A comprehensive study on the role of the Yersinia pestis virulence markers in an animal model of pneumonic plague.

We determined the role of Yersinia pestis virulence markers in an animal model of pneumonic plague. Eleven strains of Y. pestis were characterized using PCR assays to detect the presence of known virulence genes both encoded by the three plasmids as well as chromosomal markers. The virulence of all Y. pestis strains was compared in a mouse model for pneumonic plague. The presence of all known virulence genes correlated completely with virulence in the Balb/c mouse model. Strains which lacked HmsF initially exhibited visible signs of disease whereas all other strains (except wild-type strains) did not exhibit any disease signs. Forty-eight hours post-infection, mice which had received HmsF(-) strains regained body mass and were able to control infection; those infected with strains possessing a full complement of virulence genes suffered from fatal disease. The bacterial loads observed in the lung and other tissues reflected the observed clinical signs as did the cytokine changes measured in these animals. We can conclude that all known virulence genes are required for the establishment of pneumonic plague in mammalian animal models, the role of HmsF being of particular importance in disease progression.

The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins.

We describe the cloning and characterisation of the BiP gene homologues of the filamentous fungi Aspergillus niger and Aspergillus awamori. The BiP genes of these black Aspergilli encode an identical protein of 672 amino acids, which has a high homology with the BiP protein from Saccharomyces cerevisiae and contains a putative signal sequence of 38 amino acids. The DNA sequences of the Aspergillus BiP genes diverge in particular in the three intronic sequences and the 5'- and 3'- noncoding regions. Sequences resembling Heat Shock Elements (HSE) and Unfolded Protein Response (UPR) elements, as found in the yeast KAR2 promoter, are present in the 5' non-transcribed regions of both genes. The expression of the A. niger bipA gene is increased by heat shock and tunicamycin treatment.

Secretion of heterologous proteins by Aspergillus niger: production of active human interleukin-6 in a protease-deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein.

To develop improved methods for heterologous protein production in Aspergillus niger, we studied the secretion of human interleukin-6 (hIL6). Since in vitro experiments with culture medium revealed that hIL6 was rapidly degraded, several protease-deficient strains of A. niger were isolated and tested for reduced degradation of hIL6 compared with the wild-type strain. The mutant strain giving the least degradative effect on hIL6 (designated AB1.13) was transformed with several hIL6-expression plasmids. Initially, hIL6 was expressed using various signal sequences fused to the sequence of mature hIL6. The resulting transformants did not produce detectable amounts of hIL6, despite high transcription levels in one transformant. We hypothesized that hIL6 was not efficiently processed during passage along the secretion pathway. Therefore, hIL6 was expressed as a fusion protein with glucoamylase, a protein which is efficiently secreted by A. niger and expression of which can easily be measured enzymatically. To obtain mature hIL6, a sequence encoding the KEX2 cleavage-site (Lys-Arg) was inserted between glucoamylase and hIL6 sequences. Mature active hIL6 was found to be secreted in the extracellular medium. Using this combined approach of transforming a protease-deficient strain with a fusion construct containing the KEX2 site, up to 15 mg l-1 active hIL6 was obtained in shake-flask culture. A fusion construct without the KEX2 site resulted in substantially higher production of the fusion protein, but hIL6 was not active in the fused form. These results indicate that A. niger contains a protease with similar specificity as the KEX2 protease from yeast.

Fusion proteins with multiple copies of the major antigenic determinant of foot-and-mouth disease virus protect both the natural host and laboratory animals.

Proteins consisting of one, two or four copies of the amino acid sequence 137 to 162, which contains the major immunogenic site of VP1 of foot-and-mouth disease virus, attached to the N-terminus of beta-galactosidase have been expressed in Escherichia coli cells. In guinea-pigs the protein containing one copy (P71) of the viral determinant elicited only low levels of neutralizing antibody whereas protective levels were elicited by the proteins containing two (P72) or four (P74) copies of the determinant. Single inoculations of the P72 and P74 proteins containing as little as 2 micrograms or 0.8 micrograms of peptide respectively were sufficient to protect all the animals against challenge infection. Moreover, the equivalent of 40 micrograms of peptide in P74 protected pigs against challenge infection after one inoculation.

Synthesis of fusion proteins containing antigenic determinants of foot-and-mouth disease virus.

Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence encoding the capsid polypeptide VP1, was cloned in Escherichia coli following a new cloning strategy. The clone containing the VP1 sequence was used for the construction of two expression plasmids encoding VP1 fusion proteins. Subsequently, substantial amounts of the two VP1-beta-galactosidase fusion proteins, containing either one (amino acid region 140-160) or two (amino acid regions 140-160 and 200-213) antigenic determinants of the virus, were synthesized by E. coli bacteria. The protein containing the amino acid region 140-160 of VP1 fused to beta-galactosidase efficiently induced antibodies in rabbits specifically reacting with FMDV type 01,BFS. The same protein was also capable of eliciting neutralizing antibodies. The fusion protein containing both antigenic determinants did not efficiently induce antibodies reacting with FMDV.

Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus.

A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease virus (FMDV) sequences was constructed. The fusion proteins contain a large part of beta-galactosidase from Escherichia coli preceded (N-terminal) by 1, 2, 4 or 8 repeats of the antigenic determinant of FMDV consisting of amino acids 137-162 of the capsid polypeptide VP1. All four fusion proteins were efficiently produced in E. coli host bacteria. Immunization of rabbits resulted in FMDV-specific, neutralizing antibodies, the response being dependent on the number of repeats. With enzyme-linked immunosorbent-assay techniques it was shown that the FMDV antigenic determinants are exposed on the surface of the fusion proteins under non-denaturing conditions.