The Differential Phosphorylation-Dependent Signaling and Glucose Immunometabolic Responses Induced during Infection by Salmonella Enteritidis and Salmonella Heidelberg in Chicken Macrophage-like cells

Salmonella is a burden to the poultry, health, and food safety industries, resulting in illnesses, food contamination, and recalls. Salmonella enterica subspecies enterica Enteritidis (S. Enteritidis) is one of the most prevalent serotypes isolated from poultry. Salmonella enterica subspecies enterica Heidelberg (S. Heidelberg), which is becoming as prevalent as S. Enteritidis, is one of the five most isolated serotypes. Although S. Enteritidis and S. Heidelberg are almost genetically identical, they both are capable of inducing different immune and metabolic responses in host cells to successfully establish an infection. Therefore, using the kinome peptide array, we demonstrated that S. Enteritidis and S. Heidelberg infections induced differential phosphorylation of peptides on Rho proteins, caspases, toll-like receptors, and other proteins involved in metabolic- and immune-related signaling of HD11 chicken macrophages. Metabolic flux assays measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) demonstrated that S. Enteritidis at 30 min postinfection (p.i.) increased glucose metabolism, while S. Heidelberg at 30 min p.i. decreased glucose metabolism. S. Enteritidis is more invasive than S. Heidelberg. These results show different immunometabolic responses of HD11 macrophages to S. Enteritidis and S. Heidelberg infections.


Introduction
Salmonellae are rod-shaped, Gram-negative, facultative anaerobes [1] and are the number one cause of foodborne gastroenteritis [2,3]. The species Salmonella enterica is a highly diverse bacterial species consisting of six subtypes and over 2500 serovars [1,4]. Salmonella enterica subspecies enterica Enteritidis (S. Enteritidis) and Salmonella enterica subspecies enterica Heidelberg (S. Heidelberg) are two of the many serovars under the subspecies that cause nontyphoidal salmonellosis (NTS) resulting in gastroenteritis [5][6][7][8]. Salmonellae that can be transmitted from animals, humans, and plants are referred to as nonspecific serovars (nonrestricted) [1,4,9]. Reported cases of NTS poisoning include acute gastroenteritis and watery diarrhea that occur approximately 6-12 h after ingestion or contraction of bacteria in humans [4,9]. Other common symptoms may include nausea, vomiting, abdominal pain, and fever [4]. NTS symptoms usually last 4 to 10 days and may resolve on their own with time [10]. NTS can also become invasive in humans, that is, Salmonella can enter cells, replicate, and spread quickly. Sometimes Salmonella enters the bloodstream and other organs of the host, causing severe illness [5]. This happens when the pathogen is contracted by an individual that is immunocompromised [11] or has a weak/undeveloped immune system (such as in the case of children and elderly people) [4,8,11].
The study described here focuses on the intracellular responses of chicken hosts to infection by these two different serovars of Salmonella at different timepoints in vitro. With the kinome peptide array technology, we defined the changes in phosphorylation of key immune and metabolic response proteins and how these changes affect cellular function. We validated the indicated changes in cellular responses using other molecular-based techniques. Understanding the immunometabolic changes that the bacteria induces on the host and subsequent host responses highlights key mechanisms of infection in the avian immune system. This understanding would serve as a basis for potential intervention strategies toward treatment of infections by various serovars of Salmonella in poultry.

Cell Line and Maintenance
HD11 cells are referred to as chicken macrophage-like cells because they represent an immortalized bone marrow derived cell line that is transformed with the avian myelocytomatosis type MC29 virus [18]. The cells were maintained in cell culture media containing Iscove's Modified Dulbecco's Media (IMDM) (GE Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (Midsci, Valley Park, MO, USA) and 1% 1.5 mM L-glutamine (containing penicillin and streptomycin) (Gibco, Grand Island, NY, USA) at 37 • C, 5% CO 2 , and 95% humidity. When required, cells were counted using a hemocytometer and a dilution factor of 10 in trypan blue (Sigma-Aldrich, St. Louis, MO, USA). HD11 cells were obtained from the laboratory of Dr. Mark Parcells, University of Delaware.

Bacteria Serovars
Serotyped S. Enteritidis and serotyped S. Heidelberg from infected chickens were obtained from Dr. Haiqi He, US Department of Agriculture Research Service. Both S. Enteritidis and S. Heidelberg were designed to be resistant to nalidixic acid and novobiocin [37]. The Salmonella stocks were stored in tryptic soy broth (TSB (Becton, Dickinson and Company, Sparks, MD, USA)) and 20% glycerol (Sigma-Aldrich, St. Louis, MO, USA) at −80 • C. Stocks were thawed, and 100 µL was cultured in a shaker at 37 • C in an Erlenmeyer flask containing 30 mL of TSB with antibiotics (25 µg/mL novobiocin and 20 µg/mL nalidixic acid (Sigma-Aldrich, St. Louis, MO, USA)) overnight. One hundred microliters of each overnight culture was then added to a separate Erlenmeyer flask containing 30 mL of TSB with antibiotics (25 µg/mL novobiocin and 20 µg/mL nalidixic acid) and cultured under the same conditions for 4 h. Only the 4 h cultures were used for infections. The optical density of the 4 h cultures were determined using a Molecular Devices Microplate Reader SpectraMax Plus (Molecular Devices, San Jose, CA, USA) at 600 nm endpoint absorbance. The measurement derived from a spectrophotometer was converted to bacteria count using the Agilent OD600 online calculator for Escherichia coli. To calculate the number of bacteria required for each assay, n (the required number of bacteria for each sample) was divided by bacteria OD600 (optical density measured at 600 nm converted to bacteria cell count) multiplied by 1000 (i.e., (n/OD600) × 1000).

Infection of Cells with Salmonella
Using HD11 cell counts, appropriate volumes of cell suspension to obtain 1 × 10 6 cells were plated in 12-or 24-well VWR multiwell cell culture plates (VWR, Radnor, PA, USA) for approximately 2 h to adhere to the wells. These cells were then infected for 1 h with a multiplicity of infection (MOI) of 100 Salmonella per HD11 cell (100:1) for each serovar in three well replicates plus control. Infected cells were treated with 100 µg/mL of gentamicin (Sigma-Aldrich, St. Louis, MO, USA) for 30 min (to yield 1.5 h) or 1 h postinfection (p.i.) and incubated in gentamicin-free media for an additional 1 h for a total of 3 h p.i. Gentamicin is an antibiotic, targeting mostly Gram-positive bacteria [38]. However, studies have shown that treatment with gentamicin after Salmonella infection kills extracellular bacteria [38]. Therefore, treatment with gentamicin ensures that only changes due to intracellular Salmonella are being measured. The infected cells and control cells were used for experiments using different techniques as described below.

Gentamicin Protection Assay
One million cells were plated in 12-or 24-well VWR multiwell cell culture plates (VWR, Radnor, PA, USA) for approximately 2 h to adhere to the wells. These cells were then infected for 1 h with a multiplicity of infection (MOI) of 100:1 for each serovar in three well replicates plus control. Infected cells were treated with 100 µg/mL of gentamicin (Sigma-Aldrich, St. Louis, MO, USA) for 30 min (to yield 1.5 h) or 1 h postinfection (p.i.) and incubated in gentamicin-free media for an additional 1 h for a total of 3 h p.i. To evaluate the role of mTOR in Salmonella infections, 100 ng/mL final concentration of rapamycin (Sigma-Aldrich, St. Louis, MO, USA) or 2 µM of MHY1485 (Millipore Sigma, Burlington, MA, USA) was added to incubation media before infection.
After infection and treatment with gentamicin, the cells were lysed in 0.01 M of phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) containing 1% triton X100 (Sigma-Aldrich, St. Louis, MO, USA). After lysis, 100 µL from each well was added to 15 mL centrifuge tubes containing 9.9 mL of 0.01 M PBS (10 2 ) and mixed gently. One thousand microliters of each 10 2 mix was added to a separate 15 mL centrifuge tube containing 9 mL of PBS (10 3 ) and mixed gently. One thousand microliters of 10 3 was added to a centrifuge tube containing 9 mL of PBS, mixed gently, and plated in tryptic soy agar plates containing antibiotics (25 µg/mL novobiocin and 20 µg/mL nalidixic acid). After 12-18 h incubation at 37 • C, the bacteria colonies formed on the plates were counted and recorded as colony-forming units (CFUs).

Kinome Peptide Array Analysis
The kinome peptide array protocol performed for HD11 cells is briefly described below. The detailed protocol can be found in a publication by Arsenault et al. [39]. Cells were lysed in 100 µL of specially made lysis buffer containing protease inhibitors. The lysates were centrifuged, and 70 µL of the supernatant was mixed with 10 µL of activation mixture containing 500 µM of ATP to activate the kinases in the samples. Approximately 80 µL of each sample was applied to a glass peptide array (JPT Peptide Technologies, Berlin, Germany) and incubated in a sealed container placed in a 5% CO 2 incubator at 37 • C for 2 h. After incubation, sample residues were washed off the arrays and the arrays were stained in phospho-specific fluorescent ProQ Diamond Phosphoprotein Stain (Life Technologies, Carlsbad, CA, USA) for 1 h. The arrays were submerged in a destain solution containing 20% acetonitrile (EMD Millipore Chemicals, Billerica, MA, USA) and 50 mM sodium acetate (Sigma-Aldrich, St. Louis, MO, USA) to remove non-phospho-specific binding. The arrays were scanned in a Tecan PowerScanner microarray scanner (Tecan Systems, San Jose, CA, USA) at 532 to 560 nm with a 580-nm filter to detect dye fluorescence.
The images of the scanned array were gridded manually to fit the phospho-specific spots and extract signal intensity using GenePix Pro software (version 7.2.29 1, Molecular Devices, CA, USA). Microsoft Excel 2016 (Redmond, WA) files containing kinomic data were generated and further analyzed using the online normalization and analysis tool known as Platform for Intelligent, Integrated Kinome Analysis (PIIKA2) [40]. The kinome peptide array data generated from PIIKA2 were analyzed by using other online databases like STRING [41], KEGG color and search pathway [42], UniProt [43][44][45], and PhosphosSitePlus [46].
The human UniProt accession and site information orthologous to chickens are used in the results and discussion of this paper because they are easily accessible. The chicken sites corresponding to their human identifiers used in this paper are reported in Appendix A. Appendix A also contains full names of all the proteins abbreviated in the result tables.

Seahorse XFp Metabolic Assay
The Seahorse XFp Analyzer (Agilent Technologies, Santa Clara, CA, USA) is technology that measures functional metabolic data, namely the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) [47,48]. The seahorse XFp analyzer was used to perform metabolic analysis of HD11 cells before and after infection (with S. Enteritidis or S. Heidelberg).
Cells were prepared for plating in a Seahorse mini culture plate (Agilent Technologies, Santa Clara, CA, USA) by adding 5 mL of trypsin to each T75 flask of cells. The cell-trypsin suspension was transferred to a 15 or 50 mL centrifuge tube. Each flask was washed with 5 mL of IMDM media. The washes were transferred into the cell-trypsin suspension tubes and centrifuged at 300 × g for 5 min. After centrifugation, the supernatant was discarded, and the cell pellet was collected and resuspended in 2-5 mL of IMDM media. The cells were counted to determine the volume of suspension needed to plate 50,000 cells per well. The desired volume of cells (50 µL of IMDM-cell mixture) was added to the wells of the mini culture plate (excluding wells A and H). The wells on the side of the plate were moated with 400 µL of double-deionized water. After the plating process, the plates were incubated in 5% CO 2 for at least 2 h. It is strongly advised to calibrate the seahorse machine before each run; therefore, calibration plates were prepared at least 8 h before each experiment. The calibration plates were prepared by adding 400 µL of double-deionized water to the moat wells and 200 µL of Agilent Seahorse calibrant to the eight wells that held the cartridge. The cartridge was placed into the wells and incubated at 37 • C in a non-CO 2 incubator. Bacteria serovars used for infection were prepared as described in the Section 2.2.
After 2-5 h of mini culture plate incubation at 37 • C with CO 2 , 200 µL of Seahorse media (XF DMEM medium, pH 7.4, with 5 mM HEPES (Agilent Technologies, Santa Clara, CA, USA)) containing 1% 200 mM glutamine and 1% 100 mM sodium pyruvate was added to each well (including blank wells A and H). After addition of seahorse media to the mini culture plates, the mini plates were incubated at 37 • C in a non-CO 2 incubator for 30 min to 1 h. During this time, the calibration plate was removed from non-CO 2 incubator and placed into the Seahorse XFp to calibrate the machine. After the machine was fully calibrated, bacteria were added to the designated wells of the mini culture plate. The calibration plate was then replaced by the mini culture plate with the infected cells to start the assay.

Statistics
For gentamicin protection assays, analysis of variance (ANOVA) and Tukey-Kramer post hoc tests were performed for all groups at the 30 min and 2 h p.i. timepoints, i.e., control (HD11 cells without infection or treatments), infected HD11 cells, HD11 cells with treatment only (rapamycin or MHY1485), and infected HD11 cells with treatments. For the Seahorse metabolic flux assays, analysis of variance (ANOVA) and Tukey-Kramer post hoc tests were performed for the control groups and infected cell groups at 30 min and 2 h p.i. Analysis of variance (ANOVA) and Tukey-Kramer post hoc tests were performed to compare within and amongst groups using JMP pro 14.0.0. For the kinome peptide array, a one-sided paired t-test between infected (treatment) and control values was performed for each peptide via PIIKA2 [40].

Salmonella Alters Host (HD11 Cells) Immunometabolism
Analysis of the kinome peptide array performed on chicken macrophage-like cells infected with S. Enteritidis and S. Heidelberg showed significant changes in peptides phosphorylated in immune and metabolic pathways compared to control. A majority of these proteins belong to immunometabolic pathways, including the 5 adenosine monophosphate-activated protein kinase (AMPK), hypoxia inducible factor (HIF), insulin, and mammalian target of rapamycin (mTOR) signaling pathways. Table 1 highlights the number of signaling proteins that are similarly altered in chicken macrophages infected with S. Enteritidis and S. Heidelberg at 30 min and 2 h p.i. HIF-1α signaling 16 21 The number of proteins that showed the same changes in phosphorylation (increased or decreased phosphorylation) in immunometabolic signal transduction pathways for both S. Enteritidis and S. Heidelberg infected HD11. This information was derived from the STRING database's [41] top 20 KEGG pathway [42] output of the kinome peptide array data.

S. Enteritidis and S. Heidelberg Induce Differential Phosphorylation of Cytoskeletal Proteins
When Salmonella invades, it engages cellular responses and injects its proteins into the hosts' cells; these proteins can alter host kinase activities [49][50][51]. To determine the different effects S. Enteritidis and S. Heidelberg each have on the kinase activity of cytoskeleton proteins in chicken macrophages, an in-depth analysis of the pathways observed in the kinome peptide array data (Table 1) was performed. The effector proteins produced by Salmonella can alter the cell cytoskeleton by affecting the activities of Rho proteins [52]. The kinome peptide array showed that there was an increase in phosphorylation of RhoA on its inhibitory site (S188) in S. Enteritidis infected cells (Table 2), inhibiting RhoA activity. For S. Heidelberg, there was a decrease in phosphorylation on this site. An increase in phosphorylation of the inhibitory site indicates inhibition of the ability of RhoA to drive the stabilization of cytoskeletal filaments [53], thus allowing bacteria to easily invade the cell [54]. There is also a decrease in phosphorylation of ROCK2 on its inhibitory site (Y722). ROCK is activated by binding to RhoA in its active GTP-bound state [55]; however, because RhoA is being inhibited in S. Enteritidis infected cells, RhoA downstream signaling to ROCK proteins should also be inhibited. Therefore, this phosphorylation could be due to other kinases.
The changes in the phosphorylation states of key proteins in cell cytoskeletal control and stability as shown in the kinome peptide array data. The UniProt accession and site information belong to human proteins that are orthologues of chicken proteins and were collected from the PhosphoSite and UniProt databases. The orthologous chicken phosphorylation sites are reported in Appendix A. The arrows represent the sites discussed or considered for the respective proteins mentioned. ↑, significantly more phosphorylated and consequence of phosphorylation unknown; ↑, significantly more phosphorylated on an inhibitory site; ↓, significantly less phosphorylated and consequence of phosphorylation unknown; ↓, significantly less phosphorylated on an inhibitory site; ∅, not significant in the indicated dataset.

S. Enteritidis and S. Heidelberg Induce Differential Phosphorylation of Metabolic Proteins to Promote Their Survival
Once in the gut, Salmonella is capable of initiating and utilizing host immunometabolic responses for its benefit [56,57]. It is hypothesized that Salmonella induces and promotes metabolic activities in the gut to promote its survival and growth [18,58,59]. Interestingly, the kinome peptide array revealed that these metabolic activities may be different for S. Enteritidis and S. Heidelberg. A greater increase in glycolytic protein activity was observed 30 min after S. Enteritidis infection than was observed in S. Heidelberg infected cells at this time. S. Enteritidis infected cells showed increased phosphorylation of both phosphofructokinases (PFK1 and PFK2), while S. Heidelberg infected cells did not (Table 3). Although the effects of phosphorylation are not known for many of these sites, we observed that S. Enteritidis infection initiated more glycolytic kinase activity than S. Heidelberg infection. At 30 min after S. Enteritidis infection, increased phosphorylation of the energy sensor protein AMPK on its active site (T183) was observed, indicating increased activity of AMPK (Table 3). S. Heidelberg infected cells showed the inhibition of AMPK via increased phosphorylation on its inhibitory site (S496). The increased activity of AMPK indicates a decrease in energy availability or decrease in the ratio of ATP to ADP/AMP [60]. This agrees with the observed increase in kinase activity of the glycolytic enzymes for S. Enteritidis at 30 min p.i. (Table 3).
The changes in the phosphorylation states of immune-and metabolic-related peptides involved in AMPK, mTOR, and HIF-1α signaling as shown in the kinome peptide array data. The arrows represent significant changes in phosphorylation at different kinase target sites. ↑, significantly more phosphorylated and consequence of phosphorylation unknown; ↑, significantly more phosphorylated on an active site; ↑, significantly more phosphorylated on an inhibitory site; ↓, significantly less phosphorylated and consequence of phosphorylation unknown; ↓, significantly less phosphorylated on an active site; ↓, significantly less phosphorylated on an inhibitory site; ∅, not significant in the indicated dataset. PhK, phosphorylase kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerate kinase 1; PGM, phosphoglucomutase; S6K, ribosomal protein S6 kinase beta-1; 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1.
Some of these metabolic changes are directly linked to immune or inflammatory responses. HIF-1α, which induces the transcription of inflammatory factors as well as genes involved in glucose metabolism, was increasingly phosphorylated on its inhibitory site (S247) in S. Heidelberg infected cells ( Table 3). The decrease in glycolytic activity appeared unique to S. Heidelberg 30 min postinfection. Comparison of the changes in the phosphorylation of peptides on proteins involved in glycolysis of S. Heidelberg 30 min p.i. and S. Heidelberg 2 h p.i. revealed that kinase activity was not detected for phosphoglucose isomerase, PFK, and aldolase in the kinome peptide array data of S. Heidelberg 30 min p.i. However, there were kinase activities detected for the enzymes downstream of aldolase. There are instances where the products of phosphoglucose isomerase, PFK, and aldolase can be made available without the involvement of these three enzymes via an alternative substrate provider to continue the breakdown of glucose. The pentose phosphate pathway is an alternative substrate provider for glycolysis [61]. Specifically, undergoing the pentose phosphate pathway makes available fructose-6-phosphate and glyceraldehyde-3-phosphate [61], end products of the enzymes mentioned above. The pentose phosphate pathway also generates NADPH, which serves as a cofactor for inducible nitric oxide synthase (iNOS) for the production of nitric oxide (NO) [61,62]. This is supported in a publication by Haiqi et al. 2018, where the researchers observed a significant decrease in iNOS phosphorylation on its inhibitory site, thus inducing its activity [18]. The researchers also performed a nitrite assay, and the results showed a gradual increase in nitrite concentration which is comparable to NO generation in S. Heidelberg infected HD11 cells [18].
The increased activity of glycolytic proteins and the induction of glycolysis is an indication of proinflammation. Based on these results, S. Enteritidis infection induces more proinflammatory responses in chicken macrophages than S. Heidelberg. Besides the decrease in glycolytic activity in early S. Heidelberg infections, these results also indicate that S. Heidelberg may induce pentose phosphate pathway activity which may promote NO metabolism that is beneficial for bacteria growth and survival.

S. Enteritidis Infection Induces an Early Increase in Glucose Metabolism, and Early S. Heidelberg Infection Dampens Glucose Metabolism
Increased glycolysis is an indication of proinflammatory responses [24,63]. The kinome peptide array results showed an increase in glycolytic activities for S. Enteritidis and a reduction in glycolytic activities for S. Heidelberg. To determine changes in the metabolic states of HD11 cells during S. Enteritidis or S. Heidelberg infections at 30 min and 2 h p.i., a gentamicin-free metabolic flux assay measuring ECAR was performed on HD11 cells infected with each serovar. S. Enteritidis infected cells at 30 min p.i. showed an increase in ECAR (15.365 mpH/min) followed by a larger decrease

S. Enteritidis and S. Heidelberg Induce Differential Phosphorylation of Inflammatory Proteins
After invading host cells using the type III secretion system, Salmonella effector proteins also stimulate the activation of the NLRC4 inflammasome to induce pyroptosis to invade other cells [64]. S. Enteritidis infected cells showed early induction of caspase-1 activity via the increased phosphorylation on site S227. However, there was decreased phosphorylation of S227 for S. Enteritidis infected cells at 2 h p.i., while no significant data supported the phosphorylation of caspase-1 in S. Heidelberg infected cells. Caspase-1 is the final protein stimulated in the NLRC4 inflammasome to induce proinflammatory responses like pyroptosis. Therefore, its kinase activity in The metabolic assay also showed that Salmonella infections induce increased oxygen metabolism in HD11 cells. OCR measurements indicating the oxygen consumption of the HD11 cells showed an increase in OCR in cells infected with S. Enteritidis (164.525 pmol/min) and S. Heidelberg (164.19 pmol/min) at 30 min compared to control (56.44 pmol/min) ( Figure 1B). This increase in OCR was sustained past the 2 h timepoint, with the OCR of S. Enteritidis infected cells being 165 pmol/min and that of S. Heidelberg infected cells being 165 pmol/min; meanwhile, the OCR of the control cells remained relatively low at 51.83 pmol/min ( Figure 1B). These measurements comparing the OCR of infected cells to that of control cells showed statistical significance at both timepoints (p ≤ 0.0001), indicating that increased oxygen metabolism may be common to both serovars and Salmonella in general.

S. Enteritidis and S. Heidelberg Induce Differential Phosphorylation of Inflammatory Proteins
After invading host cells using the type III secretion system, Salmonella effector proteins also stimulate the activation of the NLRC4 inflammasome to induce pyroptosis to invade other cells [64]. S. Enteritidis infected cells showed early induction of caspase-1 activity via the increased phosphorylation on site S227. However, there was decreased phosphorylation of S227 for S. Enteritidis infected cells at 2 h p.i., while no significant data supported the phosphorylation of caspase-1 in S. Heidelberg infected cells. Caspase-1 is the final protein stimulated in the NLRC4 inflammasome to induce proinflammatory responses like pyroptosis. Therefore, its kinase activity in S. Enteritidis at 30 min p.i. suggests induction of its activity. Although kinase activities of other proteins involved in the NLRC4 inflammasome were observed in the kinome peptide array data of S. Heidelberg infected cells, there is no evidence to support caspase-1 activity.
Besides signs of pyroptosis, kinome data from early S. Enteritidis infection also showed stimulation of cell death signaling via the tumor necrosis factor (TNF) receptor-associated factor and proteins downstream of the receptor including Jun N-terminal kinase 1 (JNK1) and mitogen-activated protein kinase (MAPK)-interacting kinase 1 (MNK1). These signs were observed in chicken macrophages 30 min p.i. with S. Enteritidis and 2 h p.i. with S. Heidelberg. In detail, JNK1 (which is known to induce apoptosis [65,66]) was phosphorylated at site T183 (Table 4), thus stimulating early cell death in S. Enteritidis infected cells. MNK, which is also downstream of TNF-alpha-MAPK signaling was significantly less phosphorylated (Table 4) than control for S. Enteritidis at 30 min and S. Heidelberg at 2 h p.i. on the site T255, responsible for the inhibition of apoptosis [66]. In addition, the NFkB inhibitor IkB-alpha was also significantly more phosphorylated on its active site (Y42) in S. Heidelberg at 30 min p.i. (Table 4). Y42 also plays a role in the inhibition of apoptosis [67].
The changes in the phosphorylation states of some key proteins involved in immune signaling and immune regulation as shown in the kinome peptide array data. The UniProt accession and site information belong to human proteins that are orthologues of chicken proteins and were collected from the PhosphoSite and UniProt databases. The orthologous chicken phosphorylation sites are reported in Appendix A. The arrows represent the sites discussed or considered for the respective proteins mentioned. ↑, significantly more phosphorylated and consequence of phosphorylation unknown; ↑, significantly more phosphorylated on an inhibitory site; ↓, significantly less phosphorylated and consequence of phosphorylation unknown; ↓, significantly less phosphorylated on an inhibitory site; ↑, significantly more phosphorylated on an active site; ↓, significantly less phosphorylated on an active site; ∅, not significant in the indicated dataset.
The kinome peptide array data also showed the decreased phosphorylation of the proapoptotic factor caspase-3 on its inhibitory site for both serovars at 30 min p.i. (Table 4). These changes in phosphorylation to positively affect apoptosis were commonly observed in S. Heidelberg at 2 h p.i. Thus far, the kinome peptide array data suggest that cell death is reduced in the initial stages of S. Heidelberg infections compared to the initial stages of S. Enteritidis infections. The changes in phosphorylation of toll-like receptors (TLRs) and cytokine receptors such as interleukin (IL)-6 indicate inflammatory stress. Moreover, S. Enteritidis may pose a significant challenge to the host cells during initial infection which results in the stimulation of programmed cell death early on. Meanwhile, apoptosis is not induced during the early stages of S. Heidelberg infection because less inflammation is induced in the host upon initial infection.
The energetic demands of these infections can affect the hosts' immune capacity by suppressing activity of immunometabolic proteins like mTOR [68]. As energy demand increases, AMPK is activated, and this leads to the deactivation or inhibition of mTOR by AMPK [68]. mTOR is an immunometabolic protein that regulates the translation and synthesis of proteins, including those involved in immune responses [69,70]. Activity of mTOR was not detected via phosphorylation of mTOR on its active site S2448 in both S. Enteritidis and S. Heidelberg infected cells early on. Active mTORc1 phosphorylates 4EBP1 and S6K to promote protein synthesis [36,70] and cell growth and survival [36], respectively. At 2 h after S. Enteritidis and S. Heidelberg infection, mTOR is increasingly phosphorylated on its active site S2448 to induce activity in the cells. The initial absence of mTOR activity could mean that Salmonella targets mTOR for invasion or this is just a cellular response to prioritize cellular activities during infection.

S. Enteritidis Is More Invasive Than S. Heidelberg
The kinome peptide array data showed changes in the phosphorylation of cytoskeletal proteins that are more favorable to invasion by S. Enteritidis than S. Heidelberg (Table 2). Therefore, gentamicin protection assays were performed to quantify the ability of S. Enteritidis and S. Heidelberg to invade HD11 cells at 30 min and 2 h p.i. The kinome peptide array data also showed changes in mTOR activity in both serovars at different timepoints (Table 4). To determine the role of mTOR in Salmonella invasions, the cells were treated with the mTOR inhibitor rapamycin and the mTOR activator MHY1485 before infection, and gentamicin protection assays were performed. Results of the gentamicin protection assays showed that there was a significant increase in invasion by S. Enteritidis, as shown by the colony-forming unit (CFU) count, compared to S. Heidelberg at 30 min p.i. (Figure 2A). Meanwhile, at 2 h p.i., S. Enteritidis had higher plate counts than S. Heidelberg; however, this difference was not significant ( Figure 2B). The overnight colony count for S. Enteritidis at 30 min was 37.44 × 10 4 , while that of S. Heidelberg at 30 min was 17.44 × 10 4 (p-value of 0.0081). Thus, S. Enteritidis is better at invading HD11 cells compared to S. Heidelberg at 30 min p.i. There was a trend observed at 2 h after S. Enteritidis infection only (19 × 10 4 CFUs) that showed an increase in plate counts in S. Enteritidis infection with rapamycin treatment (24 × 10 4 CFUs) and a decrease in S. Enteritidis with MHY1485 treatment (12 × 10 4 CFUs) ( Figure 2B). However, the statistical analysis for these plate counts showed no significant difference between S. Enteritidis and its treatment groups at 2 h p.i. (Figure 2B). There were also no significant differences between S. Heidelberg and its treatment groups at 2 h p.i. (Figure 2B).

Discussion
In this study, we demonstrated the immunometabolic difference between S. Enteritidis and S. Heidelberg infections in chicken HD11 macrophages (Table 5). Although S. Enteritidis and S. Heidelberg are serovars belonging to the same subspecies of Salmonella, we observed that each induce different changes in energy metabolism and display different invasiveness in these macrophages. For successful survival in the host, Salmonella must first invade the host cell. The kinome peptide array data suggested that S. Enteritidis may be more efficient at invading the host cell than S. Heidelberg (Table 5) by inhibiting the activity of RhoA (Table 2) which activates ROCK2, responsible for regulating the cell cytoskeletal and actin filament stabilization [71]. Although the effects of

Discussion
In this study, we demonstrated the immunometabolic difference between S. Enteritidis and S. Heidelberg infections in chicken HD11 macrophages (Table 5). Although S. Enteritidis and S. Heidelberg are serovars belonging to the same subspecies of Salmonella, we observed that each induce different changes in energy metabolism and display different invasiveness in these macrophages. For successful survival in the host, Salmonella must first invade the host cell. The kinome peptide array data suggested that S. Enteritidis may be more efficient at invading the host cell than S. Heidelberg (Table 5) by inhibiting the activity of RhoA (Table 2) which activates ROCK2, responsible for regulating the cell cytoskeletal and actin filament stabilization [71]. Although the effects of phosphorylation for the sites of RGH06 and RGH17 are unknown (Table 2), these two proteins are known to inactivate GTPase by converting it to a GDP-bound state [72]. With the decrease in RHOA and ROCK2 activity, S. Enteritidis can easily affect other host cytoskeletal proteins using effectors from its T3SS to invade cells. This efficient invasiveness of S. Enteritidis over S. Heidelberg was supported by the gentamicin assay (Figure 2) that showed a greater average CFU count for S. Enteritidis in HD11 cells at 30 min p.i. Moreover, we also observed that different immunometabolic activities are induced in S. Enteritidis and S. Heidelberg infections upon invasion. The metabolic activities observed in both serovars are crucial components of immune responses needed to clear infections, but studies have shown that Salmonella can use some host inflammatory and metabolic activities to create a suitable niche for survival [58,59]. Early S. Enteritidis infection induced an increase in glycolysis (Table 3, Figure 1) known to be a trademark of a proinflammatory response [24,63], while no signs of increased glycolytic activities were observed in HD11 cells infected with S. Heidelberg until the 2 h p.i. timepoint (Table 3, Figure 1). The kinome peptide array data suggested that the pentose phosphate pathway may have been induced in S. Heidelberg infected cells during initial infection ( Table 3). The purpose of this response in the cell is not fully understood; however, it may be that the decrease in glycolysis is to dampen the inflammation induced during glucose metabolism and increase the production of NO via the pentose phosphate route [61,62]. The significance of this action is that it converts NO to nitrate, which Salmonella bacteria are known to metabolize for growth and survival [73]. Results from Haiqi et al. (2018) showed that S. Heidelberg infections induce significantly more NO than S. Enteritidis [18], yet further testing is required to validate the higher rate of the pentose phosphate pathway in S. Heidelberg infected cells than in those infected by S. Enteritidis to fully support this hypothesis. In short, these results revealed that S. Enteritidis and S. Heidelberg each induce different metabolic activities ( Table 5) that influence the immune responses of the host cells via the AMPK, HIF, mTOR, and insulin pathways. We also demonstrated that S. Enteritidis and S. Heidelberg have similar effects on oxygen metabolism (Table 1,  Table 5, and Figure 1B).
Studies have shown that Salmonella induces virulent mechanisms in host cells to stimulate cell death [59,74]. In this study, we identified changes in phosphorylation of programmed cell death inducing proteins during S. Enteritidis and S. Heidelberg infections. Caspase-3 and caspase-8 are both involved in the initiation of programmed cell death. S150 of caspase-3 is a site that has been found to inhibit apoptosis [75]. In the kinome peptide array dataset, we observed a decrease in phosphorylation of this inhibitory site (Table 4), which indicates a positive regulation of apoptosis. The same can be said for caspase-8 site S347 [75] (Table 4). Besides the decreased inhibition of caspase-3 at 30 min after S. Heidelberg infection, there was not much evidence in the kinome peptide array results supporting the occurrence of increased cell death in HD11 cells at 30 min p.i. However, at the S. Heidelberg 2 h p.i. point, we observed an increase in positive regulation of apoptotic factors via the decreased activity of proteins involved in the inhibition of apoptosis, such as IkB-alpha (Y42), MNK1 (T255), caspase-3, and caspase-8, and the increased activity of proapoptotic factors like JNK1 and IRF1 (Table 4). S. Enteritidis induces changes in the phosphorylation of the proinflammatory and pyroptosis-inducing protein caspase-1 (Table 4). S. Enteritidis also induced changes in phosphorylation of other proteins involved in the signaling of the NLRC4 inflammasome. This sign of inflammatory cell death, considered along with the changes in phosphorylation of JNK1 and caspase-3 and the absence of kinases that regulate cell death inhibitors like IkB-alpha and MNK at 30 min p.i. (Table 4), suggests that S. Enteritidis may cause more cell death than S. Heidelberg during initial infection (Table 5). Further testing to measure cell death is required before accepting this hypothesis.
Moreover, the inflammatory stress induced by S. Enteritidis and S. Heidelberg via the changes in phosphorylation of TLR and IL-6R (Table 4) may also play a role in cell death and invasion [76]. The gentamicin protection assays showed a significant difference between the invasiveness of S. Enteritidis and S. Heidelberg at 30 min p.i. (Figure 2A). At 2 h p.i., although not significant, S. Enteritidis plate counts were also higher than S. Heidelberg ( Figure 2B). The high number of intracellular S. Enteritidis in HD11 macrophages highlights its ability to invade and form a suitable niche in the host. S. Enteritidis invades cells, causing a change to the M1 profile. This induces the proinflammatory response [24], including the expression of proinflammatory cytokines [77] and increased downstream and feedback activity of pathogen recognition receptors (PRR) like TLR [76]. The ability of S. Enteritidis to invade more host cells increases due to this increase in proinflammatory factors [78]. This increased inflammation can ultimately lead to cell death and rupture, enabling the bacteria to invade neighboring healthy cells (Table 5).
Although apoptosis is a naturally occurring noninflammatory process carried out in a cell, it is also a mechanism to clear infected cells. Salmonella uses this response to its advantage. Apoptotic bodies formed from infected cells contain vacuoles of Salmonella, causing phagocytic cells to become infected as a result of ingesting such bodies [23,79]. Thus, induction of programmed cell death early on would enable S. Enteritidis to carry out cellular invasion. The colony plate count for S. Heidelberg remained low throughout both timepoints, yet S. Heidelberg is still capable of inducing substantial changes in the immune and metabolic signaling of host cells. This implies that S. Heidelberg does not require a large-scale invasion to survive and create a suitable niche in host cells.
These inflammatory responses can be energetically draining and may affect other processes in host cells [60,68]. mTOR complex 1 has been of major interest in the study of host response to bacterial infections because of its role in regulation and synthesis of proteins, many of which are involved in immune regulation [36,70,80]. The kinome peptide array showed a lack of mTORC1 kinase activity specifically at the active site S2448 at 30 min p.i. but showed an increase in the phosphorylation of this site at 2 h p.i. for both S. Enteritidis and S. Heidelberg. The role of mTOR in cell survival and protein synthesis raises questions about its involvement in bacterial invasion and survival during infection. To determine whether inhibition or activation of mTOR would increase or decrease Salmonella invasiveness, HD11 cells were treated with 100 g/mL of rapamycin or 2 µM of MHY1485 before Salmonella infection. The results showed no difference in invasion of HD11 macrophages treated with MHY1485 and infected by S. Enteritidis or S. Heidelberg at 30 min or 2 h p.i. (Figure 2). Although MHY1485 showed a decrease in S. Enteritidis count at 2 h and rapamycin showed an increase in S. Enteritidis count at 2 h, statistical analysis showed no significant difference between the treatment groups and S. Enteritidis infection alone (Figure 2). Comparing S. Enteritidis 2 h p.i. MHY1485 and rapamycin treatment groups to each other showed that increasing mTOR activation over time may improve clearance of the bacteria compared to decreasing or inhibiting mTOR (Table 5, Figure 2B).

Conclusions
In summary, S. Enteritidis and S. Heidelberg induce different changes in the phosphorylation of immunometabolic signaling peptides compared to control in vitro. As shown by the kinome peptide array, there are key differences in phosphorylation of peptides on proteins involved in energy metabolism, protein regulation, apoptosis, cytoskeletal regulation, and inflammation. These proteins include PFK 1 and 2; AMPK; caspases 1, 3, and 8; HIF-1α; TLR; RhoA; and more. These findings were further validated using the metabolic flux assays and gentamicin protection assays. The metabolic flux assays which measured ECAR and OCR demonstrated that (i) S. Enteritidis at 30 min p.i. resulted in increased glucose metabolism, (ii) S. Heidelberg at 30 min p.i. resulted in decreased glucose metabolism, and (iii) both Salmonella serovar infections induce increased oxygen metabolism compared to control. Gentamicin protection assays performed at 30 min and 2 h postinfection revealed that S. Enteritidis bacteria are more invasive than S. Heidelberg.
Overall, these results support the observations of invasiveness of S. Enteritidis and persistence of S. Heidelberg in poultry and an understanding of the immunometabolic activities that may contribute to such differences. That is, the immunometabolic responses that S. Enteritidis exploits in hosts for increased invasion only present short-term benefits to the bacteria. Meanwhile, the delayed host immunometabolic response to S. Heidelberg, at the cost of decreased invasiveness, offers the long-term benefits of increased persistence to the bacteria. The tradeoff for S. Enteritidis in increasing its invasiveness is the increased immune response produced by the host to clear the infection, hence the decrease in prevalence of S. Enteritidis. The persistence of S. Heidelberg infection is evident in the increasing isolation and prevalence of this bacteria in poultry over S. Enteritidis in the past decade. This project reveals the difficulty associated with efficiently treating Salmonella infections because different serovars of Salmonella may induce different immunometabolic responses in hosts. Therefore, an immune or metabolic target for the treatment of one serovar may benefit another serovar.
Funding: This research received no external funding.

Acknowledgments:
The authors would like to acknowledge Mark Parcells, University of Delaware, for providing the HD11 cell line and Haiqi He, USDA-ARS, for providing the Salmonella serovars.

Conflicts of Interest:
The authors declare no conflict of interest. Table A1. Protein names and chicken phosphorylation sites corresponding to the human sites reported in this paper.

Protein Name
Human UniProt Accession Human Site