title: Mechanisms for Pseudoalteromonas piscicida-Induced Killing of Vibrios and Other Bacterial Pathogens.
abstract: Pseudoalteromonas piscicidais a Gram-negative gammaproteobacterium found in the marine environment. Three strains of pigmentedP. piscicidawere isolated from seawater and partially characterized by inhibition studies, electron microscopy, and analysis for proteolytic enzymes. Growth inhibition and death occurred around colonies ofP. piscicidaon lawns of the naturally occurring marine pathogensVibrio vulnificus,Vibrio parahaemolyticus,Vibrio cholerae,Photobacterium damselae, andShewanella algaeInhibition also occurred on lawns ofStaphylococcus aureusbut not onEscherichia coliO157:H7 orSalmonella entericaserovar Typhimurium. Inhibition was not pH associated, but it may have been related to the secretion of a cysteine protease with strong activity, as detected with a synthetic fluorogenic substrate. This diffusible enzyme was secreted from all threeP. piscicidastrains. Direct overlay of thePseudoalteromonascolonies with synthetic fluorogenic substrates demonstrated the activity of two aminopeptidase Bs, a trypsin-like serine protease, and enzymes reactive against substrates for cathepsin G-like and caspase 1-like proteases. In seawater cultures, scanning electron microscopy revealed numerous vesicles tethered to the outer surface ofP. piscicidaand a novel mechanism of direct transfer of these vesicles toV. parahaemolyticusVesicles digested holes inV. parahaemolyticuscells, while theP. piscicidacongregated around the vibrios in a predatory fashion. This transfer of vesicles and vesicle-associated digestion of holes were not observed in other bacteria, suggesting that vesicle binding may be mediated by host-specific receptors. In conclusion, we show two mechanisms by whichP. piscicidainhibits and/or kills competing bacteria, involving the secretion of antimicrobial substances and the direct transfer of digestive vesicles to competing bacteria.IMPORTANCEPseudoalteromonasspecies are widespread in nature and reduce competing microflora by the production of antimicrobial compounds. We isolated three strains ofP. piscicidaand characterized secreted and cell-associated proteolytic enzymes, which may have antimicrobial properties. We identified a second method by whichP. piscicidakillsV. parahaemolyticusIt involves the direct transfer of apparently lytic vesicles from the surface of thePseudoalteromonasstrains to the surface ofVibriocells, with subsequent digestion of holes in theVibriocell walls. Enzymes associated with these vesicles are likely responsible for the digestion of holes in the cell walls.Pseudoalteromonas piscicidahas potential applications in aquaculture and food safety, in control of the formation of biofilms in the environment, and in food processing. These findings may facilitate the probiotic use ofP. piscicidato inactivate pathogens and may lead to the isolation of enzymes and other antimicrobial compounds of pharmacological value.
authors.affiliation: U.S. Department of Agriculture, Agricultural Research Service, Dover, Delaware, USA gary.richards@ars.usda.gov.
authors.forename: Gary P
authors.initials: GP
authors.lastname: Richards
authors.affiliation: U.S. Department of Agriculture, Agricultural Research Service, Dover, Delaware, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Watson
authors.affiliation: U.S. Department of Agriculture, Agricultural Research Service, Wyndmoor, Pennsylvania, USA.
authors.forename: David S
authors.initials: DS
authors.lastname: Needleman
authors.affiliation: U.S. Department of Agriculture, Agricultural Research Service, Wyndmoor, Pennsylvania, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Uknalis
authors.affiliation: University of Delaware, Department of Biological Sciences, Newark, Delaware, USA.
authors.forename: E Fidelma
authors.initials: EF
authors.lastname: Boyd
authors.affiliation: University of Delaware, Department of Biological Sciences, Newark, Delaware, USA.
authors.forename: Johnna P
authors.initials: JP
authors.lastname: Fay
mesh_terms: D000898:Antibiosis; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D031161:Pseudoalteromonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D012623:Seawater / Q000382:microbiology*; D014736:Vibrio parahaemolyticus / Q000187:drug effects* / Q000254:growth & development / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D057056:Cysteine Proteases
keywords: Pseudoalteromonas piscicida; Vibrio cholerae; Vibrio parahaemolyticus; Vibrio vulnificus; bacterial inhibition; enzymes; lytic vesicles; predatory bacteria; probiotics; proteases