miR-143 inhibits intracellular salmonella growth by targeting ATP6V1A in macrophage cells in pig

Accepted Manuscript miR-143 inhibits intracellular salmonella growth by targeting ATP6V1A in macrophage cells in pig

Tinghua Huang, Xiali Huang, Min Yao PII: DOI: Reference:

S0034-5288(17)30678-1 doi:10.1016/j.rvsc.2017.12.006 YRVSC 3478

To appear in:

Research in Veterinary Science

Received date: Revised date: Accepted date:

20 June 2017 16 October 2017 14 December 2017

Please cite this article as: Tinghua Huang, Xiali Huang, Min Yao , miR-143 inhibits intracellular salmonella growth by targeting ATP6V1A in macrophage cells in pig. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yrvsc(2017), doi:10.1016/j.rvsc.2017.12.006

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

miR-143 inhibits intracellular salmonella growth by targeting ATP6V1A in macrophage cells in pig Tinghua Huang*, Xiali Huang*, Min Yao* #

IP

T

*College of Animal Science, Yangtze University, Jingzhou, Hubei 434025, China.

CR

#Corresponding author, College of Animal Science, Yangtze University, Jingzhou, Hubei 434025,

AC

CE

PT

ED

M

AN

US

China. E-mail address: [email protected] Phone: +86-13117191157,

ACCEPTED MANUSCRIPT Abstract Salmonella infects many vertebrate species, and animals such as pigs can be colonized with Salmonella and become established carriers. Analyzing the roles of microRNA in intracellular proliferation is important for understanding the process of Salmonella infection. The objective of

IP

T

this study is to verify the regulation effect of miR-143 on ATP6V1A and its functions in the

CR

intracellular growth of Salmonella. A new miR-143 binding site was discovered in the 3’ UTR of ATP6V1A using a newly developed prediction tool. The binding site was confirmed by binding

US

site deletion assay. Real-time PCR results indicated that ATP6V1A was predominantly expressed in bone-marrow-derived macrophages, and the expression of miR-143 in different tissues was

AN

negatively correlated with ATP6V1A. The Salmonella proliferation assay showed that the

M

expression of miR-143 could inhibit intracellular Salmonella growth in macrophages by target ATP6V1A. The results strongly suggest that miR-143 plays important regulatory roles in the

ED

development of Salmonella infection in animals.

AC

CE

PT

Keywords: Salmonella, miR-143, intracellular growth

ACCEPTED MANUSCRIPT Introduction Salmonella colonization of pigs can lead to enterocolitis, with the bacteria often establishing a carrier status in the host (Huang et al., 2011). Salmonella-carrying status is subclinical but can decrease the quality of animal products, which has a negative economic impact on the swine

IP

T

industry (Akao et al., 2006; Funk et al., 2001). Most importantly, Salmonella-carrying animals

CR

such as pigs shed a large number of salmonella bacteria in feces, which can contaminate the pig carcass and pose a significant threat to human health (Hurd et al., 2001; Loynachan et al., 2004).

US

In our previous study, we identified miR-143 as a candidate gene that reduces the incidence

AN

and severity of salmonellosis, which is responsible for enhanced disease resistance and pathogen clearance (Yao et al., 2016). It has been reported that miR-143 is down-regulated in sporadic

M

colon cancer (Akao et al., 2006; Chen et al., 2009). The loss of miR-143 function induces

ED

pro-inflammatory signals in the innate immune system (Starczynowski et al., 2010). A recent study also showed that miR-143 is down-regulated in ulcerative colitis, putatively regulates

PT

inflammation genes, and contributes to malignant transformation of the colonic epithelium in

CE

longstanding ulcerative colitis (Pekow et al., 2012). In our previous study, miR-143 was quantified by real-time PCR, which indicated that the expression of miR-143 was also

AC

significantly down-regulated as in ulcerative colitis (Yao et al., 2016). MiR-143 appeared to have a role in the regulation of the development of the Salmonella infection process (Yao et al., 2016). Previous reports have suggested or confirmed several important targets of miR-143, including PAK4, API5, CDK6, ERK5, IRS-1, K-RAS, and MEK-2 (Akao et al., 2006; Wang et al., 2009; Wang et al., 2012; Yang et al., 2010; Zhu et al., 2010). Those targets are involved in several important signaling pathways, such as MAPK pathway, KRAS signaling, and TGFβ

ACCEPTED MANUSCRIPT Signaling (Kent et al., 2013; Lee et al., 2005; Wang et al., 2012). In our previous analysis, we discovered that ATP6V1A is a target of miR-143. ATP6V1A is involved in cellular alterations induced by VacA cytotoxin during H. pylori infection (Montecucco and Rappuoli, 2001; Rieder et al., 2005). ATP6V1A plays a major role in the electronic vacuolar ATPase (ATP6V1A and

T

other v-ATPase) proton pump, where it increases the permeability of the endosome and leads to

IP

water influx, vesicle swelling (an essential step in vacuole formation), and intracellular bacterial

CR

growth. During Salmonella inoculation, the expression of ATP6V1A increased threefold (on day 2 versus day 0 post inoculation). Compared to the RNA samples before Salmonella inoculation,

US

the expression levels of miR-143 varied inversely with the levels of ATP6V1A. These findings

AN

are in agreement with miRNA-target regulation models (Bartel, 2009). ATP6V1A is believed to be involved in infectious disease and in factors related to the immune response (Montecucco and

M

Rappuoli, 2001; Rieder et al., 2005). We therefore speculated that the down-regulation of

ED

miR-143 might increase the risk of Salmonella infection and the establishment of carrier status.

PT

In this study, a new miR-143 biding site for ATP6V1A was discovered and confirmed using a binding-site deletion assay. The functions of miR-143 and ATP6V1A were investigated using a

CE

Salmonella invasion assay. The results show that the expression of miR-143 could inhibit

AC

intracellular Salmonella growth in macrophages by targeting ATP6V1A. Materials and methods

Animal ， bacterial strain, cells, and culture media Six Salmonella fecal- negative crossbreed pigs (Duroc × Landrace × Yorkshire, two weeks of age) were housed in an animal room that was controlled under standardized conditions, allow free access to standard food and water, at least 1 week before the experiment. Pigs were

ACCEPTED MANUSCRIPT euthanized by intravenous injection of pentobarbital sodium (150 mg/kg body weight). Within 5 min after death tissue samples were excised and washed with ice-cold PBS, then cut into small pieces and snap- frozen in liquid nitrogen, stored at -80 ℃ until use. The animal use protocol for this study was approved by the Animal Care and Use Committee of Hubei Province (China,

T

YZU-2016-0026). The total RNA were extracted using RNAprep pure Tissue Kit according to

IP

the manufacturer’s instructions (TIANGEN, DP431). Real-time PCR primers were designed for

CR

MS4A8B and the reference gene GAPDH using the PrimerSelect 7.1 software (MS4A8: ACGTAAGCATTTCCCCTCTGTCAA,

GGTCAGATCCACAACCGACA).

US

TCCATGACCACTTCGGCATC,

AGCTCTGCGGGCCAAGTCATAAAA;

GAPDH:

Amplification

and

AN

detection were carried out using 100 ng of cDNA and the SYBR Green Real- time PCR master mix (TAKARA) according to the supplied protocol. The obtained values were normalized based

M

on the expression of the endogenous control GAPDH. Relative expression levels were calculated

ED

according to the 2(-ΔΔCT) method (Yao et al., 2016). Each Real-time PCR assay was repeated three

PT

times.

Salmonella typhimurium strain LT2 (ATCC700720) is virulent to humans and animals.

CE

Luria broth and M9 minimal medium were used to culture Salmonella bacteria. Carbon sources

AC

such as glucose, glycerol, citrate, or acetate were added to the M9 minimal medium to a final concentration of 0.2%. To establish calcium- or magnesium- limited conditions, CaCl2 or MgSO 4 was omitted from the M9 minimal medium. Mouse macrophages (RAW 264.7) were cultured in Dulbecco’s minimal essential medium containing 10% fetal bovine serum (FBS, DMEM). Proteose-peptone-elicited macrophages were harvested 3 days after elicitation with 1.5 ml of 10% proteose peptone. HeLa cells (ATCC-CCL2) were grown in Eagle's minimal essential medium (MEM) with

ACCEPTED MANUSCRIPT Earle's salts and 10% FBS at 37°C in an atmosphere containing 5% CO 2 . The bone marrow-derived macrophages were isolated using Ronan’s method (Kapetanovic et al., 2012). The frozen cells were thawed rapidly in a water bath at 37°C and then slowly diluted by dropwise addition of complete medium over 2-3 min to avoid shock from the sudden dilution of

10%

heat-inactivated

FBS

(Gibco),

penicillin-streptomycin

IP

medium (Sigma-Aldrich),

T

DMSO. After washing to remove the DMSO, the cells were cultured in RPMI 1640 complete

CR

(Invitrogen), and GlutaMAX-I supplement (Invitrogen). Bone- marrow-derived macrophages were obtained by culturing bone marrow cells for 5-7 d in the presence of CSF-1 (0.5 ng/ml),

US

essentially as described previously for mouse macrophages (Sester et al., 1999). Invasion and

AN

intracellular replication assays were set up by seeding 1×105 cells into each well of a 24-well

M

microtiter dish and incubating overnight.

ED

Invasion and intracellular replication assays

A 1- ml standing-overnight culture was prepared by inoculating L-broth with Salmonella

PT

bacteria from frozen glycerol stocks and incubating at 37°C. Two hours prior to infection,

CE

Salmonella was diluted to 1:20 in fresh L-broth and incubated until reaching the mid- logarithmic growth phase. Bacterial cells were pelleted and washed in PBS before adding them to each

AC

microtiter well. When the microRNA and target gene were tested, they were transfected to the cells 24 h prior to bacterial addition. To begin the transfection assay, the cells were treated with a transfection mixture consisting of 250 μL of serum- free medium, 2.0 μL of TransFastT M Transfection Reagent (Promega), and 0.15 μg of plasmid or synthetic microRNA per well. After one hour of incubation, 600 μL of serum-containing medium was added to the wells, followed by overnight culture.

ACCEPTED MANUSCRIPT The bacteria inoculation was carried out using 5 μl of Salmonella culture, and the microtiter plates were centrifuged (162×g, 10 min, 23°C), followed by 2 h of incubation at 37°C in a 5% CO2 atmosphere. Monolayers were washed 3 times with PBS and then incubated for another 2 h in fresh media containing 100 μg/ml of gentamicin. This treatment kills extracellular bacteria but

T

does not affect the viability of intracellular organisms (Finlay and Falkow, 1988). Monolayers

IP

were washed three times with PBS, and 0.2 ml of a 1% Triton X-100 solution was added. This

CR

was followed by a 5- min incubation period to release intracellular bacteria. L-broth (0.8 ml) was

counted. The experiment was repeated three times.

AN

Statistical analysis

US

added, appropriate dilutions were spread onto L-agar plates, and colony- forming units (cfu) were

M

Results are shown as means ± standard deviation (S.D.) of at independently duplicated

ED

experiments. Data were analyzed using one-way ANOVA followed by a multiple comparison test (Tukey honest significant differences) to determine which conditions were significantly different

PT

from each other. All statistical analyses were performed using R 3.02 software. Probability

Results

AC

CE

values ≤ 0.05 and ≤ 0.01 were taken to indicate statistical significance.

Identification of a new miR-143 binding site in 3’ UTR of ATP6V1A The multiple sequence alignment of the 3’ UTR sequence of ATP6V1A from 17 species was constructed. Prediction using a newly developed microRNA target prediction tool (https://sourceforge.net/projects/mirt3) indicated a new binding site located between 3006 and 3014 bp in human ATP6V1A, which has not been reported previously (the new binding site is located between 2612 and 2621 bp in mouse ATP6V1A and between 2732 and 2740 bp in pig).

ACCEPTED MANUSCRIPT The sequences of the binding site along with 50-bp flanking were sliced out and used for motif discovery with the MEME suite (Bailey et al., 2009). The top-ranked motifs within the clusters of the 3’ UTR sequence of ATP6V1A confirmed that the b inding site was enriched for miR-143 pairing. The binding site had striking complementarity in 10 nucleotides within the seed region,

IP

T

and tolerance to G::U wobbles was observed at various positions.

CR

The ATP6V1A plasmid containing the 3’ UTR sequence was used for further in vivo studies (wild type). To show a direct effect of the microRNA, the binding site of the miR-143 in

US

ATP6V1A was deleted (mutated type; sequence information is available in a supplemental document). Reporter vectors containing the wild type and mutated 3’ UTR and expressing

AN

Renilla luciferase were transfected into HeLa cells. The results showed that binding site deletion

M

significantly enhanced the expression of the upstream reporter luciferase (p<0.05), indicating that the microRNAs could repress the expression of the reporter gene by pairing with the

ED

predicted target sites (Figure 1).

PT

The mutated vectors showed significant induction of the expression level of upstream

CE

Renilla luciferase at 40, 44, and 48 h post transfection (p<0.05). This indicates there is a regulation effect between the microRNA and the target. The effects of endogenic miR-143 on the

AC

expression of ATP6V1A are unknown and may introduce bias to the experiment. The synthetic miR-143 precursor was thus co-transfected with the wild-type vector to show the effects changes to miR-143 on the expression of ATP6V1A. The results showed that the transfection of miR-143 significantly decreased the expression level of upstream Renilla luciferase at 40, 44, and 48 h post transfection (p<0.05), again indicating that miR-143 has a regulating effect on ATP6V1A. However, it is problematic to use one human HeLa cell line to validate the potential target

ACCEPTED MANUSCRIPT of miR-143 identified from a multiple sequence alignment from different species. Thus, another experiment was performed using mouse macrophage cells (RAW 264.7) to further validate the hypothesis. Again, mutations to the miR-143 binding sites in ATP6V1A in the 3’ UTR of mRNAs significantly induced the expression of upstream Renilla luciferase at 40, 44, and 48 h

T

post transfection (Figure 2). Compared with the HeLa assay, the luciferase reporter activity after

IP

transfection with the wild-type vector was significantly lower than that in macrophage cells.

CR

Most importantly, there was no significant difference between the luciferase reporter activity in cells transfected with the wild-type vector and the cells co-transfected with the wild-type plasmid

US

and synthetic miR-143. This suggests that the expression level of endogenous miR-143 in mouse

AN

microphage cells was high and that the synthetic miR-143 co-transfected with the wild-type

M

vector thus does not enhance the effect of endogenous microRNAs.

ED

Expression correlation of miR-143 to ATP6V1A As shown in Figure 3, the porcine ATP6V1A was highly expressed in bone- marrow-derived

PT

macrophages. The highest expression levels occurred in the brain, conceptus, placenta, and

CE

bone- marrow-derived macrophages (in decreasing order). The expression levels in the spleen, alveolar macrophages, and mesenteric lymph nodes were at least two times lower than those

AC

observed in tissues with high expression. The expression was very low in the monocyte-derived macrophages, kidney, ileum, jejunum, and peripheral blood mononuclear cells. In other tissues, the expression of ATP6V1A was almost undetectable. The expression levels of miR-143 in each sample were quantified along with ATP6V1A using a pincer probe microRNA assay that was established in previous research (Yao et al., 2016). A total of 120 samples have been tested (six animals and 20 tissues for each). A correlation

ACCEPTED MANUSCRIPT analysis was performed to determine whether there is a relationship between miR-143 levels and ATP6V1A expression in tissues collected from pigs (Pearson correlation use “cor” and “cor.test” functions in R). A significant positive correlation was found between the expression levels of miR-143 and ATP6V1A (p < 0.05). In other words, higher miR-143 levels corresponded to lower

IP

T

ATP6V1A expression in pigs (Figure 4).

CR

mir-143 inhibits intracellular Salmonella growth

A pTARGETT M mammalian expression vector was constructed using the full- length cDNA

US

sequence of ATP6V1A (wild type). A mutated ATP6V1A vector was also created by deleting

AN

the miR-143 binding site in the 3’ UTR region. The wild-type vector, and mutated vector, and synthetic microRNA were first transfected into bone- marrow-derived macrophage cells and then

M

treated with Salmonella bacteria to show how miR-143 would affect intracellular salmonella

ED

growth (Supplemental document 4AB). The results showed that after transfection with the mutated ATP6V1A vector, the intracellular bacteria growth continually increased over time and

PT

was significantly higher at 4, 12, and 24 h post transfection compared with the wild-type vector

CE

(p<0.05, Figure 5). Interestingly, co-transfection with synthetic miR-143 does not show any effect on the mutated vector (there was no difference in the intracellular bacterial growth), but it

AC

significantly attenuated the effects of the wild-type vector at 12 and 24 h post transfection (p<0.05, Figure 5). This indicates that miR-143 can inhibit intracellular Salmonella growth by targeting ATP6V1A (by pairing with the predicted target sites). The intracellular bacteria growth of cells transfected with the wild-type vector alone was significantly lower than that with the mutated vector, indicating that the endogenic miR-143 attenuated the effects of ATP6V1A. Again, another experiment was performed using mouse macrophage cells (RAW 264.7) to

ACCEPTED MANUSCRIPT further validate the hypothesis (Supplemental document 4CD). The results showed that after transfection with the mutated ATP6V1A vector, the intracellular bacteria growth continually increased over time and was significantly higher at 4, 12, and 24 h post transfection compared to the results obtained with the wild-type vector, which is the same as in the bone- marrow-derived

T

macrophage cells (p<0.05, Figure 6). The difference is that co-transfection with synthetic

IP

miR-143 showed an attenuation effect on not only the wild-type vector (decreasing intracellular

CR

bacterial growth) but also on the mutated vector at 12 and 24 h post transfection (p<0.05, Figure

US

6).

AN

Discussion

Salmonella infections result in two major clinical outcomes: gastroenteritis and typhoid

M

disease (Coburn et al., 2007; Ibarra and Steele-Mortimer, 2009). In both types of infections, the

ED

recruitment of polymorph nuclear phagocytes and macrophage apoptosis has been reported to occur in Peyer ’s patches (Mastroeni et al., 2009). From these patches, the bacteria disseminate

PT

through the blood stream by either entering the blood directly or passing through lymphatic

CE

vessels and the mesenteric lymph nodes (Mastroeni et al., 2009). The bacteria then travel to the reticuloendothelial system and colonize the liver and spleen (Mastroeni et al., 2009;

AC

Vazquez-Torres et al., 1999). These organs represent permissive sites for Salmonella intracellular proliferation in cases of systemic disease. At 24 h after ingestion of Salmonella by susceptible mice, approximately 10–102 organisms are observed in both the liver and spleen (Bowe et al., 1998). Five to seven days later, 107 –108 bacteria are registered, and the infected animals become moribund and die (Garcia-del Portillo, 2001). The exact type of cells where Salmonella are located in the liver and spleen are

ACCEPTED MANUSCRIPT CD18-containing phagocytes (polymorphonuclear cells and macrophages) that infiltrate the infection loci (Matsui et al., 2000). Other studies have shown that macrophages are permissive cells for Salmonella proliferation in vivo (Achouri et al., 2015; Richardson, 2015). Thus, if macrophages are immunodepleted, the morbidity and mortality of the infection are severely

T

diminished (Wijburg et al., 2000). Infection of cultured phagocytic or epithelial cells is assumed

IP

to mimic relevant bacteria–host interactions occurring in processes in vivo, the bacterial invasion,

CR

and intracellular survival and proliferation. Salmonella invasion in cultured epithelial cells is followed by an explosive growth phase after a few hours (Leung and Finlay, 1991). This

US

characteristic growth phase is also observed in macrophage cell lines. Under these permissive

AN

conditions, a 10- to 500-fold increase in intracellular bacteria is observed after 24 h.

M

Since Salmonella multiplies inside the vacuoles of host cells, it is conceivable that they could modify the vacuole membrane in some manner that allows them to extract their growth

ED

requirements from the host cell’s cytoplasm (Garcia-del Portillo, 2001). There is precedence for

PT

this in reports on several parasites such as Plasmodium lophurae and Chlamydia (Leung and Finlay, 1991). The vacuole membrane surrounding these parasites grows proportionally as the

CE

parasites multiply (similar to Salmonella) and actively participates in the uptake of nutrients

AC

(Leung and Finlay, 1991). Alternatively, Salmonella may produce special factors that scavenge nutrients from the host. We have demonstrated that ATP6V1A is required for Salmonella growth inside microphage cells and is controlled by miR-143. It is thus associated with survivability within phagocytic cells. The results suggest that intracellular Salmonella growth is an indispensable adaptation that requires special host gene ATP6V1A in addition to those needed for extracellular growth, and that this gene is controlled by microRNAs. The increased intracellular growth of ATP6V1A

ACCEPTED MANUSCRIPT mutants was due to attenuated inhibition of miR-143, which was indicated by the identical 3’ UTR sequence for the APT6V1A mutants and wild type except for the miR-143 binding site being deleted. These findings will lead to a better understanding of how Salmonella bacteria function inside host cells, and they may lead to new ways to treat Salmonella infections.

IP

T

Acknowledgments

CR

This project was funded by the National Natural Science Foundation of China (NSFC Grant No. 31402055), the Yangtze Youth Talents Fund (Grant No. 2015cqr12), the Yangtze Youth

US

Fund (Grant No. 2015cqn39), and the Scientific Research Starting Foundation for the Returned

AN

Overseas Chinese Scholars of the Ministry of Education of China.

M

References

Achouri, S., Wright, J.A., Evans, L., Macleod, C., Fraser, G., Cicuta, P., Bryant, C.E., 2015. The

ED

frequency and duration of Salmonella-macrophage adhesion events determines infection efficiency. Philosophical transactions of the Royal Society of London. Series B, Biological

PT

sciences 370, 20140033.

CE

Akao, Y., Nakagawa, Y., Naoe, T., 2006. MicroRNAs 143 and 145 are possible common onco-microRNAs in human cancers. Oncology reports 16, 845-850.

AC

Bailey, T.L., Boden, M., Buske, F.A., Frith, M., Grant, C.E., Clementi, L., Ren, J., Li, W.W., Noble, W.S., 2009. MEME SUITE: tools for motif discovery and searching. Nucleic acids research 37, W202-208.

Bartel, D.P., 2009. MicroRNAs: target recognition and regulatory functions. Cell 136, 215-233. Bowe, F., Lipps, C.J., Tsolis, R.M., Groisman, E., Heffron, F., Kusters, J.G., 1998. At least four percent of the Salmonella typhimurium genome is required for fatal infection of mice.

ACCEPTED MANUSCRIPT Infection and immunity 66, 3372-3377. Chen, X., Guo, X., Zhang, H., Xiang, Y., Chen, J., Yin, Y., Cai, X., Wang, K., Wang, G., Ba, Y., Zhu, L., Wang, J., Yang, R., Zhang, Y., Ren, Z., Zen, K., Zhang, J., Zhang, C.Y., 2009. Role of miR-143 targeting KRAS in colorectal tumorigenesis. Oncogene 28, 1385-1392.

T

Coburn, B., Grassl, G.A., Finlay, B.B., 2007. Salmonella, the host and disease: a brief review.

IP

Immunology and cell biology 85, 112-118.

CR

Finlay, B.B., Falkow, S., 1988. Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells:

US

endosome acidification is not required for bacterial invasion or intracellular replication. Biochimie 70, 1089-1099.

AN

Funk, J.A., Davies, P.R., Nichols, M.A., 2001. Longitudinal study of Salmonella enterica in growing pigs reared in multiple-site swine production systems. Veterinary microbiology 83,

M

45-60.

ED

Garcia-del Portillo, F., 2001. Salmonella intracellular proliferation: where, when and how? Microbes and infection 3, 1305-1311.

PT

Huang, T.H., Uthe, J.J., Bearson, S.M., Demirkale, C.Y., Nettleton, D., Knetter, S., Christian, C., Ramer-Tait, A.E., Wannemuehler, M.J., Tuggle, C.K., 2011. Distinct peripheral blood RNA

CE

responses to Salmonella in pigs differing in Salmonella shedding levels: intersection of

AC

IFNG, TLR and miRNA pathways. PloS one 6, e28768. Hurd, H.S., McKean, J.D., Wesley, I.V., Karriker, L.A., 2001. The effect of lairage on Salmonella isolation from market swine. Journal of food protection 64, 939-944. Ibarra, J.A., Steele-Mortimer, O., 2009. Salmonella--the ultimate insider. Salmonella virulence factors that modulate intracellular survival. Cellular microbiology 11, 1579 -1586. Kapetanovic, R., Fairbairn, L., Beraldi, D., Sester, D.P., Archibald, A.L., Tuggle, C.K., Hume, D.A., 2012. Pig bone marrow-derived macrophages resemble human macrophages in their response to bacterial lipopolysaccharide. Journal of immunology 188, 3382-3394.

ACCEPTED MANUSCRIPT Kent, O.A., Fox-Talbot, K., Halushka, M.K., 2013. RREB1 repressed miR-143/145 modulates KRAS signaling through downregulation of multiple targets. Oncogene 32, 2576 -2585. Lee, F.T., Mountain, A.J., Kelly, M.P., Hall, C., Rigopoulos, A., Johns, T.G., Smyth, F.E., Brechbiel, M.W., Nice, E.C., Burgess, A.W., Scott, A.M., 2005. Enhanced efficacy of radioimmunotherapy with 90Y-CHX-A''-DTPA-hu3S193 by inhibition of epidermal growth factor receptor (EGFR)

T

signaling with EGFR tyrosine kinase inhibitor AG1478. Clinical cancer research : an official

IP

journal of the American Association for Cancer Research 11, 7080s-7086s.

CR

Leung, K.Y., Finlay, B.B., 1991. Intracellular replication is essential for the virulence of Salmonella typhimurium. Proceedings of the National Academy of Sciences of the United

US

States of America 88, 11470-11474.

AN

Loynachan, A.T., Nugent, J.M., Erdman, M.M., Harris, D.L., 2004. Acute infection of swine by various Salmonella serovars. Journal of food protection 67, 1484-1488.

M

Mastroeni, P., Grant, A., Restif, O., Maskell, D., 2009. A dynamic view of the spread and

ED

intracellular distribution of Salmonella enterica. Nature reviews. Microbiology 7, 73 -80. Matsui, H., Eguchi, M., Kikuchi, Y., 2000. Use of confocal microscopy to detect Salmonella

PT

typhimurium within host cells associated with Spv-mediated intracellular proliferation. Microbial pathogenesis 29, 53-59.

CE

Montecucco, C., Rappuoli, R., 2001. Living dangerously: how Helicobacter pylori survives in

AC

the human stomach. Nature reviews. Molecular cell biology 2, 457-466. Pekow, J.R., Dougherty, U., Mustafi, R., Zhu, H., Kocherginsky, M., Rubin, D.T., Hanauer, S.B., Hart, J., Chang, E.B., Fichera, A., Joseph, L.J., Bissonnette, M., 2012. miR-143 and miR-145 are downregulated

in

ulcerative

colitis:

putative

regulators

of

inflammation

and

protooncogenes. Inflammatory bowel diseases 18, 94-100. Richardson, L.A., 2015. How Salmonella survives the macrophage's acid attack. PLoS biology 13, e1002117. Rieder, G., Fischer, W., Haas, R., 2005. Interaction of Helicobacter pylori with host cells:

ACCEPTED MANUSCRIPT function of secreted and translocated molecules. Current opinion in microbiology 8, 67-73. Sester, D.P., Beasley, S.J., Sweet, M.J., Fowles, L.F., Cronau, S.L., Stacey, K.J., Hume, D.A., 1999. Bacterial/CpG DNA down-modulates colony stimulating factor-1 receptor surface expression on murine bone marrow-derived macrophages with concomitant growth arrest

T

and factor-independent survival. Journal of immunology 163, 6541-6550.

IP

Starczynowski, D.T., Kuchenbauer, F., Argiropoulos, B., Sung, S., Morin, R., Muranyi, A., Hirst, M., Hogge, D., Marra, M., Wells, R.A., Buckstein, R., Lam, W., Humphries, R.K., Karsan, A., 2010.

CR

Identification of miR-145 and miR-146a as mediators of the 5q- syndrome phenotype.

US

Nature medicine 16, 49-58.

Vazquez-Torres, A., Jones-Carson, J., Baumler, A.J., Falkow, S., Valdivia, R., Brown, W., Le, M.,

AN

Berggren, R., Parks, W.T., Fang, F.C., 1999. Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes. Nature 401, 804-808.

M

Wang, C.J., Zhou, Z.G., Wang, L., Yang, L., Zhou, B., Gu, J., Chen, H.Y., Sun, X.F., 2009. Clinicopathological significance of microRNA-31, -143 and -145 expression in colorectal

ED

cancer. Disease markers 26, 27-34.

PT

Wang, Z., Zhang, X., Yang, Z., Du, H., Wu, Z., Gong, J., Yan, J., Zheng, Q., 2012. MiR-145 regulates PAK4 via the MAPK pathway and exhibits an antitumor effect in human colon

CE

cells. Biochemical and biophysical research communications 427, 444-449. Wijburg, O.L., Simmons, C.P., van Rooijen, N., Strugnell, R.A., 2000. Dual role for

AC

macrophages in vivo in pathogenesis and control of murine Salmonella enterica var. Typhimurium infections. European journal of immunology 30, 944-953. Yang, Y., Chaerkady, R., Kandasamy, K., Huang, T.C., Selvan, L.D., Dwivedi, S.B., Kent, O.A., Mendell, J.T., Pandey, A., 2010. Identifying targets of miR-143 using a SILAC-based proteomic approach. Molecular bioSystems 6, 1873-1882. Yao, M., Gao, W., Tao, H., Yang, J., Liu, G., Huang, T., 2016. Regulation signature of miR-143 and miR-26 in porcine Salmonella infection identified by binding site enrichment analysis.

ACCEPTED MANUSCRIPT Molecular genetics and genomics : MGG 291, 789-799. Zhu, H., Dougherty, U., Mustafi, R., Robinson, V.L., Pekow, J., Fichera, A., Joseph, L.J., Bissonnette, M., 2010. 679 Putative Tumor Suppressors miR-143 and miR-145 Inhibit HCT116 Colon Cancer Cell Growth in Tumor Xenografts: Roles of K-RAS, MYC, Ccnd2 and

T

Cdk6. Gastroenterology 138, S-93.

CE

PT

ED

M

AN

US

CR

IP

Tables and Figures

AC

Figure 1. Experimental validation of the predicted targets in HeLa cells. One wild-type construct (containing the predicted miRNA binding site in the 3’ UTR fragment of the target gene), one mutant construct (mutated and not containing the binding site), and antisense microRNA were investigated. The line graphs show the luciferase activity after the reporter plasmids and antisense microRNA were transfected into HeLa cells. The error bars represent the mean ± standard deviation of three duplicate samples.

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

M

Figure 2. Experimental validation of the predicted targets in macrophage cells (Raw 264.7). In

ED

the wild-type construct (red), the 3’ UTR fragment of the target gene containing the predicted miRNA binding sites was inserted within the 3’ UTR of Renilla luciferase. The mutated

PT

construct (black) was identical to the wild-type construct except that the microRNA binding site

CE

was deleted. The line graphs show the luciferase activity from after the reporter plasmids and antisense microRNA were transfected into macrophages. The error bars represent the mean ±

AC

standard deviation of three duplicate samples.

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

Figure 3. Expression profile of porcine ATP6V1A mRNA in different tissues quantified by

PT

real-time PCR. A total of six animals were tested by Real-time PCR, and each Real-time PCR

CE

assay was repeated three times. The obtained values were normalized based on the expression of the endogenous control GAPDH. Relative expression levels were calculated according to the

AC

2(-ΔΔCT ) method (Yao et al., 2016).

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

ED

Figure 4. Relationship between expression levels of ATP6V1A and miR-143 in different tissues. The expression levels of miR-143 in each sample were quantified along with ATP6V1A using

PT

Real-time PCR assay. A total of 120 samples have been tested (six animals and 20 tissues for

CE

each). Correlation analysis was performed to determine whether there is a relationship between

AC

miR-143 levels and ATP6V1A expression in tissues using R 3.02 software.

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

ED

Figure 5. Intracellular growth of Salmonella typhimurium strain LT2 in bone- marrow-derived macrophages. Macrophage cells were first transfected with miR-143, wild-type, or mutated

PT

ATP6V1A vector, followed by infection with Salmonella bacteria, incubation for 2 h, treatment

CE

with gentamicin (100 ug/ml) for 2 h to kill extracellular bacteria, and lysing immediately or incubation for an additional 8, 12, or 24 h. The line graphs show the intracellular Salmonella

AC

counts from after the macrophage cells were treated with Salmonella bacteria. The error bars represent the mean ± standard deviation of three duplicate samples.

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

ED

Figure 6. Intracellular growth of Salmonella typhimurium strain LT2 in mouse macrophages (RAW 264.7). Three treatments were tested: synthetic miR-143, wild-type ATP6V1A vector, and

PT

mutated ATP6V1A vector. The treated cells were infected with Salmonella bacteria, incubated

CE

for 2 h, treated with gentamicin (100 ug/ml) for 2 h to kill extracellular bacteria, and lysed immediately or incubated for an additional 8, 12, or 24 h. The line graphs show the intracellular

AC

Salmonella counts from after the macrophages were treated with Salmonella bacteria. The error bars represent the mean ± standard deviation of three duplicate samples.

ACCEPTED MANUSCRIPT Highlights A new miR-143 binding site was discovered in the 3’ UTR of ATP6V1A.



Expression of miR-143 in different tissues was negatively correlated with ATP6V1A.



miR-143 could inhibit intracellular Salmonella growth in macrophages by target ATP6V1A.

AC

CE

PT

ED

M

AN

US

CR

IP

T

