Study on inhibitory effect of cinnamaldehyde on Salmonella based on iTRAQ technology

Salmonella is one of the most concerned pathogenic bacterium worldwide. In our previous experiments, cinnamaldehyde showed prominent antibacterial effects on Salmonella Typhimurium (S. Typhimurium), indicating that it could be developed as a novel therapeutic drugs for Salmonella. In this assay, the mechanism of cinnamaldehyde inhibiting S. Typhimurium was investigated. We studied the antibacterial mechanism of cinnamaldehyde using isobaric tags for relative and absolute quantification (iTRAQ) with two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS), combining with bioinformatics. There were 2,212 proteins detected by iTRAQ, of which 73 proteins were up-regulated, and 82 of proteins were down-regulated. The differently expressed proteins were connected with 10 cellular components, 9 molecular functions and 13 biological processes as well as 57 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. There were some differently expressed proteins involved into biosynthesis and metabolism of amino, redox reaction, energy metabolism, toxins and drug-resistance. Cinnamaldehyde may have the potential to become a novel drug for Salmonella infection. digs the action, lays theoretical for further exploration, and new development of may the potential to be used combined with antibiotics.


Abstract
Background Salmonella is one of the most concerned pathogenic bacterium worldwide. In our previous experiments, cinnamaldehyde showed prominent antibacterial effects on Salmonella Typhimurium (S. Typhimurium), indicating that it could be developed as a novel therapeutic drugs for Salmonella.

Results
In this assay, the mechanism of cinnamaldehyde inhibiting S. Typhimurium was investigated. We studied the antibacterial mechanism of cinnamaldehyde using isobaric tags for relative and absolute quantification (iTRAQ) with two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS), combining with bioinformatics. There were 2,212 proteins detected by iTRAQ, of which 73 proteins were up-regulated, and 82 of proteins were down-regulated. The differently expressed proteins were connected with 10 cellular components, 9 molecular functions and 13 biological processes as well as 57 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. There were some differently expressed proteins involved into biosynthesis and metabolism of amino, redox reaction, energy metabolism, toxins and drug-resistance. Cinnamaldehyde may have the potential to become a novel drug for Salmonella infection.

Conclusions
Based on the iTRAQ technology, this paper explores the mechanism of cinnamaldehyde on Salmonella typhimurium at the protein level, digs the target of action, lays a theoretical foundation for further exploration, and provides new ideas for the clinical use and development of cinnamaldehyde.Therefore, cinnamaldehyde may have the potential to be used combined with antibiotics.

Background
Salmonella is a significant pathogenic bacterium, which can be related with septicemia, gastroenteritis and local infection both in human and animals [1]. As the most prevalent source of food poisoning, Salmonella could contaminate multiple foods, such as raw meat, eggs, raw milk, water and vegetables. People mainly were infected by eating contaminated foods. In the United States, Salmonella infection is a leading cause of bacteria foodborne disease outbreaks, with an estimated 1 million infections occurring annually [2]. In France, Salmonella was also a major cause of foodborne pathogen-associated deaths, ranked first in 2008-2013 [3]. It was reported that there were 88715 bromatoxism cases in 28 members of the European Union during the past 15 years [4]. There were 1,300 million people suffering severe Salmonella poisoning and 3 million died worldwide [5].
Salmonella can induce abortion of pregnant female animals [6]. Besides, hens with bacteria in the body would lay contaminated eggs, which would hatch into infected chicks [7]. There are more than 2,500 serotypes of Salmonella in the world [8]. Salmonella infections have made great economic loss and treatment burden in the world. To treat Salmonella infections, antibiotics are generally used in modern clinical treatment. However, with the overuse of antibiotics, bacteria had developed resistance against almost all kind of drugs [9]. Therefore, new therapeutic drugs are urgently needed for clinical treatment.
Cinnamaldehyde, an aldehyde compound, exists in spice plant cinnamon. Cinnamon essential oil showed good antibacterial effect in food preservation [10]. It was reported that cinnamon essential oil extended the shelf-life of vacuum-packaged common carp fillets by about 2 days, and decreased the relative abundance of Macrococcus compared with control samples [11]. Solart. et al found cinnamon essential oil could efficiently decrease the MIC of enrofloxacin from 2 to 0.031 µg/mL [12]. Gadotti and colleagues found that cinnamaldehyde treatment can reduce Salmonella populations in queso fresco by inhibiting Salmonella growth [13]. Cinnamaldehyde is the major compound in cinnamon essential oil, approximately 60%-70% [14]. Cinnamaldehyde is extensively used in the fields of medicine, food and cosmetics, and has properties of anti-inflammation, anti-oxidant, anti-bacteria and lowering blood pressure [15,16]. Hancı et al. found that cinnamaldehyde alleviated vascular congestion, and reduced plasma cell, eosinophil, as well as inflammatory cell infiltration in rat allergic rhinitis model [17]. It was reported that cinnamaldehyde could exhibit antimicrobial properties in vitro against yeasts and bacteria, such as filamentous molds, Clostridium perfringens, Escherichia coli, Listeria monocytogenes, Salmonella enterica and Bacillus cereus [18,19]. After fumigation with transcinnamaldehyde, the Salmonella enteritidis on embryonated egg shells was detected negative, indicating the potential of becoming an effective fumigation treatment for eggs [20]. Cinnamaldehyde has a good antibacterial effect. However, the antibacterial mechanism and targets of cinnamaldehyde are still unclear.
Proteomic techniques, such as isobaric tags for relative and absolute quantification (iTRAQ), liquid chromatography tandem mass spectrometry (LC-MS/MS), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), have paved new pathways toward finding drug mechanism, exploring disease process, evaluating toxicological effect of drug, and discovering new action site. Currently, studies have been conducted on essential oils and their constituents on bacteria by proteomic methods. It was reported that 1, 8-cineole changed energy metabolism of Salmonella both in transcription level and translation level. This result may indicate the potential of 1,8-cineole of becoming a novel therapeutic drugs for Salmonella infections [21]. Xu and colleagues studied the potential antifungal mechanism of tea tree oil by means of proteomics [22].They demonstrated that tea tree oil inhibited glycolysis, disrupted the TCA cycle, and induced mitochondrial dysfunction that disrupted energy metabolism of Botrytis cinerea. Therefore, proteomics technology provides a new research direction for the antibacterial mechanism of essential oils.
In this assay, we explored the mechanism of cinnamaldehyde against Salmonella using iTRAQ technology. And differently expressed proteins between control group and drug-treated group were analyzed to find relative pathways and functions according to bioinformatics.

Results
The global proteomic profile of Salmonella The MIC of cinnamaldehyde against S. Typhimurium was 128 µg/mL, and the MBC was 512 µg/mL.
Cinnamaldehyde was used to observe the effect in iTRAQ analysis at a concentration of 64 µg/mL. GO function analysis of proteins All proteins detected by iTRAQ analysis were annotated to three function classifications (GO) as followed: biological process (BP), cellular component (CC) and molecular function (MF). The data was shown in Fig. 1. The proteins were annotated to 13 BP terms, 10 CC terms and 9 MF terms. Differently expressed proteins were also annotated to three function classifications (GO). GO enrichment bubble diagram was shown in Fig. 2. In these data, most of differently expressed proteins were classified to several terms, such as cellular process and metabolic process in BP, cell and cell part in CC, and binding and catalytic activity in MF.
KEGG pathway analysis of differently expressed proteins As shown in Fig. 3, there were 73 up-regulated and 68 down-regulated proteins, and all differently expressed proteins were divided into 57 KEGG pathways. In Fig. 4, proteins were enriched to 20 KEGG pathway terms. The up-or down-regulated proteins were mainly connected with two-component system, ABC transporters, amino acids, pantothenate and CoA biosynthesis. Other pathways were not significantly affected by cinnamaldehyde: cationic antimicrobial peptide (CAMP) resistance, 2oxocarboxylic, carbon, glycine, alanine, aspartate glutamate serine and threonine metabolism, valine, leucine and isoleucine biosynthesis, bacterial chemotaxis, for example.

Cinnamaldehyde effected toxins transcription
The transcription of toxins in Salmonella co-cultured with cinnamaldehyde was quantified using realtime PCR. The results of cinnamaldehyde against toxins were shown in Fig. 5. After co-cultured with cinnamaldehyde, transcription levels of sipA, prgI and fimW were up-regulated, while levels of invB, fimA and CR079_01855 were attenuated.

Discussion
Cinnamaldehyde has been proved that is an antimicrobial agent. Previous study in our laboratory indicated that cinnamaldehyde had inhibitory effect on proteins synthesis of Salmonella at a concentration of 64 µg/mL (data not shown) by SDS-PAGE. Therefore we applied a proteomic approach to detect worldwide proteins in S. Typhimurium. And results showed cinnamaldehyde undoubtedly affect multiple functions of S. Typhimurium.
Compared with control group, biosynthesis and metabolism process of amino were obviously influenced by cinnamaldehyde (Table 1, Fig. 6 and Fig. 7). It was reported that branched-chain amino acids (BCAAs) were considered as an indicator of general nutritional status of the bacteria, while Leucine, isoleucine and valine are essential elements of BCAAs [23,24]. Acetolactate synthase is necessary for biosynthesis of isoleucine and valine, and Salmonella strains lacking acetolactate synthase requires an additional supplement of isoleucine and valine to grow properly in culture [25]. CR079_08520 and ilvH, acetolactate synthase regulatory subunit I and III, were down-regulated cocultured with cinnamaldehyde. ilvD and ilvC mediates the synthesis of BCAAs in Salmonella, and the latter promotes the synthesis of coenzyme A [26]. rpiA plays a significant role in energy carbohydrate anabolism and catabolism [27]. RpiA interconverts ribose-5-phosphate and ribulose-5-phosohate. In this assay, repressed rpiA would affect the energy metabolism level of the bacteria. carB regulates the synthesis of large chains of aminoacylphosphatase, which catalyzes the synthesis of carbamyl phosphate, a necessary precursor for the synthesis of arginine and pyrimidine [28]. In arginine synthesis pathway, carbamyl phosphate and ornithine are synthesized to obtain citrulline, which is converted to arginine by enzymatic reaction [29]. DD95_13265 is an arginine decarboxylase, which mediates the synthesis of arginine. It is generally believed that Salmonella cannot tolerate an acid condition of pH 2.5, but the arginine-dependent acid-resistance mechanism activated under hypoxia conditions allows Salmonella to survive for a long time at pH2.5 [30]. Lacking BCAAs may repress biosynthesis and metabolism of Salmonella, therefore may reduce resistance against adverse environment, for example, antibiotics. In this study, it was found that cinnamaldehyde can affect the synthesis and metabolism of various amino acids, which may be one of the targets of cinnamaldehyde inhibiting bacteria.   Until now, the clinical treatment strategy for bacterial infections has been antibiotics, but the abuse of antibiotics has accelerated bacterial resistance. Drug-resistance has become a global problem in recent years. In our results, we found some drug-resistance proteins (Table 4) were down-regulated compared with control group. arnA mediated polymyxin resistance by catalyzing UDP-glucuronic acid to pentose sugar 4-amino-4-deoxy-L-arabinose (L-Ara4N), which can interact with lipid A to reduce the magnetism between lipid A and polymyxin. D-alanyl-D-alanine dipeptidase pcgL catalyzes the hydrolysis of D-Ala-D-Ala, essential for resistance to vancomycin [37]. In Mycobacterium abscessus, Dalanyl-D-alanine dipeptidase MAB1843 potentially stimulated the DC maturation via toll-like receptor 4 to regulate host immune response [38]. blaSHV-12 can be found in a wide variety of bacteria, such as Salmonella, escherichia coli and enterobacter cloacae [39]. Beta-lactamase blaSHV-12, mediating resistance to beta-lactam drug, was reduced to 0.771 (fold change) by cinnamaldehyde. Drug resistance has been a great problem worldwide, but the resistance may be reversed with the application of cinnamaldehyde.  [40]. It was found that type I fimbria of S. enterica could bind to host membrane plasminogen and activate plasminogen to plasmin [41]. Type I fimbria is a vital component in early infection stage for binding to epithelial cells and invading into host.  (Table 5) is another down-regulated system. Under the stress of cinnamaldehyde, needle structure protein and effector proteins were decreased in protein level.
Needle protein PrgI plays a significant role in delivery toxins into host cells. PrgI is not only the needle structure protein, also can induce host inflammation in gut. It was reported that YscF could activate NAIP inflammasome in human macrophages [42]. InvB is defined as chaperone protein of SipA, and mediate the translocation of SipA from bacteria to host cells. InvB-mutant strain can only secret less than 50% of SipA compared with wild type [43]. SipA is one of effectors of SPI-1, which could induce membrane ruffling of host cell and have pro-inflammatory activity [44]. After endocytosis, SipA can also activate caspase-3 to promote infection in early phase. As a conclusion, cinnamaldehyde may have the property of inhibiting SPI-1 of Salmonella [45]. From the above results, we may come to a conclusion that cinnamaldehyde could attenuate the pathogenicity of S. typhimurium. In RT-PCR assay, however, the transcription levels of toxic proteins were up-regulated. This may because translational level is influenced negatively, or protein degradation is accelerated.

Conclusion
In this study, we detected the effect of cinnamaldehyde against S. typhimurium at a concentration of 64 µg/mL using iTRAQ technology and proteomic analysis. After co-cultured with cinnamaldehyde, biosynthesis and metabolisvm of amino of Salmonella was seriously disrupted especially BCAAs, inducing BCAA auxotrophy, and then the resistance against active nitrogen was damaged. Redox reaction of S. typhimurium was also inhibited. Moreover, pentose phosphate pathway was affected via down-regulated rpe. And some drug-resistance proteins as well as toxins were suppressed by cinnamaldehyde. Therefore, cinnamaldehyde may have the potential to be used combined with antibiotics. However, further studies are needed to ascertain the mechanism of cinnamaldehyde at different targets.

Susceptibility testing
The minimum inhibitory concentration (MIC) of cinnamaldehyde was confirmed by microdilution method in 96-well polystyrene plates [46]. The MIC was determined as the lowest concentration of cinnamaldehyde with which bacterial growth was inhibited [47]. Protein Identification 2D-LC-MSMS Labeled samples were re-suspended with HPLC buffer A (98% H 2 O, pH10) and loaded. Then samples were washed by buffer B (98% acetonitrile, pH10) at a flow rate of 700 µL/min, and the elution gradient was shown in Table 6. The process was conducted with the monitoring of absorbance value (OD 214nm ). And the elution products were collected every 1.5 min starting with the fifth minute. After elution, products were vacuum freeze-dried, re-dissolved with 0.5% formic acid (FA), and combined into several products.     [48]. All experiments were repeated three times. The primer sequences of target genes and housekeeping gene 16 s RNA are shown in Table 8.

Statistics
The results of real-time PCR were analyzed using GraphPad Prism6.0 for significance. P < 0.05 was considered significant, and P < 0.01 was considered extremely significant.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.