable Rs-2 cells from capsules was positively associated with the swelling properties of the capsules. The release of Rs-2 cells occurred through a Case II diffusion mechanism. In summary, this work indicates that alginate-starch-bentonite blends are a viable option for the development of efficient controlled-release formulations of Rs-2 biofertilizer, and which could have a promising application in natural field conditions.     

Morphology of the viable but nonculturable Vibrio cholerae as determined bt the freeze fixation technique

Abstract The morphology of the nonculturable Vibrio cholerae strain TSI-4 was examined by the freeze fixation technique of electron microscopy and subsequently four unique structures were found in the fine structure s of this bacterium. The size of the cell was about 2 3 of the growing cell. Although the cell was observed to have an outer membrane as well as the cell membrane and cytoplasm, the outer membrane was undulated and had a surface layer of fine fibers. The peptidoglycan layer was thick and more electron dense than that of normal cells.     

Adaptations to Physical Stresses in the Intertidal Zone: The Egg Capsules of Neogastropod Molluscs

The encapsulation of eggs within benthic egg capsules or gelatinousegg masses is a common phenomenon among many marine invertebrategroups, yet the functional significance of many aspects of theseegg coverings remains unexplored. In this paper I review whatis known about the effectiveness of neogastropod egg capsulesin protecting embryos from physical stresses associated withthe marine intertidal environment. Egg capsules spawned by intertidalneogastropod molluscs can provide embryos with significant protectionfrom desiccation, osmotic stress, and ultraviolet (UV) radiation,relative to embryos devoid of such coverings. Despite this,capsules desiccate rapidly in air, are highly permeable to smallsolute molecules, and are not impervious to incident UV radiation.Egg capsules of intertidal gastropods are also substantiallymore permeable to water molecules than the well studied eggcases and egg shells of insects and terrestrial vertebratesand may be no more effective in protecting embryos from suchphysical stresses than the capsules of exclusively subtidalgastropods. Hence, capsular cases appear to be poorly adaptedto protecting embryos from environmental stresses associatedwith periodic exposure to air. The degree to which the encapsulatedembryos of intertidal neogastropods are protected from environmentalstresses thus may be more reflective of adult spawning siteselection and tolerances of encapsulated embryos to these stresses,than properties of the capsular case, per se. Clearly, however,there is much still to be learned about the protective natureof capsule walls and the tremendous diversity of egg coveringsthat exists within the Gastropoda.     

Conversion of biological and immunological properties during a process of decapsulation in a strain of Staphylococcus hyicus

E. YOSHIDA, Y. ICHIMAN, M. SUGANUMA AND K. YOSHIDA. 1991. Repeated subculture at 42°C of Staphylococcus hyicus strain ST67P, which exhibits streaming-type growth in a soft-agar medium, yielded three variants, ST67L, ST67S and ST67C, which had different colonial morphologies; small compact colonies possessing long and short tails and perfect compact colonies. The parent strain and ST67L respectively gave strong and weak positive intensity when stained by rabbit antisera prepared by capsular type I and II strains of Staph. epidermidis conjugated with fluorescein isothiocyanate. Variant ST67L gave a positive result with antiserum prepared by capsular type I strain and no staining was observed with variants ST67S and ST67C against these antisera preparations. Strain ST67C had the lowest virulence although no remarkable difference was shown between the parent strain and variants ST67L and ST67S. The cell volume index of the parent strain was 1.35, 2.43 and 3.71 times larger than those of ST67L, ST67S and ST67C, respectively. The converting activity of rabbit anti-ST67P strain serum absorbed by strain ST67C required four times more of the organisms than strain ST67P, changing the colonial morphology of the strain from diffuse to compact type by the addition of antiserum to soft agar medium. Positive coagulase and false positive clumping factor reaction were shown in variants ST67C, but no remarkable alteration was observed with 19 biochemical properties determined by a conventional identification kit. In ulta-thin sections of the parent strain labelled with rabbit anti-strain ST67P serum conjugated with ferritin, large capsules surrounded by ferritin granules were demonstrated by electron microscopy. In variants ST67L and ST67S, but not ST67C medium size capsules surrounded by ferritin granules and only ferritin granules located around the cell wall, respectively, were observed.     

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: I. Production of the Adjuvant Polysaccharide by Noncapsulated Mutant

In culture fluid, Klebsiella pneumoniae type 1 Kasuya strain produces polysaccharide exhibiting a strong adjuvant effect. The active substance responsible for the strong adjuvant effect of the polysaccharide is not its acidic polysaccharide fraction (the type-specific capsular antigen) but the neutral polysaccharide fraction. In the present study, a mutant which did not produce the type-specific capsular polysaccharide was isolated from ultraviolet-irradiated cells of K. pneumoniae type 1 Kasuya strain which had been labeled with leucine-requiring marker by selecting unagglutinable cells with the antiserum to the type-specific capsular polysaccharide. Serological tests showed that the type-specific acidic capsular polysaccharide was present neither on the cells surface nor in the culture fluid of the mutant. Electron microscopically, the mutant did not possess any capsular material. On the other hand, nearly an equal amount of neutral polysaccharide antigen was produced in culture fluids of the noncapsulated mutant and the parent strain. The neutral polysaccharide antigen produced by the noncapsulated mutant exhibited the same degree of strong adjuvant effect on antibody response to bovine gammaglobulin in mice as that produced by the parent strain. The relationship between the neutral polysaccharide antigen in culture fluid and the O antigen of K. pneumoniae was discussed.     

Escherichia coli Capsule Bacteriophages II. Morphology

The Escherichia coli capsule bacteriophages (K phages) described herein are specific for certain capsular strains of E. coli, all of them test strains for different E. coli K antigens. The phages are not adsorbed to the acapsular mutants of their host organisms nor to similar strains with serologically and chemically different capsular polysaccharides. Thirteen E. coli (and one Klebsiella) K phages were visualized in the electron microscope. Most viruses are similar to P22 and thus belong to Bradley group C; however, one each of group A (long, contractile tail) and group B (long, noncontractile tail) was also found. All K phages were seen to carry spikes but no tail fibers were detected. These results suggest that the structures responsible for the recognition of the thick (about 400 nm or more) capsular polysaccharide gels are located in these spikes.     

Scanning electron microscopy of the renal corpuscle of the mesonephros in the lamprey,entosphenus japonicus martens

Summary The renal corpuscle of the lamprey mesonephros was studied under the scanning electron microscope.Bowman's capsules with individual spaces are chockshaped sacs closely packed together along a medial artery. The lateral walls of the capsules are apposed to those of neighbouring capsules.Glomerular capillaries from the medial artery extend radially between the apposed walls of neighbouring Bowman's capsules. Bulgings of capillaries into the capsular space are associated with mesangial folds of the capsular epithelium.The transitional zone of the visceral layer with podocytes and the parietal layer of squamous epithelium is bounded by linearly arranged rod-shaped epithelial cells. Apertures of the urinary tubule are lined by cells equipped with a fascicle of cilia.     

The stroma of the thymus of the rat: morphology and antigen diffusion, a reconsideration

This work reconsiders aspects of the morphology of the capsule, of the blood vasculature, of the distribution of reticular fibers, and of the diffusion of intramediastinally injected antigens in the stroma of the thymus of the rat. This was done by an analysis of standard sections of normal thymuses, of sections of thymuses perfused with colloidal carbon, of silver-impregnated sections, and of sections of thymuses of rats injected intramediastinally with a fluorescent antigen or intravenously with Trypan blue, and by electron microscopy of the thymic capsule. The capsule consisted of two layers: an outer layer covering the entire periphery of a thymic lobe, and an inner layer which outlined the entire convoluted peripheral cortex of a lobe. Cortical vessels entered the capsule and septa in which they formed a capillary network. These capsular capillaries were fenestrated and leukocytes were often present near them. Adipocytes were also seen near these vessels in some areas of the capsule, and often at the bases of septa and trabeculae. Furthermore, much of the medulla had a dense network of coarse reticular fibers, whereas the remainder of the medulla and the cortex contained a loose network of fine fibers stretching out from the capsule, septa, and trabeculae. Intramediastinally injected fluorescent antigens were observed to spread in the capsule and septa and to diffuse in the fiber networks stretched across the cortex and the medulla. Fluorescence also highlighted cortical reticular cells but not the thymocytes. Intravenously injected Trypan blue stained the capsule, the septa, the cortical reticular cells, and the autofluorescent cells outlining the corticomedullary junction of each lobule. The unusual penetration of capillaries from the thymic parenchyma into the thymic capsule suggested that the capsular capillaries participate in peculiar thymic events, such as the recruitment of blood stem-cells. It is concluded that small amounts of blood antigens normally exude from capsular capillaries and diffuse into the fibers extending from the capsule across the cortex. The phenomenon would be increased under conditions causing thymic involution. An explanation is proposed to account for the development of involution which involves the exudation of antigens from the capsular capillaries. A comparable mechanism could also account for the development of a particular experimental immune tolerance.     

Evidence for a Multiplicity of Capsular Types Among Staphylococcus aureus Strains

The Smith diffuse variant and the wound mucoid strain of Staphylococcus aureus were shown to exhibit serologically distinct capsules. The Welwood and K-6 strains of S. aureus were tested to determine their capsular types. Both Welwood and K-6 were found to be representative of the Smith capsular type. An additional 13 isolates of S. aureus from mice were tested. Gel double-diffusion tests and immunoelectrophoresis of staphylococcal antigens disclosed the possible existence of at least two additional capsular types. Passive hemagglutination tests carried out with cells sensitized with 1 mg of antigen per ml showed a multiplicity of cross-reacting antigens. However, cells sensitized either with 0.1 or 0.05 mg of antigen per ml and reacted with antisera absorbed with 10 or 1 mug/ml showed the presence of a specific antigen in each strain of S. aureus. Corroborative evidence for a multiplicity of capsular types was obtained by the specific capsular reaction. At least four capsular types of S. aureus were found. The prototypic strains for these antigens are the RLM or wound strain, the Smith diffuse strain, and mouse strains designated 36T and 43R. We propose to designate these types 1, 2, 3, and 4, respectively.     

Embryogenesis of an embryonic lethal, ‘kidney-shaped egg’ in Bombyx mori,with special reference to mesoderm differentiation

Summary

Within the kidney-shaped egg laid by the homozygous female, the embryo develops only ectodermal organs, but no mesodermal organs. The early embryogenesis of this strain was examined with light and electron microscopy. Until the early germband stage no remarkable differences are distinguishable between the normal and the kidney-shaped eggs. Afterwards, in the kidney-shaped egg large amount of cytoplasmic masses and often several cells are extruded from the presumptive head region into the yolk at the ‘pyriform’ stage, resulting in inhibiting the formation of a normal wide head lobe and an invagination occurring in its central region, the first sign of the mesoderm differentiation. This phenomenon proceeds gradually toward the posterior part and thus the differentiation of mesoderm becomes impossible throughout the entire length of germband. The results are discussed in the context of the mesoderm differentiation in Bombyx.     

ACCLIMATION TO LOW TEMPERATURE OF TWO ARTHROSPIRA PLATENSIS (CYANOBACTERIA) STRAINS INVOLVES DOWN‐REGULATION OF PSII AND IMPROVED RESISTANCE TO PHOTOINHIBITION1

This study aimed to compare the ability of two Arthrospira platensis (Nordst.) Gomont strains, M2 and Kenya, isolated from two different habitats, to acclimate to low temperature (15°C). Both strains had similar growth rates at 30°C, but once acclimated to low temperature, M2 showed a greater decline in growth (59% vs. 41% in the Kenya strain). We suggest that the Kenya strain acclimated better to low temperature by down‐regulating its photosynthetic activity through (i) decreasing antenna size and thus reducing energy flux into the photosystems; (ii) decreasing reaction center density (RC/CS_X) and the performance index, thus decreasing the trapping probability and electron transport rate while maintaining electron transport probability for electron transport beyond Q_A^? unchanged; (iii) increasing the energy dissipation flux. In contrast, the M2 strain showed no difference in antenna size and exhibited a much lower decrease in RC/CS_X and a lower dissipation rate. Hence, the Kenya strain minimized potential damage on the acceptor side of PSII compared to the M2 cells. Furthermore, acclimation to low temperature was accompanied by an improved mechanism for handling excess energy resulting in an enhanced ability of the Kenya strain to rapidly repair damaged PSII RCs and withstand a high photon flux density (HPFD) stress; this finding might be defined as a cross‐adaptation phenomenon. This study may provide a tool to identify strains suitable for outdoor mass‐production in different regions characterized by different climate conditions.     

牛源流行性出血病病毒(EHDV)血清10型毒株在我国的分离鉴定

我国存在多种血清型流行性出血热病毒(Epizootic haemorrhagic disease virus,EHDV)的流行,但尚未有关于EHDV-10型毒株的分离报道。为了解云南省EHDV的流行情况,2012～2015年,本研究在云南省设立江城、师宗、芒市三个监控点,定期采集监控动物血液,接种幼仓鼠肾细胞(Baby hamster kidney cell,BHK-21)进行病毒分离;通过PCR检测、血清中和试验、琼脂糖凝胶电泳和电镜观察等方法对分离病毒进行鉴定;对分离毒株的Seg-2/VP2与Seg-3/VP3基因节段进行克隆、测序与序列分析。2013年在云南省师宗县的哨兵牛上分离出一株EHDV毒株(YNSZ-V277-2013),病毒可引起BHK-21细胞出现圆缩、裂解的细胞病变(Cytopathic effect,CPE);电镜下病毒粒子呈球形,无囊膜,表面有大量纤维突,直径在70～80nm之间;病毒基因组dsRNA的琼脂糖凝胶电泳显示分离毒株与其他血清型EHDV一致,呈现"3-3-3"的电泳带型;序列分析显示YNSZ-V277-2013毒株的Seg-2/VP2与Seg-3/VP3序列与日本EHDV-10型毒株(ON-4/N/98)相似度最高,分别为97.5%/98.5%与98.1%/99.8%,证实分离毒株为EHDV-10型;系统发育分析显示YNSZ-V277-2013毒株的Seg-2与日本EHDV-10型毒株(ON-4/N/98)的亲缘关系最近,Seg-3与分离至日本和澳大利亚的EHDV毒株同属Eastern型。本研究首次报道了EHDV-10型毒株在我国的分离以及分离毒株的Seg-2与Seg-3基因序列特征,为进一步开展中国EHDV-10型的流行病学调查与致病性研究提供了基础。     

Cryptococcus neoformans responds to mannitol by increasing capsule size in vitro and in vivo

The polysaccharide capsule of the fungus Cryptococcus neoformans is its main virulence factor. In this study, we determined the effects of mannitol and glucose on the capsule and exopolysaccharide production. Growth in mannitol significantly increased capsular volume compared with cultivation in glucose. However, cells grown in glucose concentrations higher than 62.5 mM produced more exopolysaccharide than cells grown in mannitol. The fibre lengths and glycosyl composition of capsular polysaccharide from yeast grown in mannitol was structurally different from that of yeast grown in glucose. Furthermore, mannitol treatment of mice infected intratracheally with C. neoformans resulted in fungal cells with significantly larger capsules and the mice had reduced fungal dissemination to the brain. Our results demonstrate the capacity of carbohydrate source and concentration to modify the expression of a major virulence factor of C. neoformans. These findings may impact the clinical management of cryptococcosis.     

Characterization of CMP-N-acetylneuraminic acid synthetase of group B streptococci.

The capsular polysaccharide is a critical virulence factor for group B streptococci associated with human infections, yet little is known about capsule biosynthesis. We detected CMP-Neu5Ac synthetase, the enzyme which activates N-acetylneuraminic acid (Neu5Ac, or sialic acid) for transfer to the nascent capsular polysaccharide, in multiple group B streptococcus serotypes, all of which elaborate capsules containing Neu5Ac. CMP-Neu5Ac synthetase isolated from a high-producing type Ib strain was purified 87-fold. The enzyme had apparent Km values of 7.6 for Neu5Ac and 1.4 for CTP and a pH optimum of 8.3 to 9.4, required magnesium, and was stimulated by dithiothreitol. This is the first characterization of an enzyme involved in group B streptococcus capsular polysaccharide biosynthesis.     

Structural changes in single muscle fibers after stimulation at a low frequency

Direct stimulation of single muscle fibers from Xenopus laevis at a frequency of 1 Hz results in a decline of the peak isometric twitch tension after about 200 twitches. Fibers were chemically fixed in glutaraldehyde after a varying number of twitches and at several fatigue levels, and the ultrastructural appearance was compared with that of resting fibers treated by identical fixation methods. No gross structural abnormalities were observed but subtle changes occurred. The mitochondria of stimulated fibers contain granules of normal size and number. The inner crista width is constant but the matrix width is increased on stimulation. These changes would not compromise ATP production. The myofibrils are normal except for a slight swelling in the myosin lattice. The transverse system (T system) and sarcoplasmic reticulum are intact. The minor diameter of the transverse tubule (T tubule) is increased slightly in stimulated fibers. The gap between the T-TC membranes stays constant at about 110 A, but tiny connecting pillars are seen to cross this gap more frequently in stimulated fibers (21 +/- 5% triads) than in resting fibers (8 +/- 6%). In stimulated fibers there is a marked increase in the electron dense content of the terminal cisternae (TC). Inasmuch as the observed structural changes correlate with the number of twitches but not with the fatigue level, it is concluded that TC density and T-TC pillar formation are related to the normal mechanisms of excitation-contraction coupling.