Correlation between the susceptibility of E. coli to phagocytosis and their ability to invade HeLa cells in vitro.

The susceptibility of several strains of E. coli to phagocytic killing by polymorphonuclear lencocytes and the ability of the same strains to invade HeLa cells were studied. It was found that only the strains resistant to killing by leucocytes were able to penetrate and multiply within HeLa cells.     

Correlation between the susceptibility of E. coli to phagocytosis and their ability to invade HeLa cells in vitro

Summary The susceptibility of several strains of E. coli to phagocytic killing by polymorphonuclear leucocytes and the ability of the same strains to invade HeLa cells were studied. It was found that only the strains resistant to killing by leucocytes were able to penetrate and multiply within HeLa cells.Acknowledgments. We thank Dr Bianca Pani who supplied us with HeLa cells. Supported by grant No. 75.00695.04 from the National Research Council of Italy (CNR).     

The inability of nuclear dehydrogenating clostridia to oxidize bile salt hydroxyl groups.

In a survey of the intracellular bile salt oxidoreductase activity in fecal bacteria, 16 strains of nuclear dehydrogenating clostridia and 2 strains of non-nuclear dehydrogenating C. paraputrificum were demonstrated unable to oxidize cholate at any of the 3 OH groups. Since nuclear dehydrogenation at the delta-1 and delta-4 position requires a 3-oxo precursor steroid, it appears that these organisms require the presence of a 3 alpha-hydroxysteroid dehydrogenating organism for nuclear dehydrogenation.     

Olfactory ensheathing cells are attracted to, and can endocytose, bacteria

Olfactory ensheathing cells (OECs) have been shown previously to express Toll-like receptors and to respond to bacteria by translocating nuclear factor-kappaB from the cytoplasm to the nucleus. In this study, we show that OECs extended significantly more pseudopodia when they were exposed to Escherichia coli than in the absence of bacteria (p=0.019). Co-immunoprecipitation showed that E. coli binding to OECs was mediated by Toll-like receptor 4. Lyso-Tracker, a fluorescent probe that accumulates selectively in lysosomes, and staining for type 1 lysosome-associated membrane proteins demonstrated that endocytosed FITC-conjugated E. coli were translocated to lysosomes. They appeared to be subsequently broken down, as shown by transmission electron microscopy. No obvious adherence to the membrane and less phagocytosis was observed when OECs were incubated with inert fluorescent microspheres. The ability of OECs to endocytose bacteria supports the notion that OECs play an innate immune function by protecting olfactory tissues from bacterial infection.     

Recently discovered functions of glucosylceramides in plants and fungi

Glycosphingolipids are ubiquitous membrane lipids of eukaryotic organisms and a few bacteria. Whereas inositol-containing glycosphingolipids are restricted to plants and fungi, galactosylceramide occurs only in fungi and animals. In contrast, glucosylceramide is the unique glycosphingolipid which plants, fungi and animals have in common. However, there are specific differences in the structure of the ceramide backbone of glucosylceramides from these organisms. A comparison of the structural features and the biosynthesis of glucosylceramides from plants, fungi and animals will contribute to our understanding of their functions, which so far have been analysed mainly in animals. The availability of nearly all genes involved in the biosynthesis of glucosylceramides enables the specific manipulation of glycosphingolipid metabolism by techniques of forward and reverse genetics. Application of this approach to unicellular organisms like yeasts, multicellular filamentous fungi, as well as to complex organisms like plants will reveal common and different glucosylceramide functions in these organisms. These glycolipids play a role both in intracellular processes and in cell-to-cell interactions. These interactions may occur between cells of a multicellular organism or between cells of different species, as in host-pathogen interactions.     

Molecular adaptations to cold in psychrophilic enzymes

Psychrophiles or cold-loving organisms successfully colonize cold environments of the Earth's biosphere. To cope with the reduction of chemical reaction rates induced by low temperatures, these organisms synthesize enzymes characterized by a high catalytic activity at low temperatures associated, however, with low thermal stability. Thanks to recent advances provided by X-ray crystallography, protein engineering and biophysical studies, we are beginning to understand the molecular adaptations responsible for these properties which appear to be relatively diverse. The emerging picture suggests that psychrophilic enzymes utilize an improved flexibility of the structures involved in the catalytic cycle, whereas other protein regions if not implicated in catalysis may or may not be subjected to genetic drift.     

Impact of biofilm matrix components on interaction of commensal Escherichia coli with the gastrointestinal cell line HT-29

Commensal Escherichia coli form biofilms at body temperature by expressing the extracellular matrix components curli fimbriae and cellulose. The role of curli fimbriae and cellulose in the interaction of commensal E. coli with the intestinal epithelial cell line HT-29 was investigated. Expression of curli fimbriae by the typical commensal isolate E. coli TOB1 caused adherence and internalization of the bacteria and triggered IL-8 production in HT-29 cells. In particular, induction of IL-8 production was complex and involved curli-bound flagellin. While cellulose alone had no effect on the interaction of TOB1 with HT-29 cells, co-expression of cellulose with curli fimbriae decreased adherence to, internalization and IL-8 induction of HT-29 cells. Investigation of a panel of commensal isolates showed a partial correlation between expression of curli fimbriae and enhanced internalization and IL-8 production. In addition, a high immunostimulatory flagellin was identified. Thus, the consequences of expression of extracellular matrix components on commensal bacterial-host interactions are complex.     

Antifungal proteins: targets,mechanisms and prospective applications

All organisms have evolved several defence systems in order to protect themselves against bacteria, fungi and viruses. Higher organisms have developed a complex network of humoral and cellular responses, called adaptive immunity. A second defence system, innate immunity, was discovered in the early 1980s, consisting of small cationic peptides with a broad antimicrobial spectrum. These proteins act immediately at sites of infection or inflammation. The production of proteins with antimicrobial activity was not limited to higher organisms but was also found in insects, plants and microorganisms. During the last 2decades a broad range of proteins with very different structural features have been isolated and characterised from differing organisms ranging from bacteria to human beings. Over 500cationic membrane-acting proteins with antimicrobial and antifungal activities have been identified to date. Apart from these proteins, a very large number of antifungal proteins active on the fungal cell wall, on enzymes of the cell wall synthesis machinery, the plasma membrane and on intracellular targets have been characterised.Received 17 June 2003; received after revision 4 August 2003; accepted 18 August 2003     

Development of DNA probes for cytotoxin and enterotoxin genes in enteric bacteria

DNA probes to identify the genes encoding toxins in enteric bacteria have been developed. Use of these probes reduces the number of animals required for toxicity testing, as suspect bacteria can be directly tested for the presence of toxin. We have augmented the gene probes available by developing probes against the Escherichia coli enterotoxin LTII and shiga toxin from Shigella dysenteriae 1. The LTII gene from E. coli 357900 was identified and characterised and a suitable internal probe was obtained. The LTII gene was found not to be common among enterobacteriae from various geographical locations. Isolates predominately of animal origin from Nigeria and Thailand hybridized with the probe. The shiga toxin gene was isolated from S. dysenteriae 1 by a combination of in vivo and in vitro methods. An internal probe was identified and used against different serogroups of Shigella and E. coli isolates. The probe was found to hybridize with S. dysenteriae 1 isolates and also some S. flexneri and S. sonnei strains. Representatives were tested for toxin production and found to produce toxin at low levels.     

The sigmaS subunit of RNA polymerase as a signal integrator and network master regulator in the general stress response in Escherichia coli

The sigmaS (RpoS) subunit of RNA polymerase in Escherichia coli is a key master regulator which allows this bacterial model organism and important pathogen to adapt to and survive environmentally rough times. While hardly present in rapidly growing cells, sigmaS strongly accumulates in response to many different stress conditions, partly replaces the vegetative sigma subunit in RNA polymerase and thereby reprograms this enzyme to transcribe sigmaS-dependent genes (up to 10% of the E. coli genes). In this review, we summarize the extremely complex regulation of sigmaS itself and multiple signal input at the level of this master regulator, we describe the way in which sigmaS specifically recognizes "stress" promoters despite their similarity to vegetative promoters, and, while being far from comprehensive, we give a short overview of the far-reaching physiological impact of sigmaS. With sigmaS being a central and multiple signal integrator and master regulator of hundreds of genes organized in regulatory cascades and sub-networks or regulatory modules, this system also represents a key model system for analyzing complex cellular information processing and a starting point for understanding the complete regulatory network of an entire cell.     

Effects of the virulence plasmid ColV, I-K94 on the sensitivity of Escherichia coli to putative environmental inhibitory agents

Derivatives of Escherichia coli carrying the virulence plasmid, ColV, I-K94 were more resistant than the ColV- parents to phage Mel but were more sensitive to the hydrophobic inhibitors deoxycholate, erythromycin and lysozyme. The basis for these changes in sensitivity has been examined in ColV+ mutants with altered colicin or VmpA protein levels and in ColV+ strains with repressed transfer properties.     

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.     

Isolation of an antifreeze peptide from the Antarctic sponge <Emphasis Type="Italic">Homaxinella balfourensis</Emphasis>

Polar plants and animals survive in subzero waters (-2 degrees C) and many of these marine organisms produce antifreeze proteins (AFPs) to better adapt themselves to these conditions. AFPs prevent the growth of ice crystals which disrupt cellular membranes and destroy cells by inhibiting crystallization of water within the organism. The hydrophilic extract of an Antarctic sponge Homaxinella balfourensis exhibited a non-colligative freezing point depression effect on the crystal morphology of water. The extract was purified by repeated reverse phase high-pressure liquid chromatography, then assayed and shown to contain several AFPs. The major peptide was isolated, analyzed using matrix-assisted laser desorption ionization mass spectrometry and the partial structure of the peptide identified through amino acid sequencing. AFPs have potential applications in agriculture, medicine and the food industry.     

Procaine, a local anesthetic, interacting with the cell membrane and inhibiting the processing of exported protein precursors in Escherichia coli]

In E. coli cells grown in the presence of procaine (0.55% w/v), precursor forms of alkaline phosphatase and of glutamine binding protein accumulate besides mature forms synthesized prior to procaine addition. An experimental technique, of general application, for isolation and purification of mature and precursor forms obtained under these conditions, is described.     

Two classes of metabolites fromTheonella swinhoei are localized in distinct populations of bacterial symbionts

The marine spongeTheonella swinhoei (lithistid Family Theonellidae, Order Astrophorida) has yielded many important, bioactive natural products, most of which share structural features with bacterial natural products. The presence of microbial symbionts inT. swinhoei has been reported, and it was originally suggested that the cytotoxic macrolide swinholide A and many of the bioactive cyclic peptides fromT. swinhoei were all produced by symbiotic cyanobacteria. By transmission electron microscopy, we found four distinct cell populations to be consistently present inT. swinhoei: eukaryotic sponge cells, unicellular heterotrophic bacteria, unicellular cyanobacteria and filamentous heterotrophic bacteria. Purification and chemical analyses of each cell type showed the macrolide swinholide A to be limited to the mixed population of unicellular heterotrophic bacteria, and an anti-fungal cyclic peptide occurred only in the filamentous heterotrophic bacteria. Contrary to prior speculation, no major metabolites were located in the cyanobacteria or sponge cells.     

Multifunctional cationic host defence peptides and their clinical applications

With the rapid rise in the emergence of bacterial strains resistant to multiple classes of antimicrobial agents, there is an urgent need to develop novel antimicrobial therapies to combat these pathogens. Cationic host defence peptides (HDPs) and synthetic derivatives termed innate defence regulators (IDRs) represent a promising alternative approach in the treatment of microbial-related diseases. Cationic HDPs (also termed antimicrobial peptides) have emerged from their origins as nature’s antibiotics and are widely distributed in organisms from insects to plants to mammals and non-mammalian vertebrates. Although their original and primary function was proposed to be direct antimicrobial activity against bacteria, fungi, parasites and/or viruses, cationic HDPs are becoming increasingly recognized as multifunctional mediators, with both antimicrobial activity and diverse immunomodulatory properties. Here we provide an overview of the antimicrobial and immunomodulatory activities of cationic HDPs, and discuss their potential application as beneficial therapeutics in overcoming infectious diseases.     

Differential sensitivity to tributyltin of cytochrome-containing and cytochrome-deficient cells of Escherichia coli SASX76

The effect of tributyltin (TBT) chloride on the growth of cytochrome-deficient and cytochrome-containing cells of Escherichia coli SASX76 was examined. The former cells were found to be at least 20 times more sensitive to TBT. It is proposed that the differential sensitivity of these two cell types to the biocide, TBT, may be due to a different mode of energy generation by cytochrome-deficient and cytochrome-sufficient cells. In addition to the energy state, the pH change caused by the presence and absence of cytochromes which occurred during growth also resulted in a differential sensitivity of these cells.     

Inhibitory effect of halocyamine,an antimicrobial substance from ascidian hemocytes,on the growth of fish viruses and marine bacteria

Summary Halocyamine A, an antimicrobial substance isolated from hemocytes of the solitary ascidianHalocynthia roretzi, inhibited in vitro the growth of fish RNA viruses (infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus). Pretreatment of RNA virus with halocyamine A reduced the infectivity of the virus toward host cells. The growth of marine bacteria,Achromobacter aquamarinus andPseudomonas perfectomarinus, was also inhibited by halocyamine A but that ofAlteromonas putrefaciens andVibrio anguillarum was not. These results suggest that halocyamine may have a role in the defense mechanisms ofH. roretzi against marine viruses and bacteria.     

Circulating Trypanosoma cruzi from the same cloned population show differences in the ability to infect cells and to cause lethal infection in mice

Two subpopulations of circulating parasites displaying different abilities to infect mammalian cells and to cause lethal infection when inoculated into normal mice were demonstrated in the blood of mice acutely infected with T. cruzi. Parasites of one subpopulation rapidly penetrated mouse fibroblasts and were readily phagocytized by normal mouse peritoneal macrophages whereas parasites of the other subpopulation showed little ability to invade non-phagocytic cells and resisted phagocytosis. Inoculation of organisms of this latter population into mice resulted in infections with lower parasitemias and longer time to death as compared to controls inoculated with organisms from a population containing both types of parasites. When a population of parasites containing both types of trypanosomes was cultured in acellular medium at 28 degrees C a decrease in the number of parasites was noted to occur in the initial days of culture. This decrease was not noted when parasites of the subpopulation of trypanosomes resistant to phagocytosis were cultured similarly.     

Host cell reactivation capacity of different strains of E. coli B resistant or sensitive to ozone

Host cell reactivation capacity for ozonated or irradiated phage was determined for different strains of E. coli either more sensitive or resistant to ozone than the wild type strain. The results suggest that the ozr gene product could be involved in the same repair pathway for ozone-induced lesions on DNA as the polA gene. The possible involvement of a specific endonuclease for these lesions is also considered.