Mechanisms for Pseudoalteromonas piscicida-Induced Killing of Vibrios and Other Bacterial Pathogens
Authors: Richards Gary P. , Watson Michael A. , Needleman David S. , Uknalis Joseph , Boyd E. Fidelma , Fay Johnna P. 
Published: April 2017

ABSTRACT

Pseudoalteromonas piscicida
is a Gram-negative gammaproteobacterium found in the marine environment. Three strains of pigmented
P. piscicida
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
P. piscicida
on lawns of the naturally occurring marine pathogens
Vibrio vulnificus
,
Vibrio parahaemolyticus
,
Vibrio cholerae
,
Photobacterium damselae
, and
Shewanella algae
. Inhibition also occurred on lawns of
Staphylococcus aureus
but not on
Escherichia coli
O157:H7 or
Salmonella enterica
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
P. piscicida
strains. Direct overlay of the
Pseudoalteromonas
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
P. piscicida
and a novel mechanism of direct transfer of these vesicles to
V. parahaemolyticus
. Vesicles digested holes in
V. parahaemolyticus
cells, while the
P. piscicida
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
P. piscicida
inhibits and/or kills competing bacteria, involving the secretion of antimicrobial substances and the direct transfer of digestive vesicles to competing bacteria.

IMPORTANCE
Pseudoalteromonas
species are widespread in nature and reduce competing microflora by the production of antimicrobial compounds. We isolated three strains of
P. piscicida
and characterized secreted and cell-associated proteolytic enzymes, which may have antimicrobial properties. We identified a second method by which
P. piscicida
kills
V. parahaemolyticus
. It involves the direct transfer of apparently lytic vesicles from the surface of the
Pseudoalteromonas
strains to the surface of
Vibrio
cells, with subsequent digestion of holes in the
Vibrio
cell walls. Enzymes associated with these vesicles are likely responsible for the digestion of holes in the cell walls.
Pseudoalteromonas piscicida
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
P. piscicida
to inactivate pathogens and may lead to the isolation of enzymes and other antimicrobial compounds of pharmacological value.