Relative Rates of Lysis of Staphylococcal Cell Walls by Lytic Enzymes from Various Bacteriophage Types

Lytic enzymes were isolated from 14 strains of phage-infected Staphylococcus aureus. Cell walls were prepared from the same uninfected strains of bacteria. Comparison of the lytic rates was made for each enzyme, with each of the cell walls as substrate. Differences in the rate of substrate utilization of the various cell wall types exceeded 10-fold. Cell walls from strains 42E, 29, and 77 were the best substrates, whereas cell walls from strains 3C, 80, and 187 were the poorest substrates. The cell wall amino acid composition is discussed as related to lytic enzyme specificity. A possible explanation of phage typing of staphylococcal cells, based on enzyme activity and cell wall composition, is presented.     

Effect of Glucose on Glycine Requirement of Acanthamoeba castellanii*

SYNOPSIS. Acanthamoeba castellanii grows in a minimal medium (AMLIV) containing only arginine, methionine, leucine, isoleucine, and valine as sole nitrogen sources, other than vitamins, when glucose is the carbon source. With acetate as the carbon source, glycine must be added to AMLIV. Doubling time in AMLIV varies according to the ratio of amino acids concentrations. Several combinations yield T_d values of ～ 70 hr.     

Effect of Bacteria on Survival and Growth of Acanthamoeba castellanii

The growth and survival of Acanthamoeba castellanii in the presence of Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, and Stenotrophomonas maltophilia varied with the densities and species of bacteria. All species of bacteria suspended in a buffered saline at densities of 10⁵ to 10⁶/ml supported the growth and survival of 10⁶/ml trophozoites of Acanthamoeba castellanii in a buffered saline solution. At densities of bacteria to amoebae of 100:1 or greater, growth and survival of A. castellanii were suppressed, particularly by P. aeruginosa. In an enrichment medium, the rapid growth of most co-inoculated bacteria inhibited the growth and survival of the amoeba. Received: 12 September 1996 / Accepted: 20 September 1996     

Occurrence of Cell Lytic Enzymes in Blue-Green Bacteria

Detergent extracts of three blue-green bacteria (Agmenellum quadruplicatum strain BG1, Anacystis nidulans strain TX20, and Nostoc sp. strain MAC) contained enzymes capable of lysing suspensions of Micrococcus lysodeikticus. The enzyme preparation from A. quadruplicatum released soluble reducing fragments from purified peptidoglycan. The lytic activity exhibited a pH optimum between 6 and 7, was relatively heat stable, and was susceptible to attack by proteolytic enzymes. These results extend the range of bacterial types exhibiting cell lytic activity as well as confirm the existence of the lytic system commonly observed in "water blooms".     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Anaerobiosis-induced differentiation of Acanthamoeba castellanii

Acanthamoeba castellanii is a free living amoeba ubiquitous in soil and also commonly found in aquatic environments. In waterlogged soils, anoxia is quickly established as the dissolved oxygen is consumed by the organisms present. We were interested in the effects of anoxic conditions upon this organism. Batch cultures degassed with N₂ during mid-exponential growth, induced encystation within 12 h of anoxia, and mature cysts were formed within 2–3 days. Excystation (99%) was achieved by subsequent aeration of these cultures after 3–6 days. Anoxia-induced cysts, maintained in anoxic conditions for up to four months, remained viable. Difference spectra, during anaerobiosis, revealed that cytochromes were not lost, suggesting that the organism retains its respiratory components. The growth rate of trophozoites, grown in a chemostat, was dependent on the concentration of O₂ in the head space and glucose uptake increased at lower dissolved O₂ tensions. The results obtained suggest that A. castellanii has a complex adaptive strategy enabling it to cope with microaerobic and anoxic conditions which may be experienced in the environment.     

Chemiluminescence of Acanthamoeba castellanii.

We have investigated ferrocytochrome c-induced proton ejection from reconstituted cytochrome c oxidase-containing vesicles using careful control of the number of enzyme turnovers. Ferrocytochrome c caused the appearance of protons at the vesicle exterior, and this could be abolished by using a protonophore. In addition, its decay was dependent on the permeability of the vesicle membranes to protons and the number of turnovers of the oxidase. These observations indicate that the ejection of protons was the result of genuine translocation. The possibility of this translocation occurring via a Mitchellian loop as a result of the presence of a reduced hydrogen carrier contaminating the enzyme was considered and excluded. Proton-translocating activity in this reconstituted system depended critically on the ratio of enzyme to lipid used in the reconstitution process and we propose a rationale to account for this. We conclude that our data provide strong support for the proposal that cytochrome c oxidase acts as a proton pump and that approx. 0.9 H+ is excluded per ferrocytochrome c molecule oxidized.     

Stable transfection of Acanthamoeba castellanii

A simple method for stable transfection of Acanthamoeba castellanii using plasmids which confer resistance to neomycin G418 is described. Expression of neomycin phosphotransferase is driven by the Acanthamoeba TBP gene promoter, and can be monitored by cell growth in the presence of neomycin G418 or by Western blot analysis. Transfected cells can be passaged in the same manner as control cells and can be induced to differentiate into cysts, in which form they maintain resistance to neomycin G418 for at least several weeks, although expression of neomycin phosphotransferase is repressed during encystment. Expression of EGFP or an HA-tagged EGFP-TBP fusion can be driven from the same plasmid, using an additional copy of the Acanthamoeba TBP gene promoter or a deletion mutant. The TBP-EGFP fusion is localized to the nucleus, except in a small proportion of presumptive pre-mitotic cells. EGFP expression can also be driven by the cyst-specific CSP21 gene promoter, which is completely repressed in growing cells but strongly induced in differentiating cells. Transfected cells maintain their phenotype for several weeks, even in the absence of neomycin G418, suggesting that transfected genes are stably integrated within the genome. These results demonstrate the utility of the neomycin resistance based plasmids for stable transfection of Acanthamoeba, and may assist a number of investigations.     

Salt-induced Contraction of Bacterial Cell Walls

Intact Bacillus megaterium cells were found to contract as much as 26% in terms of dextran-impermeable volume when transferred from water to unbuffered, non-plasmolyzing NaCl solutions. This shrinkage appeared to be primarily due to electrostatic wall contraction rather than to any osmotic response of the cells. A variety of salts (but not sucrose) added to water suspensions of isolated cell walls caused protons to be released from the walls with resultant lowering of suspension pH and contraction of the structures. In effect, B. megaterium walls behaved as flexible, amphoteric polyelectrolytes, and their compactness in aqueous suspensions was affected by changes in environmental ionic strength and pH. Isolated walls were most compact in low ionic strength media with a pH of about 4, a value close to the apparent isoelectric pH of wall peptidoglycan. Electrostatic attractions appeared to play a major role in determining the compactness of highly contracted walls, and the walls responded to increased environmental ionic strength by expanding. In contrast, electrostatic repulsions were dominant in highly expanded walls, and increased environmental ionic strength induced wall contraction. Walls of whole bacteria also shrank when the cells were plasmolyzed. This second type of contraction seemed to result from relief of wall tension during plasmolysis, and it could be induced with nonionic solutes. Thus, cell wall tone in B. megaterium appeared to be set both by mechanical tension and by electrostatic interactions among wall ions.     

The Flexibility of Bacterial Cell Walls

S ummary . There are considerable differences in the extensibility of the cell walls of bacteria in different genera. Gram negative spp. examined were more flexible than the Gram positive ones.     

Effect of Monovalent Thallium Ions on Differentiation and Multiplication of Acanthamoeba castellanii

The vegetatively multiplying Acanthamoeba castellanii cells are transformed into cysts under unfavourable feeding conditions. The cyst formation may also be induced by treatment of the cells with DNA-synthesis inhibitors or by placing the cells into special ionic medium containing magnesium and calcium at pH 9, with aeration. During Acanthamoeba encystment the morphology of the cells changes significantly, namely a cellulose-protein cyst wall appears which is easily seen under the light and electron microscope. The process of encystment in Acanthamoeba castellanii is considered as a useful simple model of cytodifferentiation of eukaryotic cells.
This communication describes the effects of monovalent thallium ions on the differentiation and multiplication of Acanthamoeba cells growing in optimal feeding conditions. Thallium ions being potassium analogues are readily accumulated by cells. On the other hand, thallium ions, unlike potassium ions, are able to form complexes with some anions, which results in disturbances of some cellular functions.
Thallium ions, added to the growth medium of 2–3-days old Acanthamoeba culture at a concentration of 0.05–1.0 mM inhibit the population growth inducing the differentiation of cells into cysts. The increase of the thallium ion concentration up to 5 or 10 mM in the growth medium causes the very fast multiplication of Acanthamoeba cells. However, at these thallium ion concentrations no cysts can be observed.
Thus, on the basis of the experimental data it seems likely that thallium ions play some role in increasing the rate of multiplication and in switching on the differentiation process (encystment) in Acanthamoeba cells.     

The cytochromes of Acanthamoeba castellanii.

1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and 'microsomal' membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s).     

X-Ray Diffraction Studies on Selected Bacterial Cell Walls

The cell walls of selected bacteria were studied by X-ray diffraction analysis to determine and characterize crystalline components. The walls were isolated by mechanical disruption and purified by enzymatic and washing procedures. The X-ray diffraction lines which appeared from the gram-positive cell walls were shown to be due to the constituent "mucopeptide" fraction. No diffraction lines could be obtained from the gram-negative bacterium studied. The results show that crystallinity is associated with mucopeptide.     

Effects of Mannose on Pathogenesis of Acanthamoeba castellanii

Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.     

Effect of ribosome-inactivating proteins on ribosomes from Tetrahymena pyriformis and Acanthamoeba castellanii

The effect of ribosome-inactivating proteins type 1 (single-chain) and type 2 (two-chain, toxins) on polyphenylalanine polymerization by Tetrahymena pyriformis and Acanthamoeba castellanii ribosomes has been studied. The reaction catalysed by tetrahymena ribosomes was inhibited by two ribosome-inactivating proteins type 1 (dianthin 32 and, less effectively, momordin) whereas the reaction catalysed by amoeba ribosomes was inhibited, in a decreasing order of activity, by three ribosome-inactivating proteins type 1 (dianthin 32, saporin 6 and bryodin) and by two toxins (abrin and volkensin).     

Effect of phalloidin and viroisin on Acanthamoeba castellanii after permeabilization of the cell

We have developed a new technique for the permeabilization of the membrane of Acanthamoeba castellanii. This technique involves the use of digitonin which alters neither the morphology nor the motility of the cell, but favours the penetration of phalloidin and viroisin. Treatment of permeabilized cells with phalloidin or viroisin induces, in the cortex of the cell, an intensive proliferation of filaments which have been identified as actin. This cortical filamentous layer detaches from the membrane and slowly contracts, acting as a fine mesh sieve which concentrates the organelles in the middle of the cell, causing therefore the formation of a central granuloplasm and a cortical hyaloplasm. During this process, cell motility is irreversibly lost. The results indicate that extensive proliferation and reorganization of actin filaments cannot support cell motility and they are discussed in terms of a general understanding of amoeboid movement.