DESIGN

Overview

    Our solution for AHPND (Acute Hepatopancreatic Necrosis Disease) caused by Vibrio parahaemolyticus (Vp for short) can be divided into three sections: detecting the pathogen, treating the disease and preventing the outbreak of infection.

Detection

    AHPND poses a threat to the aquaculture industry because of its high incidence rate and wide-spread range. Thus, emphasis should be put on rapid pathogen detection in order to control the disease. However, much time consumption, high financial costs and high requirements for professional skills make current detection methods unsuitable for shrimp farmers. So we design a cell-free sensing system to detect the toxin genes of Vp, for achieving rapid and precise pathogen detection.
    The pVA1 plasmids of pathogenic Vp contain genes related to host infection. The pirA and pirB genes located on this plasmid encode toxins PirA and PirB, which can target hepatopancreatic cells of shrimps and are considered to be the most important virulence factors of Vp (1). Therefore, the transcripts of the conserved region of pirA and pirB toxin genes are chosen as the input signal.
    We employ the Ribozyme-Enabled Detection of RNA (RENDR) to construct the cell-free sensing system. RENDR uses an RNA input to activate a split ribozyme, thus producing a spliced mRNA (2). The key of the RENDR platform is the split ribozyme modified by appending an RNA guide sequence, which is designed to be complementary to the RNA input, onto each of the two ribozyme fragments. These RNA guide can co-localize the two ribozyme fragments to trigger splicing, joining together the exons of the reporter gene, then the reporter gene can express (Fig. 1). In our design, two reporters are selected. One is the β-lactamase (AmpC), which can hydrolyze nitrocefin, producing a change in color from yellow to red (Fig. 2a&2b). The other is nanoluc, which can catalyze non-fluorescent substrate to produce fluorescence (Fig. 2c&2d).
1
Fig. 1 Mechanism of ribozyme-splicing based signal transform of RENDR.
2
Fig. 2 The expression of β-lactamase and Nanoluc for detection. a The gene circuit of β-lactamase (AmpC). b The function of β-lactamase. c The gene circuit of NanoLuc. d The function of NanoLuc.

The process

3
Fig. 3 Water sample processing for detection.
Nucleic acid enrichment
    Shrimps may be infected when the Vp proliferates in the aquaculture water. So we collect aquaculture water samples for pathogen detection, which will undergo three processing steps: bacteria lysis, nucleic acids enrichment and washing. In one injector, lysis buffer will destroy the bacteria membrane and denature DNA, while acetic acid will renature plasmids only. The mix is then pushed into another injector, filtered by a filter membrane. The filter membrane is plasmids-permeable, while blocking the aggregated genomic DNA. Pieces of fiber paper in this injector will enrich the nucleic acids, for their high affinity for DNA. Last, wash paper to remove impurities.
Recombinase Polymerase Amplifiation (RPA)
    To enhance the input signals and reduce background interference, Recombinase Polymerase Amplification (RPA) is implemented to amplify the targeted toxin genes specifically. RPA is an isothermal amplification technique that achieves rapid and specific amplification of nucleic acids. The nucleic acids enriched from cell lysates by using fiber papers are amplified for following steps.
Transcription
    To obtain ssRNA which can be recognized by RENDR, the sequence of T7 promoter is added to the upstream of the forward primer to enable the transcription of amplified genes by T7 RNA polymerase.
6
RPA amplification and transcription.
Cell-free system detection
    After completing the steps above, amplified dsDNA of conserved region of pirA/pirB toxin genes are obtained. Add the dsDNA into the cell-free sensing system as the input, then the signal can be transformed and easily visualized.
6
The expression of β-lactamase and Nanoluc in cell-free detection system.

Treatment & Prevention

    Outer-membrane vesicles (OMVs) are small, membrane vesicles primarily produced by Gram-negative bacteria. They can deliver a wide range of biomolecules, including proteins and nucleic acids, to other cells (3). Genetic engineering of the OMVs‐producing bacteria has been applied in many fields. We leverage and modify OMVs from Escherichia coli and colonize the genetically modified organisms (GMOs) in shrimps’ intestine to produce OMVs.
    Compared with the whole bacterial cells, the modified OMVs are selected for the following advantages. First, the size of OMVs is small, around 50-100 nm, which makes it easy to be recruited around Vp cells. Second, OMVs have good biocompatibility, which makes it possible to deliver the cargo into Vp cells by membrane fusion.
    The high-level secretion of OMVs is the key to the functionality of modified OMVs. Destabilizing the membrane may be an approach to increase the secretion of OMVs. The outer membrane protein OmpA and lipoprotein Lpp of E. coli connect the bacterial outer membrane to the peptidoglycan layer, playing an important role in maintaining the stability of the membrane. RNA interference (RNAi) is adopted to target ompA and lpp respectively, thus inhibiting the translation of the two genes and increasing the production of OMVs. The two different sequences of siRNA are designed using siRCon solftware and are controlled by rhamnose inducible promoter. Once induced, the cross-links between outer membrane and peptidoglycan layer will decrease, reducing the stability of outer membrane and increasing the secretion of OMVs (4). Rhamnose will be mixed in the fodder of shrimps (Learn more in our Proposed Implementation page). As shrimps can not metabolize rhamnose, the mixed-in rhamnose will eventually be taken into the intestine-colonized GMOs and induce the expression of siRNA, increasing the level of OMVs secretion as a result (Fig. 4a)
    In addition, the overexpression of mepS (Fig. 4b), which encodes a peptidase that can hydrolyze the proteins connecting the outer membrane to the periplasmic junction of E. coli, makes it easier for bacteria to produce high-level of OMVs as well (5). (The two methods for high-level secretion of OMVs are showed in Fig. 4)
7
Fig. 4 Design of high-level secretion of OMVs by RNAi and mepS expression. a The gene circuit of mepS. b The gene circuit of siRNAs for ompA and lpp. c The principle of high-level secretion of OMVs.
    The OMVs are modified for carrying recombinant plasmids or surface displaying fusion proteins. In Gram-negative bacteria, plasmids can be packaged through the process of outer and inner membrane vesicles (O-IMVs) formation or due to the damage of the inner membrane (4). Thus, recombinant plasmids can be carried by modified OMVs and be delivered to Vp.
    Cell-surface display system is used to display proteins on the surface of bacteria as well as OMVs. Two outer membrane proteins, ClyA (6) and INPNC (7), are chosen to construct surface display system on OMVs, for displaying the ligands targeted to Vp or the toxin. (All of the surface display system used in our design are showed in Fig. 5)
8
Fig. 5 Overview of the surface display system used in our design.

Treatment

    For pathogenic Vp, we use modified OMVs to deliver the recombinant plasmid that will express endolysin intracellularly, thus specifically killing the Vp and healing the sick shrimps.
    Lsyqdvp001 endolysin, encoded by edl060 in phage QDVP001, has a CHAP domain that contributes to the hydrolysis of peptidoglycan, which was reported to be specific to Vp (8). Additionally, the LMT signal peptide we identified before in Vibrio natriegens (https://2021.igem.org/Team:XMU-China/Results) is added to guide Lysqdvp001 to the peptidoglycan layer when induced by L-rhamnose. As a result, in the presence of rhamnose, endolysin is expressed and secreted to the peptidoglycan layer of Vp, lysing the peptidoglycan and killing Vp.
    To increase the specificity of OMVs, the tail tubular protein A (TTPA) and tail tubular protein B (TTPB) from a phage of Vp, which can target the receptor Vp0980 on Vp (9), are displayed on the surface of OMVs by ClyA and INPNC. With TTPA/TTPB on the surface, OMVs can be recruited and then accumulated around Vp. High concentration of OMVs will increase the possibility and efficiency of plasmid delivery. (The design for killing the pathogen by endolysin is showed in Fig. 6)
10
Fig. 6 Overview of the treatment part. a L-rhamnose-induced system for the expression of LMT-edl060. b The diagram of killing the pathogen by endolysin.

Prevention

    PirA/PirB toxin, the main virulence factors of Vp, can insert into the target cell membrane in the form of heterodimer and form pores, resulting in cell damage and even death (10). Our prevention section aims at defunctionalizing the PirA/PirB toxin.
    We select aminopeptidase N1 of Litopenaeus vannamei (LvAPN1) and Fetuin-B of mouse (FET) to serve as receptors of PirA/PirB toxin, inactivating PirA/PirB toxin before it could harm the shrimps.
    Research suggested that LvAPN1 is the receptor of PirA/PirB toxin in shrimp hepatopancreatic cells (11). It contains a transmembrane domain, a Cry toxin binding (CBR) domain which can bind to the toxin and so on. Truncated LvAPN1 containing CBR domain and M1 peptidase domain is served as the receptor rLvAPN1.
    FET has a strong affinity to toxin PirB. After predicting the binding sites of FET with toxin PirB, 146th-175th residues of the A chain in FET are truncated as the receptor rFET (12, 13).
    The two receptors, rLvAPN1 and rFET, are displayed on the surface of OMVs produced by the colonized GMOs into the shrimps’ intestine. They can competitively inhibit PirA/PirB toxin from binding to its original receptor in hepatopancreatic cells of shrimps, attenuating the toxicity of PirA/PirB toxin. (The design of competitively binding to the toxins is showed in Fig. 7)
13
Fig. 7 The diagram of competitively binding to the toxins by rFET and rLvAPN1 displayed by ClyA and INPNC.

Extended Prospects

    More than just alleviating the effects of PirA/PirB toxin, we also design a CRISPR/Cas system to destroy pirA/pirB gene for achieving the virulence loss of Vp, as supplement for our prevention section. Two CRISPR-arrays are designed. One targets pirA/pirB, while the other targets the antibiotic resistance gene on the plasmid to avoid introducing new antibiotic resistance to Vp. The gene cluster of Cas protein is from Vibrio alginolyticus (ATCC 33787), and the conservative repeats are from Vp strain FORC_022 (14). Since anti-CRISPR system derived from lysogenic phage in Vp may inhibit the function of designed CRISPR/Cas system, aca2 sequence is added to the plasmid to prevent CRISPR/Cas system from being defunctionalized (15). In order to recruit a high level of OMVs around Vp, we also display TTPA/TTPB on the surface of OMVs. With a high-level secretion, OMVs displaying TTPA/TTPB can deliver the plasmids of CRISPR/Cas system into Vp. (The design of CRISPR/Cas system for prevention is showed in Fig. 8)
15
4
Fig. 8 Virulence loss of Vp by CRISPR/Cas system. a The gene circuit of type Ⅰ CRISPR/Cas system. b The diagram of defunctionalizing the Vp.

Biosafety

    GMOs will be mixed in the fodder and put into the water environment of shrimp ponds, then colonize directly in shrimps’ intestine. The kill switch must be designed for some reasons. First, before harvesting and marketing mature shrimps, we must ensure that the GMOs colonized in shrimp intestine are killed. Besides, GMOs remaining in the tailwater of shrimp ponds, must be killed before the tailwater being drained to the environment out of the shrimp ponds. MazF is selected as the toxin and its expression is induced by arabinose. By cleaving the mRNA of GMOs inside, MazF can inhibit protein synthesis and kill them, guaranteeing biosafety (Fig. 9).
15
Fig. 9 Arabinose-induced mazF expression.

Reference

      1. H.-C. Wang, S.-J. Lin, A. Mohapatra, R. Kumar, H.-C. Wang, A Review of the Functional Annotations of Important Genes in the AHPND-Causing pVA1 Plasmid. Microorganisms 8, 996 (2020).
      2. L. Gambill et al. ,https://doi.org/10.1101/2022.01.12.476080 (2022)
      3. S. M. Collins, A. C. Brown, Bacterial Outer Membrane Vesicles as Antibiotic Delivery Vehicles. Front. Immunol. 12, 733064 (2021).
      4. C. Schwechheimer, C. J. Sullivan, M. J. Kuehn, Envelope control of outer membrane vesicle production in Gram-negative bacteria. Biochemistry 52, 3031-3040 (2013).
      5. C. Schwechheimer, D. L. Rodriguez, M. J. Kuehn, NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli. Microbiologyopen 4, 375-389 (2015).
      6. K. Murase, Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins (Basel) 14, 78 (2022).
      7. Z. Zhang et al., Engineering Escherichia coli to bind to cyanobacteria. J. Biosci. Bioeng. 123, 347-352 (2017).
      8. W. Wang, M. Li, H. Lin, J. Wang, X. Mao, The Vibrio parahaemolyticus-infecting bacteriophage qdvp001: genome sequence and endolysin with a modular structure. Arch. Virol. 161, 2645-2652 (2016).
      9. M. Hu, H. Zhang, D. Gu, Y. Ma, X. Zhou, Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus. Emerg. Microbes. Infect. 9, 855-867 (2020).
      10. S. J. Lin, K. C. Hsu, H. C. Wang, Structural Insights into the Cytotoxic Mechanism of Vibrio parahaemolyticus PirAvp and PirBvp Toxins. Mar. Drugs. 15, 373 (2017).
      11. W. Luangtrakul et al., Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. PLoS. Pathog. 17, e1009463 (2021).
      12. M. V. De Los Santos et al., The Vibrio parahaemolyticus subunit toxin PirBvp recognizes glycoproteins on the epithelium of the Penaeus vannamei hepatopancreas. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 257, 110673 (2022).
      13. M. Victorio-De Los Santos et al., The B Subunit of PirABvp Toxin Secreted from Vibrio parahaemolyticus Causing AHPND Is an Amino Sugar Specific Lectin. Pathogens 9, 182 (2020).
      14. P. Baliga, M. Shekar, M. N. Venugopal, Investigation of direct repeats, spacers and proteins associated with clustered regularly interspaced short palindromic repeat (CRISPR) system of Vibrio parahaemolyticus. Mol. Genet. Genomics. 294, 253-262 (2019).
      15. N. Birkholz, R. D. Fagerlund, L. M. Smith, S. A. Jackson, P. C. Fineran, The autoregulator Aca2 mediates anti-CRISPR repression. Nucleic. Acids. Res. 47, 9658-9665 (2019).