Mechanisms for Pseudoalteromonas piscicida-Induced Killing of Vibrios and Other Bacterial Pathogens

Abstract

<i>Pseudoalteromonas piscicida</i> is a Gram-negative gammaproteobacterium found in the marine environment. Three strains of pigmented <i>P. piscicida</i> were isolated from seawater and partially characterized by inhibition studies, electron microscopy, and analysis for proteolytic enzymes. Growth inhibition and death occurred around colonies of <i>P. piscicida</i> on lawns of the naturally occurring marine pathogens <i>Vibrio vulnificus</i>, <i>Vibrio parahaemolyticus</i>, <i>Vibrio cholerae</i>, <i>Photobacterium damselae</i>, and <i>Shewanella algae</i> Inhibition also occurred on lawns of <i>Staphylococcus aureus</i> but not on <i>Escherichia coli</i> O157:H7 or <i>Salmonella enterica</i> serovar 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 three <i>P. piscicida</i> strains. Direct overlay of the <i>Pseudoalteromonas</i> colonies 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 of <i>P. piscicida</i> and a novel mechanism of direct transfer of these vesicles to <i>V. parahaemolyticus</i> Vesicles digested holes in <i>V. parahaemolyticus</i> cells, while the <i>P. piscicida</i> congregated 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 which <i>P. piscicida</i> inhibits and/or kills competing bacteria, involving the secretion of antimicrobial substances and the direct transfer of digestive vesicles to competing bacteria.<b>IMPORTANCE</b><i>Pseudoalteromonas</i> species are widespread in nature and reduce competing microflora by the production of antimicrobial compounds. We isolated three strains of <i>P. piscicida</i> and characterized secreted and cell-associated proteolytic enzymes, which may have antimicrobial properties. We identified a second method by which <i>P. piscicida</i> kills <i>V. parahaemolyticus</i> It involves the direct transfer of apparently lytic vesicles from the surface of the <i>Pseudoalteromonas</i> strains to the surface of <i>Vibrio</i> cells, with subsequent digestion of holes in the <i>Vibrio</i> cell walls. Enzymes associated with these vesicles are likely responsible for the digestion of holes in the cell walls. <i>Pseudoalteromonas piscicida</i> has 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 of <i>P. piscicida</i> to inactivate pathogens and may lead to the isolation of enzymes and other antimicrobial compounds of pharmacological value.