Technical field of the invention

The present invention relates to the field of monitoring acute hepatopancreatic necrosis disease in shrimp. More specifically, the present invention discloses that detection of the presence of alkaline phosphatase Phox enzyme, or, of the PiRA ^vp and/or the PirB ^vp toxins of acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus correlates with non-virulence or virulence, respectively of said bacterium. Hence, an assay capable of detecting said Phox enzyme and/or said toxins could be very useful to monitor said disease in shrimp.

Background art

The Gram-negative marine bacterium, Vibrio parahaemolyticus is an important aquatic pathogen and several strains are capable of causing acute hepatopancreatic necrosis disease (AHPND) and other important disease in shrimp aquaculture (Jayasree et al., 2006; Li et al., 2017). The shrimp production in AHPND-affected regions has at times dropped considerably (to ~60%) and disease has caused global loss of $ 1 billion per year to the shrimp farming industry (Lee et al., 2015; Nunan et al., 2014). Several V. parahaemolyticus strains indeed contain a pVAl plasmid (63-70 kb) encoding the binary toxins named PirA ^vp and PirB ^vp homologous to the Photorhabdus luminescens insect-related (Pir) toxins PirA/ PirB (Campa-Cordova et al., 2017; Gomez-gil et al., 2014; Han et al., 2015; Lee et al., 2015). The PirA ^vp and PirB ^vp toxins are the primary virulence factor of bacteria that mediates AHPND etiology and mortality in shrimps (Campa-Cordova et al., 2017; Dong et al., 2017; Kumar et al., 2018))

However, it is completely unknown whether the latter virulent phenotype can switch into a non- virulent phenotype and whether said phenotypes can be easily distinguished via an easy-to-use assay measuring products which are secreted by each of the phenotypes. Such an assay would be very useful to monitor for example AHPND in shrimp.

Brief description of figures

Figure 1 Coomassie-stained SDS-PAGE gel of V. parahaemolyticus extracellular proteins (VPAHPND ECPS) from different culture conditions, (i) VPAHPND ECP purified from V. parahaemolyticus M0904 culture with constant agitation (120 min ¹ ) and no floccule formation show two prominent bands at 13 and 50 kDa (Lane 1 - 4, showing the results of 4 replicate cultures) (ii) VPAHPND ECP purified from V. parahaemolyticus M0904 culture with constant agitation (110 min ¹ ) and high flocculation exhibit a single prominent band at 73 kDa (Lane 1 - 4, showing the results of 4 replicate cultures). Figure 2 Expression of virulent AHPND plasmid genes: A- ORF14, B- PirB ^vp and genes responsible for floccule formation: C- alkaline phosphatase PhoX from Vibrio parahaemolyticus M0904 strain cultured under different culture conditions. The V. parahaemolyticus M0904 strain incubated overnight in 20 ml marine broth with constant agitation in either 110 min ¹ (high flocculation) or 120 min ¹ (no floccule formation). Samples from both culture conditions were collected for gene expression assay after 12 and 24 h. For the virulent AFIPND plasmid genes, the expression in the floccules formed group was set at 1. Flowever, for alkaline phosphatase PhoX gene, the expression in the non-floccules formed group was set at 1. The results are the mean ± SE (n=3) and are presented relative to V. parahaemolyticus toxR and rpoA mRNA. Asterisks represents significant difference between the V. parahaemolyticus M0904 culture with floccules (110 rpm) and no floccules (120 rpm) *(P < 0.05), **(P < 0.01), ***(P <

0.001), ****(p < 0.0001), *****(p < 0.00001).

Figure 3 (A) Survival (%) of brine shrimp larvae after 48 h of challenge with V. parahaemolyticus M0904 culture incubated overnight in 20 ml marine broth under constant agitation at 120 min ¹ or 110 min ¹ . The larvae were challenged with V. parahaemolyticus at 10 ⁷ cells/ml of rearing water. Non-challenged larvae were served as a negative control. Error bars represent the standard error of five replicates; different letters indicate significant differences (P < 0.001).

(B) Survival (%) of brine shrimp larvae after 60 h of challenge with 1.3, 1.95, 2.6 and 3.9 pg of VPAHPND extracellular protein (ECP) concentrated from V. parahaemolyticus M0904 culture incubated with constant agitation of 120 min ¹ with PirA ^vp and PirB ^vp toxin or 110 min ¹ with alkaline phosphatase PhoX. Non-challenged larvae served as a negative control. Error bars represent the standard error of three replicates; different letters indicate significant differences (P<0.001).

(C) Survival (%) of brine shrimp larvae after 60 h of challenge with 1.3, 1.95, 2.6 and 3.9 pg of VPAHPND purified PirA ^vp and PirB ^vp toxin or VPAHPND purified alkaline phosphatase PhoX. Non-challenged larvae served as a negative control. Error bars represent the standard error of three replicates; different letters indicate significant differences (P<0.001).

Figure 4 Survival (%) of Macrobrachium larvae (8-days old) after 12, 24, 36 and 48 h post challenge with V. parahaemolyticus M0904 culture incubated overnight in 20 ml marine broth (MB) under constant agitation with 120 min ¹ or 110 min ¹ . The larvae were challenged with V. parahaemolyticus at 10 ^s cells/ml of rearing water. Non-challenged larvae were served as a negative control. Values are presented as mean ± SE (n=3). Asterisks represents significant difference between the treatment groups challenged with either 110 rpm or 120 rpm min ¹ M0904 culture *(P < 0.05), **(P < 0.01), ***(P

< 0.001), ****(p < 0.0001), *****(p < 0.00001), ******(p < 0.000001), *******(p < 0.0000001). Description of invention

The present invention discloses that low shaking speed can trigger phenotype switching in V. parahaemolyticus, mediating, among others, changes in virulence toward shrimp. The present invention indeed shows that V. parahaemolyticus at lower shaking speed downregulate PirAB ^vp toxin expression while producing and secreting an alkaline phosphatase PhoX enzyme. The present invention thus shows that V. parahaemolyticus displays distinct phenotypes depending on environmental conditions: a virulent and a non-virulent phenotype.

Therefore, the present invention relates in first instance to the usage of the alkaline phosphatase Phox of Vibrio parahaemolyticus as a marker to phenotype said Vibrio parahaemolyticus as non-virulent. The term 'Vibrio parahaemolyticus' refers to all strains of a Gram-negative marine bacterium which is an important aquatic pathogen and capable of causing acute hepatopancreatic necrosis disease (AHPND) and several other important disease such as tail necrosis, shell disease, red disease, loose shell syndrome (LSS), and white gut disease (WGD) in shrimp aquaculture (Jayasree et al., 2006; Li et al., 2017). The latter term more specifically relates the V. parahaemolyticus strains containing a pVAl plasmid (63-70 kb) encoding the binary toxins named PirA ^vp and PirB ^vp homologous to the Photorhabdus luminescens insect-related (Pir) toxins PirA/ PirB (Campa-Cordova et al., 2017; Han et al., 2015) (Gomez-Gil et al. 2014; Soto-Rodriguez et al. 2015; Lee et al. 2015). The PirA ^vp and PirB ^vp toxins are the primary virulence factor of bacteria that mediates AHPND etiology and mortality in shrimps (Campa-Cordova et al., 2017; Dong et al., 2017; Han et al., 2015). A non-limiting example of such a V. parahaemolyticus strain is strain M0904 deposited with the Belgian Coordinated Collections of Micro-Organisms (BCCM/LMG Bacteria Collection), Laboratorium voor Microbiologie, Universiteit Gent (UGent), K.L. Ledeganckstraat 35, B-9000 Gent, Belgium and having deposit number (accession number): LMG P-31518.

The term 'non-virulent' Vibrio parahaemolyticus indicates that said bacterium is not mediating AHPND etiology and mortality in shrimps and has thus a reduced toxicity in shrimp when compared to bacteria mediating AHPND etiology and mortality in shrimps.

The term 'alkaline phosphatase Phox' refers to an enzyme that releases a free inorganic phosphate from many phosphate-containing compounds and provides bacteria with the inorganic phosphate as a nutrient as is described by Torriani (1990). More specifically, said term refers to the enzyme having the accession number - Q87JR9 (UniProt) and having the following amino acid sequence: MSKETFDATRYNQSDNKPFEEVLEASLSRRSILKGGLGISAMTAFGAFGLAGCNSSSSGT SASNGSGVSK AVLNFDSIPGSLTDAVSIPQGYTAQVLVPWGTPLNAQGSAWKNDGSNTSSDQLNALGMHH DGMHFFPLND STTDGLLCINHEYIDTSALHPNGPTVANGVRTIVDEVRKEINAHGVSVVRIQLEDNMWKL VDTDPLNRRY TGATVMDLSGPVAHTALTVTRFSPDGSQARGTLNNCGNGYTPWGTYLTCEENWPGYFVNA GTRTEEQDRI

GVDDKSTRYLWETLAGNSEERLDEFTRFNVAPTGTSSADDYRNEANGHGYIVEIDPY TQNSRAKKRTALG

RFRHEGCAFGKLEAGKPVVFYSGHDSRFEYLYKFESAAAWDPADANPANRLATGDKY MDEGTLYVARFNE

DSTGTWLPLTLDSVTTSGGTLADHFNSLAEIIINTAGAADLVGATPMDRPEWCSVDP FTGSVYLTLTNNT

RRTDETNPANPRLNNKFGHVIRWDEGTSATDFIWDIFVFGSPENGDADTNRSGLNEL NQFASPDGLAFDG

RGILWIQTDNGADEVTSYTNDQMLAWPSKLTNENGDQAVIGADNQAELKRFFVGPNG CEVTGFTISPDY

KSLFVNIQHPGNWPYSDDAAQETPTGTTVRPRAATVVIRREDGGEIAV (SEQ ID N°l)

The terms 'a marker to phenotype' refers to the usage of the presence of the alkaline phosphatase PhoX in a substrate such as shrimp culture water to indicate that the Vibrio parahaemolyticus is non- virulent.

Furthermore, the present invention discloses the usage as described above to monitor acute hepatopancreatic necrosis disease in shrimp.

The term 'shrimp' refers to any shrimp known in the art and more particularly relates to freshwater shrimp such as Macrobrachium rosenbergii and Penaeus species.

The term 'acute hepatopancreatic necrosis disease (AFIPND)' refers to a disease, originally known as early mortality syndrome (EMS), which is characterized by acute, massive sloughing of hepatopancreatic tubule epithelial cells and which is caused by a virulent strain of Gram-negative marine bacterium, Vibrio parahaemolyticus ubiquitous in estuarine and coastal waters (Li et al., 2017; Tran et al., 2013).

Moreover, the present invention relates to the usage of means to detect the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus, or -in other words-, relates to the usage of molecules which bind to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus (such as in an ELISA/antibody-based assay) to phenotype said Vibrio parahaemolyticus as non-virulent.

The term 'molecules which bind to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus' relates to any molecule known in the art (such as peptides, antibodies or fragments thereof and the like) which can be used to specifically detect the presence of a particular protein such as the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus in a matrix such as water.

More specifically, the present invention relates to the usage of means as describe above wherein said molecules are antibodies or fragments thereof specifically binding to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus.

The term 'antibody or a fragment thereof' relates to an antibody characterized as being able to specifically bind to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus or any part thereof, or, an antigen-binding fragment thereof, particularly of the F(ab')2, F(ab) or single chain Fv (scFv) type, or any other type of recombinant antibody known in the art. The phrase "specifically (or selectively) detects (or binds)" the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus", when referring to an antibody, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to marker "X" from a specific species can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with marker "X" and not with other proteins, except for polymorphic variants and alleles of marker "X". This selection may be achieved by subtracting out antibodies that cross-react with marker "X" molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Flarlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

Furthermore, the present invention relates to an assay (for example a lateral flow immunochromatographic assay as described by Delmulle et al. (2005) comprising molecules which bind to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus and comprising molecules which bind to the PirA ^vp and/or the PirB ^vp toxins of Vibrio parahaemolyticus.

The term 'PirA ^vp and/or PirB ^vp toxins of Vibrio parahaemolyticus' relates to extracellular secreted toxins produced by AHPND-causing V. parahaemolyticus which are homologous to the insecticidal PirA and PirB toxins (d-endotoxins) of Photorhabdus and Xenorhabdus species (Han et al., 2015). PirA ^vp facilitates target-specific recognition by binding to a certain ligand on the cell membrane/receptors (e.g., monosaccharides like N-acetylgalactosamine (GalNAC) and oligosaccharides, while PirB ^vp (containing an N-terminal domain, PirB ^vp N, and a C-terminal domain, PirB ^vp C), is mainly responsible for cell death via pore formation and is involved in protein-protein and protein-ligand interactions (Kumar et al., 2019; Lin et al., 2017; Sirikharin et al., 2015).

The terms 'molecules which bind to the PirA ^vp and/or the PirB ^vp toxins of Vibrio parahaemolyticus' have similar meaning as described above for 'molecules which bind to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus'.

The present invention further relates to an assay as described above to phenotype said Vibrio parahaemolyticus as non-virulent or as virulent. The present invention more specifically relates to an assay as described above wherein said molecules are antibodies or fragments thereof binding to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus, and/or binding to the PirA ^vp and/or the PirB ^vp toxins of Vibrio parahaemolyticus. The terms 'antibodies or fragments thereof specifically binding to the PirA ^vp and/or the PirB ^vp toxins of Vibrio parahaemolyticus' have a similar meaning as described above for antibodies or fragments thereof binding to the alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus The present invention also relates to an assay as described above wherein said assay is an antibody- based assay such as a lateral flow immunochromatographic assay or an enzyme-linked immunosorbent assay (ELISA). A similar immunoassay-based lateral flow dipstick was -for example- developed for rapid detection of aflatoxin B1 in pig feed (Delmulle et al 2005).

Moreover, the present invention relates to a process to phenotype said Vibrio parahaemolyticus as non-virulent comprising:

-providing a sample such as a shrimp culture water sample

-bringing said water sample into contact with molecules which bind tothe alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus, and

-determining whether said alkaline phosphatase Phox enzyme of Vibrio parahaemolyticus is present in said sample, wherein said Vibrio parahaemolyticus is a non-virulent phenotype when said enzyme is present in said sample.

The present invention also relates to a process as described above wherein said sample is also brought into contact with molecules which bind to the PirA ^vp and/or the PirB ^vp toxins of Vibrio parahaemolyticus and wherein it is also determined whether PirA ^vp and/or the PirB ^vp toxins of Vibrio parahaemolyticus are present in said sample, wherein said Vibrio parahaemolyticus is a non-virulent phenotype when said enzyme is present in said sample and wherein said Vibrio parahaemolyticus is a virulent phenotype when at least one of said toxins is present in said sample.

Examples

Materials and Methods

Bacterial strains and growth conditions

Total two bacterial strains were used in the study: Vibrio parahaemolyticus M0904 (AHPND strains) and Aeromonas hydrophila LVS3 (Defoirdt et al., 2006; Kumar et al., 2018). LVS3 (autoclaved) were used as feed for Artemia larvae and M0904 were used for toxin and enzyme production for the challenge assay. The M0904 strain used in the experiment M0904 is deposited by the department of Animal Production/Lab for Aquaculture & Artemia Reference Center (Campus Coupure F, Coupure Links 653, 9000 Gent, Belgium) with the Belgian Coordinated Collections of Micro-Organisms (BCCM/LMG Bacteria Collection), Laboratorium voor Microbiologie, Universiteit Gent (UGent), K.L. Ledeganckstraat 35, B-9000 Gent, Belgium and having deposit number (accession number): LMG P- 31518. A stock culture of M0904 were streaked onto Marine agar plates (Carl Roth, Belgium) (MA) to obtain pure colonies. A single colony was inoculated into Marine broth (MB) (Carl Roth, Belgium), incubated overnight at 28 °C under constant agitation (120 min ¹ ) and the stocked culture was prepared in 40% glycerol and stored in -80 °C. The LVS3 culture (LMG 22148) was collected from the Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Belgium. The strain was streaked onto Marine agar plates (Carl Roth, Belgium), afterwards single colony was inoculated in Marine broth (Carl Roth, Belgium), and stock culture was prepared in a similar manner.

Analysis of V. parahaemolyticus cells cultured in different conditions

(a) Light and fluorescence microscopy

The V. parahaemolyticus M0904 strain culture under constant agitation at either 110 min ¹ or 120 min ¹ were observed by light and fluorescence microscope to observe the changes at cellular level. At first, 20 pi of bacterial suspension cultured at either 110 min ¹ or 120 min ¹ were put onto a clean glass slide. Than a cover glass was placed over the sample and excess liquid were removed by gently pressing the cover glass. The samples were observed in light microscope (Zeiss Axioskop 2 plus, Carl Zeiss, Germany) in lOx, 40x, 36x and lOOx magnification to detect the floccules formation by the bacterial strain. Later, the bacterial samples were stained with Calcofluor white stain (Sigma-Aldrich, USA) to detect the extracellular polysaccharides in the floccules produced by V. parahaemolyticus M0904 strain through fluorescence microscope (Zeiss Axioskop 2 plus, Carl Zeiss, Germany). Briefly, 20 mI of bacterial suspension were put onto a clean glass slide. Afterwards, 1 drop of Calcofluor white stain and 1 drop of 10 % potassium hydroxide were added to the samples (according to the manufacturer instructions). The samples were covered with cover glass and incubated for 1 minute at room temperature. Subsequently, the slides were examined under fluorescence microscope with excitation of band pass (BP) 365/12, beam splitter FT 395 and emission long pass 397 in lOx, 40x, 36x and lOOx magnification.

(b) Flow cytometry

The V. parahaemolyticus M0904 strain cells cultured at either 110 min ¹ or 120 min ¹ constant agitation was diluted to 5 x 10 ⁹ cells/ml with sterile MB using spectrophotometer (Genesys 20, Thermo Spectronic). Afterwards, 1 ml of bacterial suspension was transferred to 2 ml sterile eppendorf tubes and centrifuged for 10 min at 8000 x g in room temperature. The bacterial cells were collected and resuspended in 10 ml Tris-buffered saline (0.13 M NaCI and 10 mM Tris hydrochloride, pH 7.4). Later, 250 mI of bacterial cells in Tris-buffered saline was transferred to 2 ml sterile eppendorf tubes and 1 mI of FITC (fluorescein isothiocyanate)-lectin was added (7 different types lectins were used with different glycan specificity, Table SI). The bacterial cell and lectin mixture were then transferred to 96 well plate and to facilitate the binding of FITC-lectins to the bacteria cells the plates were incubated for 30 min in dark at room temperature.

Subsequently, the samples were analyzed with a CytoFLEX flow cytometer system (Beckman Coulter's Life sciences, France) equipped with a 37-, m (pore size) filter, a 75-, um (pore size) orifice, and a 0.3 neutral density filter and photomultiplier tube set at 500 V. The calibration and standardization of the flow cytometer were done in accordance with manufacturer specifications. The fluorescent microbeads were used as standards for fluorescence and volume and green fluorescence were measured at 525 nm (FITC) and 488 nm (FSC). The flow cytometer was set as follows: gain FSC (forward scatter)- 106, gain FITC- 113 and speed-4 (implying an event rate never exceeding 1000 events per second). Counts were recorded as logarithmic signals and were triggered on the green fluorescence channel. Data were processed with CytExpert software (Beckman Coulter's Life sciences, France), using electronic gaining to separate the desired events. Presentation of the data as FITC/count or FSC/count histogram plot allowed for optimal distinction between FITC-lectins labelled bacteria cells and instrument noise or sample background (Frossard et al., 2016; Hammes and Egli, 2010).

(c) Marine agar with congo red (MACR) plate method

The marine agar with congo red (MACR) plate method were used out to determine the production of extracellular slime by V. parahaemolyticus M0904 strain following the procedure as described by Freeman et al. (Freeman et al., 1989) and Phuoc et al. (Phuoc et al., 2009) with slight modifications. Briefly, the V. parahaemolyticus M0904 culture were incubated for 24 h at 28 °C in 20 ml marine broth (MB) with constant agitation at either 110 min ¹ or 120 min ¹ . Subsequently, the broth was discarded, and cells attached to the bottom of the erlenmeyer were collected after adding and mixing with 5 ml of FASW. The bacterial suspension in FASW were streaked onto marine agar with congo red (MACR) plates (37.4 g MB, 50 g sucrose, 15 g agar and 0.8 g congo red). The congo red was prepared as a stock solution, autoclaved and then added to the culture medium when it had cooled to 55 °C. After inoculation, the plates were incubated for 24 h at 28 °C. The bacterial strains that produce extracellular slime develop different colour colonies in MACR plates (Freeman et al., 1989). The assay was performed in triplicates and are representative of at least two independent experiment.

Purification of V. parahaemolyticus extracellular protein (VPAHPND ECP)

In total, two separate tests were performed. Briefly, the V. parahaemolyticus M0904 strain was grown overnight at 28 °C in 20 ml Marine broth (MB) (Carl Roth, Belgium) in 50 ml erlenmeyer with constant agitation either 110 min ¹ or 120 min ¹ . The overnight grown bacterial suspension was centrifuged for 15 min at 3500 x g. The supernatant was collected and filtered with a 0.2-miti sieve to obtain cell free supernatant (CFS) of V. parahaemolyticus. The CFS of V. parahaemolyticus was then concentrated and dialyzed with phosphate buffered saline (PBS) with 10 kDa amicon ^® ultra-15 centrifugal filters (Merck Millipore, USA) (Kumar et al 2019). The concentrated extracellular protein (VP _A H _PND ECP) was collected and immediately preserved at -80 °C for further analysis.

NonoLC-MS/MS analysis of PirA ^vp and PirB ^vp toxins and alkaline phosphatase PhoX enzyme from VPAHPND ECP

To identify the presumptive proteins produced by V. parahaemolyticus AFIPND strain from different culture conditions, the concentrated VPAHPND ECP samples were combined with loading buffer, vortexed, heated for 5 min at 95 °C and electrophoresed in 4-20% SDS-PAGE gel (BioRad, Belgium), with each lane receiving equivalent volume (10 mI) of protein. The gels were stained with Coomassie Biosafe (BioRad Laboratories) and the signals were detected by a ChemiDoc MP imaging system (BioRad, Belgium). The protein concentration was determined by the Bradford method (Bradford, 1976) using bovine serum albumin as standard. The possible candidate bands of PirA ^vp and PirB ^vp toxin and alkaline phosphatase PhoX enzyme on Coomassie stained SDS-PAGE gels was excised using a clean scalpel (preferentially under the laminar flow to avoid the keratin contamination) and the gel-pieces (with a clean pincet) were transferred into sterile eppendorf tubes. The gel-pieces were rinsed twice with 500 mI sterile distilled water and kept in -20 °C for LC-MS/MS analysis.

The gel pieces were then washed consecutively with water, acetonitrile (ACN) in water (50/50, v/v) and ACN and were then dried completely by vacuum drying. The dried gel bands were rehydrated with 15 mI of a trypsin-containing solution (5 ng/mI in 50 mM ammonium bicarbonate) and 50 mM ammonium bicarbonate was added until the bands were completely submerged. Next, the bands were incubated overnight at 37°C for digestion and elution of the resulting peptides from the gel. Formic acid was added to the eluted peptides to deactivate trypsin and the samples were vacuum dried. Following in-gel-digestion, the dried peptide samples were dissolved in 20 mI loading buffer (0.1% TFA in water/acetonitrile, 2/98 (v/v)) and half of each sample was analyzed on a tandem configurated Ultimate 3000 RSLC nanoLC (Thermo Scientific, Bremen, Germany) in-line connected to an LTQ- Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) equipped with a pneu-Nimbus dual ion source (Phoenix S&T) and a Butterfly nano-LC column oven (Phoenix S&T) to keep the column temperature constant at 50°C. The sample mixture was first loaded on a trapping column (made in- house, 100 pm I.D. x 20 mm length, 5 pm beads C18 Reprosil-FID, Dr. Maisch). After flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 pm I.D. x 200 mm length, 1.9 pm beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with loading buffer and were separated with a non-linear 1.5-hour gradient from 98% solvent A (0.1% formic acid in water) to 56% solvent B (0.1% formic acid in water/ACN 20:80 (v/v)) at a flow rate of 250 nl/min followed by a 5 min wash reaching 99% buffer B.

The mass spectrometer was operated in data dependent, positive ionization mode, automatically switching between MS and MS/MS acquisition for the 20 most abundant peaks in a given MS spectrum. The source voltage was 3 kV, and the capillary temperature was 275°C. In the LTQ-Orbitrap Elite, full scan MS spectra were acquired in the Orbitrap (m/z 300-2,000, AGC target 3 ^c 10 ^s ions, maximum ion injection time 100 ms) with a resolution of 60 000 (at 400 m/z). The 20 most intense ions fulfilling predefined selection criteria (AGC target 5 ^c 10 ³ ions, maximum ion injection time 20 ms, spectrum data type: centroid, exclusion of unassigned and 1 positively charged precursors, dynamic exclusion time 20 s) were then isolated in the linear ion trap and fragmented in the high-pressure cell of the ion trap. The CID collision energy was set to 35 V and the polydimethylcyclosiloxane background ion at 445.120028 Da was used for internal calibration (lock mass).

Data analysis was performed with MaxQuant (version 1.6.2.6) using the Andromeda search engine with default search settings including a false discovery rate set at 1% on both the peptide and protein level. Spectra were searched against the proteins of Vibrio parahaemolyticus strain 20130626002S01 in the database (containing 5004 protein sequences) supplemented with sequences of the PirA ^vp and PirB ^vp toxins (Gen Bank, NCBI). The mass tolerance for precursor and fragment ions was set to 4.5 and 20 x g, respectively, during the main search. Enzyme specificity was set as C-terminal to arginine and lysine, also allowing cleavage at proline bonds with a maximum of two missed cleavages. Variable modifications were set to oxidation of methionine residues, propionamidation on cysteines and acetylation of protein N-termini. Only proteins with at least one unique or razor peptide were selected. A minimum ratio count of two unique or razor peptides was required for quantification. The resulting protein lists were sorted on descending iBAQ ratio, revealing PirA ^vp , PirB ^vp and PhoX as most abundant protein in their respective protein band. The identified peptides of PirA ^vp and PirB ^vp toxins were mapped on the corresponding protein sequence.

PCR confirmation for presence of AHPND plasmid in V. parahaemolyticus cultures The V. parahaemolyticus M0904 strain cultured at either 110 min ¹ or 120 min ¹ was confirmed to harbor the AHPND plasmid by PCR using AP3 primers (Table S2). The AP3 based PCR fragment was not present in non-AHPND V. parahaemolyticus (Sirikharin et al., 2014; Suong et al., 2017).

RNA extraction and reverse transcription

The V. parahaemolyticus M0904 strain were grown overnight in triplicate in 20 ml sterile marine broth (MB) at either 110 min ¹ or 120 min ¹ . Cells were harvested after 12 and 24 h of incubation and total RNA was extracted with Qjagen RNeasy Plus Mini Kit (Cat No. 74136) from the bacterial samples in tri plicate according to the manufacturer instructions. The RNA quality and quantity were measured on NanoDrop spectrophotometer (ThermoFisher Scientific, Belgium) and RNA samples with A260/A280 ratios >2.0 and A260/A230 ratios >1.5 were used for the analysis. The RNA integrity was checked by agarose gel electrophoresis and the RNA samples were stored in -80 °C for subsequent use.

Reverse transcription was done with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Belgium) according to the manufacturer's guidelines. Briefly, 1 pg total RNA and 1 pi random hexamer primer solution was mixed first. Then, 8 mI of reaction mixture containing 4 mI of 5x reaction buffer (0.25 mol ¹ T ris-HCI pH 8.3, 0.25 mol ¹ MgC , 0.05 mol ¹ DTT), 2 mI of 0.01 mol ¹ dNTP mix, 20 units of ribonuclease inhibitor, 200 units of RevertAid™ FI minus M-MuLV reverse transcriptase was added. The reaction mixture was incubated for 5 min at 25 °C followed by 60 min at 42 °C. The reaction was terminated by heating at 70 °C for 5 min and then cooled to 4 °C. Complementary deoxyribonucleic acid (cDNA) samples were checked by PCR and stored at -20 °C for further use.

Quantitative real-time PCR (RT-qPCR) analysis

The expression of 2 VPAHPND plasmid related virulent genes including toxin and coding genes of virulent plasmid and 1 alkaline phosphatase PhoX gene associated with floccules formation were measured by RT-qPCR with pair of specific primers using StepOnePlus Real-time PCR systems (Applied Biosystems) (Table S3). The Ct values from the two reference genes rpoA (RNA polymerase A submit) and toxR mRNA, used as the internal control, were subjected to geomean and expression of the genes was calculated relative to the rpoA and toxR mRNA levels. The amplification was performed in a total volume of 20 mI, containing 10mI of 2X Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific), 1 mI of cDNA (50 ng), 8 mI of nuclease free water and 0.5 mI of each specific primer. Master mixes were prepared for each biological replicate of the sample in triplicate and RT-qPCR for target and reference genes was performed with a four-step amplification protocol: initial denaturation (10 min at 95 °C); 40 cycles of amplification and quantification (15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C); melting curve (55-95 °C) with a heating rate of 0.10 °C/s and a continuous fluorescence measurement) and cooling (4 °C). Negative control reaction was included for each primer set by omitting template cDNA. The comparative CT method (2-AACt method) following Livak and Schmittgen (Livak and Schmittgen, 2001) was used to analyse the expression level of the target genes and verified by Pfaffl relative standard curve method (Pfaffl, 2002). The Log transformed 2 ^A AACT value were subjected of a t-test, and the p values smaller than 0.05 were considered statistically significant. Axenic brine shrimp hatching

The gnotobiotic brine shrimp larvae was produced by hatching high-quality cysts axenically (germ-free) following decapsulation and hatching procedures as described by Baruah et al. (Baruah et al 2014). Briefly, 200 mg of A. franciscana cysts (EG ^® type, batch 21452, INVE Aquaculture, Dendermonde, Belgium) were hydrated in 18 ml of distilled water for 1 h. Sterile cysts and larvae were obtained via decapsulation using 660 pi NaOH (32%) and 10 ml NaOCI (50%). During the reaction, 0.2-miti filtered aeration was provided. All manipulation was carried out under a laminar flow hood and all tools were sterilized. The decapsulation was stopped after 2 min by adding 10 ml Na2S203 at lOg/l. The decapsulated cysts were washed with filtered autoclaved seawater (FASW) containing 35 g/l of instant ocean ^® synthetic sea salt (Aquarium Systems, Sarrebourg, France). The cysts were resuspended in 50 ml tube containing 30 ml FASW and hatched for 24 h on a rotor (6 min ¹ ) at 28 °C with constant illumination of approximately 27 mE/m ² s. After 28 h of incubation, hatched larvae at developmental stage instar II (mouth is opened to ingest particles) were collected, and the axenicity was verified by spread plating (100 ml) as well as by adding (500 mI) of the hatching water on Marine Agar and Marine Broth respectively, followed by incubating at 28 °C for 5 days (Baruah et al., 2012). Experiments started with non-axenic larvae were discarded.

Brine shrimp challenge assay

In total, 3 separate experiments were performed to determine the toxicity V. parahaemolyticus M0904 strain (in different culture conditions), VPAHPND ECP and purified alkaline phosphatase PhoX enzyme and PirA ^vp and PirB ^vp toxin in brine shrimp larvae. The toxicity assay were performed according to the method developed by Kumar et al. (Kumar et al., 2018). In the first experiment, the hatched brine shrimp larvae (at developmental stage II) were collected and group of 20 larvae were transferred to sterile 40-ml glass tubes containing 10 ml FASW. Subsequently, the brine shrimp larvae were challenged with pathogenic V. parahaemolyticus M0904 strain cultured in 20 ml sterile marine broth (MB) at either 110 min ¹ or 120 min ¹ constant agitation at 10 ⁷ cells/ml and fed with autoclaved LVS3. Thereafter, the glass tubes were put back on the rotor and kept at 28 °C. The survival of Artemia larvae was scored manually 48 h after the addition of pathogen. The non-challenged larvae were maintained as negative control.

In the second experiment, group of 10 brine shrimp larvae were collected and transferred to 2 ml sterile eppendorf tubes, containing 1 ml FASW. The brine shrimp larvae subsequently challenged with 1.3, 1.95, 2.6 and 3.9 pg/100 mI of VPAHPND extracellular protein (ECP) concentrated from V. parahaemolyticus M0904 culture incubated with constant agitation of 120 min ¹ with PirA ^vp and PirB ^vp toxin or 110 min ¹ with alkaline phosphatase PhoX enzyme. The survival of larvae was scored 60 h after the addition of toxins. The non-challenged larvae were maintained as negative control. In the third experiment, group of 10 brine shrimp larvae were counted and distributed into sterile 2 ml eppendorf tubes and then challenged with 1.3, 1.95, 2.6 and 3.9 pg/100 pi of VPAHPND purified PirA ^vp and PirB ^vp toxin or VPAHPND purified alkaline phosphatase PhoX enzyme as described above in the lethality test. The non-challenged larvae were maintained as negative controls. The assays were performed in quintuplicate and are representative of at least two independent experiment.

Macrobrachium rosenbergii rearing system

The experiments were carried out at the Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Belgium, in a controlled temperature room. Acclimatized adult freshwater shrimp (Macrobrachium rosenbergii) obtained from laboratory and maintained in four separate freshwater recirculation units were used as brooders. For each experiment, larvae from single ovigerous female breeder were used. Matured female with fully ripe fertilized eggs (indicated by dark grey eggs) were transferred to the hatching tanks (30 I) containing brackish water (6 g/l salinity). Twenty-four hours after hatching, the larvae were collected and stocked in two separate brackish water (12 g/l salinity) recirculation units and fed with newly hatched axenic brine shrimp larvae. The broodstock management techniques were followed as previously described (Baruah et al., 2009; Nhan et al., 2010).

Macrobrachium challenge assay

The experiments were performed to determine the toxicity V. parahaemolyticus M0904 strain incubated in different culture condition in Macrobrachium larvae (8-days old). Group of 10 Macrobrachium larvae (8-days old) were collected and transferred to 150-ml glass tubes containing 100 ml sterile brackish water (12 g/l salinity) (Rahman et al., 2004). Subsequently, the larvae were challenged with pathogenic V. parahaemolyticus M0904 strain cultured in 20 ml sterile marine broth (MB) at either 110 min ¹ or 120 min ¹ at 10 ^s cells/ml (Kumar et al., 2018). The survival of larvae was scored at 12, 24, 36 and 48 h after the addition of pathogen. Macrobrachium larvae that were not challenged with V. parahaemolyticus served as negative control. The assay was performed in quintuplicate and are representative of at least two independent experiment.

Statistical analysis

Survival data of brine shrimp larvae were arcsin transformed to satisfy normality and homoscedasticity requirements as necessary. The data were then subjected to one-way analysis of variances (ANOVA) followed by Duncan's multiple range test using statistical software statistical package for the social sciences version 24.0. P values £ 0.001 were considered significant. Gene expressions results were presented as fold expression relative to the geometrical mean of two internal control genes (toxR and rpoA). The expression level in the floccules forming group (Control) was regarded as 1.0 and thereby the expression ratio of the non-floccule group (treatment) was expressed in relation to the control. Statistical analysis for the significant differences in the expression levels between the control and treatment was performed with single-tailed Student's t-tests using the log transformed data. Survival data of M. rosenbergii were subjected to logistic regression analysis using GenStat 16 (VSN international, Hemel Hempstead, UK) to determine significant differences between the control and treatment.

Results

V. parahaemolyticus (strain M0904) grown at low shaking speed conditions (as an example of an environmental condition) flocculates

In the first experiment, the effect of shaking speed on Vibrio parahaemolyticus M0904 strain was examined considering that altered environmental conditions might modulate the bacterium and trigger a set of adaptive regulatory mechanism (Balaban et al 2004; Rossignol et al 2009; Woude, 2006).

The present results show that V. parahaemolyticus cells incubated at constant agitation of 110 min ¹ (later on called M0904/110) flocculated, whereas cells grown at 120 min ¹ (later on called M0904/120) did not produces floccules. This indicate that shaking speed at M0904/110 induce flocculation in V. parahaemolyticus culture.

Flocculating cells no longer produce PirA ^vp and PirB ^vp toxin, but rather produce and secrete an alkaline phosphatase PhoX

As M0904/110 and M0904/120 cells display distinct phenotypic features at the level of EPS production, it was verified if secretion of proteins would be affected as well (Jayaraman, 2011; Sousa et al., 2011; Thomen et al., 2017). To investigate potential changes in the secretome, the protein present in the culture medium from M0904/110 and M0904/120 cells (VPAHPND ECP) were seperated by SDS-PAGE and stained by coomassie. Strikingly, this analysis showed that M0904/120 cells secrete two main protein of 13 and 50 kDa, while M0904/110 cells secrete one prominent protein of 73 kDa, along with some other proteins that are differentially secreted by both cell types (Figure 1 A). Thus V. parahaemolyticus strain M0904 indeed changes the production of secreted proteins upon slight variation of shaking speed conditions.

Next to identify the main secreted proteins, corresponding protein bands at 13, 50 and 73 kDa where excised from the gel and subjected to mass spectrometry analysis. This analysis identified the two main proteins produced by M0904/120 cells as PirA ^vp (13 kDa) and PirB ^vp (50 kDa), the bacterial toxins encoded by a pVPAl plasmid of AHPND V. parahaemolyticus strain (Han et al., 2015).

Table 1: The identified peptides from PirA ^vp , PirB ^vp and alkaline phosphatase PhoX proteins. The corresponding protein bands at 13, 50 and 73 kDa where excised from the gel and subjected to mass spectrometry analysis. This analysis identified the two main proteins produced by M0904/120 cells as PirA ^vp (13 kDa) and PirB ^vp (50 kDa). The main protein secreted by M0904/110 cells was identified as PhoX, an alkaline phosphatase with a molecular weight of 73 kDa.

The main protein secreted by M0904/110 cells was identified as PhoX, an alkaline phosphatase with a molecular weight of 73 kDa. These results suggest that M0904/110 cells stop producing PirA/B toxins, and instead produce another secreted protein with tentative alkaline phosphatase activity.

Floccules formation attenuates the expression of toxin genes in Vibrio parahaemolyticus To provide further evidence that M0904/110 decreased expression of virulence gene (as shown above), the transcription of pVAl plasmid-bound genes, i.e., PirB ^vp (responsible for PirB ^vp toxin) and ORF-14 (coding gene of plasmid) that mediates the virulence of V. parahaemolyticus in shrimp species (Han et al., 2015; Kumar et al., 2019; Sirikharin et al., 2015) was examined. Interestingly, the expression of virulent plasmid genes was significantly downregulated in M0904/110 at 12 and 24 h as compared with M0904/120 (Figure 2 A, B). This observation is in line with the results on secreted proteins (mentioned above) and highlights that M0904/110 culture significantly decrease the transcription of virulence genes. Considering that in altered environmental condition, V. parahaemolyticus produce floccules and secrete alkaline phosphatase PhoX enzyme, the temporal expression of alkaline phosphatase PhoX genes in V. parahaemolyticus (M0904/110 or M0904/120) was investigated. The expression of alkaline phosphatase PhoX gene was significantly increased at 12 and 24 h in M0904/110, when compared with the M0904/120 (Figure 2C). Taken together, these results imply that V. parahaemolyticus in lower shaking condition switch its phenotype, which in turn leads to decreased transcription of virulence related genes and increased expression of floccules related alkaline phosphatase PhoX gene.

The AHPND Vibrio parahaemolyticus grown in lower shaking condition reduces the toxicity of AHPND strain in brine shrimp larvae

Since, environmental condition in V. parahaemolyticus culture medium plays a significant role in the expression of virulence related genes (as shown above). Next, the in vivo virulence of V. parahaemolyticus was investigated using gnotobiotic brine shrimp larvae, which is a good model for studying host-pathogen interaction (Baruah et al., 2017, 2015). At first, the toxicity of V. parahaemolyticus grown in either M0904/110 or M0904/120 on the survival of brine shrimp larvae was examined. The results showed that larvae that were challenged with M0904/110 exhibited significantly increased survival compared to the larvae challenged with M0904/120 (Figure 3 A). Flowever, it is noteworthy to mention that virulence of V. parahaemolyticus AFIPND strain is mediated by extracellular proteins (ECP) that mainly comprised of PirA ^vp and PirB ^vp toxins (Kumar et al., 2019) and since earlier in fig. 1, shows that culture of M0904/110 results in secretion of alkaline phosphatase PhoX; whereas, the M0904/120 culture produce PirA ^vp and PirB ^vp toxins. We next investigated the toxicity of V. parahaemolyticus extracellular proteins (VPAHPND ECP) purified from M0904 culture grown in either M0904/110 or M0904/120. Interestingly, the results showed that toxicity of VPAHPND ECP purified from M0904/110, decreased significantly when challenged at 1.3, 2.6, 3.9 and 5.2 pg concentration as compared with VPAHPND ECP purified from M0904/120 culture (Figure 3 B). Although, the toxicity of VPAHPND ECP purified from V. parahaemolyticus grown in either M0904/110 or M0904/120 was significantly different, it is important to assess the toxicity of pure alkaline phosphatase PhoX as compared with pure PirA ^vp and PirB ^vp toxins. Since apart from alkaline phosphatase PhoX or PirA ^vp and PirB ^vp toxin, V. parahaemolyticus may also secrete some other protein or toxins that might contribute in the toxicity of ECP in brine shrimp larvae (Sirikharin et al., 2015). To do this, the purified alkaline phosphatase PhoX or PirA ^vp and PirB ^vp toxin from M0904/110 or M0904/120 was challenged to brine shrimp larvae at the concentration of 1.3, 2.6, 3.9 and 5.2 pg/100 pi. In agreement with the previous VPAHPND ECP survival results, the results showed that brine shrimp larvae exposed to varying concentration of pure alkaline phosphatase PhoX enzyme has significantly increased survival percentage when compared with PirA ^vp and PirB ^vp toxin (Figure 3 C). Taking these results together, our in vivo assay confirms that shaking speed indeed plays an essential role in mediating virulence of V. parahaemolyticus in brine shrimp larvae. Floccule formation decreases the toxicity of AHPND Vibrio parahaemolyticus M0904 strain in Macrobrachium rosenbergii larvae

Earlier in the study, it is shown that shaking speed modulates the virulence of V. parahaemolyticus in brine shrimp larvae. This has led to the suggestion that altered shaking speed might decrease the virulence of V. parahaemolyticus in other shrimp species, e.g., freshwater shrimp, Macrobrachium rosenbergii. To test the hypothesis, additional in vivo assay was conducted to investigate the virulence of V. parahaemolyticus grown in either M0904/110 or M0904/120 on survival of 8-days old Macrobrachium larvae. The results showed that survival of Macrobrachium larvae exposed to M0904/110 was significantly higher at 12, 24, 36 and 48 h post challenge when compared with larvae challenged with M0904/120 (Figure 4). The results were consistent with brine shrimp larvae survival results and indicate that V. parahaemolyticus in lower shaking condition becomes less virulent to shrimp species.

Taken together, the above-indicated results show that the alkaline phosphatase PhoX enzyme and/or the PirA ^vp and PirB ^vp toxins which are secreted by Vibrio parahaemolyticus can be used to determine whether said bacterium is virulent or not.

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