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 co-cultured 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.
Table 1
differently expressed proteins related with metabolism of amino acids
Accession name | Gene name | Function | Fold change | P value |
A0A0M2J4K4 | asnB | L-asparaginase 2 | 1.383 | 0.002 |
A0A0J6GMN8 | aspA | Aspartate ammonia-lyase | 1.211 | 0.039 |
A0A0D6HJJ8 | carB | Carbamoyl-phosphate synthase large chain | 0.764 | 0.005 |
A0A0D6I697 | CR079_19530 | Dihydroxy-acid dehydratase | 1.263 | 0.004 |
A0A0D6GT32 | serC | Phosphoserine aminotransferase | 1.241 | 0.009 |
A0A0D6FTK7 | CR079_24500 | L-serine ammonia-lyase | 1.204 | 0.011 |
A0A0D6HM19 | thrA | Bifunctional aspartokinase/homoserine dehydrogenase | 0.695 | 0.013 |
A0A0M2J5W4 | dsdA | D-serine dehydratase | 0.540 | 0.0000227 |
A0A0J6DT19 | ilvH | Acetolactate synthase 3 regulatory subunit | 0.719 | 0.004 |
A0A0J6DTT7 | CR079_08520 | Acetolactate synthase 1 regulatory subunit | 0.718 | 0.029 |
A0A0M2IUW2 | DD95_13265 | Arginine decarboxylase | 0.522 | 0.006 |
A0A0D6IQZ8 | katG | Catalase-peroxidase | 1.400 | 0.009 |
Differently expressed proteins related with redox reaction were shown in Table 2. Iron-containing sulfur (Fe-S) proteins contribute to a variety of biological processes, including redox reactions or regulation of gene expression, and are key metal cofactors for cellular function. Besides, Fe-S protein IlvD is confirmed as active nitrogen (NO) target. In E. coli, IlvD interacts with NO, forming the dinitrosyl iron complex (DNIC), and inducing BCAA auxotrophy[31]. After cinnamaldehyde treatment, Fe-S protein assemble systems obviously declined including CR079_14145、IscU and sufE. IscU is the core component of iron sulfur cluster in the Fe-S proteins assembling process. During invasion into epithelial cells, wild-type Salmonella is much more efficiently compared with iscU mutant[32]. In E. coli, SufE binds with SurS to stimulate cysteine desulfurase, and accepts sulfane transferred from SufS[33, 34]. Therefore, cinnamaldehyde may have the property of inducing BCAA auxotrophy and NO damage against Salmonella.
Table 2
Information of differently expressed proteins relative to redox reaction
Accession name | Gene name | Function | Fold change | P value |
A0A0W5XM88 | DD95_14245 | Dimethyl sulfoxide reductase | 0.723 | 0.029 |
A0A0D6HUU0 | DD95_13310 | Dimethyl sulfoxide reductase | 0.713 | 0.047 |
A0A0D6GTP6 | CR079_22995 | Dimethyl sulfoxide reductase | 0.688 | 0.048 |
A0A0D6GC40 | sufE | Cysteine desulfuration protein | 0.811 | 0.030 |
A0A0L9JQ40 | IscU | Iron-sulfur cluster assembly scaffold protein | 0.787 | 0.020 |
A0A0D6GAI2 | CR079_14145 | Cysteine desulfurase | 0.780 | 0.047 |
A0A0M2IZP2 | grxB | Glutaredoxin | 0.704 | 0.0001 |
A0A0D6IN33 | wecF | TDP-N-acetylfucosamine:lipid II N-acetyl fucosaminyltransferase | 0.697 | 0.010 |
A0A0D6I4S1 | CR079_08080 | LPS 1,2-N-acetylglucosaminetransferase | 0.713 | 0.042 |
A0A0K6RDY0 | CR079_21580 | Trehalose-6-phosphate synthase | 0.816 | 0.030 |
A0A0D6FU80 | CR079_24700 | Hydrogenase | 0.600 | 0.007 |
A0A0D6FUX2 | CR079_24705 | Hydrogenase | 0.535 | 0.009 |
A0A0D6IM00 | CR079_24200 | Trimethylamine N-oxide reductase I catalytic subunit | 2.228 | 0.0001 |
A0A0J5H1R6 | CR079_10450 | Cytochrome b562 | 1.331 | 0.012 |
A0A0D6IKN9 | CR079_24195 | Cytochrome c-type protein | 2.499 | 0.010 |
There are several differently expressed proteins related with energy metabolism (Table 3). In Escherichia coli rpiA is an essential enzyme in the first step of pentose phosphate pathway (PPP), catalyzing the reversible conversion of D-ribose-5-phosphate to D-ribulose-5-phosphate[35]. Meanwhile rpe was reduced to 0.409 (fold change), which catalyzed the reversible conversion of D-ribulose-5-phosphate to D-xylulose-5-phosphate. Rpe-mutunt E. coli lost the capacity of utilizing single pentose sugars, and showed limited growth in complex LB medium, suggesting the importance of rpe in PPP[36]. These results suggested that cinnamaldehyde affected the energy metabolism of Salmonella.
Table 3
Information of differently expressed proteins relative to energy metabolism
Accession name | Gene name | Function | Fold change | P value |
A0A0J0XAS3 | rpiA | Ribose-5-phosphate isomerase A | 1.242 | 0.017 |
A0A0D6I769 | rpe | Ribulose-phosphate 3-epimerase | 0.409 | 0.011 |
A0A0D6I3W9 | dlgD | 2,3-diketo-L-gulonate reductase | 1.303 | 0.020 |
A0A0L9GCU5 | acpP | Acyl carrier protein | 1.307 | 0.007 |
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, D-alanyl-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.
Table 4
differently expressed proteins relative to drug-resistance
Accession name | Gene name | Function | Fold change | P value |
A0A0D6FBV2 | arnA | Bifunctional polymyxin resistance protein | 0.810 | 0.031 |
C4NZW3 | blaSHV-12 | Beta-lactamase | 0.771 | 0.003 |
A0A2D0MNI5 | OppA | Oligopeptide ABC transporter substrate-binding protein | 1.231 | 0.007 |
Q9 × 523 | pcgL | D-alanyl-D-alanine dipeptidase | 0.754 | 0.006 |
A0A0D6IPG0 | CR079_25305 | Thiol:disulfide interchange protein | 1.258 | 0.025 |
A0A0J5IUL5 | CR079_20285 | Transcriptional regulator | 0.829 | 0.030 |
A0A0M2IQW1 | CR080_14695 | Copper homeostasis protein | 0.677 | 0.008 |
Type I fimbria (Table 5) is one of the down-regulated cell parts. It was reported that fimbria is extracellular structure, and plays a crucial role in adhesion. Anna et al. demonstrated that type I fimbriae could adhere to host epithelial cells, and regulate biofilm formation according to fimH gene[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
differently expressed proteins related to toxins
Accession name | Gene name | Function | Fold change | p. value |
A0A0C5PU36 | invB | SPI-1molecular chaperone | 0.66 | 0.03 |
A0A0C5PPT0 | sipA | SPI-1effector | 0.61 | 0.0002 |
A0A0C5Q2B9 | prgI | SPI-1needle protein | 0.55 | 0.001 |
A0A0D6H7T1 | fimA | Fim subunit | 0.80 | 0.02 |
A0A0M2IX42 | CR079_01855 | Fim subunit | 0.72 | 0.01 |
A0A0D6H693 | fimW | Fim regulator | 0.73 | 0.02 |
Type III secretion system (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.