Expression of human IgE-binding factor (sCD23) inEscherichia coli

Previously, we cloned cDNA coding for type II Fc receptor for human IgE (CD23 or FcERII). A fragment of this cDNA coding for soluble IgE-binding factor fused with a fragment of human interleukin-3 gene was cloned in pBT-IL-3-sCD23 plasmid. It is demonstrated thatE. coli strain JM109 (pBT-IL-3-sCD23) expresses the hybrid protein with a yield of 10–15% total cell protein. The recombinant product is represented by equal amounts of two polypeptides with molecular weights of about 24 and 32 kD. Immunological analysis and determination of the amino acid sequence of the N-terminal ends of these polypeptides show that a protein with a molecular weight of 24 kD results from proteolysis of the full-size (32 kD) hybrid protein. The preparation obtained can be used for the development of test kits for CD23. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 6, pp. 690–694, June, 1996     

Expression of viral hemagglutinin on the surface ofE. coli

Summary Expression of a foreign protein molecule on theE. coli bacterial surface has been achieved through hybrid plasmid construction of fusion proteins using outer membrane proteinompA as a carrier system. Influenza virus hemagglutinin fusion proteins of this character have been shown to become integrated into the bacterial outer membrane and to expose their hemagglutinin moiety at the exterior surface in a conformation which is at least similar to the authentic viral antigen structure.Abbreviations bp Base pairs (DNA) - kD kiloDaltons (protein) - HA Hemagglutinin - IPTG Isopropylthiogalactoside - PBS Phosphate buffered saline - PVDF Polyvinyldifluoride Dedicated to Professor Dr. N. Zöllner on the occasion of his 65th birthday     

Experession of human interleukin-10 inEscherichia coli cells

The human interleukin-10 gene has been synthesized by the chemical enzymatic method. Vectors for cytoplasmic and periplasmic expression of recombinant interleukin-10 have been obtained inE. coli cells. A high level of protein expression was found to be characteristic of only recombinant strains producing interleukin-10 as “fused” protein (fused with the N-terminal fragment of interleukin-3). Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 3, pp. 324–327, March, 1995 Presented by Yu. A. Romanov, Member of the Russian Academy of Medical Sciences). (Address for correspondence: Nauchnyi Pr. 8, 117246 Moscow, fax 331-01-01.     

High-level Expression of the C-terminal Hydrophobic Region of HCV E2 Protein Ectodomain in E. coli

High-level expression of hepatitis C virus (HCV) E2 protein fragments encompassing its C-terminal hydrophobic region (downstream of aa 661) in Escherichia coli has been proven difficult. The extreme hydrophobicity of this region has been suspected to be detrimental to the host. In this work, we found that the C-terminal region (downstream of aa 565) of E2 ectodomain interfered with full-length expression of E2 fragments in E. coli. Nonetheless, when the central region (aa 484–622) of E2 was deleted, full-length protein was efficiently produced. C-terminal region aa 567–700 could not be efficiently expressed individually or as mouse dihydrofolate reductase (DHFR) fusion protein. However, a mutant that emerged in the cloning process was able to express full-length DHFR fusion protein. Sequencing analysis reveals the mutation to be a short frame-shift around the fusion junction, altering aa 568–571 of E2. C-terminal region of E2 ectodomain (aa 567–730) carrying this mutation was successfully expressed as hexa-histidine-tagged protein to a high level. The protein was highly insoluble and was purified under denaturing conditions. The purified protein displayed HCV E2-specific antigenicity in Western blot and specific rabbit antiserum was raised against it. These results demonstrate that hydrophobicity of the C-terminal region of E2 ectodomain is not harmful to E. coli host and has no dominative adverse effect on its bacterial expression. Other nucleotide and/or amino acid sequence properties seem to play a more important role. This finding opens up new possibilities for the development of novel bacterially-derived E2 proteins for research and clinical applications.     

A rapid phospholipase A2 bioassay using14C-oleate-labelledE. coli bacterias

Summary Two methods of phospholipase A₂ determination using¹⁴C-labelledE. coli bacterias as substrate were compared. One method works with a filter membrane for separation of cleaved¹⁴C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis. Some features of human serum phospholipase A₂ regarding pH and Ca²⁺ dependency were investigated. Possible sources of errors were discussed. It was shown that either method can differentiate between normal and pathologically elevated phospholipase A₂ levels, but that the filter method is superior in terms of sensitivity and workload.Abbreviations PLA₂ phospholipase A₂ - BSA bovine serum albumin - (F)FA (free) fatty acid - TLC thin-layer chromatography     

Inhibition of HTLV-I induction and virus-induced syncytia formation by oligodeoxynucleotides

HTLV-I is an exogenous human retrovirus that is a causative agent of adult T cell leukemia (ATL). In addition to the structural genes (gag, pol and env), a gene termed pX is postulated to be associated with leukemogenesis in ATL. Since no effective chemotherapy is currently available, it is important to find suitable therapeutic means against ATL. Here, we tested the inhibitory effect of antisense oligodeoxynucleotides (ODNs) on HTLV-I infection in different systems. ODNs were synthesized with the phosphorothioate backbone targeted to either structural genes or transactivator genes. The phosphorothioate ODNs were found to have two distinct target sites to exert their effect on HTLV-I infection: 1) Several ODNs, including sense ODNs and random oligomers, blocked syncytium formation induced by HTLV-I at a concentration of 0.1 M. Their inhibitory effect on syncytium formation seemed to be exerted in a nonantisense manner, most probably due to their interaction with the cell membrane. 2) Efficient suppression by ODNs of gag gene expression after chemical induction was observed in HTLV-I-transformed T cells in an antisense manner. In this suppression, tax-antisense ODN showed virtually complete inhibition of gag protein expression, but not RNA expression, at the concentration of 0.1 M, whereas tax-sense ODN displayed a weak inhibitory effect. Our results suggest that the influence of the phosphorothioate compound should be considered from the aspect of two separated mechanisms of antiviral activity, the effects on early (viral adsorption) and late (translation) phase infection.     

Effect of antisecretory factor on Escherichia coli STa enterotoxin-induced alkalinisation of pig jejunal acid microclimate

The effect of challenge by Escherichia coli STa enterotoxin on pig jejunal mucosal surface pH was investigated in vivo. Exposure to STa resulted in a rapid and reversible alkalinisation (P <0.001) of the jejunal mucosa from 6.27±0.11 (5) to 6.89±0.03 (5). This action of STa is probably mediated through cyclic 35-guanosine monophosphate (cGMP) since the 8-bromo analogue of cGMP induced the same effect as that observed after STa challenge. The action of STa on mucosal pH was partially inhibited by pre-administration of an antisecretory factor (ASF) preparation. The action of 8-bromo cGMP was unchanged by the presence of ASF. This implies that ASF inhibition occurs during the early stages of STa action prior to stimulation of guanylate cyclase. This effect of STa on the pig jejunal mucosal surface pH, or acid microclimate, may explain why weak acid supplementation of oral rehydration solutions can be ineffective in certain types of diarrhoeal disease.     

Influence of cloned Escherichia coli hemolysin genes, S-fimbriae and serum resistance on pathogenicity in different animal models

The virulence of the uropathogenic E. coli strain 536 (O6:K15:H31) which produces the S-fimbrial adhesin (Sfa+), is serum-resistant (Sre+) and hemolytic (Hly+) and its derivatives were assessed in five different animal models. Cloned hemolysin (hly) determinants from the chromosomes of O6, O18 and O75 E. coli strains and from the plasmid pHly152 were introduced into the spontaneous Sfa-, Sre-, Hly- mutant 536-21 and its Sfa+, Sre+, Hly- variant 536-31. As already demonstrated for the 536-21 strains (Infect. Immun. 42: 57-63) the O18-hly determinant but not the plasmid-encoded hly determinant of pHly152 transformed into 536-31 contribute to lethality in a mouse peritonitis model. Similar results were obtained with both Hly- host strains and their Hly+ transformants in a chicken embryo test and in a mouse nephropathogenicity assay in which the renal bacterial counts were measured 15 min to 8 hours after i.v. infection. S-fimbriae and serum resistance had only a marginal influence in these three in vivo systems. In contrast all three factors, S-fimbriae, serum resistance and hemolysin, were necessary for full virulence in a respiratory mouse infection assay. In a subcutaneously-induced sepsis model in the mouse restoration of S-fimbriae and serum resistance and separately chromosomally-encoded hemolysis increased virulence to a level comparable to that of the parental 536 strain.     

Identification of genomic differences between Escherichia coli strains pathogenic for poultry and E. coli K-12 MG1655 using suppression subtractive hybridization analysis

Diseases of poultry caused by Escherichia coli result in significant economic loss every year. Specific virulence factors associated with E. coli strains pathogenic for poultry have been identified, but it is likely that others remain to be identified. To identify unique DNA fragments associated with avian strains we used suppression subtractive hybridization. The genome of E. coli K-12 strain MG1655 was subtracted from the genomes of two avian E. coli strains resulting in the identification of 62 fragments specific to the two avian strains. Sequence homology analysis was done and four types of fragments were identified: plasmid sequences, phage sequences, sequences with known function and sequences without any currently known function. Two E. coli collections, a reference collection of diverse strains (ECOR) and a collection of 41 avian isolates, were screened for the presence of 25 of the 62 fragments. We identified nine fragments present in significantly more of the avian strains than of the ECOR strains. Five fragments were in significantly more of the ECOR strains than the avian strains. These results suggested that the nine fragments could play a role in the pathogenesis of E. coli as it relates to diseases of poultry.     

Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice

Systemic administration of microorganisms into tumor-bearing mice revealed preferential accumulation in tumors in comparison to clearance in organs such as spleen and liver. Here we compared the efficiency of tumor-specific colonization of pathogenic Salmonella typhimurium strains 14028 and SL1344 to the enteroinvasive Escherichia coli 4608-58 strain and to the attenuated Salmonella flexneri 2a SC602 strain, as well as to the uropathogenic E. coli CFT073, the non-pathogenic E. coli Top10, and the probiotic E. coli Nissle 1917 strain. All strains colonized and replicated in tumors efficiently each resulting in more than 1 x 10(8) colony-forming units per gram tumor tissue. Colonization of spleen and liver were significantly lower when E. coli strains were used in comparison to S. typhimurium and the non-pathogenic strains did not colonize those organs at all. Further investigation of E. coli Nissle 1917 showed that no drastic differences in colonization and amplification were seen when immunocompetent and immunocompromised animals were used, and we were able to show that E. coli Nissle 1917 replicates at the border of live and necrotic tumor tissue. We also demonstrated exogenously applied L-arabinose-dependent gene activation in colonized tumors in live mice. These findings will prepare the way for bacterium-mediated controlled protein delivery to solid tumors.     

Population characteristics of the factors of persistence ofEscherichia coli isolated from different ecological niches

The range of antilysozyme activity (the sign of persistence) ofEscherichia coli clones from subpopulations isolated from natural and artificial ecosystems is established. It is shown that heterogeneity of the microorganism population reflects the ecological status of its habitat. Therefore, it can be used as a test-indicator in biomonitoring and as an index in the standardization of eubiotics. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 9, pp. 356–358, September, 1996     

Immunocytochemical localization of the α-subunit of G-proteins in macrophages andEscherichia coli interacting with each other

The immunocytochemical localization of the α-subunit of G-proteins is established in murine macrophage-like P388D1 cells, inE. coli, and in L-forms ofE. coli. It is shown that the cytoplasmic concentration of G-proteins is increased in macrophages interacting with the bacteria. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 3, pp. 309–311, March, 1996 Presented by S. V. Prozorovskii, Member of the Russian Academy of Medical Sciences     

A rapid two dot filter assay for the detection of E. coli O157 in water samples

E. coli O157:H7 is an enterohemorrhagic bacteria that cause deadly water-borne infections implicated in outbreaks of a wide spectrum of human gastrointestinal diseases. It is therefore important to have a rapid convenient, simple and sensitive range of detection of E. coli O157:H7. A new E. coli O157 MAb designated P124 was developed for ultrasensitive detection of E. coli O157 in water, apple juice and beef for routine use. A prototype filter dot assay was designed with anti-E. coli O157 MAb bound to 0.2 microm nitrocellulose filter disk as the capture antibody. A 100 ml water sample spiked with 1-50 CFU of E. coli O157 either in the presence or absence of other non-specific bacteria were filtered for capture of the pathogen on the antibody coated nitrocellulose disk. The detection of the pathogen was successfully accomplished by the same antibody both as a capture and detecting antibody as a homosandwich. In a non-enriched format, detection of E. coli was possible with a sensitivity of 2500 CFU/100 ml. Ultrasensitive detection of ~1 CFU/100 ml sample could be achieved by a prior pathogen enrichment step before the addition of the labeled antibody. The design of this diagnostic test is based on the common architecture of all bacteria, viruses and spores, namely the manifestation of repeat lipopolysaccharide epitopes on the surface. We have developed an easy-to-use two dot visual filter assay for translation into current water testing in public health laboratories to detect E. coli O157:H7. In a 5 h assay approximately 1 CFU and approximately 5 CFU of E. coli O157 could be detected in 100 ml of water or juice and lake samples respectively. This simple homosandwich enrichment strategy can also be used to detect low levels of other water-borne pathogens.     

Invasion processes of pathogenic Escherichia coli

Pathogenic Escherichia coli causes extraintestinal infections such as urinary tract infection and meningitis, which are prevalent and associated with considerable morbidity. Previous investigations have identified common strategies evolved by pathogenic E. coli to exploit host cell function and cause extraintestinal infections, which include the invasion into non-phagocytic eukaryotic cells such as epithelial and endothelial cells and associated host cell actin cytoskeletal rearrangements. However, the mechanisms involved in pathogenic E. coli invasion of eukaryotic cells are shown to differ depending upon types of host tissues and microbial determinants. In this mini-review, invasion processes of pathogenic E. coli are discussed using E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC) as a paradigm. E. coli K1 is the most common Gram-negative organism causing neonatal meningitis, and E. coli invasion of HBMEC is shown to be a prerequisite for E. coli traversal of the blood-brain barrier in vivo. Previous studies have demonstrated that E. coli translocation of the blood-brain barrier is the result of specific E. coli host interactions including specific signal transduction pathways and modulation of endocytic pathways. Recent studies using functional genomics have identified additional microbial determinants contributing to E. coli K1 invasion of HBMEC. Complete understanding of microbial-host interactions that are involved in E. coli K1 invasion of HBMEC should help in the development of new strategies to prevent E. coli meningitis.     

Cloning, expression and characterization of the 36 KDal Salmonella typhi porin gene in Escherichia coli

A recombinant plasmid containing the gene for the 36 KDal porin of Salmonella typhi has been identified in a cosmid library of S. typhi propagated in Escherichia coli. The recombinant clone was identified by its ability to endow E. coli with susceptibility to porin specific phages, and by the appearance in the outer membrane of E. coli containing the clone of a new protein of 36 KDal. While the porin confers upon a porinless mutant of E. coli an increased susceptibility to beta-lactam antibiotics, it does not react with serum from patients with typhoid fever in immunoblotting assays.     

The lpf operon of invasive Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) strains have been shown to code for several virulence factors involved in adherence to host tissues. Here we show the existence of an additional adherence gene cluster, coding for long polar fimbriae--LPF--in several strains of serotype O78 from septicemia and newborn meningitis. The complete gene cluster was sequenced in strain 789 (lpf789), where it is located between the genes glmS and pstS, and contains four ORFs, lpfA to lpfD. The lpf operon is expressed and is important for adherence to epithelial cells. The lpf operon was found only in four of the ExPEC strains tested and is likely to have been acquired by horizontal gene transfer.     

Nucleotide sequence of a Singapore isolate of zucchini yellow mosaic virus coat protein gene revealed an altered DAG motif

A cDNA clone of zucchini yellow mosaic virus (ZYMV) RNA was mapped to the 3 terminal region. The nucleotide sequence revealed a single open reading frame of 1035 nucleotides followed by a 3 noncoding region of 215 nucleotides. The putative protease cleavage site for the release of coat protein (CP) was deduced to be between Glu-Ser (at amino acid position 66–67), which would result in a protein of 279 amino acids. This non-aphid-transmissible Singapore isolate of ZYMV showed a change of DAG to GAG triplet near the N-terminal of the CP. The CP gene was expressed as a protein fused to the -galactosidase inEscherichia coli and as an unfused protein inSaccharomyces cerevisiae.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X62662.