|Siragusa, Gregory -|
|Simmons, Ibn Abdul|
|Fouts, Derrick -|
|Kuntz, Robin -|
|Woolsey, Rebekah -|
|Schegg, Kathleen -|
|Kropinski, Andrew -|
|Ackermann, Hans -|
Submitted to: Archives of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 27, 2010
Publication Date: January 1, 2011
Citation: Seal, B.S., Siragusa, G.R., Simmons, I.M., Fouts, D.E., Garrish, J.K., Kuntz, R.L., Woolsey, R., Schegg, K.M., Kropinski, A., Ackermann, H.W. 2011. Clostridium perfringens bacteriophages FCP39O and FCP26F: genomic organization and proteomic analysis of the virions. Archives of Virology. 156(1):25-35. Interpretive Summary: The bacterium Clostridium perfringens is the third leading cause of bacterial food-borne disease among humans and is a leading cause of gastrointestinal diseases among poultry during production. Consequently bacteriophages that lyse and kill Clostridium perfringens were screened utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Bacteriophages are viruses that infect bacteria which are estimated to be the most widely distributed and diverse entities in the world. Phages can be found in all reservoirs populated by their bacterial hosts which include soil, water and the intestines of animals. These bacterial viruses have been used medically in Eastern Europe as antibacterial agents to treat diseases and there is now renewed interest in developing bacteriophages to combat bacterial diseases because of increased numbers of antibiotic resistant bacteria. However, prior to using bacteriophages or their molecular mechanisms to kill the bacterial host a thorough knowledge of the phages must be obtained so as to avoid any adverse effects on animals or humans during disease treatment. From the collection of highly lytic viruses isolated from broiler chicken offal wash and feces two isolates were designated CP39O and CP26F respectively that produced clear plaques following limit dilution plating of the virus on the host. Both viruses had morphologies typical of bacteriophages with tails as demonstrated by electron microscopy.
Technical Abstract: Initial screening for bacteriophages lytic for Clostridium perfringens was performed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Lytic phage preparations were initially characterized by transmission electron microscopy along with isolation of genomic DNA. From the collection of highly lytic viruses isolated from broiler chicken offal wash and feces two isolates were designated CP39O and CP26F respectively that produced clear plaques following limit dilution plating of the virus on the host. Both bacteriophages had icosohedral heads with approximate 100 nm tails characteristic of the Siphoviridae family in the order Caudovirales as determined by electron microscopy. The double-strand DNA genome of bacteriophage CP39O was 38,753 base pairs while the CP26F genome was 39,188 base pairs with an average 30.3% GC content encoding sixty-two (62) predicted open reading frames (ORF), of which all but one were predicted to be encoded on one strand. Predicted ORF’s included common bacteriophage elements such as the pre-neck protein with an exonuclease domain, tail protein, a tape-measure protein and portal protein along with other potential structural components. Several putative proteins including a DNA primase, single-strand DNA binding proteins, terminase, thymidylate synthase, and antirepressor or transcription factor genes were also present in the genome. Potential lytic enzymes such as a fibronectin-binding autolysin, amidase/hydrolase along with a holin and peptidase with colicin-like sequences were identified in the viral genome. Several ORF’s encoded predicted similar proteins from Clostridium spp. or other Gram-positive bacteria and bacteriophage genomes along with unknown putative Collinsella aerofaciens proteins. Proteomics analyses of the purified viruses were completed by two-dimensional gel electrophoresis followed by obtaining peptide molecular masses by MALDI-TOF-TOF MS to identify virion proteins. This resulted in identification of a putative pre-neck/exonuclease and an endopeptidase protein encoded by large open reading frames. The pre-neck/exonuclease predicted amino acid sequences had major differences in the central portion of the protein between the two phage gene products and was hypothesized to be the putative anti-receptor. Variants of the portal protein were identified and several mycobacteriophage gp6-like protein variants were detected in the greatest amounts relative to other virion proteins. Several smaller proteins with no known homologous functions were also purified with the intact viruses.