Bacterial Strain
vAh strain ML09-119 was isolated from a diseased channel catfish from a MAS outbreak in a West Alabama aquaculture facility in 2009. Molecular characterization and genome sequencing of vAh ML09-119 have been performed (19) and the complete genome sequence deposited in GenBank (Accession CP005966). Aliquots of vAh ML09-119 were cryogenically stored in 10% glycerol freeze medium at -80°C.
Catfish: Specific-pathogen free channel catfish fingerlings maintained under Auburn University IACUC-approved protocol 2018-3251 (Catfish Production and Maintenance) were used for challenges. All challenges were performed adhering to the guidelines of AU-IACUC-approved protocol 2016-2900 (Identification of toxigenic proteins of virulent Aeromonas hydrophila and evaluation of host response).
Culture Media and Culture Conditions
Tryptic soy broth (TSB) (Bacto TSB, BD) prepared according to manufacturer’s directions was used as the culture medium for planktonic growth.
Biofilm media was prepared by adding 0.2% agar powder (AlfaAesar) to TSB media prior to sterilization. Approximately 70 ml of molten biofilm agar was poured into deep well petri dishes (Fisher) and allowed to solidify. Bacterial strain vAh ML09-119 was removed from cryogenic storage and inoculated into 25 ml TSB media and grown overnight at 30°C with shaking. A 1 ml aliquot of overnight culture was transferred to 70 ml of TSB and grown at 30°C on an orbital shaker to mid-log phase, approximately 16 hours. Biofilm agar plates were inoculated from overnight culture by stab inoculation. Plates were sealed with parafilm and incubated at 30°C for 72 hours, until an adherent bacterial film covered the agar surface.
Planktonic and biofilm cultures were performed in triplicate.
Secretome Preparation
Planktonic Secretome: vAh ML09-119 was cultured as described above. Cells were pelleted by centrifugation at 20,000 x g for 15 minutes at 4°C and supernatant was decanted and retained. Cells were washed twice with cold, sterile PBS, pelleted as above, and the wash was added to the supernatant. Remaining cells were removed by passage through a low-binding 0.22 µm vacuum filter (VWR). Cell-free supernatants were used as the starting point for purification of extracellular proteins (ECPs).
Biofilm Secretome: vAh ML09-119 cells were gently removed from the biofilm media surface with a sterile cell scraper, transferred to 50 ml conical tube, and washed twice with cold, sterile PBS as described above. The cell wash was decanted and retained. To collect secreted proteins within biofilm media, the plates were disrupted using a sterile disposable probe until the soft agar had formed a slurry. The agar slurry was transferred to a sterile 50 ml conical tube and centrifuged at 20,000 x g for 15 min at 4°C to pellet the agar. Following centrifugation, the liquid media was decanted from the agar plug and retained. The agar plug was then resuspended in 20 ml cold sterile PBS, centrifuged as above, and the wash solution decanted and retained. All wash solutions and liquid media were combined and filtered, first through a low-binding 0.45 µm vacuum filter (VWR), then through a low-binding 0.22 µm vacuum filter to remove any residual agar and bacterial cells. This cell-free supernatant was used at the starting point for biofilm ECP purification.
Ammonium Sulfate Precipitation: ECPs were precipitated from cell-free supernatants by the addition of ammonium sulfate crystals (Fisher Scientific) to achieve 65% saturation, followed by incubation at 4°C on a rotary platform shaker with gently mixing for 24 hours. Precipitated proteins were collected by centrifugation at 30,000 x g for 45 min at 4°C, then dissolved in 10 ml cold Tris buffer (20mM Tris-Hcl, pH 7.6) + protease inhibitor (Complete tablets, mini, EDTA-free (Roche)). Resuspended proteins were dialyzed twice, for 18 hours and 12 hours, respectively, against the same buffer in 10 Kda dialysis cassettes (Slide-A-Lyzer (Thermo Fisher)). After dialysis, the total volume was adjusted to 20 ml by the addition of cold Tris buffer. The protein concentration of each sample was determined by the Bradford assay (Pierce Coomassie Plus Protein Assay, Thermo Fisher). These concentrated proteins were used for all assays.
Enzymatic Activity
The in vitro activity of secreted proteins was measured using specific substrates to determine the degradative and toxigenic potential of planktonic and biofilm secretomes, as described below:
Hemolysis: Hemolytic potential was measured using the method of Peatman et al. (2018) with some modifications. In brief, heparinized blood from three channel catfish was pooled and diluted 1:10 in sterile phosphate buffered saline (PBS). A suitable dilution of protein in 150 µl PBS buffer was added to 25 µl diluted blood in sterile microcentrifuge tubes. Tubes were incubated at 30°C in an orbital shaker for 2 h. Positive control tubes representing 100% hemolysis contained 150 µl sterile distilled water in place of protein samples. Negative control tubes contained 150 µl sterile PBS in place of protein samples. Controls were incubated with 25 µl diluted blood as above. Following incubation, tubes were centrifuged at 1,000 x g to pellet un-lysed cells and 150 µl of supernatant was transferred to 96-well flat bottom plates. Erythrocyte lysis was quantified by measuring absorbance of released hemoglobin at 415 nm in multi-mode plate reader (Synergy HTX, Bio-Tek) and hemolysis was reported as percent of positive control.
Universal Protease Activity: Non-specific proteolytic activity was measured using HiLyteFluor 488-labeled casein as the substrate, following manufacturer’s protocol with minor modifications (Sensolyte Green Fluorimetric Protease Assay Kit, AnaSpec, Inc.). Briefly, a suitable concentration of protein in 50 µl deionized water was added to triplicate wells of black, flat-bottom 96-well plates with non-binding surface (Greiner Bio-One). Trypsin, diluted 50-fold in deionized water, acted as a positive control and sterile deionized water served as a substrate control. Following the addition of 50 µl labeled casein substrate, plates were mixed briefly and fluorescent intensity was measured at Ex/Em = 490 nm/520 nm every five minutes for one hour in a multi-mode plate reader (Synergy HTX, Bio-Tek) with 30°C incubation temperature. Data were plotted as relative fluorescence units versus time for each sample.
Elastase Activity: Elastase-specific activity was measured using 5-FAM/QXLTM 520 labelled elastin as the substrate, following the manufacturer’s protocol with minor modifications (Sensolyte Green Fluorimetric Elastase Assay Kit, AnaSpec, Inc.). Briefly, a suitable concentration of protein in 50 µl deionized water was added to triplicate wells of black, flat-bottom 96-well plates with non-binding surface. Elastase, diluted 50-fold in assay buffer, acted as a positive control and sterile, deionized water was a substrate control. Following the addition of 50 µl labeled elastase substrate, plates were mixed briefly and fluorescent intensity was measured continuously at Ex/Em = 490 nm/520 nm, and data recorded every five minutes for one hour in a multi-mode plate reader (Synergy HTX, Bio-Tek) with 30°C incubation temperature. Data were plotted as relative fluorescence units versus time for each sample.
In vivo Proteolysis
Extracellular protein activity was measured in vivo using channel catfish fingerlings to determine potential proteolytic and cytotoxic tissue effects.
Protein Preparation: Ten microgram aliquots of secreted planktonic and biofilm-associated proteins, prepared as above, diluted in 100 µl sterile PBS were used for injection challenges.
Challenge Model: Channel catfish fingerlings were transferred to 57-liter glass aquaria containing dechlorinated municipal water and acclimated at 30°C for two days prior to challenge. Triplicate tanks containing five fish each represented planktonic ECP, biofilm-associated ECP, and injection control groups. Prior to injection, fingerlings were transferred to sedation aquaria containing 70 mg/ L tricaine methanesulfonate (MS-222) buffered to neutrality with sodium bicarbonate. Following sedation, characterized by decreased opercular movement and loss of equilibrium, 100 µl of sterile PBS containing 10 µg of total protein was injected intramuscularly just below the dorsal fin using tuberculin syringes fitted with 26 gauge needles. Control fish were injected with 100 µl sterile PBS. Fish were then returned to the appropriate aquarium and monitored until fully recovered. Fish were maintained in aquaria at 30°C for 7 days under flow-through conditions at 1 gallon per hour water replacement. Moribund fish or fish developing severe external lesions were euthanized by prolonged exposure to buffered MS-222, the tissues were collected and fixed in 10% neutral-buffered formalin. After 7 days, remaining fish were humanely euthanized and samples were collected and prepared as above.
Histology: Formalin-fixed tissues were paraffin-embedded and 4 micron sections were prepared and stained with hematoxylin and eosin according to standard methods (64). Slides were evaluated and photographed using an Olympus BX53 microscope fitted with an Olympus DP26 digital camera.
Secretome Analysis.
To determine how vAh niche occupancy might influence protein production, secreted protein profiles of vAh cultured within a biofilm and in broth were compared by liquid chromatography with tandem mass spectrometry (LC MS/MS) analysis at the UAB Mass Spectrometry/Proteomics shared facility to identify and quantify proteins present in each sample, as previously described (30), as follows.
Proteomics analysis: Samples were prepared for analysis as follows: 20µg of protein per samples was diluted to 35µl in NuPAGE LDS sample buffer (Invitrogen), reduced with dithiothreitol, and denatured at 70°C for 10 minutes prior to loading onto Novex NuPage 10% Bis-Tris protein gel (Invitrogen). The gel was separated as a short stack (10 min, 200V constant) and stained overnight with Novex Colloidal Blue Staining kit (Invitrogen). Gels were destained and each lane was cut into single molecular weight fractions and equilibrated in 100mM ammonium bicarbonate. Each plug was then digested overnight with Trypsin Gold (Mass Spectrometry grade (Promega)) following manufacturer’s instructions and peptide extracts were reconstituted to 0.1µg/µl in 0.1% formic acid.
Mass Spectrometry: Prepared peptide digests (8µl) were injected onto a 1260 Infinity nHPLC stack (Agilent Technologies) and separated using a 71µ I.D. X 15cm pulled-tip C-18 column (Jupiter C-18 300 Å, 5 micron (Phenomenex)) running in-line with a Thermo Orbitrap Velos Pro hybrid mass spectrometer, equipped with a nano-electrospray source (Thermo Fisher). All data were collected in CID mode. nHPLC was configured with binary mobile phases comprised of 0.1% formic acid (solvent A) and 0.1% formic acid in 85% acetonitrile (solvent B). Following each parent scan (300-1200 m/z at 60K resolution), fragmentation data (MS2) were collected on the top most intense 15 ions. For data-dependent scans, charge-state screening and dynamic exclusion were enabled with a repeat count of 2, repeat duration of 30s, and exclusion duration of 90s.
Mass spectrometry data conversion and searches: Xcalibur RAW files were collected in profile mode, centroided, and converted to mzXML using ReAdW v3.5.1 (IonSource). The mgf files were then created using MzXML2Search (included in Trans-Proteomics Pipeline v3.5) for all scans. The data were searched using SEQUEST (Thermo Fisher, San Jose, CA, USA; version 27), which was set for two maximum missed cleavages, a precursor mass window of 20ppm, trypsin digestion, variable modification C at 57.0293, and M at 15.9949. Searches were performed with a species-specific subset of the UniRef 100 database.
Peptide filtering, grouping, quantification and statistical analyses: Scaffold (v. 4.8.4, Proteome Software Inc., Portland, Oregon) was used to validate MS/MS based peptide and protein identifications. The list of peptide IDs generated based on SEQUEST search results were filtered using Scaffold, which filters and groups all peptides to generate and retain only high confidence IDs while also generating normalized spectral counts across all samples to allow for relative quantification. Filter cut-off values required to accept peptide identifications were set with minimum peptide length of >5 amino acids, with no MH+ charge states, peptide probabilities of >80% C.I., and with the number of unique peptides per protein ≥2. Peptide probabilities were assigned by the PeptideProphet algorithm (65). Protein identifications were accepted if proteins probabilities could be established at >99% C.I., contained at least 4 identified peptides, and with false discovery rate <1.0. Protein probabilities were assigned by the ProteinProphet algorithm (66). Scaffold incorporates the two most common methods for statistical validation of large proteome data sets, the false discovery rate (FDR) and protein probability. Relative quantification across samples were then performed via spectral counting and, when relevant, spectral count abundances were normalized between samples. Proteins present in at least two experimental replicates were included in analyses. To determine statistical significance, two non-parametric statistical analyses were performed between each pair-wise comparison, including reproducibility-optimized test statistic (ROTS) (bootstrapping value = 1000) combined with single-tail t-test (p < 0.05) (67, 68). These were then sorted according to the highest statistical relevance in each comparison. For protein abundance ratios determined by normalized spectral counts, a fold change threshold ≥1.5 was set for significance. Protein abundance of proteins present in only one experimental group was set as the average of the normalized quantitative value.
Protein Function: To define the potential function of secreted proteins, major biological processes of statistically significant proteins were determined from gene ontology annotation in UniProt (Consortium, T.U. 2018) and QuickGO (69). Predicted protein function was assessed by determining major biological processes through gene ontology. Using these data, eight functional groups were established, and proteins were sorted into these groups based on their primary biological function. A further comparison was made by compiling all proteins in each functional group from both biofilm and planktonic secretomes and expressing as parts of a whole, with side-by-side comparisons for each secretome type.
Statistical Analyses
Reproducibility-optimized test statistic (ROTS) analysis of differentially secreted proteins was performed in R (70). All other statistical analyses were performed in Prism 8.2.0 (Graphpad). One-way ANOVA followed by Tukey’s multiple comparisons post-test were performed on triplicate data with significance set at p < 0.05. Graphical representations of data were produced in Prism 8.2.0.