ABSTRACT
Endolysins have garnered significant attention as a potential alternative to antibiotics in aquaculture, mainly for combating Vibrio spp., Gram-negative pathogens responsible for infectious outbreaks. However, endolysin effectiveness against Gram-negative bacteria is limited due to the outer membrane's poor permeability. The combat against marine pathogens poses an additional challenge of finding endolysins that retain their activity in high ionic strength conditions. Thus, this study aimed to demonstrate that certain endolysins retain muralytic activity in seawater and also evaluated outer membrane permeabilizers as endolysin adjuvants. The effectiveness of KZ144 and LysPA26 endolysins, along with EDTA and oregano essential oil, was evaluated against Vibrio parahaemolyticus ATCC-17802 in natural seawater. Results revealed the muralytic activity of both endolysins in seawater. However, the endolysins appeared to counteract the permeabilizers' effect during the initial bactericidal assays. Further investigations revealed that the observed effect was not antagonistic. After the permeabilizer action, V. parahaemolyticus likely used endolysins as a growth substrate. Endolysins may not play an indifferent role if they fail to exert a bactericidal effect. Instead, they can serve as a substrate for fast-growing bacteria, such as V. parahaemolyticus, increasing bacterial density. It should be considered a potential drawback of endolysins' proteinaceous nature as bactericidal agents.
Subject(s)
Bacteriophages , Vibrio parahaemolyticus , Endopeptidases/pharmacology , Gram-Negative Bacteria , Bacteria , Anti-Bacterial Agents/pharmacologyABSTRACT
Endolysins have been proposed as a potential antibacterial alternative for aquaculture, especially against Vibrio; the bacterial-agents that most frequently cause disease. Although multiple marine vibriophages have been characterized to date, research on vibriophage endolysins is recent. In this study, biochemical characterization of LysVpKK5 endolysin encoded by Vibrio parahaemolyticus-infecting VpKK5 phage was performed. In silico analysis revealed that LysVpKK5 possesses a conserved amidase_2 domain with a zinc-binding motif of high structural similarity to T7 lysozyme (RMSD = 0.107 Å). Contrary to expectations, the activity was inhibited with Zn2+ and was improved with other divalent cations, especially Ca2+. It showed optimal muralytic activity at pH 10, and curiously, no lytic activity at pH ≤ 7 was recorded. As for the thermal stability test, the optimal activity was recorded at 30 °C; the higher residual activity was recorded at 4 °C, and was lost at ≥ 50 °C. On the other hand, increasing NaCl concentrations reduced the activity gradually; the optimal activity was recorded at 50 mM NaCl. On the other hand, the enzymatic activity at 0.5 M NaCl was approx 30% and of approx 50% in seawater. LysVpKK5 endolysin exhibited a higher activity on V. parahaemolyticus ATCC-17802 strain, in comparison with AHPND + strains.
Subject(s)
Bacteriophages/chemistry , Endopeptidases/metabolism , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Peptidoglycan/metabolism , Vibrio parahaemolyticus/virology , Viral Proteins/metabolism , Amino Acid Sequence , Aquatic Organisms , Bacteriophages/classification , Bacteriophages/genetics , Bacteriophages/metabolism , Binding Sites , Calcium/chemistry , Calcium/pharmacology , Cations, Divalent , Endopeptidases/chemistry , Endopeptidases/genetics , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , N-Acetylmuramoyl-L-alanine Amidase/chemistry , N-Acetylmuramoyl-L-alanine Amidase/genetics , Phylogeny , Protein Binding/drug effects , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Sodium Chloride/chemistry , Sodium Chloride/pharmacology , Substrate Specificity , Viral Proteins/chemistry , Viral Proteins/genetics , Zinc/chemistry , Zinc/pharmacologyABSTRACT
Vibrio parahaemolyticus is the causative bacterium of acute hepatopancreatic necrosis disease (AHPND) in white shrimp Litopenaeus vannamei. This bacterium secretes protein toxins whose genes are encoded in an auto-transmissible plasmid called pVA1. The presence of this plasmid in V. parahaemolyticus is determinant for disease development. Its propagation is not only linked to bacterial colonisation capacity but also to horizontal gene transfer mechanisms. Nevertheless, the active uptake of plasmid, which is known as natural genetic transformation (NGT), has not yet been proposed as a possible acquisition mechanism of the pVA1 plasmid among Vibrio species. Previous studies suggest that some Vibrio species have the ability to undergo NGT in the presence of chitin. Therefore, the objective of this study was to evaluate the induction of NGT mediated by chitin in V. parahaemolyticus (ATCC-17802) through its ability to incorporate and express the pVA1 plasmid. The results showed that a reference strain that does not initially contain the plasmid can incorporate the plasmid under the appropriate transformation conditions, and cause mortality in white shrimp similar to that observed for pathogenic strains isolated from infectious outbreaks. Given the management and conditions of a shrimp farm with large amounts of chitinous exoskeletons, it is feasible that NGT could be a possible acquisition mechanism of plasmid pVA1 among Vibrio species, turning a non-causative strain of V. parahaemolyticus into a causative strain. With this study, we have expanded the knowledge of the pathogenesis process mediated by NGT and the understanding of the possible propagation mechanisms of emerging diseases in the aquaculture sector.
