Characterization of mechanisms involved in uptake of Streptococcus dysgalactiae by bovine mammary epithelial cells

Bovine mammary epithelial cells were pretreated with inhibitors of protein kinase activity, actin polymerization and receptor-mediated endocytosis. In addition, mammary epithelial cells and Streptococcus dysgalactiae were pretreated with inhibitors of protein synthesis. Results showed that activity of tyrosine protein kinases, intact microfilaments and de novo eukaryotic protein synthesis was required for uptake of S. dysgalactiae by bovine mammary epithelial cells; a process that appeared to occur via receptor-mediated endocytosis. In contrast, de novo bacterial protein synthesis was not required for uptake of S. dysgalactiae by MAC-T cells. This study provides insight into bacterial and cellular mechanisms involved in early host-pathogen interactions, putting into perspective the role of mammary epithelial cells in the development and establishment of intramammary infections by S. dysgalactiae.     

Effect of ruminal degradability of crude protein and nonstructural carbohydrates on the efficiency of bacterial crude protein synthesis and amino acid flow to the abomasum of dairy cows

Four lactating Israeli Holstein cows that were ruminally and abomasally cannulated were used in an experiment with a 4 x 4 Latin square experimental design to study the effects of different amounts of ruminally degradable crude protein (CP) and ruminally degradable nonstructural carbohydrates on ruminal fermentation and efficiency of bacterial CP synthesis. Four diets were formulated to contain the following percentages (percentage of respective fraction) of ruminally degradable protein (RDP) and ruminally degradable nonstructural carbohydrates, respectively: 1) 73.8 and 85.3%, 2) 72.4 and 75.3%, 3) 67.7 and 86.0%, and 4) 66.3 and 76.0%. The 2 x 2 factorial effects of high and low concentrations of RDP or nonstructural carbohydrates were examined. Intakes of DM and organic matter (OM) were similar among treatments, and apparent and true ruminal digestibilities of OM were also similar. Apparent digestibility of CP in the total tract was higher for diets containing high concentrations of ruminally degradable nonstructural carbohydrates. Efficiency of microbial CP synthesis was higher for diets supplemented with low concentrations of RDP and averaged 196 g of microbial CP/kg of OM truly digested in the rumen. Total and bacterial CP flows were higher for diets containing low concentrations of RDP. Therefore, greater amounts of amino acids (AA) of bacterial origin reached the abomasum. The abomasal flow of AA was higher for diets containing low concentrations of RDP. Most of the profiles for essential AA in the abomasum were influenced and balanced by profiles for bacteria. When diets contained a high concentration of RDP (73% of total dietary CP), the supplementation of a high concentration of ruminally degradable nonstructural carbohydrates had no positive influence on bacterial yield or efficiency of bacterial CP synthesis. Other factors, such as AA and peptides included in the RUP fraction, may be important to maximize the efficiency of bacterial CP synthesis.     

Identification and characterization of an immunogenic outer membrane protein of Campylobacter jejuni

We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428. The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed. The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18. Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E. coli. Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum. All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18. The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C. jejuni. PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C. jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species. In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues. Expression of this construct in E. coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography. Sera from patients with past C. jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen. Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C. jejuni and is highly immunogenic. It is therefore a good candidate as an antigen for the serological diagnosis of past C. jejuni infections.     

Tetracyclines: antibiotic action, uptake, and resistance mechanisms

Tetracyclines probably penetrate bacterial cells by passive diffusion and inhibit bacterial growth by interfering with protein synthesis or by destroying the membrane. A growing number of various bacterial species acquire resistance to the bacteriostatic activity of tetracycline. The two widespread mechanisms of bacterial resistance do not destroy tetracycline: one is mediated by efflux pumps, the other involves an EF-G-like protein that confers ribosome protection. Oxidative destruction of tetracycline has been found in a few species. Several efflux transporters, including multidrug-resistance pumps and tetracycline-specific exporters, confer bacterial resistance against tetracycline. Single amino acids of these carrier proteins important for tetracycline transport and substrate specificity have been identified, allowing the mechanism of tetracycline transport to begin to emerge.     

Tumor suppressor genes: prospects for cancer therapies

Penicillins have been shown to inhibit bacterial cell wall synthesis, and interact with penicillin binding proteins, leading to bacterial lysis. These two mechanisms, the former more than the latter are believed to be responsible for their therapeutic potential. It has further been demonstrated that only actively multiplying cells are susceptible to bactericidal effects of the antibiotic, which is in accordance with the suggested mechanism of action. Bacterial growth takes place in terms of size and number, both requiring additional cell wall. An increase in bacterial size is due to an increase in the volume of cytosol and area of the cell wall. Presently there is no proof that the former is the cause of the latter or vice versa. Penicillin by inhibiting cell wall synthesis would inhibit both growth and multiplication. Since the antibiotic is bactericidal to rapidly multiplying cells, its effect on cell wall would interfere with its bactericidal action. As per the present understanding penicillin acts principally by inhibiting cell wall synthesis. There is however a discrepancy between its observed effects and what should logically be expected, which forces us to reexamine the mechanism of action of penicillin. We believe that the present understanding of the action of penicillin is incomplete if not outright faulty. It would be expedient to radically modify the same in view of its implication, for example on drug development.     

Structure-based design of inhibitors specific for bacterial thymidylate synthase

Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.     

Lysine synthesis and catabolism are coordinately regulated during tobacco seed development

The regulation of synthesis and accumulation of the essential amino acid lysine was studied in seeds of transgenic tobacco plants expressing, in a seed-specific manner, two feedback-insensitive bacterial enzymes: dihydrodipicolinate synthase (EC 4.2.1.52) and aspartate kinase (EC 2.7.2.4). High-level expression of the two bacterial enzymes resulted in only a slight increase in free lysine accumulation at intermediate stages of seed development, while free lysine declined to the low level of control plants toward maturity. To test whether enhanced catabolism may have contributed to the failure of free lysine to accumulate in seeds of transgenic plants, we analyzed the activity of lysine-ketoglutarate reductase (EC 1.5.1.7), an enzyme that catabolizes lysine into saccharopine. In both the control and the transgenic plants, the timing of appearance of lysine-ketoglutarate reductase activity correlated very closely with that of dihydrodipicolinate synthase activity, suggesting that lysine synthesis and catabolism were coordinately regulated during seed development. Notably, the activity of lysine-ketoglutarate reductase was significantly higher in seeds of the transgenic plants than in the controls. Coexpression of both bacterial enzymes in the same plant resulted in a significant increase in the proportions of lysine and threonine in seed albumins. Apparently, the normal low steady-state levels of free lysine and threonine in tobacco seeds may be rate limiting for the synthesis of seed proteins, which are relatively rich in these amino acids.     

Fusarin C: isolation and identification of two microsomal metabolites

Expression vectors for surface display and production of single-chain (Fv) antibodies (scAb) have been constructed based on the phagemid pSEX, which expresses DNA encoding a scAb fused to the gene III product of filamentous phage [Breitling et al., Gene 104 (1991) 147-153]. A smaller version of this phagemid, pSEX20, was made by removing an unnecessary cat. To produce a vector for the surface display of other proteins and peptides, the scAb of pSEX20 was substituted by a polycloning site (MCS) to give pSEX40. For the presentation of Ab on the surface of Escherichia coli, phagemid pAP10 was derived from pSEX20 by substituting gene III with a gene encoding the peptidoglycan-associated lipoprotein (PAL). Vectors for producing scAb that can be purified by antibody and metal affinity chromatography were constructed by substituting gene III in the vector pSEX20 with DNA encoding a peptide with a C-terminal epitope recognised by a monoclonal antibody (phagemid pOPE40) or with five C-terminal histidines (pOPE 90).     

Anandamide synthesis is induced by arachidonate mobilizing agonists in cells of the immune system

The hypothesis that the capability of agents to mobilize arachidonic acid (AA) could predict increased anandamide (ANA) synthesis in a macrophage cell line has been examined. Lipopolysaccharide (LPS), platelet-activating factor (PAF) and cannabinoids such as Delta9-tetrahydrocannabinol (THC) and anandamide were all found to be agonists for the release of AA and led to increased ANA synthesis in RAW264.7 mouse macrophage cells. Nitric oxide, in contrast, stimulated AA release without raising ANA levels. ANA stimulation of its own synthesis indicates the existence of a positive feedback mechanism. The possible involvement of the CB2 receptor in THC-mediated AA release and ANA synthesis is addressed using the antagonist SR144528. ANA synthesis is also increased by the combination of calcium ionophore and indomethacin, suggesting that ANA is metabolized by a cyclooxygenase in this system. The data imply that ANA could play a role in the response of the immune system to cannabinoids and bacterial endotoxins and that AA mobilization is a predictor for increased ANA synthesis.     

Design of a novel P450: a functional bacterial-human cytochrome P450 chimera

We report the construction of a functional chimera from approximately 50% bacterial (cytosolic) cytochrome P450cam and 50% mammalian (membrane-bound) cytochrome P450 2C9. The chimeric protein shows a reduced CO-difference spectrum absorption at 446 nm, and circular dichroism spectra indicate that the protein is globular. The protein is soluble and catalyzes the oxidation of 4-chlorotoluene using molecular oxygen and reducing equivalents from bacterial putidaredoxin and putidaredoxin reductase. This chimera provides a novel method for addressing structure-function issues and may prove useful in the design of oxidants for benign and stereospecific synthesis, as well as catalysts for bioremediation of polluted areas. Furthermore, these results provide the first evidence that bacterial P450 enzymes and mammalian P450 enzymes are likely to share a common tertiary structure.     

Bacterial steroidogenesis by periodontal pathogens and the effect of bacterial enzymes on steroid conversions by human gingival fibroblasts in culture

Studies were performed to investigate the effect of microbial culture supernatants of periodontal pathogens on the metabolism of radiolabelled testosterone in the presence or absence of human gingival fibroblasts. Subgingival plaque samples were obtained on paper points from 3 sites with probing depth values of 6-8 mm. Samples were incubated with 14C-testosterone for 24 h in brain heart infusion (BHI) broth. Similar incubations were also carried out with strains of A. actinomycetemcomitans, P. Intermedius and P. gingivalis to study the metabolism of radiolabelled testosterone by these periodontal pathogens. At the end of a 24 h incubation period with fibroblasts and supernatants or sonicates, the radioactive metabolites were extracted with ethyl acetate, evaporated and subjected to thin layer chromatography. The separated metabolites were quantified by scanning the radioactive plates using a Berthold linear analyser. When three sub-gingival plaque samples were incubated with radiolabelled testosterone there were 50-fold, 10-12-fold and 15-17-fold increases in 5 alpha-dihydrotestosterone (DHT) synthesis over 4-androstenedione production in these mixed microbial cultures. The two strains of P. intermedius produced 3- and 20-fold increases in 4-androstenedione production and DHT synthesis respectively. Both strains of A. actinomycetemcomitans and P. gingivalis showed 3-4-fold and 12-28-fold increases respectively in 4-androstenedione synthesis over that of DHT. Culture supernatants of P. intermedius and P. gingivalis caused 3-fold and 2-fold increases in DHT synthesis by fibroblasts over controls. There was little change in the case of the third pathogen. Since DHT has implications on matrix synthesis by fibroblasts in the environment of plaque associated inflammatory periodontal disease, bacterial metabolism and the effect of bacterial supernatants on human gingival fibroblasts can influence the degree of inflammatory repair.     

Virulence of enterohemorrhagic Escherichia coli: role of molecular crosstalk

Many bacterial exotoxins, originally defined by cytopathic effects, may also possess additional biological activities. The capacity of exotoxins to elicit the synthesis and secretion of pro- or anti-inflammatory cytokines may be as important as their direct toxic effects in pathogenesis. One example of such 'molecular crosstalk' occurs between Shiga toxins and the cytokines made in response to these toxins during the development of disease.     

Promotion of met-tRNAiMet binding to ribosomes by yIF2, a bacterial IF2 homolog in yeast

Delivery of the initiator methionine transfer RNA (Met-tRNAiMet) to the ribosome is a key step in the initiation of protein synthesis. Previous results have indicated that this step is catalyzed by the structurally dissimilar translation factors in prokaryotes and eukaryotes-initiation factor 2 (IF2) and eukaryotic initiation factor 2 (eIF2), respectively. A bacterial IF2 homolog has been identified in both eukaryotes and archaea. By using a combination of molecular genetic and biochemical studies, the Saccharomyces cerevisiae IF2 homolog is shown to function in general translation initiation by promoting Met-tRNAiMet binding to ribosomes. Thus, the mechanism of protein synthesis in eukaryotes and prokaryotes is more similar than was previously realized.     

Collagen synthesis by liver stellate cells is released from its normal feedback regulation by acetaldehyde-induced modification of the carboxyl-terminal propeptide of procollagen

Acetaldehyde stimulates collagen synthesis in stellate cells and forms adducts with procollagen in the liver of alcoholics. To assess the possibility that modification of the carboxyl-terminal propeptide by acetaldehyde affects its capacity to exert a feedback inhibition of collagen synthesis after splitting from procollagen, the propeptide was prepared by gel filtration of the bacterial collagenase digests of procollagen type I (obtained from 10(9) calvaria fibroblasts of newborn rats) and reacted with either 250 mM acetaldehyde and 100 mM CNBH3 or with 170 microM acetaldehyde without reducing agents, to mimick in vivo conditions. The unmodified propeptide produced a concentration-dependent inhibition of collagen synthesis by Ito cells. By contrast, the acetaldehyde-modified propeptide produced a lesser inhibition of procollagen synthesis in the cells, associated with a greater accumulation of collagen in the media. The incubation with 170 microM acetaldehyde and, to a lesser extent, 50 mM ethanol produced collagenase-digestible adducts in stellate cells. Thus, the formation of acetaldehyde adducts with the carboxyl-terminal propeptide of procollagen may account, at least in part, for the stimulatory effect of acetaldehyde on collagen synthesis by stellate cells and may lead to collagen accumulation through a decrease of the normal feedback regulation of collagen synthesis by the propeptide.     

Folate synthesized by bacteria in the human upper small intestine is assimilated by the host

BACKGROUND & AIMS: Some intestinal flora are known to synthesize folate. The aim of this study was to determine whether folate synthesized by small intestinal flora is assimilated by the human host. METHODS: Subjects with atrophic gastritis and healthy volunteers were studied before and after omeprazole administration. A double-lumen perfusion tube was placed in the duodenum. 3H-labeled P-aminobenzoic acid, a precursor substrate for bacterial folate synthesis, was perfused. Downstream intestinal aspirates and a 48-hour urine collection were obtained. RESULTS: Atrophic gastritis and omeprazole administration were associated with increases in duodenal pH and in small intestinal flora. Bacterially synthesized folates were isolated from the intestinal aspirates. Tritiated 5-methyltetrahydrofolate, a major metabolite of folate, was isolated from the urine of omeprazole-treated subjects in greater quantities than from drug-free subjects (P<0.01); the quantity of tritiated 5-methyltetrahydrofolate in the urine of the subjects with atrophic gastritis was similarly elevated. CONCLUSIONS: (1) Mild bacterial overgrowth caused by atrophic gastritis and administration of omeprazole are associated with de novo folate synthesis in the lumen of the small intestine; (2) the human host absorbs and uses some of these folates; and (3) the contribution to folate nutriture from this source remains unclear.     

Attempts to purify a membrane attached chromoid of bacteriophage lambda

Using methods which proved successful for the isolation of E. coli chromosome in a folded form (the E. coli chromoid), we have attempted to purity the "native" form of bacteriophage gamma chromosome from gamma infected cells. Upon sedimentation of lysates we find that phage DNA separates into two fractions, one of which cosediments with the bacterial chromoid ; the other sediments nearer to the top of gradient. Both fractions probably contain membrane-bound phage DNA, and both support the in vivo synthesis of phage DNA. The heavier fraction contains more closed circular parental DNA molecules than the lighter fraction. Formation of the latter is blocked by certain phage mutations. Being relatively free of bacterial DNA, the lighter fraction is suitable for further analysis.