Method for controlling covalent colonization of bacteria by thiolation of chemical reactions and applications

Abstract

The application discloses a method for controlling thiolated bacterial covalent colonization by chemical reaction and application. The application provides a unique in-situ chemical reaction mediated bacterial covalent colonization strategy. Through a simple one-step iminothiolane ring-opening reaction, the primary amino group on the surface of bacteria can be converted into a free thiol group under cell-compatible conditions. Surface thiolation can be used for modification of different strains, and the number of thiol groups introduced by each bacterium can be adjusted by different feeding ratios. These chemically reactive bacteria can spontaneously bind to the mucus layer through a sulfur-sulfur exchange reaction between the newly converted non-native thiol group on the surface of the bacteria and the disulfide bond contained in mucin without catalyst. The application converts the bacteria through surface modification to form a "living chemical reagent", and the surface-converted living reactive bacteria provide an unprecedented method for chemical control of bacterial colonization, which has guiding significance for the development of the next generation of living bacterial biological agents.

Description

Technical Field

[0001] This invention relates to a method and application of chemically controlled covalent colonization of thiolized bacteria, belonging to the field of biomedical technology. Background Technology

[0002] Microbiota-host interactions play a vital role in human health and profoundly influence microbial behavior (1-3). Sufficient colonization of beneficial and symbiotic bacteria is crucial for maintaining microbial homeostasis to sustain host metabolism and immune function, thereby preventing and treating diseases (4-7). In nature, microbial colonization primarily depends on interactions between bacterial surfaces and various in vivo biological interfaces (8-10). For example, bacteria have evolved and express a variety of surface adhesins to promote interfacial adhesion, which in turn promotes extracellular mucopolysaccharide and biofilm formation to enhance colonization (11-13). Therefore, methods capable of manipulating interfacial interactions between bacteria and their environment are of great significance for controlling microbial colonization.

[0003] The use of synthetic bioengineering to express specific membrane ligands or receptors has been extensively explored to improve bacterial interactions with host extracellular matrix proteins (such as fibrinogen and collagen) (14-17). However, this approach still faces several challenges, such as the limitation of genetic modification to certain model cells or strains, and the potential for gene proliferation and mutation when used as therapeutic agents. For example, genetically engineered E. coli can be used to controllably express surface capsular polysaccharides and increase bacterial colonization in target tissues by intervening in interactions with immune cells (18). Other examples include using genetically modified biomembrane structures to modulate the adaptive and responsive nature of Bacillus subtilis's dynamic interactions with the environment (19, 20). On the other hand, in previous studies, we, along with other research groups, employed physical encapsulation or surface modification of bacteria to reduce their affinity for unfavorable environmental substances (21-26), while simultaneously increasing bacterial retention or colonization in vivo through hydrogen bonding (27-30), electrostatic interactions (31, 32), hydrophobic interactions (33, 34), and π-π conjugation interactions (35). However, the attachment of encapsulated living cells to the host / tissue surface remains unstable because these modifications are easily disrupted by adverse factors in the microenvironment such as proteins / lipids, pH, or salt. Furthermore, although some methods allow for the formation of functional films around cells, such as layer-by-layer assembly or surface-initiated polymerization around cells (36, 37), these techniques can completely alter the surface biology of cells. For example, complete encapsulation or blocking of cell surface proteins / receptors significantly weakens bacterial-host interactions. In addition, previous surface modification methods often introduced polymers or engineered materials, and the in vivo immune responses evoked by these newly introduced materials have largely hindered the clinical translation of these methods. To our knowledge, previous methods relied solely on non-covalent bonds between bacteria and their surroundings, inevitably leading to insufficient interactions. Therefore, alternative methods are in high demand, particularly those that enhance interfacial interactions to improve the colonization capacity of both beneficial and symbiotic bacteria.

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[0042] The purpose of this invention is to address the deficiency in the non-covalent bonding interactions between bacteria and their surrounding interfaces in existing technologies by providing a covalently assisted colonization method for bacteria in vivo, achieved through a catalyst-free in-situ chemical reaction. The abundant primary amino residues on the surface of various bacteria can be converted into free thiols under cytocompatibility conditions via a simple one-step imine thiol alkylation reaction. Surface thiolation produces chemically reactive live bacteria capable of forming new disulfide bonds through dynamic sulfur-sulfur exchange reactions, spontaneously connecting to a mucin-rich layer of polydisulfide bonds. Due to their ability to form strong covalent bonds, surface-thiolated bacteria show advantages in targeting and colonizing mucin-containing tissues / interfaces. Colonization of intestinal microorganisms in the jejunum has always been a challenging problem. Compared to unmodified naked bacteria, reactive bacteria containing thiol groups can achieve up to 170-fold increased colonization in the mucin-rich jejunum. Thiolation of the surface of therapeutic *Escherichia coli* Nissel 1917 (EcN) and subsequent animal experiments showed that thiolated EcN significantly alleviated inflammation associated with jejunal mucositis. This work reveals how bacterial colonization behavior can be controlled by manipulating the chemical interactions at the bacterial-host interface. Given the unique properties of covalently assisted colonization, surface-reactive transformation of bacteria can be performed, providing strong support for the preparation of various functional in vivo biopharmaceuticals.

[0043] To achieve the above objectives, the present invention provides a chemically controlled method for covalent colonization of thiolized bacteria for non-diagnostic and therapeutic purposes, comprising: incubating thiolized bacteria with proteins or protein-containing tissues in vitro under catalyst-free conditions, wherein the thiol groups on the bacterial surface undergo an exchange reaction with the disulfide bonds in the protein to form a covalent bond.

[0044] Preferably, the protein is a mucin.

[0045] Preferably, the thiolated bacteria include thiolated bacteria prepared by any one of the following methods:

[0046] Method 1: Incubate bacteria with a thiolation reagent and buffer at room temperature. The thiolation reagent reacts with the primary amino groups of the N-terminus and / or lysine residues in peptides or proteins on the bacterial surface, thereby introducing free thiolation groups and obtaining surface-thiolated bacteria.

[0047] Method 2: Surface-thiolized bacteria are prepared by electrostatically adsorbing thiolized biodegradable polymers onto the bacterial surface or by reacting them with primary amino or carboxyl groups on the bacterial surface;

[0048] Method 3: Surface-thiated bacteria are formed by expressing polypeptides or proteins containing cysteine ​​(containing thiol groups) on the surface of bacteria (expression can be achieved through methods such as plasmid transfection).

[0049] Preferably, the thiolated reagent in Method 1 includes 2-iminothionecyclopentane hydrochloride; the buffer solution is a phosphate buffer with a pH of 7-8; and the biodegradable polymer material in Method 2 is at least one of hyaluronic acid, chitosan, polydopamine, and PEG.

[0050] Preferably, the concentration of the 2-iminothiocyclopentane hydrochloride is 10–100 μg / mL, and the incubation time is ≤150 minutes.

[0051] The present invention also provides a thiolated bacterium, which is obtained by co-incubating bacteria with a thiolation reagent and a buffer at room temperature, wherein the thiolation reagent reacts with the primary amino groups of the N-terminus and / or lysine residues in peptides or proteins present on the bacterial surface, thereby introducing free thiolation groups, thus obtaining surface thiolated bacteria.

[0052] The present invention also provides a probiotic preparation, the active ingredient of which includes surface-thiolated live probiotics.

[0053] Preferably, the surface-thiolized live probiotics are prepared by any one of the following methods:

[0054] Method 1: Live probiotics are co-incubated with a thiolation reagent and buffer at room temperature. The thiolation reagent reacts with the primary amino groups of the N-terminus and / or lysine residues in peptides or proteins on the bacterial surface, thereby introducing free thiolation groups and obtaining surface-thiolated bacteria.

[0055] Method 2: Surface-thiolized bacteria are prepared by electrostatically adsorbing thiolized biodegradable polymers onto the bacterial surface or by reacting them with primary amino or carboxyl groups on the bacterial surface;

[0056] Method 3: Surface-thiated bacteria are formed by expressing polypeptides or proteins containing cysteine ​​(containing thiol groups) on the surface of bacteria (expression can be achieved through methods such as plasmid transfection).

[0057] Preferably, the live probiotics include at least one of Escherichia coli EcN, Gram-negative Akkermansia myxophilus AKK, Salmonella typhimurium STM, and Gram-positive Enterococcus faecalis EF, Bacillus megaterium BMB, and Bacillus cereus BC.

[0058] The present invention also provides the use of a probiotic preparation in the preparation of a medicament for the treatment or adjunctive treatment of inflammation related to small intestinal mucositis, wherein the active ingredient of the probiotic preparation is surface-thiolized Escherichia coli EcN.

[0059] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0060] This invention utilizes a simple one-step iminothionylpentane decyclization reaction to convert primary amino groups on the bacterial surface into free thiols under cellular compatibility conditions. Surface thiolation can be used to modify different bacterial strains, and the number of thiols introduced into each bacterium can be adjusted by different feed ratios. These chemically reactive bacteria can spontaneously bind to the mucus layer by undergoing a sulfur-sulfur exchange reaction with newly converted, non-native thiols on the bacterial surface via disulfide bonds contained in mucin, without a catalyst. This binding is dependent on the level of bacterial surface thiolation. Jejunal colonization has always been a challenging aspect of microbial colonization, but when a single bacterium contains 9.3 × 10⁻⁶ thiols on its surface... 7 When thiol groups are present, thiolized bacteria colonize the mucus-rich jejunum 170-fold more frequently. Animal experiments show that oral administration of thiolized probiotics increases covalent reaction-mediated colonization in the jejunum and significantly improves the symptoms of jejunal mucositis. The results of this invention demonstrate that by surface-modifying bacteria to make them chemically reactive, forming "living chemical reagents," this surface-transformation of living reactive bacteria provides an unprecedented method for chemically controlling bacterial colonization, which has certain guiding significance for the development of next-generation living bacterial biopharmaceuticals. Attached Figure Description

[0061] Figure 1 The structure of mucin;

[0062] Figure 2This study aims to prepare, characterize, and validate the reactivity of surface-thiolized bacteria. Specifically, a represents the survival rate of EcN after reaction with 2-iminothiacyclopentane for different times (60, 90, 120, and 150 minutes); b–d represent the survival rate, growth curve, and surface thiol content of EcN after reaction with different concentrations of 2-iminothiacyclopentane in the range of 0–400 μg / mL for 90 minutes, respectively; e represents the Zeta potential of EcN after thiolization with different concentrations of 2-iminothiacyclopentane; and f represents the surface thiol content of native EcN. Flow cytometry histograms and MFI values ​​of EcN@SH after 90 min of reaction with Cy5.5-maleimide; g is a typical LSCM image of GFP-expressing EcN and EcN@SH after reaction with Cy5.5-maleimide; scale bar: 2 μm; error bars represent standard deviation (n=3); significance was assessed using one-way ANOVA, followed by Fisher's LSD multiple comparisons, p-values ​​are given, *p<0.05, **p<0.01, ****p<0.0001;

[0063] Figure 3 For use with 2-iminothionecyclopentane (IC) 50 Bacterial survival curves after reaction (152.3 μg / mL); the number of viable bacteria was calculated by bacterial plate counting;

[0064] Figure 4 The bacterial survival rate after treatment with 0.1 mM DTNB for 2 hours;

[0065] Figure 5 A standard curve for quantitatively measuring the sulfhydryl content in L-cysteine ​​at different concentrations using Traut's reagent;

[0066] Figure 6 Representative curves (EC50) showing the number of sulfhydryl groups in each bacterium after reacting with 0–400 μg / mL of 2-iminothiacyclopentane for 90 minutes. 50 88.3 μg / mL);

[0067] Figure 7 This is a schematic diagram of chemically mediated bacterial covalent colonization; where a represents the preparation of surface-thiolized bacteria through a simple one-step 2-iminothionecyclopentane decyclization reaction under cell compatibility conditions; and b represents the binding of chemically reactive surface-thiolized bacteria to disulfide-rich mucins located at various tissue interfaces via a catalyst-free dynamic sulfur-sulfide exchange reaction.

[0068] Figure 8 The amino levels of native EcN and EcN@SH were characterized by flow cytometry using the NH2-sensitive clickable label Cy5-NHS; significance was assessed using a t-test (two-tailed), ns, no significance;

[0069] Figure 9 Size distribution of natural EcN and EcN@SH as measured by DLS;

[0070] Figure 10 LSCM images of unmodified EcN and thiolized EcN@SH; green and red channels show EcN's intrinsic endogenous GFP and Cy5.5-maleimide-labeled thiol layers, respectively. Scale bar: 10 μm.

[0071] Figure 11 The mean fluorescence intensity (MFI) of Cy5,5-maleimide-labeled thiol groups on EcN and EcN@SH was analyzed by flow cytometry; error bars represent standard deviation (n=5), significance was assessed using Student's t-test (two-tailed), and p-values ​​are given, ***p<0.001;

[0072] Figure 12 Coomassie blue staining of total protein extracted from natural and thiolized EcN cells;

[0073] Figure 13 To assess the ability of thiolated bacteria to degrade tryptophan to indole via the production of tryptophanase by indole assay; where, a) is a digital photograph of the indole assay for tryptophan metabolism in native and thiolated EcN; the black arrow indicates the purple indole ring of a positive result; b) is the quantitative detection of indole production by HPLC analysis; significance was assessed using a t-test (two-tailed), ns, no significance.

[0074] Figure 14 Zeta potentials of natural and thiolized bacteria AKK(a), STM(b), EF(c), BMB(d), and BC(e);

[0075] Figure 15Surface thiolization of different bacterial species; where a, e, i, m, and q are flow cytometry histograms after 90 min of reaction of thiolized (a)AKK, (e)STM, (i)EF, (m)BMB, and (q)BC with Cy5,5-maleimide, and unmodified bacteria serve as controls; b, f, j, n, and r are the MFI values ​​of unmodified bacteria and the corresponding thiolized (b)AKK, (f)STM, (j)EF, (n)BMB, and (r)BC after reaction with Cy5,5-maleimide. c, g, k, o, s are typical LSCM images of thiolized (c)AKK, (g)STM, (k)EF, (o)BMB, and (s)BC, scale bar: 5 μm; d, h, l, p, t are growth curves of thiolized (d)AKK, (h)STM, (l)EF, (p)BMB, and (t)BC, with natural bacteria used as controls; error bars represent standard deviation (n=3); significance was assessed using a t-test (two-tailed), p-values ​​are given, ****p<0.0001;

[0076] Figure 16 Typical LSCM images of unmodified and thiolized Gram-negative bacteria STM(a) and AKK(b) after reaction with Cy5,5-maleimide, scale bar: 2 μm;

[0077] Figure 17 Typical LSCM images of unmodified and thiolized Gram-positive bacteria EF(a), BMB(b), and BC(c) after reaction with Cy5,5-maleimide, scale bar: 2 μm;

[0078] Figure 18 The reaction of surface-thiolized bacteria with mucin is shown in Figure a. Figure a illustrates the reaction between thiolized bacteria and mucin via dynamic sulfur-sulfur exchange in the absence of a catalyst. Figure b shows the MFI values ​​of unmodified EcN and EcN@SH. Each cell was reacted with 0.03 mg / mL Ly5.5_mucin for 1 hour, and each cell contained 8.5 × 10⁻⁶ cells. 7The flow cytometry histograms (c) and MFI values ​​(d) of bacteria with different surface thiol densities after reacting with Cy5.5_mucin for 1 hour at 0.1 mg / mL are shown. The flow cytometry histograms (e) and MFI values ​​(f) of EcN@SH with mucin attachment after reaction with Cy5.5-maleimide are shown. The diagram illustrates the blocking of sulfur-sulfur exchange by the reducing agent. The flow cytometry histograms (h) and MFI values ​​(i) of EcN@SH with 0.03 mg / mL L-ascorbic acid in the presence of 0, 0.3, or 1 mg / mL L-ascorbic acid are shown. The figure (j) shows the flow cytometry histograms (h) and MFI values ​​(i) of EcN@SH with 0.03 mg / mL L-ascorbic acid in the presence or absence of 1 mg / mL L-ascorbic acid. Representative LSCM images of EcN@SH after reaction with Cy5.5_mucin in the case of L-ascorbic acid; scale bar: 3 μm; error bars represent standard deviation (n=3); significance was assessed using t-test (two-tailed) or one-way ANOVA analysis, followed by Fisher's LSD multiple comparisons, p-values ​​are given, *p<0.05, **p<0.01, ****p<0.0001, ns, no significance;

[0079] Figure 19 To measure the decay of SH levels over time in native EcN and EcN@SH using the SH-sensitive and clickable label Cy5.5-maleimide;

[0080] Figure 20 After reacting with Cy5.5_mucin (0.1 mg / ml) for 1 hour, natural EcN and EcN@SH were analyzed by flow cytometry; error bars represent standard deviation (n=3); significance was assessed using a t-test (two-tailed), and p-values ​​are given, **p<0.01;

[0081] Figure 21 EcN was measured by DLS and the size of EcN after mucin attachment; significance was assessed using Student's t-test (two-tailed), ns, no significance;

[0082] Figure 22 Zeta potentials of EcN and EcN attached to mucin, measured by DLS; significance was assessed using Student's t-test (two-tailed), ns, no significance;

[0083] Figure 23 The MFI values ​​were obtained after EcN@SH reacted with 0.15 mg / mL Ly5.5_mucin in the presence of 0.1 mg / mL LTT. Significance was assessed using one-way ANOVA, followed by Fisher's LSD multiple comparisons. p-values ​​were given, with ***p < 0.001.

[0084] Figure 24The MFI values ​​were obtained after EcN@SH reacted with 0.15 mg / mL LCy5.5_mucin in the presence of 0.3 mg / mL SeCA. Significance was assessed using one-way ANOVA, followed by Fisher's LSD multiple comparisons. p-values ​​were given, with ***p<0.001.

[0085] Figure 25 This section describes the covalent colonization of thiolized bacteria in the mucus layer. Image a shows a schematic diagram of the experimental design used to examine the reaction between thiolized bacteria and the jejunal mucus layer. Images b and c are IVIS images (b) and fluorescence intensity (c) of the corresponding everted mouse jejunal segments after culture with equal amounts of unmodified EcN or EcN@SH (expressing mCherry). Scale bar: 0.5 cm. Image d shows the number of bacteria attached to the mouse jejunal mucus layer by LB agar counting. Images e and f are IVIS images (e) and corresponding fluorescence intensity (f) of porcine jejunal segments after incubation with equal amounts of native EcN or EcN@SH (expressing mCherry). Scale bar: 3 cm. Image g shows the number of bacteria attached to the porcine jejunal mucus layer by agar counting. Image h shows the number of bacteria attached to the porcine jejunal mucus layer after oral administration. Representative LSCM images of mouse mucus layers collected 1 hour after gavage administration of PBS, unmodified EcN, or EcN@SH, where EcN expresses endogenous mCherry. Scale bar: 10 μm; i is a typical agar plate image containing EcN or EcN@SH collected from mouse jejunal mucus 1 hour after oral gavage administration of unmodified EcN or EcN@SH; j and k are the counts of EcN and EcN@SH collected from jejunal mucus (j) and jejunal tissue (k) at 1, 4, and 24 hours after oral gavage administration, respectively; error bars represent standard deviations (n ​​= 3–4); significance was assessed using a two-tailed t-test, and p-values ​​are given: *p < 0.05, **p < 0.01, ***p < 0.001.

[0086] Figure 26 Representative agar plates containing EcN or EcN@SH were collected from jejunal mucus 4 hours after oral administration of equal amounts of EcN and EcN@SH (1.0 × 10⁸ CFU per mouse).

[0087] Figure 27 The combined use of L-ascorbic acid significantly reduced EcN@SH adhesion; where a) is a typical agar plate image containing EcN or EcN@SH collected from mouse jejunal mucus 1 hour after oral gavage administration of unmodified EcN or EcN@SH or EcN@SH+L-ascorbic acid (1 mg / ml); b) is the count of EcN and EcN@SH 1 hour after oral gavage administration; significance was assessed using one-way ANOVA, followed by Fisher's LSD multiple comparisons, and p-values ​​were given, **p<0.01. ns, no significance;

[0088] Figure 28 The mean intestinal length after administration of equal amounts of EcN or EcN@SH (1.0 × 10⁻⁶ per mouse) was calculated. 8 CFU); the error bar represents the standard deviation (n=4); a t-test (two-tailed) is used to assess significance. ns, meaningless;

[0089] Figure 29 H&E stained images of sections of mouse liver, kidney, spleen, lung, and heart tissue;

[0090] Figure 30 Thioylated EcN was used to alleviate jejunal mucositis; where a is a schematic diagram of how covalently mediated thioylated EcN colonization promotes the relief of jejunal mucositis; daily administration of PBS, 1×10 8 Mice were orally administered CFUs-unmodified EcN or an equivalent amount of EcN@SH via gavage for 5 consecutive days. On day 3, mice were intraperitoneally injected with 150 mg / kg of 5-FU. On day 6, mice were euthanized and samples were collected. Healthy mice were used as controls. b is the average intestinal length after treatment; c is the serum TNF-α level measured by an ELISA kit; d is a typical image of jejunal H&E staining, with blue arrows indicating inflammation and red arrows indicating vacuolation and edema. Scale bar: 100 μm. e, f, and g represent quantitative analyses of villus length (e), crypt depth (f), and villus / crypt ratio (g) in the jejunum; h shows immunofluorescence images of ZO-1 and occludin staining of the jejunum, scale bar: 100 μm; error bars represent standard deviations (n ​​= 5); significance was assessed using one-way ANOVA, followed by Fisher's LSD multiple comparisons, with p-values ​​given: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, no significance;

[0091] Figure 31 A schematic diagram illustrating the localized and enhanced treatment of jejunal mucositis via covalently mediated thiolated EcN.

[0092] Figure 32 To measure serum IL-6 levels using an ELISA kit, significance was assessed using one-way ANOVA, followed by Fisher's LSD multiple comparisons. p-values ​​are given, *p<0.05, **p<0.01.

[0093] Figure 33 To assess inflammation in mouse jejunal tissue, myeloperoxidase (MPO) staining was performed; where a represents the mean number of MPO-positive cells counted in the jejunum; b represents a typical image of MPO staining in the jejunum; significance was assessed using one-way ANOVA followed by Fisher's LSD multiple comparisons, with p-values ​​given, **p<0.01, ***p<0.001. Detailed Implementation

[0094] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0095] The materials and methods used in the embodiments of this invention are listed below:

[0096] 1. Chemical and biological products involved

[0097] 2-Iminothiacyclopentane hydrochloride (Traut's reagent, molecular weight 137.63, Thermo Scientific) TM The study included swine gastric mucin (type III, Sigma-Aldrich), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's recipe, Thermo Scientific™), L-ascorbic acid (BBI Life Sciences), 5-fluorouracil (5-FU, Adamas), Cy5.5-maleimide (Cy5.5-maleimide, Aladdin), and Cy5.5-N-hydroxysuccinimide (Cy5.5-NHS, Aladdin). Cy5.5-labeled mucin (Cy5.5_mucin) was prepared by reacting Cy5.5-NHS at 37°C for 3 hours, with residual free Cy5.5-NHS removed by ultrafiltration. LB liquid medium was prepared using 25 g LB broth in 1 L deionized (DI) water and used after autoclaving. LB agar plates are prepared by adding 8 mL of LB agar solution (containing 25 g of LB broth and 15 g of agar in 1 L of deionized water) to a bacterial culture dish.

[0098] 2. Bacterial strains and plasmids

[0099] *Escherichia coli* Nissle 1917 (EcN), *Akkermansia muciniphila* (AKK), *Salmonella typhimurium* (STM), *Enterococcus faecalis* (EF), *Bacillus megaterium de Bary* (BMB), and *Bacillus cereus* (BC) were purchased from the China Integrated Microbial Culture Collection (GMCC, China). Plasmids pBBR1MCS2-Tac-GFP and pBBR1MCS2-Tac-mCherry were purchased from a domestic supplier and were used for intracellular expression of GFP and mCherry in EcN cells.

[0100] 3. Methods for converting primary amine groups to thiol groups on bacterial surface

[0101] An EcN colony was removed from an LB agar plate and incubated overnight in LB liquid medium (200 rpm, 37°C). The EcN cells were then washed twice with DI water and resuspended in 987.5 μL of ice-cold phosphate-buffered saline (PBS, pH 7.4). 12.5 μL of 2-iminothiacyclopentane solution (2 mg / mL) was then added to the suspension, followed by incubation at room temperature for 90 min to induce efficient thiolation on the bacterial surface. Reaction conditions were optimized using final reaction concentrations of 2-iminothiacyclopentane (10, 25, 50, 100, 200, and 400 μg / mL) and reaction times (60, 90, 120, and 150 min). The thiolated EcN (EcN@SH) was further washed twice with PBS to remove residual 2-iminothiacyclopentane. The prepared EcN@SH and native EcN suspended in PBS were stored at 4°C.

[0102] 4. Method for determining the number of thiol groups

[0103] Ellman's reagent (DTNB) is used to quantify the number of thiol groups modified on the surface of bacteria. Bacteria are then subjected to a concentration of 3 × 10⁻⁶. 8 CFUs / mL were resuspended in PBS, and DTNB was added to achieve a final working concentration of 0.1 mM. The reaction was carried out at room temperature for 2 hours. After removing the precipitated bacteria by centrifugation (6,000 g, 5 min), 200 μL of the supernatant was transferred to a microtiter plate, and the absorbance was immediately measured at 412 nm (microplate reader, BioTek). The amount of thiol groups was calculated based on a standard curve prepared using L-cysteine ​​hydrochloride. Native EcN was used as a control for thiol group determination. Simultaneously, the number of viable cells after treatment with 0.1 mM Ellman reagent and EcN@SH was determined by counting on selective LB plates.

[0104] 5. Characterization methods for thiol-modified bacteria

[0105] The particle size and zeta potential of the thiolized and mucin-attached bacteria were determined by dynamic light scattering (DLS) in double-distilled water (ddH2O) at room temperature. Thiolized bacteria were also characterized by laser scanning confocal microscopy (LSCM, Leica TCS SP8, Germany) and flow cytometry (Beckman CytoFlex, USA). Briefly, the prepared thiolized bacteria were mixed with a Cy5,5-maleimide solution (0.01 mg / mL) and incubated at room temperature for 20 min to allow for a thiol-maleimide click reaction to label thiol groups. The reactivity and position of the newly formed thiol groups on EcN@SH were then analyzed by LSCM or flow cytometry. Native bacteria were used as controls for the above characterization. Flow cytometry was also used to determine NH2 levels (Cy5-NHS probe). Coomassie blue staining (P0017F, BeyoBlue) was also performed. TM The Super Fast Staining Solution analyzed the total protein content of EcN and EcN@SH.

[0106] 6. Methods for determining the growth curves of natural and thiolized bacteria

[0107] After reacting with 2-iminothiacyclopentane, the bacteria were diluted and placed in 96-well plates containing 5 × 10⁻⁶ mcg of culture medium. 5 Cells / well. Incubate the plate with gentle shaking at 37°C. Record the absorbance (OD600) at 600 nm using a microplate reader (BioTek, USA) for 11 hours, at 0.5-hour intervals. Use natural bacteria as a control.

[0108] 7. Methods for determining the metabolic activity of natural and thiolized bacteria

[0109] Indole metabolism assays were used to qualitatively and quantitatively assess the metabolic activities of native and thiolized bacteria. Equal amounts of native and thiolized EcN (2 × 10⁻⁶) were used. 8 CFU was added to 3 mL of sterile peptone medium (0.5% NaCl and 1% peptone dissolved in distilled water, pH adjusted to 7.6) and incubated at 37°C for 48 hours. At the set time, a few drops of diethyl ether were added to the peptone medium. Then, two drops of Kovac reagent were added to qualitatively observe indole production. The amount of indole produced was determined by high-performance liquid chromatography (HPLC) (Agilent 1260 Infinity II, USA) under the following conditions: Diamonsil C18 column (5 μm, 150 × 4.6 mm), column temperature 45°C, detection wavelength 260 nm, and mobile phase (0.1% formic acid acetonitrile solution and 0.1% formic acid aqueous solution).

[0110] 8. The reaction process between surface-thiolized bacteria and mucin in vitro.

[0111] In vitro experiments demonstrated the dynamic sulfur-sulfur exchange reaction between EcN@SH and mucin. Native and thiolized EcN were resuspended in ice-cold buffer (3 × 10⁻⁶). 8 To a final concentration of 0.03 mg / mL Cy5.5_mucin, 10 μL of 3 mg / mL Cy5.5_mucin was added to a solution containing 0.99 mL of CFUs / mL. The reaction was allowed to proceed at room temperature with shaking at 750 RPM / min for 1 hour. At specified time points, both native and thiolized EcN were centrifuged (6,000 g, 5 min) and washed twice with PBS to completely remove unattached Cy5.5_mucin. Bacteria reacted with Cy5.5_mucin were then sent for flow cytometry analysis. Experiments were performed under the same procedure, but the final concentration of Cy5.5_mucin was increased to 0.1 mg / mL. To investigate the effect of thiolization levels on mucin attachment, the density of thiol groups on the bacterial surface was increased from 5.5 × 10⁻⁶ per cell. 7 Increased to 9.3 × 10 7 Following the same protocol, bacterial suspensions with different levels of thiolization were further incubated and reacted with Cy5.5_mucin (0.1 mg / mL). Simultaneously, to verify the involvement of thiol groups in the reaction, Cy5.5-maleimide, a thiol-sensitive reagent, was used for labeling, and the changes in the number of thiol groups after reaction with mucin were recorded by flow cytometry. Finally, to verify the formation of disulfide bonds between thiolized bacteria and mucin, different concentrations of L-ascorbic acid (0, 0.3, and 1 mg / mL), dithiothreitol (DTT, 0.1 mg / mL), or selenocysteine ​​(SeCA, 0.3 mg / mL) were added to the reaction system during the reaction between thiolized EcN and Cy5.5_mucin. The binding of Cy5.5_mucin to thiolized EcN was observed by LSCM imaging and quantified by flow cytometry.

[0112] 8. Adhesion of thiol-modified bacteria to isolated rat and pig jejunum

[0113] Fresh rat jejunum was divided into segments approximately 1.5 cm long and everted to expose the intestinal mucosa. The two ends of the everted intestine were carefully ligated. The cut jejunal segments were randomly divided into two groups (n = 3 per group) and inoculated with equal amounts of EcN or EcN@SH (1 × 10⁻⁶). 8CFU / mL of mCherry (expressing anti-kanamycin properties) was soaked in PBS for 1 hour with gentle agitation (10 RPM / min). At specified time points, the intestinal segment was washed once with PBS and then soaked twice in distilled water to completely remove unattached bacteria. The intestinal segment was then imaged using an in vivo imaging system (IVIS) and counted using selective LB plates after homogenization in PBS. For validation of the effectiveness of covalently mediated attachment to isolated porcine jejunal segments, the procedure was slightly modified due to the larger size of the porcine jejunum. The cut porcine jejunum was fixed to a foam board with the mucosa facing upwards using a needle. Prepared EcN or EcN@SH (3 × 10⁻⁶) was then applied. 8 CFU / mL, expressing mCherry with anti-kanamycin properties, 20 μL / mL 2 Add the mucosal side and allow it to contact for 1 hour. After the time is up, gently rinse the tissue with PBS for 10 seconds. Send the porcine jejunum segment for IVIS imaging, and then homogenize it in PBS for further plate counting.

[0114] 9. Animal experiments

[0115] All animal protocols were approved by the Laboratory Animal Management and Use Committee of Shanghai Jiao Tong University School of Medicine.

[0116] (1) Covalent localization of thiol-modified bacteria in mouse jejunal mucus layer

[0117] Mice (ICR, female, 6-8 weeks old) were fasted for 15 hours before the experiment. EcN was transfected with a plasmid expressing mCherry and labeled with anti-kanamycin. Mice were administered 3 × 10⁻⁶ mcN in 150 μL PBS orally via gavage. 8 CFU of EcN or EcN@SH. Mice administered PBS served as controls. Mice were euthanized at predetermined time points (1, 4, and 24 hours) and jejunal tissue was harvested. To assess bacterial adhesion and colonization, jejunum and their respective mucus layers were collected. Mucus (10 μL) was smeared onto a glass slide and sent for LSCM imaging. Jejunal tissue and mucus were further ground and diluted with 1 mL PBS. A suspension (40 μL) was taken from the tissue and mucus and smeared onto a selective solid LB plate (100 μg / mL kanamycin). The bacteria were incubated overnight at 37°C before counting. To verify the formation of disulfide bonds between thiolized bacteria and mucin in vivo, L-ascorbic acid (1 mg / mL) and thiolized EcN 3 × 10⁻⁶ were further administered orally by gavage in 150 μL PBS. 8 CFU. Mice given an equal amount of naked bacteria served as controls. One hour after gavage, the mice were euthanized, and jejunal tissue was collected, homogenized, and counted on LB agar plates.

[0118] (2) 5-FU-induced mouse model of jejunal mucositis

[0119] Mice (Balb / c, male, 6-8 weeks old) were randomly assigned to four groups and orally administered EcN or EcN@SH (1×10⁻⁶) via gavage. 8 5-FU (150 mg / kg mouse) or PBS was administered intraperitoneally for 5 days. Mice were euthanized on day 3 to collect samples. Healthy mice served as controls. Intestinal length was measured. Serum samples were obtained by centrifugation at 4000g for 5 minutes. TNF-α and IL-6 concentrations were detected using an enzyme-linked immunosorbent assay (ELISA) kit (MultiSciences Biotech, China). Jejunal tissue samples were collected for blinded histopathological analysis. Jejunal samples were fixed with 4% paraformaldehyde, processed according to standard paraffin embedding procedures, sectioned to 4 μm, and then stained with hematoxylin and eosin (H&E) and myeloperoxidase (MPO), as well as immunofluorescence staining with ZO-1 (anti-ZO-1 Alexa Fluor 647) and occludin (anti-occludin Alexa Fluor 488). Villus length and crypt depth in each intestinal region were measured simultaneously based on the HE section results.

[0120] (3) Statistical Analysis

[0121] All statistical analyses were evaluated using GraphPad Prism 8. Statistical differences were determined using Student's t-test (two-tailed) or one-way ANOVA with Fisher's LSD multiple comparisons. Differences between the experimental and control groups were considered statistically significant: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns: not significant.

[0122] Example 1

[0123] Design, preparation, and characterization of surface-thiolized bacteria:

[0124] Mucin is an important, highly glycosylated protein and a major organic component of the mucus layer. It is present at various tissue interfaces and serves as a protective layer covering epithelial cells in many organs, including the nose, trachea, eyes, vagina, and intestines. Mucin possesses cysteine-rich domains at its N and C terminals, and its chain extension is mediated by end-to-end disulfide bonds of mucin monomers. Figure 1Simultaneously, the cysteine-rich regions of mucin are abundant as internal structural domains, facilitating the formation of disulfide bond side chains between cysteine ​​thiol groups within the mucin. Exogenous free thiol groups can exchange with disulfide bond groups present in mucin to form new disulfide bonds. As a proof-of-concept study, we chose the EcN strain as a model bacterium because it can regulate the homeostasis of the host microbiome and, along with other probiotics, regulate the secretion of related enzymes, local immunity, and digestion. Surface thiolation of EcN was carried out via a simple one-step chemical reaction with 2-iminothiacyclopentane (Traut's reagent), a water-soluble cyclic thioimine ester. After mixing with phosphate-buffered saline (PBS, pH 7.4) at room temperature, 2-iminothiacyclopentane readily reacts with the primary amino groups of N-terminal and lysine residues in peptides / proteins present on the bacterial surface, thereby introducing free thiol groups. To ensure that the effect of the reaction reagents on bacterial viability and proliferation was negligible, the reaction time for surface thiolation was first optimized. The cytotoxicity of 2-iminothiacyclopentane to EcN was assessed by bacterial counting on selective LB agar plates after different time intervals of reaction with 2-iminothiacyclopentane. Figure 2 As shown in figure a, the activity of EcN decreased with reaction time, and a significant decrease in bacterial activity was observed after the reaction time was extended to 150 minutes. Further screening was conducted at 2-iminothionecyclopentane concentrations ranging from 0 to 400 μg / mL using a reaction time of 90 minutes. Figure 2 b and Figure 3 The trend of viability changes plotted in the figure shows dose-dependent cytotoxicity. It is noted that almost no effect on bacterial viability was observed at concentrations below 100 μg / mL. To examine whether surface thiolation affects EcN growth, thiolated EcN (EcN@SH) was cultured with 100% LB and monitored at 600 nm (OD). 600 The absorbance values ​​at the specified point recorded the growth curve over 11 hours, indicating that concentrations below 50 μg / mL had no effect on bacterial proliferation. Figure 2 c). Furthermore, we determined the bacterial thiolization level using 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB, Ellman's reagent). The biocompatibility of DTNB was first assessed by incubating cells at a working concentration (0.1 mM DTNB) for 2 hours. Figure 4 As shown, the number of viable cells remained unchanged after DTNB treatment, indicating that the cells did not lyse and possessed intact bacterial membranes during DTNB treatment. The amount of surface thiol groups was calculated based on a standard curve obtained from serially diluted solutions of L-cysteine ​​hydrochloride hydrate. Figure 5 ).like Figure 2As shown in d, even under the action of a low concentration of 2-iminothionecyclopentane (10 μg / mL), the number of sulfhydryl groups on a single bacterium increased by 26.5%, with each bacterium adding up to 1.4 × 10⁻⁶ sulfhydryl groups. 7 The level of thiolation increased in a dose-dependent manner, increasing by 50% at a concentration of 88.3 μg / mL for 2-iminothiacyclopentane. Figure 6 With increasing concentrations to 25 and 50 μg / mL, while having almost no effect on bacterial viability and growth, the number of thiol groups on EcN@SH increased significantly by 1.7 and 2.4 times compared to natural EcN, with newly generated thiol groups increasing to 3.8 × 10⁻⁶. 7 and 7.6×10 7 Count / bacterium. Therefore, to balance the relationship between thiolation level and bacterial viability, unless otherwise specified, subsequent experiments were conducted under suitable conditions of 25 μg / mL 2-iminothiocyclopentane and reaction in PBS at room temperature for 90 minutes. The reaction with 2-iminothiocyclopentane maintained charge characteristics similar to the original amino group. Figure 7 a). Flow cytometry was used to determine amine levels using Cy5-N-hydroxysuccinimide (Cy5-NHS), further verifying that the number of amines on thiol-modified bacteria remained unchanged. Figure 8 Simultaneously, dynamic light scattering (DLS) was applied to measure the size after thiolation. Figure 9 The results showed no significant change in particle size and a slight decrease in zeta potential of 2 to 4 mV, depending on the level of surface thiolation. Figure 2 e). The decrease in surface potential and the newly generated S - The negative charge is consistent with that of thiol groups attached to the bacterial surface. Overall, the above results indicate that, under cytocompatibility conditions, 2-iminothiacyclopentane can effectively convert primary amino groups on the bacterial surface into thiol groups.

[0125] Example 2

[0126] The reactivity of introducing thiol groups and the universality of surface thiolation:

[0127] The reactivity and location of newly added thiol groups were analyzed using flow cytometry and laser scanning confocal microscopy (LSCM), with Cy5,5-maleimide (Cy5,5-maleimide) used as a selective click labeling reagent. Figure 2As shown in f (left figure), after reacting with Cy5.5-maleimide at room temperature for 90 minutes, native EcN exhibited a slight fluorescence shift, which is due to the reaction of the intrinsic thiol group with the maleimide group of the fluorescent dye. In contrast, EcN treated with 25 μg / mL 2-iminothiacyclopentane underwent a near-complete reaction and shift with Cy5.5-maleimide, and the average fluorescence intensity (MFI) of Cy5.5 increased by approximately 4.1 times. Figure 2 (f. Right figure). More importantly, the LSCM image clearly shows that a Cy5.5-maleimide-labeled thiol base layer was formed on the surface of a single EcN@SH expressing GFP. Figure 2 g and Figure 10 The thiol signal was 12.3 times higher than that of naked bacteria. Figure 11 This result clarifies that thiolation mediated by 2-iminothiacyclopentane does indeed occur primarily on the bacterial surface. Notably, although intrinsically free thiols were detected in bacteria, based on flow cytometry and LSCM results, the intrinsic thiols showed relatively lower reactivity to Cy5,5-maleimide compared to chemically transformed thiols. This finding can be explained by the fact that most naturally occurring thiols in bacteria are embedded in complex three-dimensional protein structures, while chemically transformed thiols have a different reactivity with bacteria. The distance between the groups allows for greater flexibility in the reaction of these converted thiol groups. Total protein extracted from EcN and EcN@SH was visualized and analyzed using Coomassie staining. The overall bacterial protein composition remained unaffected, and no additional products were observed after thiolization. Figure 12 We further selected tryptophan metabolism to explore the metabolic capabilities of thiolated bacteria. Many bacteria can metabolize tryptophan into specific metabolites, such as indole and its derivatives, serotonin, and D-tryptophan, which are bioactive substrates that play many functions in the gastrointestinal system, including immune regulation and intestinal barrier protection. The indole assay was used to assess the ability of thiolated bacteria to degrade tryptophan into indole by producing tryptophanases, such as... Figure 13 As shown, thiolated EcN exhibited similar positive results to the native bacteria, producing a purple indole ring on the ether layer upon addition of Kovac reagent. The concentration of the generated indole was also quantified by high-performance liquid chromatography (HPLC). Interestingly, no difference was shown in the metabolic activity, i.e., indole production, between native and thiolated bacteria, confirming that the effect of thiolation on bacterial tryptophan metabolism is negligible. Figure 13 ).

[0128] We further validated the universality and cell compatibility of this method for thiolation of other bacteria. Several strains, including Gram-negative Akkermansia myxophilus (AKK) and Salmonella typhimurium (STM), and Gram-positive Enterococcus faecalis (EF), Bacillus megaterium (BMB), and Bacillus cereus (BC), were thiolated under optimized conditions. Compared to EcN@SH, the zeta potentials of AKK, STM, EF, BMB, and BC decreased from -32.0±0.3, -26.2±1.6, -26.0±0.4, -36.0±0.4, and -32.8±0.7 to -33.3±0.8, -28.3±0.8, -27.3±1.1, -42.0±0.4, and -40.2±0.3 mV, respectively. Figure 14 After reaction with Cy5,5-maleimide, the MFI values ​​of thiolated AKK, STM, EF, BMB, and BC increased significantly by 3.1, 4.1, 35.9, 39.6, and 31.1 times, respectively. Figure 15 a, b, e, f, i, j, m, n, q, and r). Interestingly, after reaction with Cy5,5-maleimide, the fluorescence signal heterogeneity among different bacteria was relatively low, with MFI ranging from 2000 to 8000, which is attributed to the varying numbers of intrinsic thiol groups ( Figure 15 b, f, j, n, and r). LSCM imaging also revealed the presence and location of thiol groups, showing a uniform Cy5.5 labeling layer around each thiolized bacterium. Figure 15 c, g, k, o, and s), while the signal on natural bacteria is negligible. Figure 15 , 16 And 17). It was found that the sulfhydrylation level of Gram-positive bacteria was approximately 8 to 20 times higher than that of Gram-negative bacteria. Figure 15 b, f, j, n, and r). This is likely because Gram-positive strains have more primary amines on their surface, as previously reported. Similarly, no negative impact on the viability and growth of these thiolated bacteria was observed, further supporting the high biocompatibility of this thiolation strategy. Figure 15 (d, h, l, p, and t). In summary, this biocompatible thiolation method is applicable to a variety of bacterial species, and its surface can be endowed with a large number of reactive thiols for further reactions.

[0129] Example 3

[0130] The reaction of surface-thiolized bacteria with mucin:

[0131] After confirming the reactivity of thiol groups on the surface of thiolized bacteria, we then investigated the catalyst-free reaction between EcN@SH and mucin through dynamic sulfur-sulfide exchange. Figure 18First, flow cytometry was used to explore the time-dependent decay of free thiol groups on the surface of PBS. Figure 19 As shown, half of the newly formed thiols disappear rapidly within 2 hours, but the thiolation level decreases more slowly after 2 hours and recovers to the same level as natural EcN within 12 hours. Figure 19 This study revealed that newly formed thiol groups can be retained on EcN@SH for a relatively long time. For ease of detection, the mucin was first labeled with N-hydroxysuccinimide-activated Cy5.5 (Cy5.5_mucin), and the product was purified by ultrafiltration. After incubating EcN@SH with 0.03 mg / mL Cy5.5_mucin for 1 hour, the MFI of Cy5.5_mucin in single bacteria was significantly increased compared to that in native bacteria. Figure 18 b). As the mucin concentration increased to 0.1 mg / mL ( Figure 20 A similar increase was also observed, indicating increased binding of Cy5.5_mucin to thiolized bacteria. To investigate the effect of thiolization levels on mucin attachment, we varied the density of thiol groups on the bacterial surface and found that as the number of thiol groups increased from 5.5 × 10⁻⁶ per cell... 7 Increased to 9.3 × 10 7 The MFI of Cy5.5_mucin attached to the surface of EcN is significantly increased ( Figure 18 (c and d). Thiolization-level dependent mucin attachment indicates that the interaction between thiolized bacteria and mucin can be easily modulated by altering the number of surface thiol groups. To further verify the involvement of thiol groups in chemically mediated mucin attachment, we used thiol-sensitive Cy5.5-maleimide to record changes in the number of thiol groups after reaction with mucin. The results were as expected; after incubation with mucin, the amount of thiol represented by the Cy5.5 fluorescence intensity of thiolized EcN was significantly reduced, demonstrating the consumption of free thiol groups during the reaction. Figure 18 e and f). Furthermore, DLS measurements of changes in bacterial size and surface potential after mucin attachment showed an increase in particle size compared to native EcN. Figure 21 ), and the zeta potential decreased slightly by about 1-2 mV ( Figure 21 and 22 This further demonstrates the covalent bonding of mucin to thiolized bacteria. To verify the formation of disulfide bonds between thiolized bacteria and mucin, considering that the thiol-disulfide bond exchange reaction can be inhibited by a reducing agent... Figure 18 Therefore, L-ascorbic acid was added to the reaction system during the reaction. At an L-ascorbic acid concentration of 0.3 mg / mL, the MFI on the bacterial surface of thiolated EcN significantly decreased after reacting with Cy5.5_mucin, and further decreased with increasing concentration to 1 mg / mL (g). Figure 18These results indicate that the binding of Cy5_mucin to thiolated bacteria is affected by L-ascorbic acid, which is attributed to the blocking of the thio-disulfide bond exchange reaction by the reducing agent. Simultaneously, similar results were obtained by LSCM imaging, showing that the attachment of fluorescently labeled Cy5_mucin to thiolated EcN was significantly inhibited after the addition of L-ascorbic acid. Figure 18 j). These results are highly consistent with previous reports, such as the ability of reducing agents to inhibit thiol-disulfide bond exchange-mediated cellular uptake. Furthermore, dithiothreitol (DTT), which specifically reduces disulfide bonds, was used to cleave newly formed disulfide bonds between EcN@SH and mucin. Figure 23 As shown, the mucin attached to EcN@SH can be completely cleaved by DTT (0.1 mg / mL), further verifying that the attachment of mucin to EcN@SH is mediated by covalent binding through thiol-disulfide bond exchange. Besides the reducing agent, selenocystamine (SeCA) contains a diselenocysteine ​​bond (-Se-Se), which can act as a substrate mimic and compete with the disulfide bond (-SS-) for thiol exchange, thereby inhibiting the reaction between EcN@SH and mucin. Figure 24 As shown, 0.3 mg / mL SeCA can largely block the binding of mucin to EcN@SH, further indicating the existence of thiol-disulfide bond exchange between EcN@SH and mucin. In short, these data confirm the reactivity of thiol-modified bacteria with cysteine-rich mucin to form new disulfide bonds through sulfur-sulfide exchange.

[0132] Example 4

[0133] Covalently assisted colonization of thiolized bacteria in the mucus layer:

[0134] The jejunal microbiota has been found to be closely associated with diabetes, obesity, and mucositis induced by alcohol, radiation, and chemotherapy. However, due to unfavorable microenvironment and physical factors, the jejunum has always been a challenging site for microbiota colonization, as evidenced by its extremely low 10-1 3 -10 5 As reflected by the CFUs / mL bacterial abundance level, this is consistent with 10 in the colon. 11 -10 12 The high bacterial abundance (CFUs / mL) contrasts sharply with the jejunal colonization barrier. Therefore, overcoming the barrier to bacterial colonization in the jejunum is crucial for enhancing the effectiveness of intestinal microbiota transplantation. Inspired by the abundant disulfide-rich mucus layer covering the jejunal wall, we hypothesized that the covalent bonds between thiolized bacteria and mucins might promote bacterial adhesion and colonization. To test this hypothesis, mouse jejunum samples were everted and exposed to thiolized EcN expressing mCherry for 1 hour, and the fluorescence intensity of bacteria attached to the jejunal mucosa was measured using an in vivo imaging system (IVIS). Figure 25 a). Compared to native EcN, the jejunal segment exposed to EcN@SH exhibited a stronger fluorescence signal, indicating enhanced adhesion of thiol-modified bacteria to the mucosal layer. Figure 25 b). Quantitative analysis also showed a significant increase in EcN@SH adhesion compared to natural EcN. Figure 25 c) This demonstrates that EcN@SH can adhere to the mucus layer via a sulfur-sulfur exchange reaction. Further homogenization of the cut jejunum segment for bacterial counting showed a 6.9-fold increase in the number of EcNs attached to the jejunal mucosa. Figure 25 d). Encouraged by previous results, we also evaluated the effectiveness of covalently mediated attachment in isolated porcine jejunum. Pigs are arguably one of the most suitable models to mimic human organ systems, particularly the skin and mucosal systems, offering numerous translational advantages in preclinical models. The adhesion of EcN@SH to the jejunal mucosa was evaluated using corresponding tissues from Bama miniature pigs weighing approximately 20 kg. Significantly higher retention of fluorescence signal was detected in thiolized EcN compared to native EcN. Figure 25 e and f), further plate counting revealed a 3.5-fold increase in thiol-modified bacterial attachment (e and f). Figure 25 g). Then, we explored the in vivo adhesion and colonization of EcN@SH in mice after oral administration. Using 1.0 × 10⁻⁶ g⁻¹… 8 Jejunal tissue and mucus were collected at 1, 12, and 24 hours after EcN@SH administration of CFU. PBS and unmodified native EcN served as controls. Jejunal mucus collected 1 hour after gavage was observed by LSCM. To distinguish between administered EcN and endogenous bacteria, EcN was transfected with a plasmid containing the mCherry gene and kanamycin resistance. As expected, no EcN was observed in the mucus samples taken from mice administered PBS. Figure 25 Although fluorescent EcN was observed in unmodified EcN gavage mouse samples, the corresponding bacterial counts were extremely low. In contrast, abundant red fluorescent EcN@SH was observed in the jejunal mucus of mice administered EcN@SH. The localization of EcN and EcN@SH in the jejunal tissue and mucus was quantified by plate counting at different time intervals. Figure 25 i). like Figure 25 As shown in Figure j, at all specified time points, the number of EcN@SH retained in the jejunal mucus significantly exceeded the number of unmodified EcN. One hour after gavage, the number of EcN@SH colonized in the jejunal mucus reached as high as 1.7 × 10⁻⁶. 7The CFUs / g concentration was 171.3 times that of mice with natural EcN. Even after a prolonged post-administration time of 24 hours, the localization increment of thiolized EcN in jejunal mucus remained as high as 44.0-fold compared to mice treated with natural EcN. Comparable increment trends were obtained from sampled jejunal tissue, with increments of 40.6, 15.9, and 9.6-fold at 1, 4, and 24 hours post-gavage, respectively. Figure 25 k and Figure 26 This further demonstrates that EcN@SH can increase adhesion and colonization in the mouse jejunum through a sulfur-sulfur exchange reaction. When L-ascorbic acid (1 mg / mL) was administered orally with thiolated EcN, the reducing agent blocked the sulfur-sulfur exchange reaction. Similarly, EcN@SH localized in mouse jejunal mucus was significantly increased compared to native EcN, and the adhesion of EcN@SH was significantly reduced by approximately 88.1 times when L-ascorbic acid was used in combination. Figure 27 This further validated that covalent bonding is involved in the adhesion of thiolized bacteria to the jejunal mucus layer. Furthermore, mouse intestinal length and hematoxylin and eosin (H&E) staining demonstrated that thiolized bacterial transplantation did not induce detectable damage or inflammatory responses. Figure 28 and Figure 29 ).

[0135] Example 5

[0136] Thioylated EcN alleviates enterocolitis:

[0137] Finally, we turned our attention to assessing whether thiol-modified bacteria could be used to alleviate inflammation associated with jejunal mucositis, a side effect of chemotherapy, radiotherapy, and antimetabolites. EcN is already used clinically to treat inflammatory bowel disease, and we hypothesized that transplanting EcN@SH could increase adhesion and colonization by enhancing mucus chemistry, thereby promoting the relief of jejunal mucositis. Figure 30 To demonstrate this, thiolated EcN was evaluated in a 5-fluorouracil-induced (5-FU-induced) mouse model of jejunal mucositis, characterized by shortened intestinal and villus length, cytokine abnormalities, and histopathological damage to intestinal tissue. Figure 31 ). 1×10 mmol / L per mouse per day 8 Five days after administration of CFUs to EcN@SH, mice were euthanized to collect samples. Healthy mice served as positive controls, while PBS and equivalent unmodified EcN served as negative controls. Figure 30As shown in b, the beneficial effects of EcN administration alone were negligible, reflected in the lack of a significant increase in intestinal length compared to PBS mice. Notably, the intestinal length of mice administered EcN@SH by gavage was comparable to the positive control and significantly longer than both the PBS and EcN groups. Compared to the two negative controls, the expression levels of inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were also significantly reduced in mice administered EcN@SH. Figure 30 c and Figure 32 Furthermore, myeloperoxidase (MPO) staining was performed to assess inflammation in mouse jejunal tissue, and after EcN@SH treatment, the number of MPO-positive cells observed in jejunal lesions was significantly reduced. Figure 33 Furthermore, based on H&E staining of jejunal sections, EcN@SH more effectively alleviated inflammatory responses, vacuolation, and edema in the jejunum. Figure 30 d). Histopathological analysis showed that, in all treatment groups, mice treated with EcN@SH exhibited lower disturbances in villus length and crypt depth. Figure 30 e.g., in two negative control groups, immunofluorescence staining of jejunal tissue showed reduced and then ceased expression of ZO-1 and occludin, but this could be significantly reversed by EcN@SH. Figure 30 (h) Elevated tight junction protein levels revealed the protective role of EcN@SH in restoring intestinal barrier integrity. Similar to healthy mice, restoration of crypt depth and intestinal and villus length, relief of inflammation, reduction of histopathological damage, and repair of the intestinal barrier confirmed the value of thiol-modified bacteria in covalently mediated microbial therapy.

[0138] In summary, we have described a chemical strategy for manipulating bacterial-environment interactions through covalent bonding. This approach involves several unique features: (1) the concept of chemically-mediated live bacterial agents; (2) the concept of covalently-mediated bacterial-environment interactions; (3) the first example of in vivo in situ chemical reactions leading to bacterial adhesion and colonization; and (4) the first successful targeted transplantation of microorganisms into the jejunum. In short, our work opens a new window for chemically modulating microbiome-host interactions, providing methods and scientific justification for the preparation of various innovative live bacterial formulations for a wide range of biomedical applications.

[0139] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for chemically controlled covalent colonization of thiol-modified bacteria, characterized in that, For non-diagnostic and therapeutic purposes, including: after incubating thiolized bacteria with proteins or protein-containing tissues in vitro under catalyst-free conditions, the thiol groups on the surface of the bacteria undergo an exchange reaction with the disulfide bonds in the protein to form a covalent bond; The method for preparing the thiolized bacteria is as follows: bacteria are co-incubated with the thiolizing agent 2-iminothiacyclopentane and buffer at room temperature. The thiolizing agent reacts with the primary amino groups of the N-terminus and / or lysine residues in peptides or proteins on the bacterial surface, thereby introducing free thiol groups and obtaining surface thiolized bacteria. The bacteria include at least one of Escherichia coli (EcN), Gram-negative Akkermansia myxophilus (AKK), Salmonella typhimurium (STM), and Gram-positive Enterococcus faecalis (EF), Bacillus megaterium (BMB), and Bacillus cereus (BC).

2. The method for chemically controlled covalent colonization of thiol-modified bacteria as described in claim 1, characterized in that, The protein in question is a mucin.

3. The method for chemically controlled covalent colonization of thiol-modified bacteria as described in claim 1, characterized in that, The buffer solution is a phosphate buffer with a pH of 7-8.

4. A thiol-modifying bacterium, characterized in that, The thiolized bacteria are obtained by co-incubating bacteria with the thiolizing agent 2-iminothiacyclopentane and buffer at room temperature. The thiolizing agent reacts with the primary amino groups of the N-terminus and / or lysine residues in peptides or proteins on the bacterial surface, thereby introducing free thiol groups. The bacteria include at least one of Escherichia coli (EcN), Gram-negative Akkermansia myxophilus (AKK), Salmonella typhimurium (STM), and Gram-positive Enterococcus faecalis (EF), Bacillus megaterium (BMB), and Bacillus cereus (BC).

5. A probiotic preparation, characterized in that, Its active ingredients include live probiotics with surface thiolized groups; The preparation method of the surface-thiolized live probiotics is as follows: live probiotics are co-incubated with thiolizing reagent 2-iminothiacyclopentane and buffer at room temperature. The thiolizing reagent reacts with the primary amino groups of N-terminal and / or lysine residues in peptides or proteins on the bacterial surface, thereby introducing free thiol groups and obtaining surface-thiolized bacteria. The probiotics include at least one of Escherichia coli EcN, Gram-negative Akkermansia myxophilus AKK, Salmonella typhimurium STM, and Gram-positive Enterococcus faecalis EF, Bacillus megaterium BMB, and Bacillus cereus BC.

6. The use of a probiotic preparation in the preparation of a medicament for the treatment or adjunctive treatment of inflammation related to small intestinal mucositis, wherein the active ingredient of the probiotic preparation is surface-thiolated Escherichia coli EcN; the preparation method of the surface-thiolated Escherichia coli EcN is as follows: Escherichia coli EcN is co-incubated with a thiolation reagent 2-iminothiacyclopentane and a buffer at room temperature, and the thiolation reagent reacts with the primary amino groups of the N-terminus and / or lysine residues in the peptides or proteins on the surface of Escherichia coli EcN, thereby introducing free thiolation groups to obtain surface-thiolated Escherichia coli EcN.

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