Age-Stratified Seroprevalence of Respiratory Syncytial Virus: Analysis Using Prefusion F and G Protein Antibodies.

This is a cross-sectional serosurveillance study for RSV. Between June and September of 2021, a total of 150 sera were collected from 30 individuals in each age group (<5, 5-18, 19-49, 50-64, and ≥65 years). Seroprevalence was estimated using enzyme-linked immunosorbent assays targeting two stabilized prefusion F (preF; DS-Cav1 and SC-TM) and G proteins. The overall seroprevalence was low in young children and older adults, despite them having a higher risk of severe RSV infection. There was a remarkable difference in age-stratified seroprevalence rates between anti-preF and anti-G protein antibodies. Given the high disease burden and low seroprevalence in both infants and old adults, RSV vaccination would be crucial for pregnant women and people aged over 60 years.

Development and Validation of an Enzyme-Linked Immunosorbent Assay-Based Protocol for Evaluation of Respiratory Syncytial Virus Vaccines.

Recently, respiratory syncytial virus (RSV) vaccines based on the prefusion F (pre-F) antigen were approved in the United States. We aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based protocol for the practical and large-scale evaluation of RSV vaccines. Two modified pre-F proteins (DS-Cav1 and SC-TM) were produced by genetic recombination and replication using an adenoviral vector. The protocol was established by optimizing the concentrations of the coating antigen (pre-F proteins), secondary antibodies, and blocking buffer. To validate the protocol, we examined its accuracy, precision, and specificity using serum samples from 150 participants across various age groups and the standard serum provided by the National Institute of Health. In the linear correlation analysis, coating concentrations of 5 and 2.5 µg/mL of DS-Cav1 and SC-TM showed high coefficients of determination (r > 0.90), respectively. Concentrations of secondary antibodies (alkaline phosphatase-conjugated anti-human immunoglobulin G, diluted 1:2000) and blocking reagents (5% skim milk/PBS-T) were optimized to minimize non-specific reactions. High accuracy was observed for DS-Cav1 (r = 0.90) and SC-TM (r = 0.86). Further, both antigens showed high precision (coefficient of variation < 15%). Inhibition ELISA revealed cross-reactivity of antibodies against DS-Cav1 and SC-TM, but not with the attachment (G) protein.

Gamma Irradiation-Inactivated Respiratory Syncytial Virus Vaccine Provides Protection but Exacerbates Pulmonary Inflammation by Switching from Prefusion to Postfusion F Protein.

Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes lower respiratory diseases among infants and elderly people. Moreover, formalin-inactivated RSV (FI-RSV) vaccine induces serious enhanced respiratory disease (ERD). Radiation has been investigated as an alternative approach for producing inactivated or live-attenuated vaccines, which enhance the antigenicity and heterogeneous protective effects of vaccines compared with conventional formalin inactivation. In this study, we developed an RSV vaccine using gamma irradiation and analyzed its efficacy against RSV vaccine-induced ERD in a mouse model. Although gamma irradiation-inactivated RSV (RI-RSV) carbonylation was lower than FI-RSV carbonylation and RI-RSV showed a significant antibody production and viral clearance, RI-RSV caused more obvious body weight loss, pulmonary eosinophil infiltration, and pulmonary mucus secretion. Further, the conversion of prefusion F (pre-F) to postfusion F (post-F) was significant for both RI-RSV and FI-RSV, while that of RI-RSV was significantly higher than that of FI-RSV. We found that the conversion from pre- to post-F during radiation was caused by radiation-induced reactive oxygen species. Although we could not propose an effective RSV vaccine manufacturing method, we found that ERD was induced by RSV vaccine by various biochemical effects that affect antigen modification during RSV vaccine manufacturing, rather than simply by the combination of formalin and alum. Therefore, these biochemical actions should be considered in future developments of RSV vaccine. IMPORTANCE Radiation inactivation for viral vaccine production has been known to elicit a better immune response than other inactivation methods due to less surface protein damage. However, we found in this study that radiation-inactivated RSV (RI-RSV) vaccine induced a level of immune response similar to that induced by formalin-inactivated RSV (FI-RSV). Although RI-RSV vaccine showed less carbonylation than FI-RSV, it induced more conformational changes from pre-F to post-F due to the gamma radiation-induced reactive oxygen species response, which may be a key factor in RI-RSV-induced ERD. Therefore, ERD induced by RSV vaccine may be due to pre-F to post-F denaturation by random protein modifications caused by external stress. Our findings provide new ideas for inactivated vaccines for RSV and other viruses and confirm the importance of pre-F in RSV vaccines.

Low-dose radiation therapy suppresses viral pneumonia by enhancing broad-spectrum anti-inflammatory responses via transforming growth factor-ß production.

Low-dose radiation therapy (LDRT) can suppress intractable inflammation, such as that in rheumatoid arthritis, and is used for treating more than 10,000 rheumatoid arthritis patients annually in Europe. Several recent clinical trials have reported that LDRT can effectively reduce the severity of coronavirus disease (COVID-19) and other cases of viral pneumonia. However, the therapeutic mechanism of LDRT remains unelucidated. Therefore, in the current study, we aimed to investigate the molecular mechanism underlying immunological alterations in influenza pneumonia after LDRT. Mice were irradiated to the whole lung 1 day post-infection. The changes in levels of inflammatory mediators (cytokines and chemokines) and immune cell populations in the bronchoalveolar lavage (BALF), lungs, and serum were examined. LDRT-treated mice displayed markedly increased survival rates and reduced lung edema and airway and vascular inflammation in the lung; however, the viral titers in the lungs were unaffected. Levels of primary inflammatory cytokines were reduced after LDRT, and transforming growth factor-ß (TGF-ß) levels increased significantly on day 1 following LDRT. Levels of chemokines increased from day 3 following LDRT. Additionally, M2 macrophage polarization or recruitment was increased following LDRT. We found that LDRT-induced TGF-ß reduced the levels of cytokines and polarized M2 cells and blocked immune cell infiltration, including neutrophils, in BALF. LDRT-induced early TGF-ß production was shown to be a key regulator involved in broad-spectrum anti-inflammatory activity in virus-infected lungs. Therefore, LDRT or TGF-ß may be an alternative therapy for viral pneumonia.

Radioprotection of deinococcal exopolysaccharide BRD125 by regenerating hematopoietic stem cells.

There is a substantial need for the development of biomaterials for protecting hematopoietic stem cells and enhancing hematopoiesis after radiation damage. Bacterial exopolysaccharide (EPS) has been shown to be very attractive to researchers as a radioprotectant owing to its high antioxidant, anti-cancer, and limited adverse effects. In the present study, we isolated EPS from a novel strain, Deinococcus radiodurans BRD125, which produces EPS in high abundance, and investigated its applicability as a radioprotective biomaterial. We found that EPS isolated from EPS-rich D. radiodurans BRD125 (DeinoPol-BRD125) had an excellent free-radical scavenging effect and reduced irradiation-induced apoptosis. In addition, bone-marrow and spleen-cell apoptosis in irradiated mice were significantly reduced by DeinoPol-BRD125 administration. DeinoPol-BRD125 enhanced the expression of hematopoiesis-related cytokines such as GM-CSF, G-GSF, M-CSF, and SCF, thereby enhancing hematopoietic stem cells protection and regeneration. Taken together, our findings are the first to report the immunological mechanism of a novel radioprotectant, DeinoPol-BRD125, which might constitute an ideal radioprotective and radiation mitigating agent as a supplement drug during radiotherapy.

Anti-allergic function of the cell wall (DeinoWall) from Deinococcus radiodurans.

Deinococcus radiodurans is an extremophile, well known to be extremely resistant to external stresses due to its unique physiological system and structure of cellular components. Although the proportion of D. radiodurans has been reported to be negatively correlated with atopic dermatitis, the exact function of D. radiodurans in allergic diseases and its precise mechanisms have not been studied. In the present study, we hypothesize that D. radiodurans or its cellular constituents play a critical role in the skin to prevent allergic inflammatory responses by modulating immunity. Heat-killed D. radiodurans inhibited the production of Th2 cytokines, such as IL-4 and IL-5, induced by ovalbumin (OVA) stimulation in splenocytes from OVA-sensitized mice. Among the cellular constituents of D. radiodurans, such as cell wall (DeinoWall), cell membrane (DeinoMem), and exopolysaccharide (DeinoPol), only DeinoWall inhibited the production of Th2 cytokines and 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD), a Th2-predominant allergic disease in mice. Moreover, serum IgE levels and infiltration of mast cells into skin lesions, the markers of Th2 response induced by DNCB application, were significantly inhibited by treatment with DeinoWall. Remarkably, DeinoWall induced the maturation of bone marrow-derived dendritic cells (BMDCs) that promote Th1-biased immunity, which might balance Th1/Th2 and regulate allergic inflammatory responses. Collectively, these results suggest that DeinoWall acts as a major cellular constituent in the negative regulation of allergic inflammatory responses by D. radiodurans and might be a viable candidate for the treatment of allergic diseases.

Development of Live Attenuated Salmonella Typhimurium Vaccine Strain Using Radiation Mutation Enhancement Technology (R-MET).

Salmonella enterica is a leading cause of food-borne diseases in humans worldwide, resulting in severe morbidity and mortality. They are carried asymptomatically in the intestine or gallbladder of livestock, and are transmitted predominantly from animals to humans via the fecal-oral route. Thus, the best preventive strategy is to preemptively prevent transmission to humans by vaccinating livestock. Live attenuated vaccines have been mostly favored because they elicit both cellular and humoral immunity and provide long-term protective immunity. However, developing these vaccines is a laborious and time-consuming process. Therefore, most live attenuated vaccines have been mainly used for phenotypic screening using the auxotrophic replica plate method, and new types of vaccines have not been sufficiently explored. In this study, we used Radiation-Mutation Enhancement Technology (R-MET) to introduce a wide variety of mutations and attenuate the virulence of Salmonella spp. to develop live vaccine strains. The Salmonella Typhimurium, ST454 strain (ST WT) was irradiated with Cobalt60 gamma-irradiator at 1.5 kGy for 1 h to maximize the mutation rate, and attenuated daughter colonies were screened using in vitro macrophage replication capacity and in vivo mouse infection assays. Among 30 candidates, ATOMSal-L6, with 9,961-fold lower virulence than the parent strain (ST454) in the mouse LD50 model, was chosen. This vaccine candidate was mutated at 71 sites, and in particular, lost one bacteriophage. As a vaccine, ATOMSal-L6 induced a Salmonella-specific IgG response to provide effective protective immunity upon intramuscular vaccination of mice. Furthermore, when mice and sows were orally immunized with ATOMSal-L6, we found a strong protective immune response, including multifunctional cellular immunity. These results indicate that ATOMSal-L6 is the first live vaccine candidate to be developed using R-MET, to the best of our knowledge. R-MET can be used as a fast and effective live vaccine development technology that can be used to develop vaccine strains against emerging or serotype-shifting pathogens.

Complete Genome Sequence of Streptococcus oralis SF100, Isolated from Blood Cultures from a Patient with Infective Endocarditis.

Streptococcus oralis is a commensal viridans group streptococcus of the human oral cavity and a frequent cause of endovascular infection. Here, we report the complete whole-genome sequence of S. oralis strain SF100, which was originally isolated from the blood of a patient with infective endocarditis. This strain contains the lysogenic bacteriophage SM1, which enhances the virulence of SF100 in animal models of endocardial infection.

Salmonella Vaccine Vector System for Foot-and-Mouth Disease Virus and Evaluation of Its Efficacy with Virus-Like Particles.

Foot-and-mouth disease virus (FMDV) causes a highly contagious and devastating disease in livestock animals and has a great potential to cause severe economic loss worldwide. The major antigen of FMDV capsid protein, VP1, contains the major B-cell epitope responsible for effectively eliciting protective humoral immunity. In this study, irradiated Salmonella Typhimurium (KST0666) were used as transgenic vectors containing stress-inducible plasmid pRECN-VP1 to deliver the VP1 protein from FMDV-type A/WH/CHA/09. Mice were orally inoculated with ATOMASal-L3 harboring pRECN-VP1, and FMDV virus-like particles, where (VLPFMDV)-specific humoral, mucosal, and cellular immune responses were evaluated. Mice vaccinated with attenuated Salmonella (KST0666) expressing VP1 (named KST0669) showed high levels of VLP-specific IgA in feces and IgG in serum, with high FMDV neutralization titer. Moreover, KST0669-vaccinated mice showed increased population of IFN-γ (type 1 T helper cells; Th1 cells)-, IL-5 (Th2 cells)-, and IL-17A (Th17 cells)-expressing CD4+ as well as activated CD8+ T cells (IFN-γ+CD8+ cells), detected by stimulating VLPFMDV. All data indicate that our Salmonella vector system successfully delivered FMDV VP1 to immune cells and that the humoral and cellular efficacy of the vaccine can be easily evaluated using VLPFMDV in a Biosafety Level I (BSL1) laboratory.

Inhibition of Fibrinolysis by Streptococcal Phage LysinSM1.

Expression of bacteriophage lysinSM1 by Streptococcus oralis strain SF100 is thought to be important for the pathogenesis of infective endocarditis, due to its ability to mediate bacterial binding to fibrinogen. To better define the lysinSM1 binding site on fibrinogen Aα, and to investigate the impact of binding on fibrinolysis, we examined the interaction of lysinSM1 with a series of recombinant fibrinogen Aα variants. These studies revealed that lysinSM1 binds the C-terminal region of fibrinogen Aα spanned by amino acid residues 534 to 610, with an affinity of equilibrium dissociation constant (KD) of 3.23 × 10-5 M. This binding site overlaps the known binding site for plasminogen, an inactive precursor of plasmin, which is a key protease responsible for degrading fibrin polymers. When tested in vitro, lysinSM1 competitively inhibited plasminogen binding to the αC region of fibrinogen Aα. It also inhibited plasminogen-mediated fibrinolysis, as measured by thromboelastography (TEG). These results indicate that lysinSM1 is a bi-functional virulence factor for streptococci, serving as both an adhesin and a plasminogen inhibitor. Thus, lysinSM1 may facilitate the attachment of bacteria to fibrinogen on the surface of damaged cardiac valves and may also inhibit plasminogen-mediated lysis of infected thrombi (vegetations) on valve surfaces. IMPORTANCE The interaction of streptococci with human fibrinogen and platelets on damaged endocardium is a central event in the pathogenesis of infective endocarditis. Streptococcus oralis can bind platelets via the interaction of bacteriophage lysinSM1 with fibrinogen on the platelet surface, and this process has been associated with increased virulence in an animal model of endocarditis. We now report that lysinSM1 binds to the αC region of the human fibrinogen Aα chain. This interaction blocks plasminogen binding to fibrinogen and inhibits fibrinolysis. In vivo, this inhibition could prevent the lysis of infected vegetations, thereby promoting bacterial persistence and virulence.

Induction of Apoptotic Cell Death by Oral Streptococci in Human Periodontal Ligament Cells.

Initiation and progression of oral infectious diseases are associated with streptococcal species. Bacterial infection induces inflammatory responses together with reactive oxygen species (ROS), often causing cell death and tissue damage in the host. In the present study, we investigated the effects of oral streptococci on cytotoxicity and ROS production in human periodontal ligament (PDL) cells. Streptococcus gordonii showed cell cytotoxicity in a dose- and time-dependent manner. The cytotoxicity might be due to apoptosis since S. gordonii increased annexin V-positive cells, and the cytotoxicity was reduced by an apoptosis inhibitor, Z-VAD-FMK. Other oral streptococci such as Streptococcus mitis, Streptococcus sanguinis, and Streptococcus sobrinus also induced apoptosis, whereas Streptococcus mutans did not. All streptococci tested except S. mutans triggered ROS production in human PDL cells. Interestingly, however, streptococci-induced apoptosis appears to be ROS-independent, as the cell death induced by S. gordonii was not recovered by the ROS inhibitor, resveratrol or n-acetylcysteine. Instead, hydrogen peroxide (H2O2) appears to be important for the cytotoxic effects of streptococci since most oral streptococci except S. mutans generated H2O2, and the cytotoxicity was dramatically reduced by catalase. Furthermore, streptococcal lipoproteins are involved in cytotoxicity, as we observed that cytotoxicity induced by the lipoprotein-deficient S. gordonii mutant was less potent than that by the wild-type and was attenuated by anti-TLR2-neutralizing antibody. Indeed, lipoproteins purified from S. gordonii alone were sufficient to induce cytotoxicity. Notably, S. gordonii lipoproteins did not induce H2O2 or ROS but cooperatively induced cell death when co-treated with H2O2. Taken together, these results suggest that most oral streptococci except S. mutans efficiently induce damage to human PDL cells by inducing apoptotic cell death with bacterial H2O2 and lipoproteins, which might contribute to the progression of oral infectious diseases such as apical periodontitis.

Characterization of humoral and cellular immune features of gamma-irradiated influenza vaccine.

The most widely used influenza vaccines are prepared by chemical inactivation. However, chemical, especially formalin, treatment-induced modifications of the antigenic structure of the virus are frequently associated with adverse effects including low efficacy of protection, unexpected immune responses, or exacerbation of disease. Gamma-irradiation was suggested as an alternative influenza virus inactivation method due to its great features of completely inactivating virus while not damaging the structures of protein antigens, and cross-protective ability against heterologous strains. However, immunological features of gamma radiation-inactivated influenza vaccine have not been fully understood. In this study, we aimed to investigate the humoral and cellular immune responses of gamma radiation-inactivated influenza vaccine. The gamma irradiation-inactivated influenza vaccine (RADVAXFluA) showed complete viral inactivation but retained normal viral structure with functional activities of viral protein antigens. Intranasal immunization of RADVAXFluA provided better protection against influenza virus infection than formalin-inactivated influenza virus (FIV) in mice. RADVAXFluA greatly enhanced the production of virus-specific serum IgG and alveolar mucosal IgA, which effectively neutralized HA (hemagglutinin) and NA (neuraminidase) activities, and blocked viral binding to the cells, respectively. Further analysis of IgG subclasses showed RADVAXFluA-immunized sera had higher levels of IgG1 and IgG2a than those of FIV-immunized sera. In addition, analysis of cellular immunity found RADVAXFluA induced strong dendritic cells (DC) activation resulting in higher DC-mediated activation of CD8+ T cells than FIV. The results support improved immunogenicity by RADVAXFluA.

Development and Validation of Enzyme-Linked Immunosorbent Assay for Group B Streptococcal Polysaccharide Vaccine.

Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of neonatal sepsis and meningitis in infants. Limitations of prenatal GBS screening and intrapartum antibiotic prophylaxis render developing GBS vaccines a high priority. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for the practical and large-scale evaluation of GBS capsular polysaccharide (PS) vaccine immunogenicity against three main serotypes, Ia, III, and V. GBS-ELISA was developed and subsequently validated using a standardized curve-fitting four-parameter logistic method. Specificity was measured using adsorption of serum with homologous and heterologous PS. Homologous adsorption showed a ≥75% inhibition of all three serotypes, whereas with heterologous PS, IgG GBS-ELISA inhibited only ≤25% of serotypes III and V. However, with serotype Ia, IgG antibody levels decreased by >50%, even after adsorption with heterologous PS (III or V). In comparison, the inhibition opsonophagocytic killing assay (OPA) of serotypes Ia GBS exhibited a reduction in opsonophagocytic activity of only 20% and 1.1% for serotypes III and V GBS, respectively. The precision of the GBS-ELISA was assessed in five independent experiments using four serum samples. The coefficient of variation was <5% for all three serotypes. This standardized GBS-ELISA would be useful for GBS vaccine development and its evaluation.

Deinococcus radiodurans Exopolysaccharide Inhibits Staphylococcus aureus Biofilm Formation.

Deinococcus radiodurans is an extremely resistant bacterium against extracellular stress owing to on its unique physiological functions and the structure of its cellular constituents. Interestingly, it has been reported that the pattern of alteration in Deinococcus proportion on the skin is negatively correlated with skin inflammatory diseases, whereas the proportion of Staphylococcus aureus was increased in patients with chronic skin inflammatory diseases. However, the biological mechanisms of deinococcal interactions with other skin commensal bacteria have not been studied. In this study, we hypothesized that deinococcal cellular constituents play a pivotal role in preventing S. aureus colonization by inhibiting biofilm formation. To prove this, we first isolated cellular constituents, such as exopolysaccharide (DeinoPol), cell wall (DeinoWall), and cell membrane (DeinoMem), from D. radiodurans and investigated their inhibitory effects on S. aureus colonization and biofilm formation in vitro and in vivo. Among them, only DeinoPol exhibited an anti-biofilm effect without affecting bacterial growth and inhibiting staphylococcal colonization and inflammation in a mouse skin infection model. Moreover, the inhibitory effect was impaired in the Δdra0033 strain, a mutant that cannot produce DeinoPol. Remarkably, DeinoPol not only interfered with S. aureus biofilm formation at early and late stages but also disrupted a preexisting biofilm by inhibiting the production of poly-N-acetylglucosamine (PNAG), a key molecule required for S. aureus biofilm formation. Taken together, the present study suggests that DeinoPol is a key molecule in the negative regulation of S. aureus biofilm formation by D. radiodurans. Therefore, DeinoPol could be applied to prevent and/or treat infections or inflammatory diseases associated with S. aureus biofilms.

Radiation-Inactivated S. gallinarum Vaccine Provides a High Protective Immune Response by Activating Both Humoral and Cellular Immunity.

Salmonella enterica subsp. enterica serovar Gallinarum (SG) is a common pathogen in chickens, and causes an acute systemic disease that leads to high mortality. The live attenuated vaccine 9R is able to successfully protect chickens older than six weeks by activating a robust cell-mediated immune response, but its safety and efficacy in young chickens remains controversial. An inactivated SG vaccine is being used as an alternative, but because of its low cellular immune response, it cannot be used as a replacement for live attenuated 9R vaccine. In this study, we employed gamma irradiation instead of formalin as an inactivation method to increase the efficacy of the inactivated SG vaccine. Humoral, cellular, and protective immune responses were compared in both mouse and chicken models. The radiation-inactivated SG vaccine (r-SG) induced production of significantly higher levels of IgG2b and IgG3 antibodies than the formalin-inactivated vaccine (f-SG), and provided a homogeneous functional antibody response against group D, but not group B Salmonella. Moreover, we found that r-SG vaccination could provide a higher protective immune response than f-SG by inducing higher Th17 activation. These results indicate that r-SG can provide a protective immune response similar to the live attenuated 9R vaccine by activating a higher humoral immunity and a lower, but still protective, cellular immune response. Therefore, we expect that the radiation inactivation method might substitute for the 9R vaccine with little or no side effects in chickens younger than six weeks.

Molecular Characteristics of IS1216 Carrying Multidrug Resistance Gene Cluster in Serotype III/Sequence Type 19 Group B Streptococcus.

Streptococcus agalactiae is the leading cause of meningitis in newborns and a significant cause of invasive diseases in pregnant women and adults with underlying diseases. Antibiotic resistance against erythromycin and clindamycin in group B streptococcus (GBS) isolates has been increasing worldwide. GBS expresses the Srr1 and Srr2 proteins, which have important roles in bacterial infection. They have been investigated as novel vaccine candidates against GBS infection, with promising results. But a recent study detected non-srr1/2-expressing clinical isolates belonging to serotype III. Thus, we aimed to analyze the genotypes of non-srr1/2 GBS clinical isolates collected between 2013 and 2016 in South Korea. Forty-one (13.4%) of the 305 serotype III isolates were identified as non-srr1/2 strains, including sequence type 19 (ST19) (n = 16) and ST27 (n = 18) strains. The results of the comparative genomic analysis of the ST19/serotype III/non-srr1/2 strains further revealed four unique gene clusters. Site 4 in the srr1 gene locus was replaced by an lsa(E)-lnu(B)-aadK-aac-aph-aadE-carrying multidrug-resistant gene cluster flanked by two IS1216 transposases with 99% homology to the enterococcal plasmid pKUB3007-1. Despite the Srr1 and Srr2 deficiencies, which resulted in reduced fibrinogen binding, the adherence of non-srr1/2 strains to endothelial and epithelial cells was comparable to that of Srr1- or Srr2-expressing strains. Moreover, their virulence in mouse models of meningitis was not significantly affected. Furthermore, additional adhesin-encoding genes, including a gene encoding a BspA-like protein, which may contribute to colonization by non-srr1/2 strains, were identified via whole-genome analysis. Thus, our study provides important findings that can aid in the development of vaccines and antibiotics against GBS. IMPORTANCE Most previously isolated group B streptococcus (GBS) strains express either the Srr1 or Srr2 glycoprotein, which plays an important role in bacterial colonization and invasion. These glycoproteins are potential protein vaccine candidates. In this study, we first report GBS clinical isolates in which the srr1/2 gene was deleted or replaced with foreign genes. Despite Srr1/2 deficiency, in vitro adherence to mammalian cells and in vivo virulence in murine models were not affected, suggesting that the isolates might have another adherence mechanism that enhanced their virulence aside from Srr1/2-fibrinogen-mediated adherence. In addition, several non-srr1/2 isolates replaced the srr1/2 gene with the lnu(B) and lsa(E) antibiotic resistance genes flanked by IS1216, effectively causing multidrug resistance. Collectively, we believe that our study identifies the underlying genes responsible for the pathogenesis of new GBS serotype III. Furthermore, our study emphasizes the need for alternative antibiotics for patients who are allergic to ß-lactams and for those who are pregnant.

ptsI gene in the phosphotransfer system is a potential target for developing a live attenuated Salmonella vaccine.

Salmonella enterica serovar Typhimurium causes invasive non­typhoidal Salmonella diseases in animals and humans, resulting in a high mortality rate and huge economic losses globally. As the prevalence of antibiotic­resistant Salmonella has been increasing, vaccination is thought to be the most effective and economical strategy to manage salmonellosis. The present study aimed to investigate whether dysfunction in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), which is critical for carbon uptake and survival in macrophages, may be adequate to generate Salmonella­attenuated vaccine strains. A Salmonella strain (KST0555) was generated by deleting the ptsI gene from the PTS and it was revealed that this auxotrophic mutant was unable to efficiently utilize predominant carbon sources during infection (glucose and glycerol), reduced its invasion and replication capacity in macrophages, and significantly (P=0.0065) lowered its virulence in the setting of a mouse colitis model, along with a substantially decreased intestinal colonization and invasiveness compared with its parent strain. The reverse transcription­quantitative PCR results demonstrated that the virulence genes in Salmonella pathogenicity island-1 (SPI-1) and -2 (SPI-2) and the motility of KST0555 were all downregulated compared with its parent strain. Finally, it was revealed that when mice were immunized orally with live KST0555, Salmonella­specific humoral and cellular immune responses were effectively elicited, providing protection against Salmonella infection. Thus, the present promising data provides a strong rationale for the advancement of KST0555 as a live Salmonella vaccine candidate and ptsI as a potential target for developing a live attenuated bacterial vaccine strain.

Irradiation-Induced Intestinal Damage Is Recovered by the Indigenous Gut Bacteria Lactobacillus acidophilus.

The intestinal tract is one of the most sensitive organs following irradiation. The protective effect of specific indigenous microbiota on irradiation-induced damage to intestinal epithelial cells has not been reported. Mice were irradiated with a single dose of 6 Gy of gamma rays. The intestinal damage was analyzed by histopathology. Intestinal stemness and differentiation were determined by intestinal organoid culture. Microbiota community was observed by high-throughput 16S rRNA gene sequencing and oligotyping analysis. We showed that distal small intestine was damaged by sublethal dose of gamma irradiation. Intestinal organoids derived from the irradiated mice showed defects in budding and mucin expression, suggesting the detrimental effect of irradiation on the intestinal stemness and differentiation. In addition, irradiation reduced intestinal immunoglobulin A level, concomitant with decreased microbiota diversity based on our high-throughput 16S rRNA gene sequencing data. Especially, the relative abundance of Lactobacillus was reduced at early time point post-irradiation; however, it was recovered at late time point. Oligotyping analysis within the Lactobacillus genus indicated that Lactobacillus-related oligotype 1 (OT1) including Lactobacillus acidophilus might drive recovery after irradiation as it was associated with increased long-term numbers post-exposure. We showed that treatment with heat-killed L. acidophilus rescued the budding-impaired organoids and induced sufficient differentiation in epithelial cells, and particularly mucin-producing cells, in intestinal organoids. This study provides the first evidence that the indigenous gut bacteria L. acidophilus enhance intestinal epithelial function with respect to irradiation-induced intestinal damage by improving intestinal stem cell function and cell differentiation.

Development of Oxytolerant Salmonella typhimurium Using Radiation Mutation Technology (RMT) for Cancer Therapy.

A critical limitation of Salmonella typhimurium (S. typhimurium) as an anti-cancer agent is the loss of their invasive or replicative activities, which results in no or less delivery of anti-cancer agents inside cancer cells in cancer therapy. Here we developed an oxytolerant attenuated Salmonella strain (KST0650) from the parental KST0649 (ΔptsIΔcrr) strain using radiation mutation technology (RMT). The oxytolerant KST0650 strain possessed 20-times higher replication activity in CT26 cancer cells and was less virulent than KST0649. Furthermore, KST0650 migrated effectively into tumor tissues in mice. KST0650 was further equipped with a plasmid harboring a spliced form of the intracellular pro-apoptotic protein sATF6, and the expression of sATF6 was controlled by the radiation-inducible recN promoter. The new strain was named as KST0652, in which sATF6 protein expression was induced in response to radiation in a dose-dependent manner. This strain was effectively delivered inside cancer cells and tumor tissues via the Salmonella type III secretion system (T3SS). In addition, combination treatment with KST0652 and radiation showed a synergistic anti-tumor effect in murine tumor model with complete inhibition of tumor growth and protection against death. In conclusion, we showed that RMT can be used to effectively develop an anti-tumor Salmonella strain for delivering anti-cancer agents inside tumors.

Antioxidant Activities of an Exopolysaccharide (DeinoPol) Produced by the Extreme Radiation-Resistant Bacterium Deinococcus radiodurans.

Deinococcus radiodurans shows extreme resistance to a range of remarkable environmental stresses. Deinococcal exopolysaccharide (DeinoPol) is a component of the cell wall, but its role in stress resistance has not yet been well-described. In this study, we isolated and characterized DeinoPol from Deinococcus radiodurans R1 strain and investigated its application as an antioxidant agent. Bioinformatic analysis indicated that dra0033, encoding an ExoP-like protein, was involved in DeinoPol biosynthesis, and dra0033 mutation significantly decreased survival rates in response to stresses. Purified DeinoPol consists of different monosaccharides and has a molecular weight of approximately 80 to 100 kDa. DeinoPol also demonstrates highly protective effects on human keratinocytes in response to stress-induced apoptosis by effectively scavenging ROS. Taken together, these findings indicate that DeinoPol is the first reported deinococcal exopolysaccharide that might be used in cosmetics and pharmaceuticals as a safe and attractive radical scavenger.