Aeromonas hydrophila infection induces Toll-like receptor 2 (tlr2) and associated downstream signaling in Indian catfish, Clarias magur (Hamilton, 1822)

Ethical statement

This research work was carried out in compliance with the animal welfare laws in India. Guidelines of CPCSEA [(Committee for the Purpose of Control and Supervision of Experiments on Animals), Ministry of Fisheries, Animal Husbandry and Dairying, Department of Animal Husbandry and Dairying, Government of India] on care, treatment and experimental protocol of animals in scientific experimentation on fishes were followed. The animal care and experimental challenge protocol was approved by the Institutional Animal Ethics Committee (NBFGR/IAEC/2019/007) of ICAR-National Bureau of Fish Genetic Resources, Lucknow, India.

Experimental fish and maintenance

Apparently healthy farm bred juvenile of C. magur (n = 120; size-22.31 ± 1.34 cm; weight 50 ± 4.54 g) were handpicked after drying of magur rearing ponds in ICAR-NBFGR, Lucknow, Uttar Pradesh, India. Fishes were transported and stocked in lab condition in 2,000 L capacity fiber reinforced plastic (FRP) tank with sandy-clay bed provided with hideouts to avoid cannibalism. In each tank, 40 numbers of fish were kept. Fish were fed with commercially available magur feed (Growel Feed Pvt. Ltd, Lucknow, Uttar Pradesh, India) @ 2% of their body weight twice a day. Fishes were maintained for 1 month before carrying out the experiment. Half of the water was exchanged weekly.

Cloning of magur tlr2 ( mtlr2)

Initially, to amplify partial cDNA sequence of tlr2 gene, PCR primers (Table 1) were designed from conserved region of the nucleotide sequences of channel catfish, Ictalurus punctatus (GenBank accession HQ677714.1) and yellow catfish, Tachysurus fulvidraco (KU950711.1) tlr2 gene. The initial partial fragment of mtlr2 was amplified from the spleen cDNA of C. magur. The amplified PCR fragment was cloned into pTZ57R/T vector (Thermo Scientific). The recombinant plasmids containing the PCR amplified fragment was sequenced in both the direction using M13 forward and reverse sequencing primers. Likewise, primers were designed from the overlapping sequences of the newly generated sequences and PCR products were sequenced for obtaining the intermittent sequences. To amplify the full-length mtlr2 cDNA sequence, 5′ and 3′-cDNA ends were amplified using SMARTer RACE cDNA amplification kit (Clontech, San Jose, CA, USA) and the specific primers designed from the newly obtained sequences. The amplified PCR products of the cDNA ends were cloned and sequenced as mentioned earlier.

Bioinformatics analyses of mtlr2 sequence

The sequences of different amplicons amplified in PCR were read by Chromas version 2.6.5 and joined together based on consensus sequences to obtain the complete cDNA sequence of mtlr2. NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) search was carried out for obtaining the sequence homology of mtlr2 with that of other reported tlr2. The open reading frame (ORF) and the corresponding protein sequence were identified with the help of ORF finder tool of NCBI (https://www.ncbi.nlm.nih.gov/orffinder/). Multiple sequence alignment of deduced amino acid sequence of mtlr2 with vertebrates’ counterparts such as human, zebra fish, common carp, rohu, channel catfish and large yellow croaker was carried out using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) to identify the conserved features and domains. Signal peptide, transmembrane domain and TIR domain were predicted using the SMART software (http://smart.embl-heidelberg.de/) whilst the remaining LRR motifs were identified manually. LRR domain was also searched in the deduced amino acid sequence for the presence of LxxLxLxxNxL as decribed by Matsushima et al. (2007). The theoretical isoelectic point (P_i) and molecular weight (M_w) were computed with protParamExpasy programme (http://web.expasy.org/protparam/). The amino acids were deduced based on cDNA sequences using the translate program found on the ExPASy website. Microsatellites in the mtlr2 sequence were identified using microsatellite repeats finder (http://insilico.ehu.es/mini_tools/microsatellites/). Nucleotide and protein blast was carried out to determine identity percentage of mtlr2 with other related fish species. To construct phylogenetic tree, multiple-sequence alignment of tlr2 amino acid sequences of fishes, human, animals was carried out with the help of Clustal Omega. Phylogenetic tree of tlr2 was constructed by neighbour joining method of MEGA 7.0 programme bootstrapped 1,000 times using default parameters.

Bacterial strains

Our previously isolated Aeromonas hydrophila 9C strain (Muduli et al., 2020) was used as test strain, while Staphylococcus aureus (ATCC 25923) was used as positive control for this experiment. The bacteria were cultured in tryptic-soy broth (TSB) at 37 °C, overnight and total plate count was estimated individually for both the strains before in-vivo challenge.

In vivo bacterial challenge and sample collection

One hundred twenty numbers (n = 120) of apparently healthy magur (~50g) were randomly divided into three groups i.e., negative control (Phosphate buffer saline or PBS injected), treatment (A. hydrophila injected) and positive control (S. aureus injected). Each group consisted of 40 fish in 1,000 L FRP tank. Completely randomized design (CRD) was followed to set up the experiment. Before initiation of the experiment, three fish were sampled randomly for bacterial and parasitological examination to ensure that fish were free of bacterial and parasitic infection. During the experimental period, the temperature, dissolved oxygen and pH were 35 ± 2 °C, 6.8 ± 0.78 mg L⁻¹, 8.2 ± 0.45, respectively as measured by a multiparameter water quality meter (Hanna Instruments, Nușfalău, Romania), whereas, nitrite and ammonia were estimated to be 0.013 ± 0.007 mg L⁻¹ and 0.106 ± 0.02 mg L⁻¹, respectively following APHA (1998) protocol.

For bacterial infection, fishes in treatment group were intra-peritoneally (i.p.) injected with Lethal dose (LD₅₀) of A. hydrophila at 1 × 10⁵ cfu/fish in 100µl of sterile phosphate buffer saline (PBS). Fishes in positive control were intra-peritoneally injected with S. aureus at 1 × 10⁵ cfu/fish in 100 µl PBS. The negative control group were injected with100 µl of sterile PBS and kept separately. Thereafter, at 3, 8, 24, 72, 144 h post-infection (hpi), three fish from bacteria-injected and control group were randomly picked, euthanized in 300mg/L concentration of MS-222 and sacrificed. Tissues such as gill, liver, spleen, intestine and kidney tissue were dissected out in to RNAlater and stored at −80 °C until RNA extraction. At the end of the experiment, the survived fishes in A. hydrophila and S. aureus group were kept separately for further studies.

Total RNA extraction and cDNA synthesis

Total RNA was extracted from the tissues using TRI Reagent (Sigma-Aldrich) as per the manufacturer’s instructions. Tissue sample of 30–50 mg was homogenized in 1 ml of TRI reagent using lysing matrix D beads (1.4 mm) in FastPrep homogenization system (MP Biomedicals). RNA pellet was dissolved in diethyl pyrocarbonate (DEPC) treated nuclease-free water and stored at –80 °C until further use. RNA concentration and purity were analysed in Denovix UV-V is spectrophotometer (Denovix). To remove the residual genomic DNA, 1 µg total RNA was treated with 1U of DNase I, RNase free (Thermo Scientific) following manufacturer’s protocol. DNase-treated total RNA (1 µg) was reverse-transcribed using RevertAid First Strand cDNA Synthesis kit with oligo-dT primer (Thermo Scientific) as per the manufacturer’s instructions.

Quantitative real-time PCR (qRT-PCR) primers

The real-time PCR primers were designed for mtlr2, myd88, nf-kb, IL-1β and ef-1α. The details of primers used in real-time PCR were listed in Table 1. Before performing qPCR, amplification efficiency was determined. A series of four-fold dilution of cDNA ranging from 2,000 ng to 7.8 ng was prepared using cDNA synthesized from pooled spleen tissue of three healthy C. magur. Standard curve, correlation coefficient (R²) and PCR amplification efficiencies (E) were automatically generated in the ABI StepOne Plus qPCR system based on slope of standard curve produced using four-fold diluted cDNA as template. The formula used to calculate efficiency was as follows E (%) = (10^−1/slope − 1) × 100 (Kubista et al., 2006). The primers with PCR efficiencies between 90%−105% were used for relative quantification.

Basal expression analysis of mtlr2, myd88, nf-kb, il-1β

Basal expression of mtlr2 and its downstream signaling molecules myd88, nf-kb, il-1β were determined. Twelve different tissues; gill, liver, spleen, intestine, anterior kidney, posterior kidney, stomach, skin, muscle, heart, brain and accessory respiratory organ (ARO) were collected separately from three naive Indian magur (~50g). Total RNA was isolated from each sample and cDNA was prepared following the manufactures protocol. Quantitative real-time PCR as mentioned later was carried out. Tissue with lowest expression was used as calibrator with respect to which fold change expression of above genes were calculated using the 2^−ΔΔCt method (Livak & Schmittgen, 2001). In our earlier experiment on validation of reference genes in C. magur, ef-1α was found to be the most stable reference gene for normalization of relative gene expression by real-time reverse transcription PCR in the context of pathological conditions (A. hydrophila injected and PBS injected) (data unpublished). Therefore, ef-1α was used as an internal control.

qRT-PCR

The qRT-PCR was performed in Applied Biosystem StepOne Plus Real-time PCR system using TB Green™ Premix Ex Taq™ II (TAKARA) following manufacture’s instruction. Amplification was carried out in 12.5 µl reaction volume, comprising 6.25 µl SYBR green master mix, 0.25 µl of ROX, 0.5 µl of forward and reverse primer (5 µM each), 1 µl of cDNA (100 ng/ul) and 4 µl of nuclease-free water. Each sample was run in triplicate, which served as technical replicate under following cycling condition: initial denaturation at 95 °C for 30 s followed by 40 cycles of 94 °C for 10 s, 60 °C for 10 s, 72 °C for 10 s. This was subsequently followed by melt-curve analysis for 15 s at 95 °C and 1 min at 60 °C. A no template control in triplicate was run for each plate. The data in form of threshold cycle (Ct) for each sample was normalized with corresponding ef-1α. The fold-change in expression in the treatment group was compared with that of PBS-injected control group using the 2^−ΔΔCt method (Livak & Schmittgen, 2001). The data obtained from qRT-PCR analysis from nine technical replicate out of three biological replicate were subjected to statistical analysis.

Statistical analysis

All data were expressed as mean ± standard error of mean (S.E.M). Statistical analysis was performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). The significant difference in fold change expression at different time point within individual tissue was determined with time point as independent variable and fold change in expression as dependent variable at 95% significance level using statistical model ANOVA with Tukey post-hoc test (p < 0.05). The p values significant at <0.05, <0.01, <0.001 levels, respectively, compared to controls indicated by different superscripts.

Characterization of full-length cDNA of mtlr2

Full-length cDNA encoding mtlr2 was amplified from magur spleen cDNA. Initially, two set of primer (2F-3R, 3F-5R primer) yielded 626 bp (Fragment 1) and 1,129 bp (Fragment 2) amplification. Further, a new set of internal/nested primer (F3-R3) generated 694 bp (Fr3) overlapping Fragment 1 and 2. Joining these fragments together a long stretch of 1,830 bp product was obtained. Cloning, sequencing, and NCBI-BLAST search of 1,830 bp cDNA had sequence similarity with other fish tlr2 which confirmed it to be tlr2. The 3′-and 5′-RACE were performed to obtain 3′-and 5′-terminal regions of cDNA. The full-length mtlr2 cDNA was 3,066 bp, including 5′ and 3′ un-translated region (UTR) of 113 and 582 bp, respectively. The predicted single ORF of mtlr2 was 2,373 bp, located between 114–2,487 bp nucleotide and encodes 790 amino acids with a theoretical pI value of 6.11 and molecular weight 90.095 kDa. Structurally, it comprised of a signal peptide (1–42 aa), one leucine-rich repeat region (LRR) at N-terminal (LRR1-NT; 50–73 aa) and C-terminal LRR (LRR-CT; 588–608 aa), twenty LRRs in between N-and C-terminal, one trans-membrane (Tm) domain (609–631 aa) followed by one TIR domain (670–783 aa) (Figs. 1–3). The highly conserved motifs were identified in TIR domains, and wide range of diversity was observed within the LRR regions (Fig. 3). Two microsatellites were detected in the UTR region of cDNA sequence of mtlr2 i.e., pentamer penta repeat (GTTTT)₅ at 69 bp of 3′UTR region and dimer octarepeat (AC)₈ repeat at 2,862 bp of 5′UTR region. One hexamer tri-repeat (CTGACT)₃ also present in coding region at 215 bp location. Two mRNA instability motifs (ATTTA) were found in the 3′-UTR, which suggest that mtlr2 may be transiently expressed (Fig. 1). The mtlr2 nucleotide sequence was submitted to GenBank database with GenBank ID: MT625141.

Structural identity and phylogenetic relationship of mtlr2 with other species

Similarity comparison of the mtlr2 nucleic acid sequence with pangasius, channel catfish, and yellow catfish revealed identity percentage of 83.19, 79.20 and 80.69, respectively at nucleic acid level. At the deduced amino acid level, the corresponding identity % was 77.63, 76.4, and 72.83, respectively with pangasius, channel catfish, and yellow catfish. With carp and higher vertebrate family, the identity % of mtlr2 amino acids was ~60 and ~37, respectively. The identity of conserved TIR domain of mtlr2 ranged from 89.9% with pangasius catfish to lowest 57.4% with Japanese flounder. TIR domain of mtlr2 shares highest identity with catfish group i.e. 89.9% with pangasius. TIR domain of mtlr2 shares identity of 77.5% with zebrafish, 64% with mouse and 65.2% with human (Table 2).

Phylogenetic tree was constructed to understand the evolutionary relationship of mtlr2 with tlr2 reported from other teleosts. Among the fishes, tlr2 representing catfish group including magur, channel catfish, yellow catfish and pangasius were closely related and formed one cluster, and were well separated from carps viz., rohu, mrigal, common carp, grass carp and zebra fish. Within the members of catfish family, mtlr2 branched with pangasius and channel catfish, indicating close evolutionary relationship. Other group of fishes such as trout, bream, flounder, croaker, grouper, and cod formed a separate cluster from carps and catfishes. As oblivious from phylogenetic tree that fish tlr2 are well divergent from other higher animals (cluster-IV) (Fig. 4).

Basal expression of mtlr2 and its downstream molecules

The qRT-PCR amplification efficiencies (E) of primer sets fell within the suitable experimental range as per MIQE guideline (Bustin et al., 2009) i.e., from 95.81% to 104.66%; while correlation coefficient (R²) ranged from 0.989 to 0.998 respectively (Table 3). Tissue specific expression of mtlr2, myd88, nf-kb and il-1β gene in twelve different tissues including gill, liver, spleen, anterior kidney, posterior kidney, intestine, accessory respiratory organ (ARO), stomach, muscle, brain, heart and skin was evaluated by qRT-PCR assay. Their expression in various organs/tissues was normalized with the reference gene ef-1α and represented as relative fold changes from the lowest expressing tissue (calibrator). Highest basal expression of mtlr2, myd88 and il-1β in spleen, nf-kb in anterior kidney was observed. Lowest basal expression of mtlr2 in skin and myd88, nf-kb, il-1β in muscle was detected. Although, the tissue-specific expression of mtlr2 was varied but the expression was at detectable level in all the examined tissue (Figs. 5A–5D).

Inductive expression of mtlr2 in response to S. aureus

Modulations of mtlr2 gene transcripts were analyzed in gill, liver, spleen, intestine and kidney by qRT-PCR following i.p. injection of S. aureus. As shown in Fig 6, mtlr2 expression was significantly induced in various tissues at different time points.

Differential expression of mtlr2 and its downstream signaling molecules in response to A. hydrophila infection

Modulation of mtlr2 expression was studied following A. hydrophila infection in gill, liver, spleen, intestine and kidney. The kinetics of up-or down-regulation of the mtlr2 gene exhibited a similarity of general trend, rising to a high level at 3 to 24 hpi and then falling to the basal level at 72 to 144 hpi, but the up-and down-regulation differed at different time points in different tissues. A significant up-regulation of mtlr2, myd88, nf-kb gene expression was observed in gill, liver, spleen, intestine and kidney within 24 hpi (Figs. 7A–7C). Similarly, significantly up-regulated expression of il-1β, a pro-inflammatory cytokine was noticed in gill, liver, spleen, intestine and kidney within 24 hpi (Fig. 7D).