Using intestinal probiotics to promote calcium absorption
Calcium is a mineral necessary for the body to maintain healthy bones. Bone loss can lead to metabolic bone diseases, such as osteoporosis. This situation often occurs in the elderly population. Since the calcium intake of the Chinese elderly usually cannot meet the Nutrition Society's recommended standards, calcium deficiency is more common for the Chinese elderly. According to reports, the amount of calcium absorbed by the body depends on the amount of soluble calcium in the duodenum and proximal jejunum. Due to the precipitation of calcium ions in the intestines with antagonists such as oxalate, phytate, and cellulose, calcium's solubility may be reduced. In China, because vegetables account for a large proportion of the traditional diet, the rich oxalate in vegetables will prevent the elderly from absorbing calcium.
Recently, people have attracted great interest in enhancing calcium solubility and subsequent calcium absorption by food substances (especially peptides). Combining calcium with peptides to prevent calcium precipitation can effectively increase calcium absorption in the body. So far, Calcium-binding peptides have been found and selected in various food sources, including egg yolks, cow cheese protein, whey, soybeans, wheat germ, tilapia, Alaskan pollock, and shrimp processing by-products. One of the most widely known is the casein phosphopeptides (CPPs) in milk. CPPs can promote calcium absorption by chelating calcium with phosphoserine residues. Some peptides lacking phosphoserine residues also enhance intestinal calcium absorption by binding calcium to Asp and Glu residues.
Inspired by short peptides that can bind calcium, we reviewed and designed a peptide (CaAP) containing a series of short peptides that can promote calcium absorption. We let probiotics express and secrete this peptide in the intestine to promote calcium absorption in the intestine. By introducing LuxI/LuxR quorum sensing system, bacteria can first gain population advantage by expressing antimicrobial peptides, thereby colonizing and forming flora in the intestine. When the number of bacteria reaches a certain level, the antimicrobial peptide secretion system will be turned off and the secretion of CaAPs will be turned on. In this case, the antimicrobial peptides will not be secreted out but will inhibit itself so that the number of flora can be maintained in a stable range. Meanwhile, CaAPs will be continuously expressed and secreted, combined with calcium ions in the intestine, and promote the absorption of calcium ions by epithelial cells.
Through this design, we hope to develop a stable and sustainable endogenous drug delivery method that can truly improve the quality of life and health of human beings, especially the elderly. This project can also promote the progress of SDG Goal3: Good health and well-being.
Calcium Absorption Peptide (CaAP)
Casein phosphopeptides are phosphorylated casein of digest milk, so protein kinases are required. However, given that protein kinases play an essential role in signal transduction, and they may not be able to fold and function correctly in the chassis, it is challenging to phosphorylate peptides in bacteria. Therefore, we choose short peptides without phosphoserine residues to promote calcium absorption.
Through literature search and database screening, currently selected peptides are GPAGPHGPVG, FDHIVY, YQEPVIAPKL[1], NDEELNK[2], DHTKE[3]. Experiments have determined these peptides can promote higher calcium absorption.
GPAGPHGPVG, FDHIVY, and YQEPVIAPKL
GPAGPHGPVG, FDHIVY, and YQEPVIAPKL are derived from tilapia collagen hydrolysate. In terms of safety, the MTT test showed that among the three short peptides, YQEPVIAPKL at six tested concentrations had no cytotoxicity. In the concentration range of 25 to 500μg/mL, neither GPAGPHGPVG nor FDHIVY affects cell viability.
Use the o-cresol-catechol colorimetric method to verify their calcium chelating activity. The calcium chelating activity of GPAGPHGPVG, FDHIVY, and YQEPVIAPKL respectively reached 18.80±0.49, 35.73±0.74, 28.4±0.94 mg/g, indicating that the FDFDY peptide with the shortest sequence has more calcium chelating sites. For these peptides, the composition and the peptide sequence may play an essential role in the calcium chelating activity. Except for the binding site of casein phosphopeptide (CPP) “Ser(p)-Ser(p)-Ser(p)-Glu-Glu,” the most likely calcium-binding sites are Asp(D) and Glu(E )carboxyl. Besides, due to the δ-N of the imidazole ring of His(H) and the ε-amino nitrogen of Lys(K), necessary amino acids such as His(H) and Lys(K) have a significant contribution to the calcium-binding activity of the peptide.
The results further showed that compared with adding calcium and phosphorus, adding GPAGPHGPVG, FDHIVY, and YQEPVIAPKL respectively increased calcium transport by 89±9, 202±12, and 130±7%. As the transport time increases, the difference in calcium transport induced by each peptide becomes more evident. FDHIVY has the most significant effect on calcium transport, followed by YQEPVIAPKL and then GPAGPHGPVG. This result is consistent with their calcium chelating activity. Previous findings indicate that peptide chelating activity is necessary to promote calcium absorption in the Caco-2 cell monolayer.
Calcium absorption peptides with high biological activity are believed to affect calcium absorption by interacting with membranes and opening specific calcium channels. The most suitable peptide structure can bind calcium for transport and release ions quickly. calcium may be absorbed after being released from the peptide, or it may be absorbed in the form of calcium peptide chelate.
NDEELNK
Asn-Asp-Glu-Glu-Leu-Asn-Lys (NDEELNK) hepeptide is derived from the trypsin hydrolysate of sea cucumber eggs. This peptide can spontaneously bind calcium at a stoichiometric ratio of 1:1, and the calcium-binding site may involve the carboxyl oxygen and amino nitrogen atoms of two glutamic acids and one aspartic acid residues in the NDEELNK peptide. Besides, it is reported in the literature that the DEELNK-calcium complex undergoes decomposition and self-aggregation in a smaller-sized grid during the digestive process of the gastrointestinal tract, which may help to absorb calcium across the Caco-2 cell monolayer.
DHTKE
Asp-His-Thr-Lys-Glu (DHTKE) pentapeptide comes from the hydrolysis of egg white. This peptide can spontaneously bind calcium at a stoichiometric ratio of 1:1, and the calcium-binding site corresponds to the carboxyl oxygen, amino nitrogen, and imidazole nitrogen atoms of the DHTKE peptide. Besides, Caco-2 cells were used in vitro to study the DHTKE-calcium complex's effect on improving calcium absorption. Compared with the control without calcium, the results show that the DHTKE-calcium complex can promote the penetration of calcium into the cytoplasm and further increase the Caco-2 cell monolayer's calcium absorption by more than seven times.
The five peptides are connected by FR linkers to form a short peptide that promotes calcium absorption so that it can be cleaved and hydrolyzed by digestive tract proteases.
Signal peptide
To enable CAAP to be secreted into the intestinal environment, we plan to add a signal peptide to the N segment of CAAP to guide the extracellular environment's secretion. Our candidates are NSP4, OmpA, DsbA, PelB, PhoA. We hope to obtain a suitable signal peptide through comparison so that CaAP can be efficiently secreted into the periplasm.
The entire designed gene fusion fragment length is about 500 bp, and the gene loop is organized as follows.
Quorum sensing
Based on previous work, We decided to use luxI/luxR and TetR to construct a quorum sensor system with the antimicrobial peptide microcin B17 (MccB17) expression system to achieve the "on" and "off" effects of controlling the growth of engineered bacteria (schematic diagram).
MccB17 is a short peptide microcin produced by an endogenous plasmid found in Escherichia coli. It acts on DNA topoisomerase and gyrase, preventing bacterial from DNA replication and transcription and promoting double-strand breaks. McbABCDEFG jointly completes the expression, processing, and secretion of MccB17. Among them, mcbA expresses the main structure of the antimicrobial peptide, and mcbBCD is responsible for the post-translational modification of the antimicrobial peptide. McbEF expresses the channel protein to secrete the peptide antibiotic. McbG is accountable for the immunity against MccB17. McbEFG is all related to E. coli's immunity while self-producing microcin. TldD/tldE is a metalloprotease that is ubiquitous in bacteria. When the surrounding bacteria absorb MccB17 produced by Escherichia coli, TldD/tldE will activate it and kill the bacteria.
In our antimicrobial peptide expressing system, McbABCD is under the control of a constitutive promoter, but PtetR regulates the expression of McbEFG. When the number of engineered bacteria in the intestine is small, few dimers are formed by the expression products of luxI and luxR genes, which cannot effectively activate the luxpR promoter. Thus, TetR and CaAP expression systems are both silent. However, McbEFG is expressed and transports MccB17, which can inhibit the growth of surrounding bacteria. When the number of bacteria increases to a certain extent, the dimers formed by the products of luxI and luxR will accumulate and effectively activate luxpR. At this time, tetR is turned on, and PtetR is off. As a result, McbEFG stops expressing, and the CaAP system begins to work. Since the population number has reached a certain level, the engineered bacteria can express CaAP more efficiently. Meanwhile, MccB17 is still continuously expressed but cannot be effectively effluxed, which will cause the toxins to gradually accumulate in the engineered bacteria and prevent them from continuously unrestricted proliferation.
To achieve better expression efficiency, we have made an improvement in this system by optimizing the expression of an antimicrobial peptide. We rearranged the gene structure and regulation system and took a unique idea to create a new promoter fusion. As ptetR has been studied thoroughly and is well-characterized, we created promoter fusion of pl and tetO. Not only is it regulated by tetR and anhydrotetracycline, but it also inherits the expression level of pl. This method also allows us to create the desired promoter simpler.
Kill Switch
In order to ensure our engineered probiotic will not jeopardize the environment or users, this year, we integrated two kinds of Kill Switches into our project. The major kind of Kill Switch is cold triggered toxin/antitoxin Kill Switch to deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine.After carefully considering, we finally selected the toxin/ antitoxin system from killer systems and RNA thermometer from the response systems.
The toxin/antitoxin system is a mature and stable system that is extensively applied both inside and outside the iGEM competition. By regulating the strength of RBS, we could achieve a state that toxin is totally neutralized and guarantee the security on the host. Here we first chose RelE/RelB system of type II TA systems but replace it with MazF/MazE system later. RNA thermometers are apparent outstandingly because of its appropriate reaction temperature range, high sensitivity and high efficiency. Moreover, they do not rely on extra proteins or are affected by food intake. Here we selected NoChill- 06 which presents to be the most sensitive and efficient one. So, our Kill Switch consists of a toxin-antitoxin system and an RNA thermometer NoChill-06 to regulate it to deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine.
The antitoxin MazE is liable and expressed at a relatively high level. The MazF toxin is constitutively co-expressed with the antitoxin under the control of an RNA thermometer No-Chill 06. Under the body temperature (37℃)，No-Chill 06 unfolds and exposes its ribosome binding site (RBS) to express. MazE and MazF neutralize each other by protein-protein interaction and form a stable complexity in a one-to-two ratio. When the bacteria encounter a cold shock(30℃), MazE is degraded rapidly by an ATP-dependent serine protease ClpAP and releases MazF. The toxin MazF acts as a site-specific endoribonuclease to almost all cellular mRNAs, therefore resulting in cell growth arrest and finally cell death [4]. The antitoxin MazE is liable and expressed at a relatively high level. The MazF toxin is constitutively co-expressed with the antitoxin under the control of an RNA thermometer No-Chill 06. Under the body temperature (37℃)，No-Chill 06 unfolds and exposes its ribosome binding site (RBS) to express. MazE and MazF neutralize each other by protein-protein interaction and form a stable complexity in a one-to-two ratio. When the bacteria encounter a cold shock(30℃), MazE is degraded rapidly by an ATP-dependent serine protease ClpAP and releases MazF. The toxin MazF acts as a site-specific endoribonuclease to almost all cellular mRNAs, therefore resulting in cell growth arrest and finally cell death [4].
Click here to see our detailed consideration of Kill Switch in the engineering page.
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