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, and 70 ml of molten agar was poured into deep well petri dishes (Fisher), as previously described. (64). An aliquot of vAh ML09-119 was removed from cryogenic storage and cultured in TSB overnight at 30°C on an orbital shaker. This culture was then used to prepare planktonic and biofilm cultures, as previously described (64). Briefly, 70 ml of fresh TSB media was inoculated with 1 ml of the overnight culture and grown at 30°C with shaking to mid-log phase. Biofilm agar plates were inoculated from overnight culture by stab inoculation, sealed with parafilm, and incubated at 30°C for 72 hours. Planktonic and biofilm cultures were performed in triplicate.
Secretome Preparation
Planktonic Secretome: Secreted proteins of planktonically-cultured vAh ML09-119 were purified from cell-free supernatants, prepared as previously described (64). Briefly, vAh was cultured in TSB media as described above, cells were pelleted by centrifugation, and supernatant was decanted and retained. Pelleted cells were washed twice with cold, sterile PBS, pelleted as above, and the wash was added to the supernatant. Combined supernatants were then passed through a low-binding 0.22 µm vacuum filter (VWR) to remove any remaining cells.
Biofilm Secretome: Secreted proteins of biofilm-cultured vAh ML09-119 were purified from cell-free supernatants, as previously described (64). In brief, cells were removed from the biofilm media surface and washed twice with cold, sterile PBS as described above. The cell wash was decanted and retained. Secreted proteins were then collected from within the biofilm media by disrupting the biofilm media until the soft agar had formed a slurry. The slurry was transferred to conical tubes, centrifuged to pellet the agar, and the liquid media was decanted and retained. The agar plug was then resuspended in sterile PBS and centrifuged as above. The wash solutions was decanted and retained. All retained supernatants were combined and residual agar and bacterial cells were removed by filtration through a low-binding 0.45 µm and 0.22 µm vacuum filters (VWR).
Ammonium Sulfate Precipitation: Extracellular proteins (ECPs) were precipitated from cell-free supernatants by ammonium sulfate precipitation, as previously described (30, 64). Briefly, ammonium sulfate crystals (Fisher Scientific) were added to cell-free supernatants to achieve 65% saturation and incubated at 4°C with gentle mixing for 24 hours. Precipitated proteins were collected by centrifugation, resuspended in Tris buffer, and dialyzed twice against the same buffer in 10 Kda dialysis cassettes (Slide-A-Lyzer (Thermo Fisher)). Following dialysis, the volume of all protein samples were adjusted to 20 ml by the addition of cold Tris buffer. 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 enzymatic 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 Barger et al. (2020). In brief, heparinized blood from channel catfish was diluted 1:10 in sterile PBS and incubated with a suitable dilutions of protein for 2 hours at 30°C in an orbital shaker. Positive control tubes representing 100% hemolysis contained sterile distilled water in place of protein samples. Negative control tubes contained sterile PBS in place of protein samples. Following incubation, tubes were centrifuged to pellet un-lysed erythrocytes and supernatant was transferred to clear, 96-well flat bottom plates. Hemolysis 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.), as previously described (64). Briefly, a suitable concentration of protein was added to triplicate wells of black, flat-bottom 96-well plates with non-binding surface (Greiner Bio-One). Trypsin served as a positive control and sterile deionized water served as a substrate control. Labeled casein was added to each well and relative fluorescence 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.), as previously described (64). Briefly, a suitable concentration of protein was added to triplicate wells of black, flat-bottom 96-well plates with non-binding surface. Positive and negative controls were elastase and sterile, deionized water, respectively. Labeled elastin substrate was then added to each well and relative fluorescence was measured continuously at Ex/Em = 490 nm/520 nm 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 (65). Slides were evaluated and photographed using an Olympus BX53 microscope with an Olympus UPlanFL N 20X/0.50 objective, fitted with an Olympus DP26 digital camera, and captured with Olympus cellSens Entry Imagining software (Olympus Corporation). No further imaging processing or manipulation was performed on photomicrographs.
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 University of Alabama at Birmingham Mass Spectrometry/Proteomics shared facility (Birmingham, Alabama, USA) 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 sample in NuPAGE LDS sample buffer (Invitrogen) was loaded onto a Novex NuPage 10% Bis-Tris protein gel (Invitrogen), separated as a short stack, and stained overnight with Novex Colloidal Blue Staining kit (Invitrogen). Gels were then destained and lanes were cut into single molecular weight fractions. Following equilibration in 100mM ammonium bicarbonate, fractions were digested overnight with Trypsin Gold (Mass Spectrometry grade (Promega)) and peptide extracts were reconstituted in 0.1% formic acid to 0.1µg/µl.
Mass Spectrometry: Digested samples were analyzed on a 260 Infinity HPLC stack (Agilent Technologies) and chromatographic separation occurred on a C18 reverse-phase column (Jupiter C-18, 71µ x 15 cm, 300 Å, 5 micron (Phenomenex)) with an in-line Thermo Orbitrap Velos Pro hybrid mass spectrometer, equipped with a nano-electrospray source (Thermo Fisher). Binary mobile phase solvents were comprised of 0.1% formic acid (solvent A) and 0.1% formic acid in 85% acetonitrile (solvent B). All data were collected in CID mode. A parent scan range of 300 to 1200 m/z (at 60K resolution) was chosen and fragmentation data (MS2) were collected on the top 15 most intense ions. For data-dependent acquisition, 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: Data acquisition was executed using Xcalibur software. Xcalibur RAW files were collected in profile mode, converted to centroid data, and then 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 with a species-specific subset of the UniRef 100 database 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.
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. Peptides identified by SEQUEST search were filtered with Scaffold. A 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 were set as filter cut-off values required to accept peptide identification. Peptide probabilities were assigned by the PeptideProphet algorithm (66, 67). The two most common methods for statistical validation of large proteome data, False discovery rate (FDR) and protein probability, are incorporated in Scaffold. 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. Spectral counting and was performed for relative quantification across samples. Spectral count abundances were normalized between samples, when relevant. Proteins present in at least two experimental replicates were included in analyses. To identify differentially secreted proteins, two nonparametric statistical analyses including reproducibility-optimized test statistic (ROTS) (bootstrapping value = 1000) combined with single-tail t-test (p < 0.05) (68, 69) were performed between each pair-wise comparison. 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. For proteins present in only one experimental group, the average of the normalized quantitative value was designated as the protein abundance.
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 (70). 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 (71). 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.