Purification and characterization of a recombinant alpha-N-acetylgalactosaminidase from Clostridium perfringens

Clostridium perfringens alpha-N-acetylgalactosaminidase (alphaNAG) hydrolyzed the terminal N-acetyl-alpha-d-galactosamine from the blood type A(2) antigen producing H antigen, blood type O. Blood type O is universally compatible in the ABO system. Purification of the native enzyme is difficult with very low yields. To obtain the enzyme in satisfactory yield, the gene encoding the clostridial enzyme was cloned in an Escherichia coli T7 expression system. A highly purified preparation of recombinant alphaNAG was obtained from cell lysates by ion-exchange chromatography and high-pressure liquid chromatography. The final preparation was homogeneous by SDS-PAGE with a molecular mass of 71.96kDa and the native molecular weight of 72.42kDa. The enzyme was highly selective for terminal N-acetylgalactosamine residues. No other significant exoglycosidase activities, particularly neuraminidase, were detected. The pH optimum of the enzyme was between 6.5 and 7.0 and activity was relatively unaffected by ionic strength. ELISA experiments demonstrated activity against blood type A(2) epitope. These characteristics were similar to those of native alphaNAG from C. perfringens. With adequate expression in E. coli, sufficient recombinant alphaNAG enzyme mass can be obtained for potential use in enzymatic conversion of human blood type A(2) red blood cells to universally transfusable type O red blood cells.     

Identification of a novel locus that regulates expression of toxin genes in Clostridium perfringens

A novel gene that regulates the alpha-toxin (plc), kappa-toxin (colA), and theta;-toxin (pfoA) genes was identified using toxin-negative mutant strains of Clostridium perfringens. The cloned 3.2-kb fragment contained the virX gene encoding a 51-amino acid polypeptide of unknown function that seemed to be responsible for the activation of toxin genes. The virX knock out mutant of wild-type strain 13 showed a reduced expression of the plc, colA, and pfoA genes, which was complemented by the transformation of the intact virX gene. Deletion and site-directed mutagenesis studies suggested that the virX gene acts as a regulatory RNA rather than as a peptide regulator. The virX locus found in this study might play a part in the signal transduction to regulate toxin production in C. perfringens.     

Genetic analysis of the ycgJ-metB-cysK-ygaG operon negatively regulated by the VirR/VirS system in Clostridium perfringens

The 5'-flanking region of the metB-cysK-ygaG operon, whose expression is negatively regulated by the VirR/VirS system in C. perfringens, was analyzed. The region contained the ycgJ, mscL, and colA genes encoding a hypothetical protein, a large conductance mechanosensitive channel protein, and kappa-toxin (collagenase), respectively. Northern analysis revealed that the ycgJ gene was transcribed as a 4.9-kb operon together with the metB-cysK-ygaG genes and that this operon was negatively regulated by the VirR/VirS system. It is indicated that the pfoA (theta-toxin or perfringolysin O), colA, and ycgJ-metB-cysK-ygaG genes that belong to the VirR/VirS regulon are situated very close together in a 26.5-kb region of the chromosome, but do not form a pathogenic island.     

Molecular cloning and functional expression of rat liver cytosolic acetyl-CoA hydrolase.

A cytosolic acetyl-CoA hydrolase (CACH) was purified from rat liver to homogeneity by a new method using Triton X-100 as a stabilizer. We digested the purified enzyme with an endopeptidase and determined the N-terminal amino-acid sequences of the two proteolytic fragments. From the sequence data, we designed probes for RT-PCR, and amplified CACH cDNA from rat liver mRNA. The CACH cDNA contains a 1668-bp ORF encoding a protein of 556 amino-acid residues (62 017 Da). Recombinant expression of the cDNA in insect cells resulted in overproduction of functional acetyl-CoA hydrolase with comparable acyl-CoA chain-length specificity and Michaelis constant for acetyl-CoA to those of the native CACH. Database searching shows no homology to other known proteins, but reveals high similarities to two mouse expressed sequence tags (91% and 93% homology) and human mRNA for KIAA0707 hypothetical protein (50% homology) of unknown function.     

Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5

The nucleotide sequence of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 was determined. The gene consists of an open reading frame of 3453 nucleotides and encodes a protein of 1151 amino acids with a molecular mass of 126408 Da. The deduced amino acid sequence of CBPA contained one domain highly similar to a catalytic domain of glycosyl hydrolases belonging to family 9, two linker-like domains and four domains of unknown function. Among the four domains of unknown function, the domains 1 and 2 region had significant homology in amino acid sequence with the cellulose-binding domains in the family 9 glycosyl hydrolases. The cloned gene was inserted into an expression vector, pBAD-TOPO, and expressed in Escherichia coli as a fused protein. The fused protein was detected by immunoblotting using antiserum against CBPA.     

Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis

Clostridium perfringens (C. perfringens) has been developed as a potential oral delivery vehicle to deliver antigens or therapeutic compounds to Gut-Associated Lymphoid Tissue (GALT). However, this recombinant C. perfringens carries a plasmid-encoded expression system, which raises several safety concerns regarding possible horizontal plasmid transfer and spread of plasmid-associated antibiotic resistant genes. Furthermore, this bacterium produces the extracellular theta toxin, which poses a potential safety issue for general administration. Using a Clostridium-specific-targetron donor plasmid, we inserted the Simian Immunodefiency Virus (SIV) p27 gene into the theta toxin gene (pfoA) on the C. perfringens chromosome, which simultaneously inactivated the theta gene and introduced SIV p27 gene onto the bacterial chromosome. Such mutant C. perfringens without an input plasmid or antibiotic resistant gene stably produced a large amount of SIV p27 protein during sporulation and did not produce theta toxin. Upon oral feeding of the mutant bacteria to mice, intact p27 protein was detected in the lower GI tract. The re-engineered C. perfringens provides a biosafe efficient oral vehicle to deliver antigen to the gastrointestinal tract.