Production of a heterologous recombinant protein using fragments of the glyceraldehyde-3-phosphate dehydrogenase promoter from Penicillium camemberti

The biotechnological applications of cheese-ripening fungi have been limited by a lack of genetics tools, in particular the identification and characterization of suitable promoters for protein expression. In this study, the suitability of the glyceraldehyde-3-phosphate dehydrogenase (gpdP) promoter from Penicillium camemberti to drive the production of a recombinant protein was evaluated. The gpdP gene and its promoter were isolated using PCR and Genome Walker. The promoter of gpdP has two regions with high identity to the regulatory elements gpd-box and ct-box previously described in Aspergillus nidulans. Two fragments of the promoter containing the gpd- and ct-box or the ct-box alone were used to drive the in vivo production of recombinant β-galactosidase using A. nidulans as host. Our results indicate that larger fragment containing gpd-box enhances the production of β-galactosidase activity levels respect to ct-box alone, and that both boxes are necessary to obtain maximal enzymatic activity production. The smaller fragment (187 nt) containing the ct-box alone was able to trigger up to 27% of β-galactosidase activity, and to our knowledge this is the smallest fragment from a gpd gene used to produce a recombinant protein. Differences were not observed when glycerol, galactose or glucose were used as carbon sources, suggesting that the promoter activity is carbohydrate-independent. This is the first report in which a Penicillium gpd promoter is used for recombinant protein production. Our results open the way for the future development of a system for recombinant proteins expression in the biotechnologically important cheese-ripening fungus P. camemberti.     

Bovine CCL28 Mediates Chemotaxis via CCR10 and Demonstrates Direct Antimicrobial Activity against Mastitis Causing Bacteria

In addition to the well characterized function of chemokines in mediating the homing and accumulation of leukocytes to tissues, some chemokines also exhibit potent antimicrobial activity. Little is known of the potential role of chemokines in bovine mammary gland health and disease. The chemokine CCL28 has previously been shown to play a key role in the homing and accumulation of IgA antibody secreting cells to the lactating murine mammary gland. CCL28 has also been shown to act as an antimicrobial peptide with activity demonstrated against a wide range of pathogens including bacteria, fungi and protozoans. Here we describe the cloning and function of bovine CCL28 and document the concentration of this chemokine in bovine milk. Bovine CCL28 was shown to mediate cellular chemotaxis via the CCR10 chemokine receptor and exhibited antimicrobial activity against a variety of bovine mastitis causing organisms. The concentration of bovine CCL28 in milk was found to be highly correlated with the lactation cycle. Highest concentrations of CCL28 were observed soon after parturition, with levels decreasing over time. These results suggest a potential role for CCL28 in the prevention/resolution of bovine mastitis.     

Adaptation of Pandemic H2N2 Influenza A Viruses in Humans

The 1957 A/H2N2 influenza virus caused an estimated 2 million fatalities during the pandemic. Since viruses of the H2 subtype continue to infect avian species and pigs, the threat of reintroduction into humans remains. To determine factors involved in the zoonotic origin of the 1957 pandemic, we performed analyses on genetic sequences of 175 newly sequenced human and avian H2N2 virus isolates and all publicly available influenza virus genomes.     

Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma-infected Chlamydia sp. strains.

Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by the combined use of enzymatic treatment and differential centrifugation, and the use of detergents. With these methods only incubation with Triton X-100 resulted in decontamination of Mycoplasma-contaminated suspensions of several laboratory strains of Chlamydia pneumoniae, C. pecorum, and C. trachomatis. Only one C. pneumoniae strain, UZG-1, was sensitive to Triton X-100 treatment. Since 39 of 40 throat swabs from patients with symptoms of an upper respiratory tract infection had positive reactions in the Mycoplasma group-specific PCR, this procedure could also have clinical significance in attempts to propagate C. pneumoniae strains from clinical specimens.     

Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.)

Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R₂ seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP 6-benzylaminopurine - 2,4-D dichloro-phenoxyacetic acid - 2iP 6-(, ,-dimethylallyl-amino)-purine - IBA indole-3-butyric acid - IAA indole-3-acetic acid - Km kanamycin - NPTII neomycin phosphotransferase II     

Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls

In wild aquatic birds and poultry around the world, influenza A viruses carrying 15 antigenic subtypes of hemagglutinin (HA) and 9 antigenic subtypes of neuraminidase (NA) have been described. Here we describe a previously unidentified antigenic subtype of HA (H16), detected in viruses circulating in black-headed gulls in Sweden. In agreement with established criteria for the definition of antigenic subtypes, hemagglutination inhibition assays and immunodiffusion assays failed to detect specific reactivity between H16 and the previously described subtypes H1 to H15. Genetically, H16 HA was found to be distantly related to H13 HA, a subtype also detected exclusively in shorebirds, and the amino acid composition of the putative receptor-binding site of H13 and H16 HAs was found to be distinct from that in HA subtypes circulating in ducks and geese. The H16 viruses contained NA genes that were similar to those of other Eurasian shorebirds but genetically distinct from N3 genes detected in other birds and geographical locations. The European gull viruses were further distinguishable from other influenza A viruses based on their PB2, NP, and NS genes. Gaining information on the full spectrum of avian influenza A viruses and creating reagents for their detection and identification will remain an important task for influenza surveillance, outbreak control, and animal and public health. We propose that sequence analyses of HA and NA genes of influenza A viruses be used for the rapid identification of existing and novel HA and NA subtypes.     

A crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion, and virulence

Biofilms play an important role in many chronic bacterial infections. Production of an extracellular mixture of sugar polymers called exopolysaccharide is characteristic and critical for biofilm formation. However, there is limited information about the mechanisms involved in the biosynthesis and modification of exopolysaccharide components and how these processes influence bacterial pathogenesis. Staphylococcus epidermidis is an important human pathogen that frequently causes persistent infections by biofilm formation on indwelling medical devices. It produces a poly-N-acetylglucosamine molecule that emerges as an exopolysaccharide component of many bacterial pathogens. Using a novel method based on size exclusion chromatography-mass spectrometry, we demonstrate that the surface-attached protein IcaB is responsible for deacetylation of the poly-N-acetylglucosamine molecule. Most likely due to the loss of its cationic character, non-deacetylated poly-acetylglucosamine in an isogenic icaB mutant strain was devoid of the ability to attach to the bacterial cell surface. Importantly, deacetylation of the polymer was essential for key virulence mechanisms of S. epidermidis, namely biofilm formation, colonization, and resistance to neutrophil phagocytosis and human antibacterial peptides. Furthermore, persistence of the icaB mutant strain was significantly impaired in a murine model of device-related infection. This is the first study to describe a mechanism of exopolysaccharide modification that is indispensable for the development of biofilm-associated human disease. Notably, this general virulence mechanism is likely similar for other pathogenic bacteria and constitutes an excellent target for therapeutic maneuvers aimed at combating biofilm-associated infection.     

HrpA, a DEAH-box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli

Endonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for co-ordinate regulation of genes involved in F1845 fimbrial biogenesis. Cleavage occurs by an unidentified endoribonuclease, is translation dependent and involves a unique recognition mechanism. Here, we present the results of a genetic strategy used to identify factors involved in daa mRNA processing. We used a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP). A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry. To determine the location of mutations, two different genetic approaches, Hfr crosses and P1 transductions, were used. The mutation responsible for the processing defect was subsequently mapped to the 32 min region of the E. coli chromosome. A putative DEAH-box RNA helicase-encoding gene at this position, hrpA, was able to restore the ability of the mutant to cleave daa mRNA. Site-directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore the processing defect in the mutant. We propose that HrpA is a novel enzyme involved in mRNA processing in E. coli.