Hemolysin Co-regulated Family Proteins Hcp1 and Hcp2 Contribute to Edwardsiella ictaluri Pathogenesis

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC), a devastating disease resulting in significant economic losses in the U.S. catfish industry. Bacterial secretion systems are involved in many bacteria's virulence, and Type VI Secretion System (T6SS) is a critical apparatus utilized by several pathogenic Gram-negative bacteria. E. ictaluri strain 93–146 genome has a complete T6SS operon with 16 genes, but the roles of these genes are still not explored. In this research, we aimed to understand the roles of two hemolysin co-regulated family proteins, Hcp1 (EvpC) and Hcp2. To achieve this goal, single and double E. ictaluri mutants (EiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2) were generated and characterized. Catfish peritoneal macrophages were able to kill EiΔhcp2 better than EiΔevpC, EiΔevpCΔhcp2, and E. ictaluri wild-type (EiWT). The attachment of EiΔhcp2 and EiΔevpCΔhcp2 to ovary cells significantly decreased compared to EiWT whereas the cell invasion rates of these mutants were the same as that of EiWT. Mutants exposed to normal catfish serum in vitro showed serum resistance. The fish challenges demonstrated that EiΔevpC and EiΔevpCΔhcp2 were attenuated completely and provided excellent protection against EiWT infection in catfish fingerlings. Interestingly, EiΔhcp2 caused higher mortality than that of EiWT in catfish fingerlings, and severe clinical signs were observed. Although fry were more susceptible to vaccination with EiΔevpC and EiΔevpCΔhcp2, their attenuation and protection were significantly higher compared to EiWT and sham groups, respectively. Taken together, our data indicated that evpC (hcp1) is involved in E. ictaluri virulence in catfish while hcp2 is involved in adhesion to epithelial cells and survival inside catfish macrophages.


INTRODUCTION
Edwardsiella ictaluri (E. ictaluri) is the causative agent of enteric septicemia of catfish (ESC) (1). Although E. ictaluri is well-adapted to catfish, it can also infect other freshwater fish species (2)(3)(4). At the early stages of host invasion, E. ictaluri encounters the host immune system (5,6). However, E. ictaluri is capable of surviving and replicating inside catfish professional phagocytic cells, macrophages, and neutrophils (7). To replicate successfully inside the host cells, E. ictaluri must resist and overcome the bacterial killing mechanisms present in the host macrophages and neutrophils, such as oxidative and nitrosative stress (8)(9)(10).
The survival of E. ictaluri highly depends on its resistance to host stress factors and modulating the host environment. Edwardsiella ictaluri encodes urease that is activated in acidic phagosomes of macrophage to cope with low pH (11,12). Low pH and low phosphate concentration inside the phagosome can trigger the expression level of genes in both Type III and Type VI secretion systems (T3SS and T6SS), which assists E. ictaluri survival inside the host immune cells (13). It was shown that the effector proteins secreted via T3SS had an important role in virulence of E. ictaluri, and mutation of these genes caused decreased intracellular replication inside catfish head kidneyderived macrophages (14,15).
Hemolysin co-regulated family proteins (Hcp) are involved in bacteria-host interaction. Particularly, they are involved in adhesion and invasion, intracellular survival of bacteria, bacterial cytotoxicity, and virulence (16). In E. ictaluri, T6SS proteins, Eip19 (evpE), Eip18 (evpC), Eip55 (evpB), Eip20 (evpA), have been first identified during the catfish host-pathogen interaction (17). The secretion of evpC is transcriptionally controlled by two-component system regulatory protein esrC in low-pH and phosphate conditions in E. ictaluri (13). Fish pathogen E. piscicida also possesses T6SS, which is required for virulence (18). In E. tarda, evpC plays a dual role as a chaperone and T6SSdependent secreted protein (19). evpC belongs to Hcp family proteins and can bind to T6SS-dependent effector proteins in bacterial cytoplasm and guide effector proteins through the T6SS needle (20). Due to their role as a chaperone protein, evpC interacts with the T6SS-dependent effector proteins such as evpP in E. tarda (21). A recent study showed that evpP effector protein secreted via evpC could target the macrophages' inflammasome activation (22). E. ictaluri genome has a complete T6SS operon with evpC (hcp1) while hcp2 is located outside of the T6SS operon. In this study, we evaluated the role of hcp genes in E. ictaluricatfish interaction. Our study revealed roles of evpC and hcp2 in adhesion and invasion of catfish epithelial cells, survival and replication inside catfish peritoneal macrophages, adaptation to the stress factors, and virulence and efficacy in catfish.

In-frame Deletion of evpC and hcp2
The nucleotide sequences of evpC (NT01EI_RS11900) and hcp2 (NT01EI_RS14960) were obtained from the E. ictaluri 93-146 genome (GenBank accession: 95 CP001600) (29). The overlap extension PCR method was used to generate evpC and hcp2 in-frame deletion fragments. Briefly, external and internal primers were designed to amplify the regions for upstream and downstream of each gene ( Table 2). Two amplified fragments were combined through splicing by overlap extension (SOEing) (30). The overlap PCR product and the pMEG375 suicide plasmid were digested with the same restriction enzymes, and the mutated insert was ligated into the pMEG375. After electroporation and selection of the correct plasmid in CC118, the plasmid was transferred to E. coli BW19851 by electroporation, which was then used to transfer the plasmid into E. ictaluri strain 93-146 by conjugation. Two-step selection was used to obtain in-frame deletion mutants. At the first step, ampicillin-resistant E. ictaluri colonies were inoculated into BHI broth containing ampicillin and colistin. In the second step, positive colonies were streaked on the BHI agar containing colistin only. These colonies were re-streaked on the BHI agar with 5% sucrose, 0.35% D-mannitol, and colistin. Ampicillinsensitive colonies with the mutant band were in-frame deletion colonies. The deletion of each gene was confirmed by PCR and sequencing. For the construction of double mutant, E. coli BW19851 carrying pMEG375 with overlap hcp2 and Ei evpC were conjugated, and two-step selection yielded Ei evpC hcp2. PCR and sequencing confirmed the deletion of hcp2 in the double mutant.

Hemolysis Assay
Ei evpC, Ei hcp2, Ei evpC hcp2, and EiWT were streaked on sheep blood agar plates (Fisher Scientific), which were incubated at 30 • C for 48 h. Hemolytic activity of the mutants was visualized using a Stuart Colony Counter with sub-stage illumination (Cole-Parmer).

Construction of Bioluminescent Strains
pAKgfplux1 was used to construct bioluminescent Ei evpC, Ei hcp2, and Ei evpC hcp2 strains, as described previously (28). Briefly, E. coli SM10λpir carrying pAKgfplux1 and mutants were grown overnight and mixed at the ratio of 1:2 (donor: recipient). Mixture pellet was spotted on 0.45 µM filter paper placed on BHI agar and grown at 30 • C for 24 h. Filter paper containing a mixture of bacteria was washed with BHI broth containing ampicillin and colistin, and serial dilutions were spread on selective BHI agar containing ampicillin and colistin. Ampicillin resistant mutant colonies carrying pAKgfplux1 appeared on the selective plates after 30 • C for 24-48 h.

Serum Treatment
Bioluminescent Ei evpC, Ei hcp2, and Ei evpC hcp2 strains were exposed to catfish normal serum. Bioluminescent EiWT (positive control) and E. coli DH5α (negative control) were also included in each experiment. Catfish serum was collected as previously described (28

Bioluminescent Imaging
Sixteen specific-pathogen-free (SPF) catfish fingerlings (12.72 ± 1.00 cm, 24.95 ± 5.47 g) were obtained from the CVM hatchery and stocked into four tanks (4 fish/tank). Three tanks were assigned to Ei evpC, Ei hcp2, and Ei evpC hcp2 (treatments), and one tank for EiWT (positive control). After 1 week of acclimatization, the water level was reduced to 10 L, and 100 ml bacterial culture was added to each tank (final dose of 5 × 10 7 colony forming units, CFU, per ml of water). Following 1 h incubation, water flow was restored in each tank. Fish were anesthetized with 100 mg/L MS222, and bioluminescence emitted from the fish body was collected for one min by using IVIS Lumina XRMS In Vivo Imaging System Series III (PerkinElmer). Following bioluminescent imaging, fish were transferred to buckets with aerated water for recovery. Bioluminescent imaging was conducted at 0, 6, 12, and 24 h post-infection, and subsequent daily intervals until 14 days.

Bacterial Killing Assay
The bacterial killing assay was performed as previously described (12,31,32). Briefly, peritoneal macrophages were collected from a year-old channel catfish (250-300 g) injected with 1 ml squalene (Sigma). Following 4-day post-injection, peritoneal macrophages were harvested from five catfish by injecting 10 ml cold phosphate-buffered saline (1X, PBS) to the peritoneal cavity of catfish. Harvested cells were pooled and washed with PBS three times. The cells were resuspended in channel catfish macrophage medium (CCMM) including RPMI (RPMI 1640 sans phenol red & L-glutamine, Lonza) containing 1× glutamine substitute (GlutaMAX -I CTS, Invitrogen), 15 mM HEPES buffer (Invitrogen), in 0.18% sodium bicarbonate solution (Invitrogen), 0.05 mM 2-beta-mercaptoethanol (Sigma), and 5% heat-inactivated (HI) pooled channel catfish serum. Next, peritoneal macrophages (5 × 10 5 cells) were transferred into a 96well plate (Evergreen Scientific), and bioluminescent E. ictaluri strains were added at a 1:1 ratio and mixed gently by pipetting up and down. The final volume of the cell-bacteria mixture was 200 µl in each well, and the plate included four replicate wells for each treatment and negative control (cell only). The plate was centrifuged at 1,500 rpm for 5 min at 24 • C to compact the cells and bacteria at the bottom. The plate was then incubated for 1 h at 30 • C to allow the invasion of catfish peritoneal macrophages by bioluminescent mutants and EiWT. Following the first incubation, the cell-bacteria mixture was centrifuged at 2,000 rpm for 7 min, and the media was removed. After this, CCMM containing 100 µg/ml gentamicin were added, and cells were incubated an additional 1 h at 30 • C to kill nonphagocyted E. ictaluri. At the end of incubation, each well was washed three times with PBS, and peritoneal macrophages were suspended in CCMM with 10 µg/ml gentamicin. Finally, cells were transferred to black 96-well-plates (Fisher Scientific), and the plate was placed in Cytation 5 (BioTek) where the cells were incubated for 48 h under 5% CO 2 at 30 • C. Bioluminescence was captured every hour, and data were analyzed to determine the number of survived bioluminescent bacteria in catfish peritoneal macrophages.

Attachment and Invasion Assays
Attachment and invasion assays were performed by using channel catfish ovary (CCO) cell line, as described previously (33). Briefly, CCO cells were resuspended in DMEM medium (Sigma) supplemented with 10% fetal bovine serum and 4 mM L-glutamine at a final concentration of 1 x 10 7 cells ml −1 . Bioluminescent mutants and EiWT were mixed with CCO cells at a 1:1 ratio and placed in a 24-well-plate. The cell-bacteria mixture's final volume was 1 ml in each well, and the plate included four replicate wells for each treatment and negative control (no bacteria). The plate was incubated 1 h at 28 • C for the attachment of mutants and EiWT to CCO. After that, the cell suspensions were incubated in DMEM containing 100 µg/ml gentamicin for 1 h to kill the external bacteria. The plate was washed with PBS three times, and the invasion of E. ictaluri strains was determined by imaging IVIS Lumina XRMS In Vivo Imaging System Series III (PerkinElmer).

Stress Assays
The mutants' survival in oxidative stress in hydrogen peroxide (H 2 O 2 ) (Sigma) and nitrosative stress in sodium nitroprusside (SNP) (Sigma) were tested in BHI (rich medium) and low phosphate minimal medium at pH 5.5 (MM19-P) (34). Bacteria were grown overnight, and OD 600 adjusted to 0.5 for each culture.

Virulence and Efficacy of Mutants in Catfish Fingerlings and Fry
Vaccination and efficacy were performed as previously described (35). Briefly, specific-pathogen-free (SPF) channel catfish fingerlings and fry were obtained from the MSU-CVM Hatchery. Catfish fingerlings (10.46 ± 0.86 cm, 14.03 ± 3.57 g) were stocked into 15 tanks at a rate of 25 fish/tank. Catfish fry were stocked in 12 tanks at a rate of 50 fish/tank. Fish were acclimated at 26-28 • C for 1 week and fed twice a day. Chlorine, dissolved oxygen, and temperature were monitored daily. Treatments were randomly assigned to Ei evpC, Ei hcp2, Ei evpC hcp2 (vaccination), EiWT (positive control), and BHI (sham) groups. Each treatment had three replicates. Immersion vaccination was applied by lowering the water level in each tank to 10-L, and by adding 100 ml of bacterial culture (final dose of 2.4 × 10 7 CFU/ml water). After 1 h, water flow (1 liter/min) was restored to each tank. Mortalities were recorded daily for 21 days, and the percent mortalities were calculated for each group.
To assess the protective capabilities of mutants, all fish that survived the Ei evpC, Ei hcp2, and Ei evpC hcp2 vaccination were re-challenged with EiWT (2.8 × 10 7 CFU/ml) 21 days post-vaccination as described above. Fish mortalities were recorded daily, and the experiment was terminated when no fish mortalities were observed for three consecutive days.

Statistical Analysis
The significance of the differences between treatment means was established by one-way ANOVA and two-way ANOVA procedures with Tukey's test in SAS for Windows 9.4 (SAS Institute, Inc., Cary, NC). The level of significance for all tests was set at p < 0.05.

Hemolytic Activity of the Mutants
A beta-hemolysis with a narrow clear hemolytic zone around the colonies was observed, and hemolytic activity of Ei evpC, Ei hcp2, and Ei evpC hcp2 was similar to EiWT (Figure 1).

Survival of the Mutants Under Complement Stress
Channel catfish serum was used to evaluate the survival of mutants under complement stress. Ei evpC, Ei hcp2, and Ei evpC hcp2 were able to survive after 4 h of incubation in catfish serum (Figure 2A), and no significant differences have been detected between the mutants and EiWT (Figure 2B). However, significant differences in the intensity of bacterial bioluminescence were found between 0 and 4 h (p < 0.05; Figure 2C). These results indicate that EiWT and mutant strains were robust to complement killing and able to replicate in catfish serum.

Persistence of the Mutants in Catfish Fingerlings
The bioluminescent imaging was used to monitor the persistence of Ei evpC, Ei hcp2, and Ei evpC hcp2 in catfish fingerlings.
EiWT was able to kill all catfish fingerlings in 5 days shortly after ESC clinical signs were observed ( Figure 3A). Catfish fingerlings exposed to Ei evpC and Ei evpC hcp2 mutants survived, and clearance of mutants from the catfish fingerlings was observed. However, the immersion challenge of Ei hcp2 showed severe mortality of all catfish fingerlings in 8 days (Figure 3A). The bioluminescent photon counts from fingerlings showed that the number of Ei evpC and Ei evpC hcp2 had peaked at the highest point at 48 h ( Figure 3B). On the other hand, the bioluminescence of Ei hcp2 was continued to increase after 48 h post-infection ( Figure 3B). These findings demonstrated that Ei evpC and Ei evpC hcp2 were attenuated and cleared from catfish fingerlings while Ei hcp2 was not attenuated.

Bacterial Killing of the Mutants in Catfish Peritoneal Macrophages
EiWT and mutant strains were observed in phagosome/phagolysosome and cytoplasm of peritoneal macrophages by light microscopy (Figure 4A). The intensity of bacterial bioluminescence in catfish macrophages did not differ among the treatment groups at 0 h ( Figure 4B). However, the luminescence of bacteria increased in all treatments at 6 h post-treatment. The intensity of luminescence from Ei hcp2 was significantly lower than that of Ei evpC at this time point ( Figure 4B). Interestingly, bacterial luminescence decreased in all groups at 12 h post-treatment, and the luminescence of Ei hcp2 was lower significantly compared to Ei evpC at this time point. However, there were no significant differences in the intensity of luminescence between Ei hcp2 and EiWT and Ei evpC hcp2 at both 6 and 12 h post-treatment ( Figure 4B).

FIGURE 1 | Hemolytic activities of Ei evpC, Ei hcp2, Ei evpC hcp2, and EiWT on blood agar plates.
After 24 h, bacterial luminescence decreased in all treatment groups, and there were no significant differences between the treatments (Figure 4B). Our results suggest that EiWT and Hcp mutants are capable of surviving and replicating in catfish peritoneal macrophages up to 6 h post-treatment. However, peritoneal macrophages efficiently killed EiWT and Hcp mutant strains after 24 h of in vitro infection (Figure 4C).

Attachment and Invasion Capabilities of the Mutants in CCO Cells
CCO cell line was used to assess the attachment and invasion capabilities of EiWT and mutants, Ei evpC, Ei hcp2, and Ei evpC hcp2 ( Figure 5A). The attachment ability of Ei hcp2 and Ei evpC hcp2 significantly declined compared to EiWT (p < 0.05; Figure 5B). However, no significant differences were recorded between Ei evpC and EiWT (p < 0.05). In addition to the attachment, invasion of all mutants was reduced, but there were no significant differences compared to EiWT (p < 0.05; Figure 5C). These results indicate that Hcp mutants resulted in low attachment and invasion capabilities.

Survival and Stress Resistance of the Mutants in BHI and MM19-P
The survival and resistance of the EiWT and mutants to nitrite oxide and hydrogen peroxide were evaluated. The exposure of mutants and EiWT to SNP and H 2 O 2 in BHI and MM19-P showed a variation in the growth rate of bacteria (Figures 6A,B). Their resistance was increased in MM19-P compared to BHI up to 12 h (Figures 6C,E). Due to the low pH (5.5) in MM19-P, the resistance of mutants and EiWT was enhanced in 0 and 4 h. The mutants and EiWT strains grew exponentially up to 24 h in BHI whereas their growth was restricted in MM19-P at 24 h (Figures 6D,F). Ei evpC hcp2 double mutant had more resistance to SNP and H 2 O 2 stress in BHI and MM19-P up to 12 h. However, Ei evpC and Ei hcp2 showed a similar growth rate in BHI and MM19-P.

DISCUSSION
This research aimed to determine the potential roles of hcp1 (evpC) and hcp2 of T6SS in E. ictaluri virulence in catfish. To achieve this, Ei evpC, Ei hcp2, and Ei evpC hcp2 mutants  were constructed and persistence in catfish, survival and replication inside catfish peritoneal macrophages, attachment and invasion capabilities in catfish epithelial cells, adaptation and survival to stress factors, and virulence and efficacy in catfish were investigated ( Table 3).
Almost all strains of E. ictaluri show beta-type homolysis and hemolytic zone is narrow (36). Hemolytic activity can vary between strains and there is no clear correlation between hemolytic activity and virulence (37,38). Our study indicated that hemolytic activities of Ei evpC, Ei hcp2, and Ei evpC hcp2 were similar to EiWT, and deletion of evpC and hcp2 genes did not have any effect on E. ictaluri hemolytic activity. Hemolysin co-regulated family proteins (Hcp) are involved in adhesion and invasion, intracellular survival of bacteria, bacterial cytotoxicity, and virulence (16).
Edwardsiella ictaluri can evade the complement system in catfish serum and establish a systemic infection. Edwardsiella ictaluri can differentially regulate its proteins in catfish serum (39). Our study revealed that Ei evpC, Ei hcp2, and Ei evpC hcp2 were resistant to complement killing in catfish blood. Mutation in evpC, hcp2, and evpC-hcp2 did not affect E. ictaluri's resistance to complement killing, which indicates that Hcp family proteins of T6SS are not essential for E. ictaluri to survive in catfish serum.
The real-time bioluminescent imaging is a quantification method that allows detection E. ictaluri infection and persistence of mutants in catfish (40,41). The bioluminescence from Ei evpC (7.5 × 10 4 photons −1 cm −2 steradian −1 ) and Ei evpC hcp2 (3.6 × 10 4 photons −1 cm −2 steradian −1 ) was reached the peak at 48 h post-infection, after which bacterial clearance from catfish was observed. However, bioluminescence from Ei hcp2 (1.4 × 10 5 photons −1 cm −2 steradian −1 ) and EiWT (1.6 × 10 5 photons −1 cm −2 steradian −1 ) was gradually increased, even after 48 h post-infection, until the fish dies. Our result showed that Ei evpC and Ei evpC hcp2 had no mortalities for 14 days, although the bioluminescence quantity of Ei evpC and Ei evpC hcp2 started to increase earlier than Ei hcp2 and EiWT. Our bioluminescent imaging data were corroborated with our virulence and efficacy study showing that persistence and replication of Ei evpC and Ei evpC hcp2 in catfish up to 48 h post-infection and decrease afterward may stimulate catfish an immune response, hence the survival of catfish.
Hcp family proteins are secreted inside host macrophages and required for intracellular survival in host macrophages (42,43). Lack of a functional hcp reduced survival of Burkholderia pseudomallei in macrophages (44,45). In E. tarda, the deletion of evpC caused a lower replication rate in gourami phagocytes (46). In this study, we found that the mutation in hcp2 displayed a lower replication rate for the intracellular growth of E. ictaluri inside the catfish peritoneal macrophages. The numbers of macrophages with intracellular Ei evpC, Ei hcp2,   This may depend on lack of hcp2 or a hcp2-dependent effector protein, which warrant further investigation.
Hcp family proteins are involved in adherence and invasion of the host epithelial tissues. Disruption in hcp genes could cause   (50). We demonstrated that evpC and hcp2 mutants' adhesion capabilities were dissimilar while their invasion capabilities were similar in CCO cells. Mutation in evpC did not decrease adherence to CCO cells while a mutation in hcp2 and both in evpC and hcp2 did. This suggests that hcp2 is required for epithelial cell attachment of E. ictaluri whereas both evpC and hcp2 are not essential for epithelial cell invasion of E. ictaluri. T6SS facilitates the uptake of important metals under stress conditions by releasing proteinaceous metallophores into the host environment (51). The role of T6SS in manganese scavenging under oxidative stress has been revealed in Burkholderia thailandensis (52). Intracellular compartmentalization of Salmonella typhimurium inside macrophages initiates stress conditions, including nitrosative and oxidative stress, to suppress the replication of bacteria (53). T6SS effectors are involved in bacterial survival in oxidative stress (54,55). In E. ictaluri and E. piscicida, the T6SS effector EvpP enhanced resistance to oxidative stress (56,57). To investigate the role of Hcp family proteins of T6SS in E. ictaluri stress resistance, we applied nitrosative and oxidative stress with SNP and H 2 O 2 in BHI and in MM19-P to imitate stressful phagosome conditions. Our results indicated that mutants and EiWT were able to grow in nutrient rich media, but SNP and H 2 O 2 stress reduced survival of Ei hcp2 and Ei evpC hcp2 at 24 h. It seems that hcp2 is more critical for E. ictaluri to cope with SNP and H 2 O 2 stress in presence of nutrients. In nutrient restricted media, mutants and EiWT were able to grow up to 4 h, but SNP and H 2 O 2 stress suppressed EiWT growth more than mutants, which may indicate both evpC and hcp2 are not critical in phagosome conditions.
In vivo and in vitro infection models indicated that Hcp family proteins were associated with bacterial virulence and host colonization. In Aeromonas hydrophila, E. coli, and B. pseudomallei, hcp is required for virulence because hcp mutants were less virulent than wild-type (58,59). Additionally, evpC was essential for the virulence of E. tarda (18). Here, we showed that evpC contributed to the pathogenicity of E. ictaluri in catfish. Vaccination of catfish fingerlings with Ei evpC and Ei evpC hcp2 provided complete protection against ESC in catfish fingerlings. However, Ei hcp2 showed a hypervirulent phenotype causing higher mortality with severe symptoms in catfish fingerlings and was not tested in catfish fry. The mortality rates of Ei evpC and Ei evpC hcp2 in catfish fry immersion challenge indicated that Ei evpC showed significantly less mortality and better protection compared to Ei evpC hcp2.
Although evpC is located in the T6SS operon of E. ictaluri, hcp2 is founded ∼67 kilobases (kb) away from evpC (Figure 8). It is possible that putative hcp2 might be an effector protein in E. ictaluri. It is worth noting that the protein sequence alignment of evpC and hcp2 had no significant match (data not shown). Thus, these two proteins classified in the Hcp protein family may have a different role in E. ictaluri.
In conclusion, the two Hcp family proteins found in the E. ictaluri genome seems to have diverse roles in E. ictaluri pathogenesis. hcp2 is important in adherence to epithelial cells and replication within macrophages. However, evpC plays a crucial role in E. ictaluri virulence in catfish. Therefore, secretion of potential evpC and hcp2 dependent effector proteins via T6SS need more investigation.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by Institutional Animal Care and Use Committee at Mississippi State University.