Progress 07/15/19 to 07/14/23
Outputs
Target Audience:The major target audience is the bivalve aquaculture industry in the United States.We have a solid record in reducing unnecessarily duplicative efforts through collaboration with a variety of stakeholders involved in bivalve aquaculture. Coordination with stakeholders is managed by Dr. Gomez-Chiarri, a member of the Eastern Oyster Genome Consortium, Eastern Oyster Shellfish Breeding Consortium, Northeast Regional Aquaculture Center Technical Advisory Committee, the East Coast Pathology Working Group, and Northeast Bivalve Hatchery Health Collaborative. These relationships allow us to coordinate and integrate our research with other national efforts on the management of infectious diseases in bivalve aquaculture. We are also extensively involved in outreach and extension, routinely interacting with industry stakeholders and developing workshops at conferences attended by the industry, such as the Milford Aquaculture Seminar and the Northeast Aquaculture Conference and Exposition (NACE). We strive to highlight our research using news resources, publications, participation in open houses and other events. Our students take advantage of the SciWrite program at URI, developing media pieces on their research (e.g., a podcast on probiotics, a shiny app on oyster microbial communities). We are also thoroughly committed to Justice, Equity, Diversity, and Inclusion Initiatives, with five of our current graduate students are underrepresented minorities. A long-term goal of this project is the creation of new products to prevent infectious disease outbreaks in aquaculture facilities.We were successful in developing a stable, liquid formulation of our probiont S4 and a U.S. patent was awarded in 2023. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A total of seven graduate students (five women, three men) have contributed to this project. Fourof these graduate students belong to underrepresented minority groups*.Three of these students finished their PhD degrees#. Graduate students: Christopher Schuttert#, Jessica Coppersmith, Tejashree Modak#, Margaret Rosario*#, Jaypee Samson, Damian Cavanaugh*, Kira Bernabe*, Jamal Andrews* Undergraduate researchers involved in this project: Margaret Hill, Riley Sullivan, Willy Njeru, Simon Metcalf, Sean Ovalles, Sydney Charette How have the results been disseminated to communities of interest?In addition to the publications and products previously listed, numerous presentations were provided at scientific conferences by both PIs and graduate students. Recent examples include the annual CRWAD, the Annual Meeting of the National Shellfisheries Association (Baltimore, MD, March 2023),and Applied Hologenomics(Bilbao, Spain, Fall 2022). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Role of Type VI secretion systems (T6SS) in virulence of the pathogenVibrio coralliilyticus. Previous research by us and others has shown that the bivalve and coral pathogen,Vibrio coralliilyticusstrain RE22Sm, utilizes two complementary T6SSs as virulence factors (T6SS1 located on chromosome one, T6SS2 on chromosome two). Mutations in key target T6SS genes, includinghcp(hemolysin co-regulated pilus, tube of the T6SS assembly) andvgrG(valine- glycine repeat protein G, tip of the nanomachine) were created by allelic exchange mutagenesis using homologous recombination. Wild-type (wt) and mutant strains were tested for killing of other bacteria (T6SS null-E. coliSm10 cells) and larval eastern oysters. Mutation ofhcp1resulted in complete loss of virulence toward larval oysters and attenuated virulence towardE. coliSm10. Mutation ofvgrG1also resulted in the complete loss of virulence toward oyster larvae but did not affect virulence towardE. colicells. Mutation ofvgrG2significantly reduced virulence against larval oysters but maintained full virulence towardE. colicells. Complementation of mutants restored the wt phenotype against both larval oysters and preyE. colicells. These findings support the hypothesis thatV. coralliilyticusRE22Sm has two T6SSs that enable killing ofE. coliprey cells and oyster larvae.Further experiments, however, discovered that thatthe wrong gene was targeted forhcp2. Although competition experiments with the other mutants and transcriptomic studies still support the roles of T6SS1 and T6SS2 in RE22 pathogenesis, we are making newhcp2 mutants to validate these results.We will do a more complete manualannotation of ourVibrio coralliilyticusRE22 genome to aid in future transcriptomic and mutagenesis studies,and validate all mutants using genome sequencing and gene expression data. Evidence for T6SS effector-immunity gene pairs.Bacteria use their T6SS to deliver effectors (e.g., toxins) to targeted cells and produce immunity proteins to protect against self-intoxication. We identified effector-immunity (E/I) gene pairsin V. parahaemolyticusPSU5579, the causative agent of Acute Hepatopancreatic Necrosis Disease in shrimp. The antibacterial T6SS1 in PSU5579 exhibits strong homology to the T6SS2 in RE22 (71% nucleotide identity with 92% query coverage, E=0.0) across 23 genes that make up the core T6SS genes. The gene order within these two gene clusters is identical. In contrast, the T6SS2 gene clusters share little homology and conservation of gene order. Both strains (PSU5579 and RE22) possess homologs of several previously reported and validated antibacterial E/I modules that are widely distributed throughout theVibriogenus. For example, both possess a gene encoding a GHH domain nuclease followed by genes encoding hypothetical proteins, one of which is predicted to be an immunity protein and one or two others that appear to be divergent duplicated immunity proteins. These results now encourage the experimental validation of these E/I gene pairs in RE22. Some of these T6SS effectors may have potential roles in the suppression of oyster larval immune responses by RE22 that we have previously reported in 2020. Role of outer membrane vesicles (OMVs) on RE22 virulence.Another potential mechanism of delivery of virulence factors to other organisms are OMVs. RE22 OMVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and dynamic light scattering.Hemocytes from healthy adult oysters were exposed to varying concentrations of OMVs. Treated hemocytes demonstrated increased metabolic activity (resazurin metabolism).This suggests that oyster hemocytes respond to pathogen OMVs as part of the oyster innate immune response. Role of T3SS in virulence of the pathogenVibrio coralliilyticus.Transcriptomic data from competitions between probiont S4 and pathogen RE22 indicated that another virulence factor, T3SS, may have a role in RE22 virulence. We identified the structural genes of T3SS in the RE22 genome and selected several for deletion:sctE(major translocon),sctA(needle tip),sctC(secretin ring), andsctV(export gate). A mutant ofsctVdemonstrated a reduction of RE22 virulence against larval oysters. ProbiontPhaeobacter inhibinsS4 downregulates gene expression of RE22 virulence factors.Our previous research has shown that S4 has complex mechanisms of action against RE22, including antibiosis, niche exclusion through biofilm formation, and quorum quenching.To more deeply elucidate the molecular interactions between RE22, competition experiments were conducted by co-culturing probiont S4 established biofilms with pathogen RE22 for five hours and comparing gene expressions to concurrently grown monoculture controls. In co-culture, RE22 genes involved in virulence were down regulated, including genes belonging to secretion systems such as Type III and Type VI.Additionally,decreased expression of several RE22 genes for flagellar proteins, including flagellin,flgA, flgE,fliD, andfliL,was observed under co-culture conditions. Reduced RE22 motility in the presence of tropodithietic acid (TDA), an antibiotic produced by S4Sm, has been previously reported. However, this transcriptome analysis indicated that reduced RE22 motility may also be partly due to downregulation ofdisulfide bond family protein genesdsbAanddsbD. Mutations or deletions ofdsbAhavebeen linked to decreased virulence and motility in gram-negative bacteria, including otherVibriospecies. RE22 was found to upregulate efflux pump genes, likely in response to the production of TDA by S4Sm. While RE22 differentially expresses hundreds of genes when challenged with S4Sm, only a few dozen S4Sm genes were statistically differentially expressed during these experiments. Interestingly, an acyl-homoserine-lactone (AHL) synthase was highly down regulated in S4Sm during co-culture with RE22. We previously discovered that S4Sm AHLs act as quorum quenchers against RE22, and therefore it was curious to observe reduced expression of the S4Sm AHL synthase gene during exposure to RE22. P. inhibensS4 is resistant to pathogen RE22 attack.WhileE. coliis highly susceptible to T6SS-mediated killing by RE22,P. inhibensS4 is >350-fold less susceptible thanE. colito killing by RE22. Experiments with S4 targeted mutants showed that TDA (antibiotic) production and a fully functional QS network are important in its resistance to attack by RE22.This finding supports the utility ofP. inhibensS4 as a probiont to help mitigate RE22 infections. Genomic characterization of themarine probiontPhaeobacter inhibensS4Sm.A new assembly ofP. inhibensS4Sm was completed utilizing long read sequencing chemistry, closing the genome and filling in numerous gaps in the previous assembly. The genome was submitted to NCBI under accession numberGCF_030060455.1 and was annotated using theNCBI Prokaryotic Genome Annotation Pipeline (PGAP). The completed genome permitted a pangenome analysis comparing S4Sm with all the otherP. inhibensgenomes on NCBI and will improve analysis of RNA-Seq differential gene expression (DGE) analysis of S4Sm experiments described above. In summary, our data confirm that: (1)P. inhibensS4 is an excellent probiont againstV. coralliilyticusRE22; (2) the probiotic mechanisms of host protection against RE22 are multifactorial, including contributions from TDA production, biofilm formation, AHL-mediated QQ, and immune modulation of the host; and (3) the pathogenicity of RE22 relies on bacterial secretion systems to target bacterial prey cells and cause disease in oyster larvae.We have also discovered other potential mechanism of probiont S4 activity.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Takyi, E., LaPorte, J., Sohn, S., Stevick, R.J., Witkop, E.M., Gregg, L.S., Chesler?Poole, A., Small, J., White, M.M., Giray, C. and Rowley, D.C., Nelson D.R., Gomez-Chiarri M. 2023. Development and evaluation of a formulation of probiont Phaeobacter inhibens S4 for the management of vibriosis in bivalve hatcheries. Aquaculture, Fish and Fisheries, 3(3), pp.256-267.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
LaPorte, J.P., Spinard, E.J., Cavanagh, D., Gomez-Chiarri, M., Rowley, D.C., Mekalanos, J.J., Mittraparp-Arthorn, P. and Nelson, D.R., 2023. Draft Genome Sequence of Vibrio parahaemolyticus PSU5579, Isolated during an Outbreak of Acute Hepatopancreatic Necrosis Disease in Thailand. Microbiology Resource Announcements, 12(2), pp.e00873-22.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Takyi E, Stevick RJ, Witkop EM, Gregg LS, Chesler?Poole A, Small J, White MM, Hudson R, Giray C, Rowley DC, Nelson DR, Gomez-Chiarri M. (2023) Probiotic treatment modulates the bacterial microbiome of larval eastern oysters, Crassostrea virginica, in hatcheries. Aquaculture, accepted with revisions.
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Progress 07/15/21 to 07/14/22
Outputs
Target Audience:We have a solid record inreducing unnecessarily duplicativeeffortsthrough collaboration with a variety of stakeholders involved in bivalve aquaculture. Coordination with stakeholders is managed by Gomez-Chiarri, a member of the Eastern Oyster Genome Consortium, Eastern Oyster Shellfish Breeding Consortium, Northeast Regional Aquaculture Center Technical Advisory Committee, the East Coast Pathology Working Group, and Northeast Bivalve Hatchery Health Collaborative (in process of being established). These relationships allow us to coordinate and integrate our research with other national efforts on the management of infectious diseases in bivalve aquaculture. We are also extensively involved inoutreach and extension, routinely interacting with industry stakeholders and developing workshops at conferences attended by the industry, such as the Milford Aquaculture Seminar and the Northeast Aquaculture Conference and Exposition (NACE).We strive to highlight our research using news resources, publications, participation in open houses and other events (e.g., Ocean Exploration Zone at the Volvo race stopover in Newport).Our students take advantage of the SciWrite program at URI, developing media pieces on their research (e.g., a podcast on probiotics,a shiny appon oyster microbial communities). We are also thoroughly committed to Justice, Equity, Diversity, and Inclusion Initiatives, with five of our current graduate students being underrepresented minorities. Changes/Problems:The pandemic has caused some delays in accomplishing our goals, but we are on track to complete the aims of this project. What opportunities for training and professional development has the project provided?A total of seven graduate students (five women, two men) have contributed to this project. Five of these graduate students belong to underrepresented minority groups. How have the results been disseminated to communities of interest?Modak, T. H. & Gomez-Chiarri, M., 2020, Contrasting immunomodulatory effects of probiotic and pathogenic bacteria on Eastern oyster,crassostrea virginica, larvae.Vaccines8, 1-23 Schuttert, Christian W., et al. "Two Type VI Secretion Systems in Vibrio coralliilyticus RE22Sm exhibit differential target specificity for bacteria prey and oyster larvae."bioRxiv(2021). Submitted publications now in peer review: Takyi, et al., Probiotic treatment in the hatchery shifts the composition of bacterial microbiomes in larval eastern oysters, Crassostrea virginica, submitted to Frontiers in Marine Science Takyi et al.EVALUATION OF A FORMULATION OF PROBIONT PHAEOBACTER INHIBENS S4 ON LARVAL OYSTER PERFORMANCE IN THE HATCHERY, submitted to Aquaculture Rsearch Laporte et al.Draft Genome Sequence ofVibrio parahaemolyticusPSU5579, Isolated During an Outbreak of Acute Hepatopancreatic Necrosis Disease (AHPND) in Thailand, submitted to Microbial Resource Announcements What do you plan to do during the next reporting period to accomplish the goals?Future plan: Test the virulence of RE22T3SS mutants against oyster larvae. Experimentally validate T6SSE/I gene pairs in RE22.
Impacts
What was accomplished under these goals?
Probiont S4 treatment leads to immunomodulation in the host, while RE22 causes immunosuppression.We used transcriptomics to probe how S4 (probiont) and RE22 (pathogen) modulate gene expression in oyster larvae. Interestingly, larvae exposed to RE22 showed evidence of immunosuppression, mainly focused on inhibition of signaling cascades involved in immune activation in response to bacteria (e.g., MyD88, NF-kappaB), and blocking expression of immune effectors downstream of these pathways. In contrast, gene expression profiles after S4 exposure were consistent with activation of these immune signaling pathways and upregulated expression of both bacterial and viral immune effectors. Therefore, pretreatment of larvae with S4 leads to the production of immune effectors, which then are present in the system when pathogen RE22 is added, probably contributing to pathogen neutralization before it is able to cause immunosuppression. The antibacterial and pathogenic roles of T6SS inV. coralliilyticusRE22. Our sequencing of the RE22 genome revealed two T6SS gene clusters. T6SS ofV. choleraeis involved in attack on other bacteria and intestinal colonization. We constructed mutants of key genes in both the S4 T6SS1 (gene cluster on chromosome 1) and T6SS2 (on chromosome 2) andtestedwhether the mutants wereimpaired for killing eitherbacteria or oyster larvae (Table 1).Mutations toeitherhcp1orvgrG1(encodingthe tube and tip of the nanomachine, respectively) resulted in sharply reduced predation by RE22 againstE. coliprey, withhcp2removal having a more profound effect. Table 1. Effect of mutations inV. coralliilyticusRE22 T6SS genes onE. coliSm10 prey cell killing and oyster larvae survival. Attacker cell strain Prey cell survival (% of start)a Oyster larvae survival (%)b V. coralliilyticusRE22 wt 0.046% 48% RE22 hcp1 mutant 6.25% 89% RE22 hcp2 mutant 34% 74% RE22 vgrG1 mutant 0.026% 90% RE22 vgrG2 mutant 0.074% 84% aT6SS anti-bacterial assay conditions: attacker cells: prey cells = 4:1; cells incubated on a filter for 4 h, 27°C; cells resuspended, and enumerated by dilution and plating;bOyster larvae challenged with RE22 for 24 h as described in Karim et al. (2013)6;95.4% survival with no RE22 added. These results revealed that both of the RE22 T6SSs, and especially T6SS2, are involved in antibacterial activity. Just as importantly, we also discovered that the T6SS1 of RE22 isa majorvirulence factor for killing oyster larvae.Mutations in eitherhcp1orvgrG1resulted in near complete attenuation of larval killing.Mutations inhcp2andvgrG2also increased larval survival (1.5-1.75-fold), although to less of an extent as compared tohcp1orvgrG1. Additionally, mutations that disrupt quorum sensing in RE22 (e.g., delta-luxNanddelta-?vcpR) significantly reduced both antibacterial T6SS activity and pathogenicity against oysters.This finding is transformative in our understanding of the pathogenicity of RE22 against oyster larvae, and establishes the T6SS1 as a critical virulence factor in larval killing.It further suggests that the RE22 T6SS are regulated by QS, which aligns with studies from otherVibriospecies. Evidence for T6SS effector-immunity gene pairs.Bacteria use T6SS to deliver effectors (e.g., toxins) to target cells and produce immunity proteins to protect against self-intoxication.Weidentifiedeffector-immunity (E/I) gene pairs inV. parahaemolyticusPSU5579, the causative agent of Acute Hepatopancreatic Necrosis Disease in shrimp. When we compared the genomes of PSU5579 andV. coralliilyticusRE22, we found that the antibacterial T6SS1 in PSU5579 exhibited strong homology to the T6SS2 in RE22 (71% nucleotide identity with 92% query coverage, E=0.0) across 23 genes that make up the core T6SS genes. The gene order within these two gene clusters is identical. In contrast, the T6SS2 gene clusters share little homology and conservation of gene order. Both strains possess homologs of several previously reported and validated antibacterial E/I modules that are widely distributed throughout theVibriogenus. For example, both PSU5579 and RE22 possess a gene encoding a GHH domain nuclease followed by genes encoding hypothetical proteins, one of which is predicted to be an immunity protein and one or two others that appear to be divergent duplicated immunity proteins.These results now encourage the experimental validation of these E/I gene pairs in RE22. Probiont S4 isresistantto pathogen RE22 attack.WhileE. coliis highly susceptible toT6SS-mediatedkilling by RE22, we found thatP. inhibensS4 is >350-fold less susceptible thanE. colito killing by RE22, and further that antibiotic (TDA) production and a fully functional QS network are highly important in its resistance to attack. S4 downregulates gene expression of RE22 virulence factors.We have also measured global changes in gene expression for both the probiont and pathogen during co-culture competition experiments using transcriptomics (Fig.4). When RE22 was added to an established biofilm of S4, we observed downregulation of genes and pathways linked with RE22 virulence (e.g., T1SS, T3SS, T6SS, motility and adhesion pathways)and also changes in the expression of pathways that likely contribute to pathogenicity (e.g.,dsbAanddsbD, involved in the formation and maintenance of disulfide bonds that are critical for the activity of key proteins involved in virulence in other gram-negative bacteria). RE22 also upregulates expression of genes coding for efflux pumps (involved in toxin removal) in response to S4. On the other hand, gene expression of S4 was mostly unaffected by RE22.These results support our hypotheses that mechanisms of S4 probiotic activity include down-regulation of expression of virulence factors in pathogen RE22, an effectthat is modulatedby QS. Role of T3SS in virulence of the oyster and coral pathogenVibrio coralliilyticus.The above studies revealed that when knocking outa single copy of either thehcporvgrGgene of the two T6SSs, a reduction in virulence was observed. However, when the second copy of either gene was deleted, there was an increase in virulence to levels greater than observed in the single gene knockout. We therefore hypothesized that another virulence factor, T3SS, is being upregulated in the absence of a functional T6SS. We identified the structural genes of T3SS in the RE22 genome and selected several for deletion:sctE(major translocon),sctA(needle tip),sctC(secretin ring), andsctV(export gate). At this time, asctVmutant has been constructed and tested.The?sctVstrain consistently demonstrated a reduction of virulence. An average increase of 74% was observed in larval survival when challenged with the mutant strain as compared to the wild type (3 trials). In summary, our data now demonstrate that: (1)P. inhibensS4 is an excellent probiont againstV. coralliilyticusRE22; (2) the probiotic mechanisms of host protection against RE22 are multifactorial, including contributions from TDA production, biofilm formation, AHL-mediated QQ, and immune modulation of the host; and (3) the pathogenicity of RE22 relies on previously unidentified secretion systems that deliver toxins to target cells.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Schuttert, Christian W., et al. "Two Type VI Secretion Systems in Vibrio coralliilyticus RE22Sm exhibit differential target specificity for bacteria prey and oyster larvae." bioRxiv (2021).
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Laporte et al. Draft Genome Sequence of Vibrio parahaemolyticus PSU5579, Isolated During an Outbreak of Acute Hepatopancreatic Necrosis Disease (AHPND) in Thailand, submitted to Microbial Resource Announcements.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Takyi, et al., Probiotic treatment in the hatchery shifts the composition of bacterial microbiomes in larval eastern oysters, Crassostrea virginica, submitted to Frontiers in Marine Science
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Takyi et al. EVALUATION OF A FORMULATION OF PROBIONT PHAEOBACTER INHIBENS S4 ON LARVAL OYSTER PERFORMANCE IN THE HATCHERY, submitted to Aquaculture Research
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Modak, T. H., & Gomez-Chiarri, M. (2020). Contrasting immunomodulatory effects of probiotic and pathogenic bacteria on eastern oyster, crassostrea virginica, larvae. Vaccines, 8(4), 588.
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