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Arcanobacterium haemolyticum phospholipase D is genetically and functionally similar to Corynebacterium
1Department of Veterinary Science, University of Arizona, Tucson 85721.
Abstract:
Arcanobacterium haemolyticum, a pathogen of the human upper respiratory tract and other systems, has been reported to produce soluble toxins, including a phospholipase D (PLD). We confirmed production of PLD by this organism and cloned and sequenced pld. Arcanobacterial PLD (PLD-A) was found to be a protein of approximately 31.5 kDa with a pI of approximately 9.4. Cosmid cloning, followed by subcloning into phagemid pBluescriptIISK+, yielded Escherichia coli(pAh140), a recombinant with a gene product corresponding to PLD-A. Evidence of PLD activity was found by three assays in supernatant fluid of cultures of E. coli(pAh140) and A. haemolyticum, but not in E. coli(pBluescriptIISK+). Experiments to determine if this protein was secreted were not conducted, but previous work with PLD from Corynebacterium pseudotuberculosis suggested that the presence of the enzyme in culture supernatant fluids was due to lysis of E. coli rather than to active transport. Antibodies in polyclonal sera from goats immunized with native or recombinant PLD-A neutralized native and recombinant PLD-A activity, and antibodies against native or recombinant PLD from C. pseudotuberculosis (PLD-P) partially neutralized native and recombinant PLD-A. Antibodies prepared against recombinant PLD-A labelled both recombinant and native PLD-A in Western blots (immunoblots) and dot blots, but antibodies against PLD-P did not. Sequencing of the insert in pAh140 revealed an open reading frame of 930 bp coding for 309 amino acids, including a putative signal sequence of 26 amino acids (3.2 kDa, determined on the basis of homology with the 24-amino-acid signal sequence of pld from C. pseudotuberculosis bv. ovis) and the mature PLD protein (31.5 kDa). Sequence comparisons of coding regions revealed 65% DNA homology with pld genes from C. pseudotuberculosis and Corynebacterium ulcerans. Comparison of amino acid sequences revealed 64% homology of PLD-A both with PLD-P and with PLD produced by C. ulcerans.
Insights
This study confirms Arcanobacterium haemolyticum produces phospholipase D (PLD). The gene was cloned and sequenced, revealing a 31.5 kDa protein with significant homology to related bacterial PLDs.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Arcanobacterium haemolyticum is a pathogen associated with upper respiratory tract infections.
- This bacterium is known to produce soluble toxins, including phospholipase D (PLD).
Purpose of the Study:
- To confirm PLD production by Arcanobacterium haemolyticum.
- To clone, sequence, and characterize the gene encoding PLD from A. haemolyticum.
- To investigate the immunological relationship between Arcanobacterium haemolyticum PLD and PLDs from related species.
Main Methods:
- Cloning and sequencing of the pld gene from A. haemolyticum.
- Expression of recombinant PLD in Escherichia coli.
- Enzyme activity assays to confirm PLD function.
- Immunological assays including Western blots, dot blots, and neutralization tests using antibodies against native and recombinant PLD-A and PLD-P.
Main Results:
- The pld gene from A. haemolyticum was successfully cloned and sequenced, yielding a 31.5 kDa protein (PLD-A).
- Recombinant E. coli expressing the gene showed PLD activity, confirming gene function.
- Antibodies against PLD-A neutralized its activity and showed cross-reactivity with PLD from Corynebacterium pseudotuberculosis (PLD-P).
- Sequence analysis revealed 65% DNA homology and 64% amino acid homology with pld genes from C. pseudotuberculosis and Corynebacterium ulcerans.
Conclusions:
- Arcanobacterium haemolyticum produces a distinct phospholipase D (PLD-A) with characteristics similar to PLDs from related Corynebacterium species.
- The cloned and sequenced PLD-A provides a basis for further immunological and functional studies.
- The findings suggest potential cross-reactivity and shared evolutionary origins among PLDs from these bacterial species.