Investigation of Pseudoalteromonas sp. Strain JC3 as a Putative Probiotic for Shrimp Aquaculture
Margaret Hill1, Margaret Rosario1, Victoria Johnson1, Jacqueline Camm2, Damian Cavanagh2, Dr. David Nelson2, Dr. David Rowley1 1Department
of Biomedical and Pharmaceutical Sciences, Alex and Ani Positive Impact Lab, University of Rhode Island, Kingston, RI, USA 2Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI, USA
Abstract/Introduction
Early Mortality Syndrome (EMS) is a disease affecting many species of Penaeus shrimp in aquaculture systems. In 2013 the causative agent was identified as Vibrio parahaemolyticus causing Acute Hepatopancreatic Necrosis Disease (AHPND). This Gram-negative bacterium utilizes a Type 6 Secretion System (T6SS) to inject binary toxins PirA and PirB, causing deterioration of epithelial cells in the shrimp's key organs. Current forms of disease mitigation, including antibiotics, vaccines and immunostimulatory methods, either fuel antibiotic resistance, are impractical, or are costly. Probiotics hold a promising future for pathogenic bacterial elimination because they may boost host survival using multiple modes of action. Pseudoalteromonas sp. strain JC3 is a Gram-negative marine bacterium that has shown protective effects against the AHPND-causing strain V. parahaemolyticus PSU5579; however, the mechanism of action for this host protection is currently unknown. Prior to conducting this research, genomic analysis of JC3 suggested the biosynthetic potential to produce secondary metabolites, including cyclic peptides known as alterochromides that exert cytotoxic effects. In this project, we identified alterochromides and other possible antimicrobial compounds produced by JC3, such as quinolones and the antimicrobial compound darobactin. We hypothesize that these compounds decrease V. parahaemolyticus pathogenicity by limiting growth and interfering with cell-cell signaling.
Results
Figure 3. Structures of selected compounds identified by LC-MS/MS.
Figure 1. P. sp. JC3 secretes compounds that limit the growth of V. parahaemolyticus 5579. A JC3 colony of JC3 was spotted onto a marine agar plate inoculated with V. parahaemolyticus. Following incubation, a zone of no growth for 5579 was observed surrounding the JC3 colony, suggesting the secretion of an antibiotic(s). The identity of the antibiotic(s) is currently unknown but may be related to alterochromide production.
Acyl-homoserine Lactone (AHL) Variation 256
General Structure of an Alterochromide:
R1 = H, Br R2 = Isoleucine, Leucine, Valine
Quinolone (HHQ) Variation 381 (exact mass: 381.30) • 17 carbon alkyl chain • One side of desaturation- Where? • Cis or trans? • Double bond location remains unknown • Only reported in the molecular networking paper
Quinolone (HHQ) Variation 244
Figure 4. Presence of biosynthetic gene cluster1 and LCMS/MS data indicates the production of darobactin, a Gramnegative selective antibiotic.
ST: gut HP: hepato-pancreas MG: mid-gut
Conclusion/Future Directions • Chemical investigation of Pseudoalteromonas sp. JC3
Kumar R et al. Reviews in Aquaculture. DOI: 10.1111/raq.12414
Methods for Compound Identification Cultures of JC3 (10 mL each) were grown in various marine media for 5 days and then extracted using solid phase extraction. Compounds were detected in culture extracts using LC-MS. A heat map was generated based on peak intensities. A 10 L culture of JC3 was grown for 12 days in YP30 marine medium and extracted with XAD resin. Compounds were eluted with 50:50 methanol/water (0.1 % formic acid) and concentrated in vacuo. The extracted compounds will be separated using HPLC and further analyzed by LC-MS and 1H NMR spectroscopy.
• Figure 2. Chemical profiling of JC3 grown in various media types and growth Cultures conditions. (10 mL each) were centrifuged at 2,500 rpm for 10 min, desalted using a solid-phase extraction column and reconstituted to 0.25 mg/mL for analysis on LCMS/MS. The heat map was constructed using peak intensities from MS. Alterochromides were detected using UV absorbance (360 nm).
•
cultures supports the production of alterochromide secondary metabolites, supporting the previous genomic analysis suggesting this possibility. Other secondary metabolites were identified including AHLs, alkyl quinolones, and darobactin, which could also contribute to the overall antibiotic effects of this bacterium. Next steps include further purification of the compounds and testing for antimicrobial properties against pathogenic V. parahaemolyticus.
Acknowledgments Funding for this work was provided by USDA (2019-67016-29868) to DR and DN. Research reported in this presentation was further supported by the Rhode Island Institutional Development Award (IDeA) Network of Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103430.
References 1. Imai, Yu, et al. “A New Antibiotic Selectively Kills Gram-Negative Pathogens.” Nature News, Nature Publishing Group, 20 Nov. 2019, www.nature.com/articles/s41586-019-1791-1. 2. Lin, Jinshui, et al. “The Pseudomonas Quinolone Signal (PQS): Not Just for Quorum Sensing Anymore.” Frontiers, Frontiers, 1 Jan. 1AD, www.frontiersin.org/articles/10.3389/fcimb.2018.00230/full. 3. Tran, Loc, et al. “Determination of the Infectious Nature of the Agent of Acute Hepatopancreatic Necrosis Syndrome Affecting Penaeid Shrimp.” University of Arizona, Inter-Research, 16 July 2015, arizona.pure.elsevier.com/en/publications/determination-of-the-infectious-nature-of-the-agent-of-acutehepa. 4. Kumar R et al. Reviews in Aquaculture. DOI: 10.1111/raq.12414