Peptidoglycan lytic activity of the Pseudomonas aeruginosa phage phiKZ gp144 lytic transglycosylase.
The gp144 endolysin gene from the Pseudomonas aeruginosa phage phiKZ was cloned and studies of gp144 expression into Escherichia coli showed host cell lysis. The gp144 protein was purified directly from the culture supernatant and from the bacterial cell pellet and showed in vitro antibacterial lytic activity against P. aeruginosa bacteria and degraded purified peptidoglycan of Gram-negative bacteria. MS analysis identified the gp144 peptidoglycan cleavage site and confirmed a lytic transglycosylase enzyme. Studies of gp144 expression in the presence of sodium azide (NaN(3)), an inhibitor of the protein export machinery, and into an E. coli MM52 secA(ts) mutant at permissive and restrictive temperatures showed that gp144 was secreted independently of the Sec system. The solution conformation of purified gp144 analyzed by circular dichroism spectroscopy was 61% in alpha-helical content, and showed a 72% decrease when interacting with dimyristoylphosphatidylglycerol (DMPG), one of the major components of bacterial membranes and less than 10% with dimyristoylphosphatidylcholine (DMPC) found in eukaryotic membranes. Membrane vesicles of DMPG anionic lipids containing calcein indicated that gp144 caused a rapid release of fluorescent calcein when interacting with synthetic membranes. These results indicated that gp144 from phiKZ is a lytic transglycosylase capable of interacting with and disorganizing bacterial membranes and has potential as an antipseudomonal in phage therapy.
Paradis-Bleau
Catherine
C
Département de Biologie Médicale, Faculté de Médecine, Universite Laval, Sainte-Foy, Quebec, Canada.
Cloutier
Isabelle
I
Lemieux
Lise
L
Sanschagrin
François
F
Laroche
Jérôme
J
Auger
Michèle
M
Garnier
Alain
A
Levesque
Roger C
RC
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001433:Bacteriolysis; D002240:Carbohydrate Sequence; D002942:Circular Dichroism; D019295:Computational Biology; D015870:Gene Expression / Q000187:drug effects; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000494:pharmacology; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism*; D046928:Peptidoglycan Glycosyltransferase / Q000235:genetics / Q000378:metabolism*; D017105:Pseudomonas Phages / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013379:Substrate Specificity
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D010457:Peptidoglycan; D000069816:SEC Translocation Channels; C003320:sodium nitride; D046928:Peptidoglycan Glycosyltransferase; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; D009584:Nitrogen