Activating Effect of Staphylococcal Enterotoxins

The effect of enterotoxins is to induce the production of endogenous IF. St. aureus enteropathogenic proteins (enterotoxins) possess an antitumour effect. After intraperitoneal inoculation, they decrease the size and, in some cases, prevent the development of the human hypernephroma in the cheek pouch of golden hamsters. The effect of enteropathgenic proteims may possibly consist in inducing the production of endogenous immune interferon which activates the host immune system and enhances the rejection of heterologous tumour cells.

Studies on Enterotoxins and Antimicrobial Resistance in Staphylococcus aureus Isolated from Various Sources

Staphylococcus aureus represents a public health challenge all over the world. Therefore, this study aims to analyze the prevalence of five genes (sea, seb, sec, see and seg) encoding the staphylococcal enterotoxins in S. aureus isolated from different sources and to evaluate the association of these toxins in comparison to susceptibility towards 12 antimicrobials;antimicrobial susceptibility was conducted by disc diffusion method. Detection of staphylococcal enterotoxins was performed by PCR and the ability to express these genes was assessed among isolates by RT-PCR. The most common enterotoxin gene was sea gene (66%), followed by seb, sec, see and seg (38%, 23%, 19% and 5%) respectively. Expression of sea, seb and seg genes was variable. However, sec and see genes were not expressed by any of the tested isolates. No statistically significant association exists between (seb, sec and see) and isolation sources, while the sea was significantly associated with clinical isolates. High significant correlation was found between elevated sea expression and multidrug-resistance. Our findings indicate that the pathogenic potential of S. aureus may be greater than previously thought. This emphasizes the utmost need to implement proactive measures and more emphasis will be placed on the application of hygiene practices in hospitals to control S. aureus infection and enterotoxins production.

Detection and Analysis on StaphylococcalEnterotoxins in Raw Cow Milk from DifferentRegions of Jinan, China

[Objective] This study was conducted to investigate the pollution caused by staphylococcal enterotoxins(SE) in raw milk and the safety of dairy products in Jinan, and to provide a scientific basis for food safety risk analysis. [Method] A total of 130 raw milk samples were collected from different regions of Jinan, and detected for Staphylococcus aureus by referring to GB4789.10-2010. Then, enzyme-linked immunosorbent assay was performed to detect whether the S.aureus strains produced enterotoxins, and the enterotoxin type was identified using colloidal gold-based immunochromatographic test strips. [Result] Fiftyseven of the raw milk samples were polluted by S.aureus, so the detection rate of S.aureus was 43.85%; and 11 of the strains produced enterotoxins. Among the 11 enterotoxin-producing strains, seven produced SEB, only one produced SEC, and the SE type of other three strains was not identified. [Conclusion] Enzyme-linked immunosorbent assay and colloidal gold-based immunochromatographic test strips can be used in combination to rapidly detect staphylococcal enterotoxins and identify enterotoxin type, although there are some limitations. SEB is the main type of staphylococcal enterotoxin causing pollution in milk of Shandong Province.

Temperature-Induced Unfolding Pathway of Staphylococcal Enterotoxin B:Insights from Circular Dichroism and Molecular Dynamics Simulation

In this study,circular dichroism(CD)and molecular dynamics(MD)simulation were used to investigate the thermal unfolding pathway of staphylococcal enterotoxin B(SEB)at temperatures of 298–371 and 298–500 K,and the relationship between the experimental and simulation results were explored.Our computational findings on the secondary structure of SEB showed that at room temperature,the CD spectroscopic results were highly consistent with the MD results.Moreover,under heating conditions,the changing trends of helix,sheet and random coil obtained by CD spectral fitting were highly consistent with those obtained by MD.In order to gain a deeper understanding of the thermal stability mechanism of SEB,the MD trajectories were analyzed in terms of root mean square deviation(RMSD),secondary structure assignment(SSA),radius of gyration(R_(g)),free energy surfaces(FES),solvent-accessible surface area(SASA),hydrogen bonds and salt bridges.The results showed that at low heating temperature,domain Ⅰ without loops(omitting the mobile loop region)mainly relied on hydrophobic interaction to maintain its thermal stability,whereas the thermal stability of domain Ⅱ was mainly controlled by salt bridges and hydrogen bonds.Under high heating temperature conditions,the hydrophobic interactions in domain Ⅰ without loops were destroyed and the secondary structure was almost completely lost,while domain Ⅱ could still rely on salt bridges as molecular staples to barely maintain the stability of the secondary structure.These results help us to understand the thermodynamic and kinetic mechanisms that maintain the thermal stability of SEB at the molecular level,and provide a direction for establishing safer and more effective food sterilization processes.

Electrochemical and colorimetric dual-signal detection of Staphylococcus aureus enterotoxin B based on AuPt bimetallic nanoparticles loaded Fe-N-C single atom nanocomposite

Sensitive detection of Staphylococcus aureus enterotoxin B(SEB)is of importance for preventing food poisoning from threatening human health.In this work,an electrochemical and colorimetric dual-signal detection assay of SEB was developed.The probe(Ab2/AuPt@Fe-N-C)was bound to SEB captured by Ab1,where the Ab2/AuPt@Fe-N-C triggered methylene blue degradation and resulted in the decrease of electrochemical signal.Furthermore,the probe catalyzed the oxidation of 3,3’,5,5’-tetramethyl biphenyl to generate a colorimetric absorbance at 652 nm.Once the target was captured and formed a sandwich-like complex,the color changed from colorless to blue.SEB detection by colorimetric and electrochemical methods showed a linear relationship in the concentration ranges of 0.0002-10.0000 and 0.0005-10.0000 ng/mL,with limits of detection of 0.0667 and 0.1670 pg/mL,respectively.The dual-signal biosensor was successfully used to detect SEB in milk and water samples,which has great potential in toxin detection in food and the environment.

Preparation and activity of conjugate of monoclonal antibody HAb18 against hepatoma F( ab′ )_2 fragment and staphylococcal enterotoxin A

AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.

Guanylyl cyclase C signaling axis and colon cancer prevention

Colorectal cancer(CRC) is a major cause of cancerrelated mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C(GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin(GUCA2A) and uroguanylin(GUCA2B), which bind and activate GUCY2 C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2 C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2 C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C's role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2 C ligand linaclotide(Linzess^(TM)). Here we review the known contributions of the GUCY2 C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention.

Virulence of <i>Aeromonas hydrophila</i>Isolated from Fresh Water Catfish

Background: A large proportion of Nigerians consume fish as the source of protein in their meals. This may be attributed to health factors, preference and affordability for low income earners. The incidence of Aeromonas hydrophila in fresh catfish may constitute a significant health risk to the consumer if there is a horizontal transfer to man as it has been reported to be pathogenic. This study examined the possibility of fresh water catfish being a reservoir of pathogenic Aeromonas hydrophila. Method: Aeromonas hydrophila was isolated from the different organs of fresh water catfish (Clarias gariepinus and Ictalurus punctatus) obtained from Kporoko river in Lokoja. Aeromonas hydrophila was identified using both phenotypic and genotypic methods. The pathogenic traits of the Aeromonas species such as biofilm formation, production of haemolysin, enterotoxin and enzymes were determined. Results: Aeromonas hydrophila occurred in all the examined fish organs (fish, liver, kidney, skin and gut) of Clarias gariepinus but occurred only in the skin, intestine, kidney and gut of Ictalurus punctatus examined, but the incidence of Aeromonas hydrophila was prevalent in the gut of all the fishes analyzed. All the Aeromonas isolates analysed in this study produced biofilm, haemolysins and lipase enzymes. They also produced enterotoxins with values ranging between 0.069 - 1.138. Conclusion: The occurrence of Aeromonas in fresh catfish possessing these pathogenic traits is of great public health significance to man as it indicates the likelihood of man being predisposed to toxigenicity when the toxin concentration reaches a lethal value. It is therefore recommended that the internal organs of fresh catfish be thoroughly cleaned and cooked before consumption.

Studies on Pongamia pinnata (L.) Pierre leaves: understanding the mechanism(s) of action in infectious diarrhea

While data are available on the effect of medicinal plants on intestinal motility and their antibacterial action, there is a paucity of information on their mode of action on various aspects of diarrheal pathogenicity, namely colonization to intestinal epithelial cells and production/action of enterotoxins. Crude decoction of dried leaves of Pongamia pinnata was evaluated for its antimicrobial (antibacterial, antigiardial and antirotaviral) effect; and its effect on production and action of enterotoxins (cholera toxin, CT; Escherichia coli labile toxin, LT; and E. coli stable toxin, ST); and adherence of enteropathogenic E. coli and invasion of enteroinvasive E. coli and Shigella flexneri to epithelial cells. The decoction had no antibacterial, antigiardial and antirotaviral activity, but reduced production of CT and bacterial invasion to epithelial cells. The observed results indicated that the crude decoction of P. pinnata has selective antidiarrheal action with efficacy against cholera and enteroinvasive bacterial strains causing bloody diarrheal episodes.

Effect of Clostridium perfringens enterotoxin on gastric cancer cells SGC7901 which highly expressed claudin-4 protein

AIM To investigate the effects of Clostridium perfringens enterotoxin(CPE) on gastric cancer cells which highly expressed claudin-4(CL4) protein.METHODS In this study, we detected expression of CL4 protein in different gastric cancer cell lines. Then, we investigated the effects of CPE on SGC7901 cells which highly expressed CL4 protein and the effects of CPE on sub-cutaneous tumor in nude mice models.RESULTS CL4 are highly expressed in SGC7901 cells. CPE expressedsignificant cytotoxicity in SGC7901 cells. Suppression of CL4 expression significantly decreased CPE-mediated cytotoxicity. CPE also inhibited tumor growth in subcutaneous tumor xenograft models.CONCLUSION CPE showed CL4 mediated cytotoxicity on gastric cancer cells SGC7901 and inhib-ited tumor growth in nude mice models.

Occurrence and characterization of toxigenic Bacillus cereus in food and infant feces

Objective: To investigate the true incidence of Bacillus cereus(B. cereus) in food and children diarrhea cases. Methods: A total of 110 samples of various dairy products such as raw milk, long life pasteurized milk, yoghurt and infant powdered milk formulas, raw rice, and feces were examined for the presence of B. cereus by selective plating on mannitol-egg-yolk-polymyxin agar. Confirmation of B. cereus was carried out by biochemical tests and PCR. Identification of non-B. cereus isolates was carried out by 16 S r DNA sequencing. Antimicrobial susceptibility was done by disk diffusion method.Results: Overall 35 samples(31.8%, n = 110) yielded Bacillus-like growth. Of which 19 samples(54.28%) were positive for B. cereus. All isolates were positive for enterotoxin production. No psychrotolerant B. cereus strains were detected in all samples. All B. cereus isolates were resistant to penicillin G, but susceptible to vancomycin, erythromycin and clindamycin. Conclusions: The results of this study confirm the importance of including B. cereus in disease control and prevention programs, as well as in routine clinical and food quality control laboratories in both Saudi Arabia and Egypt.

Contruction of the Genetic Engineering Strain Expressed Nontoxic ST_1-LT_B Fusion Protein Against Enterotoxigenic Eschenichia coli

Thermostable enterotoxinⅠ(ST1) mutant genes and thermolabile enterotoxin B subunit (LTB)genes were amplified by PCR from plasmids of Eschenichia coli C83902. The recombinantexpression plasmid pZST3LTB containing ST1-LTB fusion gene was constructed by recombinantDNA technique and then transformed into Escherichia coli BL21(DE3). The ST1-LTB fusionprotein was highly expressed in recombinant strain BL21(DE3)(pZST3LTB) and the fusionprotein was about 38.53% of total cellular protein by SDS-PAGE and thin-layer gelscanning analysis. More important, mice immunized with crude preparation containing thefusion protein inclusion bodies or inactivated recombinant strain produced antibodiesthat were able to recognize ST1 in vitro. These sera antibodies were able to neutralizethe biological activity of native ST1 in the suckling mouse assay. Hence the ST1-LTBfusion protein was nontoxic and immunogenic, the constructed recombinant strain BL21(DE3)(pZST3LTB) could be used as a candidate of vaccine strain.

Recent developments on the role of Clostridium difficile in inflammatory bowel disease

Clostridium difficile (CD), specifically its toxins, have been implicated as a risk factor for exacerbation of the inflammatory process in up to 5% of patients with ulcerative colitis or Crohn’s disease. Typical evidence of colonic changes with CD infection, including pseudomembranous exudate, are often not present; however, a severe clinical course may result, including precipitation of toxic colitis and toxic megacolon. Recently, hypervirulent CD strains have been reported raising concern for a more severe disease process in patients with underlying inflammatory bowel disease.Moreover, small bowel involvement or CD enteritis has been increasingly described, usually in those with a history of a prior colectomy or total proctocolectomy for prior severe and extensive inflammatory bowel disease. Finally, refractory or treatment-resistant pouchitis may occur with CD infection.

Predictive Modeling for Growth and Enterotoxin Production of Staphylococcus aureus in Milk

Predictive microbiology was utilized to model Staphylococcus aureus （S. aureus） growth and staphylococcal enterotoxin A （SEA） production in milk in this study. The modifed logistic model, modifed Gompertz model and Baranyi model were applied to model growth data of S. aureus between 15℃ and 37℃. Model comparisons indicated that Baranyi model described the growth data more accurately than two others with a mean square error of 0.0129. Growth rates generated from Baranyi model matched the observed ones with a bias factor of 0.999 and an accuracy factor of 1.01, and ft a square root model with respect to temperature; other two modifed models both overestimated the observed ones. SEA amount began to be detected when the cell number reached106.4 cfu ？ mL-1, and showed the linear correlation with time. Besides, the rate of SEA production ftted an exponential relationship as a function of temperature. Predictions based on the study could be applied to indicate possible growth of S. aureus and prevent the occurrence of staphylococcal food poisoning.

Bacteroides fragilis enterotoxin upregulates heme oxygenase-1 in dendritic cells via reactive oxygen species-,mitogen-activated protein kinase-,and Nrf2-dependent pathway

BACKGROUND Enterotoxigenic Bacteroides fragilis(ETBF)causes colitis and diarrhea,and is considered a candidate pathogen in inflammatory bowel diseases as well as colorectal cancers.These diseases are dependent on ETBF-secreted toxin(BFT).Dendritic cells(DCs)play an important role in directing the nature of adaptive immune responses to bacterial infection and heme oxygenase-1(HO-1)is involved in the regulation of DC function.AIM To investigate the role of BFT in HO-1 expression in DCs.METHODS Murine DCs were generated from specific pathogen-free C57BL/6 and Nrf2−/−knockout mice.DCs were exposed to BFT,after which HO-1 expression and the related signaling factor activation were measured by quantitative RT-PCR,EMSA,fluorescent microscopy,immunoblot,and ELISA.RESULTS HO-1 expression was upregulated in DCs stimulated with BFT.Although BFT activated transcription factors such as NF-κB,AP-1,and Nrf2,activation of NF-κB and AP-1 was not involved in the induction of HO-1 expression in BFT-exposed DCs.Instead,upregulation of HO-1 expression was dependent on Nrf2 activation in DCs.Moreover,HO-1 expression via Nrf2 in DCs was regulated by mitogenactivated protein kinases such as ERK and p38.Furthermore,BFT enhanced the production of reactive oxygen species(ROS)and inhibition of ROS production resulted in a significant decrease of phospho-ERK,phospho-p38,Nrf2,and HO-1 CONCLUSION These results suggest that signaling pathways involving ROS-mediated ERK and p38 mitogen-activated protein kinases-Nrf2 activation in DCs are required for HO-1 induction during exposure to ETBF-produced BFT.

Assessment of the inhibitory effects of sodium nitrite, nisin, potassium sorbate, and sodium lactate on Staphylococcus aureus growth and staphylococcal enterotoxin A production in cooked pork sausage using a predictive growth model

This study was conducted to analyze the effects of sodium nitrite,nisin,potassium sorbate,and sodium lactate against Staphylococcus aureus(S.aureus)growth and staphylococcal enterotoxins(SEs)production in cooked pork sausage by inoculating sausage samples containing preservative with an S.aureus strain producing staphylococcal enterotoxin A(SEA)and then storing them at 37℃ for 36 h.Samples were analyzed every 3 h to count the S.aureus colonies and to detect SEA.The modified Gompertz model was used to describe S.aureus growth in the samples under various conditions,and the preservatives with a significant antimicrobial effect were selected.In addition,the antimicrobial effects of the selected preservatives under various concentrations were tested.Results showed that sodium nitrite,nisin,and potassium sorbate had a weak effect against S.aureus growth and had no effect against SEA production,whereas sodium lactate could significantly inhibit S.aureus growth and SEA production.Moreover,the antimicrobial effect of sodium lactate was concentration-dependent,wherein sodium lactate concentration<12 g/kg showed no inhibitory effect,but when the concentration was increased to 24 g/kg,sodium lactate could effectively inhibit S.aureus growth and SEA production,and at 48 g/kg,sodium lactate had a significant inhibitory effect.

Update on molecular diversity and multipathogenicity of staphylococcal superantigen toxins

Staphylococcal superantigen(SAg)toxins are the most notable virulence factors associated with Staphylococcus aureus,which is a pathogen associated with serious community and hospital acquired infections in humans and various diseases in animals.Recently,SAg toxins have become a superfamily with 29 types,including staphylococcal enterotoxins(SEs)with emetic activity,SE-like toxins(SEIs)that do not induce emesis in primate models or have yet not been tested,and toxic shock syndrome toxin-1(TSST-1).SEs and SEIs can be subdivided into classical types(SEA to SEE)and novel types(SEG to SEIY,SE01,SE02,SEI26 and SEI27).The genes of SAg toxins are located in diverse accessory genetic elements and share certain structural and biological properties.SAg toxins are heat-stable proteins that exhibit pyrogenicity,superantigenicity and capacity to induce lethal hypersensitivity to endotoxin in humans and animals.They have multiple pathogenicities that can interfere with normal immune function of host,increase the chances of survival and transmission of pathogenic baaeria in host,consequently contribute to the occurrence and development of various infeaions,persistent infeaions or food poisoning.This review focuses on the following aspeas of SAg toxins:(1)superfamily members of classic and novelty discovered staphylococcal SAgs;⑵diversity of gene locations and molecular structural characteristics;(3)biological characteristics and activities;(4)multi-pathogenicity of SAgs in animal and human diseases,including bovine mastitis,swine sepsis,abscesses and skin edema in pig,arthritis and septicemia in poultry,and nosocomial infections and food-borne diseases in humans.

Construction, Expression and Characterization of a Chimeric Protein Targeting Carcinoembryonic Antigen in Lung Cancer

The carcinoembryonic antigen（CEA） is an oncofetal glycoprotein known as an important clinical tumor marker and is overexpressed in several types of tumors, including colorectal and lung carcinomas. We constructed a chimeric protein that exhibits both specific binding and immune stimulating activities, by fusing staphylococcal enterotoxin A（SEA） to the C-terminus of an anti-CEA single-chain disulfide-stabilized Fv（scdsFv） antibody （single-chain-C-terminus/SEA, SC-C/SEA）. The SC-C/SEA protein was expressed in Escherichia coli（E. coli）, refolded, and purified on an immobilized Ni2＋ affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and Western blot analysis reveal that the target protein was expressed sufficiently. We used immunofluorescence assays to demonstrate that SC-C/SEA could bind specifically to human lung carcinoma cells（A549）, but almost human uterine cervix cells（HeLa）. We also used the L-lactate dehydrogenase（LDH） release assay to show that SC-C/SEA elicits a strong A549 tumor-specific cytotoxic T lymphocyte（CTL） response in vitro. The results suggest that SC-C/SEA shows specific activity against CEA-positive cells and has potential application in CEA-targeted cancer immunotherapy.

Effect and Mechanism of Superantigen Staphylococcal Enterotoxin Therapy for Mouse Gastric Tumor

The anti-tumor effect and mechanism of the staphylococcal enterotoxin A (SEA) were studied. The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumor ceils (MGC80-3). The experimental group was treated with SEA, and the control group was treated with normal saline. The percentage of tumor generation and tumor mass was measured. The results showed that the percentage of the tumor generation in the SEA-treated mice was lower than in the control group, but there was no significant difference (P>0. 05). However, the tumor mass in the experimental group was significantly lighter than in the control group, with the difference being very significant (P<0. 001). There were more CD4+ T cells and CD8+ T cells in the tumor of the mice treated with SEA than those of the control group. SEA has an obvious anti-tumor effect on mice gastric tumor. The mechanism might be that SEA induces the effect of superantigen-dependent cell mediated cytotoxicity to the tumor cells.

Mutants of Escherichia coli heat-labile enterotoxin and cholera toxin as mucosal adjuvants

Mucosal vaccination has been getting more and more recognition because of its compliance and low risk of spreading infectious disease by contaminated syringes used in subcutaneous immunization. However, most vaccines are unable to induce immune responses when given mucosally, and require the use of strong adjuvant for effective delivery systems. Heat-labile enterotoxin (LT) and Cholera toxin(CT) are powerful mucosal adjuvants when co-administered with soluble antigens. But high toxicity hampers their use in humans. Thanks to the fine knowledge of the structure-function relationship of LT and CT, many nontoxic or low toxic mutants have been generated, part of them retain high adjuvanticity of mucosal immunization. Among these mutants, LTS63K, LTA72R, LTR192G and CTE29H, CTE112K have been widely investigated. LTS63K and CTE112K are fully non toxic, whereas LTA72R and CTE29H are low toxic, and LTR192G is nontoxic in vitro(it remains the same toxicity as wild type LT in vivo). These mutants are extremely active as mucosal adjuvants when co-administrated with a variety of antigens in different animal models. They will be investigated more widely and deeply in the future. Some of them will be tested soon in human bodies.