The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens.

Extracellular toxin production in Clostridium perfringens is positively regulated by the two-component regulatory genes virR and virS. Northern (RNA) blots carried out with RNA preparations from the wild-type strain 13 and the isogenic virR and virS mutants TS133 and JIR4000 showed that the virR and virS genes composed an operon and were transcribed as a single 2.1-kb mRNA molecule. Primer extension analysis led to the identification of two promoters upstream of virR. Hybridization analysis of the mutants and their complemented derivatives showed that the virR/virS system positively regulated the production of alpha-toxin (or phospholipase C, theta-toxin (perfringolysin O), and kappa-toxin (collagenase) at the transcriptional level. However, the modes of regulation of these genes were shown to differ. The theta-toxin structural gene, pfoA, had both a major and a very minor promoter, with the major promoter being virR/virS dependent. The colA gene, which encodes the kappa-toxin, had two major promoters, only one of which was virR/virS-dependent. In contrast, the alpha-toxin structural gene, p1c, had only one promoter, which was shown to be partially regulated by the virR and virS genes. Comparative analysis of the virR/virS-dependent promoters did not reveal any common sequence motifs that could represent VirR-binding sites. It was concluded that either the virR/virS system modulates its effects via secondary regulatory genes that are specific for each toxin structural gene or the VirR protein does not have a single consensus binding sequence.

Chimeric potyvirus-like particles as vaccine carriers.

Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune responses. Coat protein (CP) monomers of a potyvirus (Johnsongrass mosaic virus) when produced in heterologous host expression systems (Escherichia coli, yeast and insect cells) self-polymerized to produce potyvirus-like particles (PVLPs). The N- and C-terminal regions of potyvirus CP are surface-exposed and are not required for assembly. Hybrid CP monomers containing short peptides fused to their N- and/or C-termini, or large target antigens fused to the N-terminus or replacing most of the N- or C-terminal exposed regions retained the ability to assemble into hybrid PVLPs. Such chimeric PVLPs were highly immunogenic in mice and rabbits even in the absence of any adjuvant. Potyvirus CP is highly versatile in accommodating peptides or large antigens and is able to present antigens exposed on the surface of virus-like particles. This, combined with the efficiency of high level bacterial and insect cell expression systems, makes PVLPs an attractive non-pathogenic and non-replicative vaccine carrier.

Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens.

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb HindIII fragment in the C. perfringens chromosome. A 4.3kb PstI fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb HindIII fragment, or the 4.3 kb PstI fragment, resulted in toxin and enzyme levels greater than or equal to those of the wild-type strain. The PstI fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30,140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51,274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved in virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.

High level production of potyvirus-like particles in insect cells infected with recombinant baculovirus.

The full length gene for the coat protein (CP) of the potyvirus, Johnsongrass mosaic virus, was incorporated into recombinant baculovirus and expressed in insect cells. Western blot and Coomassie-stained polyacrylamide gel electrophoresis analysis of infected insect cells demonstrated that CP was produced in large quantity. Electron microscopic examination of these cells showed the presence of numerous potyvirus-like particles in the cytoplasm. Morphologically the particles resembled potyvirus particles assembled in vitro in the absence of viral RNA and those found in Escherichia coli expressing the recombinant CP gene.

High level production of hybrid potyvirus-like particles carrying repetitive copies of foreign antigens in Escherichia coli.

Synthesis in E. coli of native coat protein of Johnsongrass mosaic virus, and hybrid protein molecules containing foreign antigens, resulted in the intracellular formation of potyvirus-like particles (PVLPs). The foreign antigens used were an octapeptide epitope from Plasmodium falciparum and a decapeptide hormone (luteinizing hormone releasing hormone) at the N- or at both N- and C-terminal regions of the coat protein molecule, and a full length protein antigen (Sj26-glutathione S-transferase of 26 kD from Schistosoma japonicum) replacing the N-terminal 62 amino acids of the coat protein. Electron microscopy of ultrathin sections of E. coli revealed that PVLPs resulting from coat protein molecules containing peptide fusions appeared in vast arrays of parallel strands within the cytoplasm sometimes extending the length of the cell and at times the cells were strung together, with "threads" of PVLPs appearing to connect individual bacterial cells. PVLPs resulting from the fusion of the 26 kD antigen Sj26 to coat protein were shorter and wider. The physical form of the high molecular weight PVLPs enabled purification by simple size exclusion column chromatography. The Sj26-PVLPs administered to mice without adjuvant elicited antibody responses comparable to monomeric Sj26 administered with Freund's Complete Adjuvant.

Production of an enzymatically inactive analog of phospholipase D from Corynebacterium pseudotuberculosis.

The gene pld, encoding the phospholipase D (PLD) of Corynebacterium pseudotuberculosis, was mutagenized using formic acid and then expressed in Escherichia coli. Mutagenesis was targeted at the coding region of pld, so as to produce only one or a limited number of point mutations. Transformants were screened for the enzymatic and immunological properties of their PLD products. One clone was found to produce a protein which was enzymatically inactive, but which was comparable to the wild-type PLD in size and antigenicity. The sequence of the pld mutant revealed a single base change. As a consequence, the codon for His20 was converted to Tyr. These results suggest that His20 forms part of the active site of the PLD molecule. If this protein is immunogenic in sheep, it would form the basis of a genetically inactivated vaccine.

Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosis of pertussis using the polymerase chain reaction.

A tandemly repeated 1,046-base-pair (bp) ClaI DNA fragment from Bordetella pertussis was cloned into Escherichia coli by using the vector pUC19. This fragment, when isolated, hybridized strongly to DNA from all 100 clinical isolates of B. pertussis tested. It was shown to have homology to single-copy sequences in Bordetella bronchiseptica but not Bordetella parapertussis and did not hybridize to lysate blots of a wide range of other bacteria, including members of the closely related genera Pasteurella, Alcaligenes, and Haemophilus. The 1,046-bp fragment was sequenced, and complementary synthetic oligonucleotides flanking a 153-bp region within the repeated element were used as primers for specific amplification of this region using the polymerase chain reaction (PCR). This procedure was then applied to the rapid (5-h) detection of B. pertussis in nasopharyngeal secretions collected from 332 children with suspected pertussis. The test yielded positive results in a total of 98 samples, compared with 66 for culture and 33 for direct immunofluorescence (IF). All of the IF-positive samples were PCR positive, as were 63 of the samples from which B. pertussis was eventually cultured. Two hundred thirty-one specimens which were negative by IF and culture were also negative in the PCR assay. However, 33 culture- and IF-negative specimens were positive by PCR assay. Several of these specimens were collected from close contacts of culture-proven pertussis patients, were follow-up specimens from such patients, or were from patients with serological evidence of pertussis and therefore may be true-rather than false-positives.

Cloning, nucleotide sequence, and expression in Escherichia coli of the phospholipase D gene from Corynebacterium pseudotuberculosis.

The phospholipase D (PLD) gene from Corynebacterium pseudotuberculosis has been cloned, sequenced, and expressed in Escherichia coli. Analysis of DNA sequence data reveals a major open reading frame encoding a 31.4-kilodalton protein, a size consistent with that estimated for the PLD protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of these data with the amino-terminal protein sequence indicates that the mature PLD protein is preceded by a 24-residue signal sequence. Expression of the PLD gene in E. coli is initiated from the corynebacterial promoter, and the resulting protein has sphingomyelinase activity. Primer extension mapping localized the 5' end of the PLD gene mRNA to a site 5 to 7 base pairs downstream of a region similar to the consensus sequence for E. coli promoters. Northern and Southern blot analyses suggest that the gene is transcribed from mRNA approximately 1.1 kilobases in length and that it is present in a single copy within the C. pseudotuberculosis genome.

Antiviral and antiproliferative activities of interferon-alpha 1: the role of cysteine residues.

Site-specific in vitro mutagenesis was used to direct serine for cysteine substitutions within the sequence of human interferon-alpha 1 (IFN-alpha 1). Antiviral specific activities and antiproliferative activities of IFN-alpha 1 analogs, expressed in M13 as fusion proteins, were assessed following purification by monoclonal antibody affinity chromatography. Based on analysis of IFN-alpha 2, IFN-alpha 1 contains two disulfide bridges between cysteine residues 29 and 139 and cysteine residues 1 and 99. IFN-alpha 1 also contains a fifth cysteine residue at position 86. The series of serine for cysteine substitutions performed indicated that IFN-alpha 1 molecules unable to form the residue 29 to residue 139 disulfide bridge have substantially reduced antiviral and antiproliferative activities, IFN-alpha 1 molecules unable to form the residue 1 to residue 99 disulfide bridge have only marginally altered antiviral and antiproliferative activities, the low antiviral activity of IFN-alpha 1 compared with other human IFN-alpha subtypes is not due to the formation of nonnative disulfide bridges involving the fifth cysteine residue at position 86, which the other subtypes lack, and (iv) the reduced biological activities of certain analogs may be due to the formation of nonnative disulfide bridges.

Single amino acid substitutions at conserved residues of human interferon-alpha can effect antiviral specific activity.

Site-specific in vitro mutagenesis was used to direct various amino acid substitutions at conserved positions within the sequence of human interferon-alpha 1 (IFN-alpha 1). The antiviral specific activity of IFN-alpha 1, expressed in M13 as a fusion protein [IFN-alpha 1 (phi WT)], could be altered by single amino acid substitutions. The substitution of glycine for tyrosine at position 123 results in a loss of more than 99% of the antiviral specific activity on human cells, but causes no significant change in the antiviral specific activity on primary bovine cells. The tyrosine at position 123 is thus implicated in determining human cell specificity. Based on analysis of IFN-alpha 2, IFN-alpha 1 contains two dulsulphide bridges between cysteine residues 29 and 139 and cysteine residues 1 and 99. IFN-alpha 1 also contains a fifth cysteine residue at position 86. IFN-alpha 1 (phi WT) carrying three serine for cysteine substitutions at positions 1, 86 and 99 retains 23% of the antiviral specific activity of IFN-alpha 1 (phi WT) on human cells. However, the antiviral activity on bovine cells is not significantly affected by this modification. The presence of the disulphide bridge between residues 1 and 99 thus appears to be required for full antiviral activity on human but not bovine cells. A single serine for cysteine substitution at position 29 reduces the antiviral specific activity on both human and bovine cells by some 95%. This data shows that the disulphide bridge between residues 29 and 139 is critical for the antiviral activity of IFN-alpha's.

Nucleotide sequence and expression in E. coli of a human interferon-alpha gene selected from a genomic library using synthetic oligonucleotides.

The complete nucleotide sequence of a human interferon-alpha gene is reported. The gene, designated IFN-alpha M1, was isolated from a human genomic library in phage lambda Charon 4A using synthetic oligonucleotides as hybridization probes. Based on a comparison of nucleotide sequence data obtained from this recombinant phage with published interferon-alpha gene sequences, a region of DNA capable of coding for a pre-interferon of 189 amino acids was identified. An AluI fragment containing the coding region for the mature interferon was inserted into the HincII site of the phage M13mp11, resulting in a fusion of portions of the IFN-alpha M1 and the beta-galactosidase genes. Antiviral activity was detected in extracts from E. coli infected with the recombinant M13 phage carrying the fused gene. The antiviral activity was completely neutralized by antibodies to human interferon-alpha.

Chlorhexidine as an effective agent against Chlamydia trachomatis in vitro and in vivo.

In a small field trial, chlorhexidine (50 micrograms/ml) was as effective against clinical trachoma as topical tetracyclines or oral co-trimoxazole; in vitro, 2.5 micrograms/ml was chlamydicidal.