Deinococcus radiodurans-derived membrane vesicles protect HaCaT cells against H2O2-induced oxidative stress via modulation of MAPK and Nrf2/ARE pathways.

BACKGROUND: Deinococcus radiodurans is a robust bacterium that can withstand harsh environments that cause oxidative stress to macromolecules due to its cellular structure and physiological functions. Cells release extracellular vesicles for intercellular communication and the transfer of biological information; their payload reflects the status of the source cells. Yet, the biological role and mechanism of Deinococcus radiodurans-derived extracellular vesicles remain unclear. AIM: This study investigated the protective effects of membrane vesicles derived from D. radiodurans (R1-MVs) against H2O2-induced oxidative stress in HaCaT cells. RESULTS: R1-MVs were identified as 322 nm spherical molecules. Pretreatment with R1-MVs inhibited H2O2-mediated apoptosis in HaCaT cells by suppressing the loss of mitochondrial membrane potential and reactive oxygen species (ROS) production. R1-MVs increased the superoxide dismutase (SOD) and catalase (CAT) activities, restored glutathione (GSH) homeostasis, and reduced malondialdehyde (MDA) production in H2O2-exposed HaCaT cells. Moreover, the protective effect of R1-MVs against H2O2-induced oxidative stress in HaCaT cells was dependent on the downregulation of mitogen-activated protein kinase (MAPK) phosphorylation and the upregulation of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. Furthermore, the weaker protective capabilities of R1-MVs derived from ΔDR2577 mutant than that of the wild-type R1-MVs confirmed our inferences and indicated that SlpA protein plays a crucial role in R1-MVs against H2O2-induced oxidative stress. CONCLUSION: Taken together, R1-MVs exert significant protective effects against H2O2-induced oxidative stress in keratinocytes and have the potential to be applied in radiation-induced oxidative stress models.

Sec62 Suppresses Foot-and-Mouth Disease Virus Proliferation by Promotion of IRE1α-RIG-I Antiviral Signaling.

Foot-and-mouth disease virus (FMDV) is highly infectious and causes a major plague in animal farming. Unfolded protein response is one of the major cellular responses to pathogenic infections, which performs a crucial role in cell survival, apoptosis, and antiviral innate immune response. In this study, we showed that FMDV infection activated two unfolded protein response branches (PERK-eIF2α and ATF6 signaling) in both baby hamster kidney cells (BHK-21) and porcine kidney (PK-15) cells, whereas it suppressed the IRE1α-XBP1 signaling by decreasing IRE1α level. Further study revealed IRE1α signaling as an important antiviral innate immune mechanism against FMDV. Sec62, the transport protein, was greatly decreased at the late stages of FMDV infection. By overexpression and knockdown study, we also found that the expression of Sec62 was positively involved in the levels of IRE1α and RIG-I and subsequent activation of downstream antiviral signaling pathways in FMDV-infected PK-15 cells. Taken together, our study demonstrates that Sec62 is an important antiviral factor that upregulates IRE1α-RIG-I-dependent antiviral innate immune responses, and FMDV evades antiviral host defense mechanism by downregulating Sec62-IRE1α/RIG-I.

Immunostimulatory Potential of Extracellular Vesicles Isolated from an Edible Plant, Petasites japonicus, via the Induction of Murine Dendritic Cell Maturation.

Extracellular vesicles (EVs) have recently been isolated from different plants. Plant-derived EVs have been proposed as potent therapeutics and drug-delivery nanoplatforms for delivering biomolecules, including proteins, RNAs, DNAs, and lipids. Herein, Petasites japonicus-derived EVs (PJ-EVs) were isolated through a series of centrifugation steps and characterized using dynamic light scattering and transmission electron microscopy. Immunomodulatory effects of PJ-EVs were assessed using dendritic cells (DCs). PJ-EVs exhibited a spherical morphology with an average size of 122.6 nm. They induced the maturation of DCs via an increase in the expression of surface molecules (CD80, CD86, MHC-I, and MHC-II), production of Th1-polarizing cytokines (TNF-α and IL-12p70), and antigen-presenting ability; however, they reduced the antigen-uptake ability. Furthermore, maturation of DCs induced by PJ-EVs was dependent on the activation and phosphorylation of MAPK and NF-κB signal pathways. Notably, PJ-EV-treated DCs strongly induced the proliferation and differentiation of naïve T cells toward Th1-type T cells and cytotoxic CD8+ T cells along with robust secretion of IFN-γ and IL-2. In conclusion, our study indicates that PJ-EVs can be potent immunostimulatory candidates with an ability of strongly inducing the maturation of DCs.

Protective Effect of Polysaccharides Extracted from Cudrania tricuspidata Fruit against Cisplatin-Induced Cytotoxicity in Macrophages and a Mouse Model.

Although cisplatin is one of most effective chemotherapeutic drugs that is widely used to treat various types of cancer, it can cause undesirable damage in immune cells and normal tissue because of its strong cytotoxicity and non-selectivity. This study was conducted to investigate the cytoprotective effects of Cudrania tricuspidata fruit-derived polysaccharides (CTPS) against cisplatin-induced cytotoxicity in macrophages, lung cancer cell lines, and a mouse model, and to explore the possibility of application of CTPS as a supplement for anticancer therapy. Both cisplatin alone and cisplatin with CTPS induced a significant cytotoxicity in A549 and H460 lung cancer cells, whereas cytotoxicity was suppressed by CTPS in cisplatin-treated RAW264.7 cells. CTPS significantly attenuated the apoptotic and necrotic population, as well as cell penetration in cisplatin-treated RAW264.7 cells, which ultimately inhibited the upregulation of Bcl-2-associated X protein (Bax), cytosolic cytochrome c, poly (adenosine diphosphateribose) polymerase (PARP) cleavage, and caspases-3, -8, and -9, and the downregulation of B cell lymphoma-2 (Bcl-2). The CTPS-induced cytoprotective action was mediated with a reduction in reactive oxygen species production and mitochondrial transmembrane potential loss in cisplatin-treated RAW264.7 cells. In agreement with the results obtained above, CTPS induced the attenuation of cell damage in cisplatin-treated bone marrow-derived macrophages (primary cells). In in vivo studies, CTPS significantly inhibited metastatic colonies and bodyweight loss as well as immunotoxicity in splenic T cells compared to the cisplatin-treated group in lung metastasis-induced mice. Furthermore, CTPS decreased the level of CRE and BUN in serum. In summation, these results suggest that CTPS-induced cytoprotective action may play a role in alleviating the side effects induced by chemotherapeutic drugs.

Bombyx batryticatus Protein-Rich Extract Induces Maturation of Dendritic Cells and Th1 Polarization: A Potential Immunological Adjuvant for Cancer Vaccine.

Bombyx batryticatus, a protein-rich edible insect, is widely used as a traditional medicine in China. Several pharmacological studies have reported the anticancer activity of B. batryticatus extracts; however, the capacity of B. batryticatus extracts as immune potentiators for increasing the efficacy of cancer immunotherapy is still unverified. In the present study, we investigated the immunomodulatory role of B. batryticatus protein-rich extract (BBPE) in bone marrow-derived dendritic cells (BMDCs) and DC vaccine-immunized mice. BBPE-treated BMDCs displayed characteristics of mature immune status, including high expression of surface molecules (CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II), increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-12p70), enhanced antigen-presenting ability, and reduced endocytosis. BBPE-treated BMDCs promoted naive CD4+ and CD8+ T-cell proliferation and activation. Furthermore, BBPE/ovalbumin (OVA)-pulsed DC-immunized mice showed a stronger OVA-specific multifunctional T-cell response in CD4+ and CD8+ T cells and a stronger Th1 antibody response than mice receiving differently treated DCs, which showed the enhanced protective effect against tumor growth in E.G7 tumor-bearing mice. Our data demonstrate that BBPE can be a novel immune potentiator for a DC-based vaccine in anticancer therapy.

Chrysin Derivative CM1 and Exhibited Anti-Inflammatory Action by Upregulating Toll-Interacting Protein Expression in Lipopolysaccharide-Stimulated RAW264.7 Macrophage Cells.

Although our previous study revealed that gamma-irradiated chrysin enhanced anti-inflammatory activity compared to intact chrysin, it remains unclear whether the chrysin derivative, CM1, produced by gamma irradiation, negatively regulates toll-like receptor (TLR) signaling. In this study, we investigated the molecular basis for the downregulation of TLR4 signal transduction by CM1 in macrophages. We initially determined the appropriate concentration of CM1 and found no cellular toxicity below 2 µg/mL. Upon stimulation with lipopolysaccharide (LPS), CM1 modulated LPS-stimulated inflammatory action by suppressing the release of proinflammatory mediators (cytokines TNF-α and IL-6) and nitric oxide (NO) and downregulated the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Furthermore, CM1 markedly elevated the expression of the TLR negative regulator toll-interacting protein (Tollip) in dose- and time-dependent manners. LPS-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II), proinflammatory cytokines (TNF-α and IL-6), COX-2, and iNOS-mediated NO were inhibited by CM1; these effects were prevented by the knockdown of Tollip expression. Additionally, CM1 did not affect the downregulation of LPS-induced expression of MAPKs and NF-κB signaling in Tollip-downregulated cells. These findings provide insight into effective therapeutic intervention of inflammatory disease by increasing the understanding of the negative regulation of TLR signaling induced by CM1.

Hymenobacter baengnokdamensis sp. nov., Isolated from the Soil of a Crater Lake in Korea.

An aerobic, Gram-stain-negative, non-motile, non-spore-forming, rod-shaped and pink-colored bacterial strain, designated BRD72T, was isolated from a crater lake (Baengnokdam) at the top of Mt. Hallasan in the Republic of Korea. Cells were catalase-positive and oxidase-negative. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the isolate was a member of the genus Hymenobacter and most closely related to Hymenobacter marinus KJ035T (96.2% similarity). The isolate was found to produce carotenoid pigment, but not flexirubin-type pigment. The predominant fatty acids of strain BRD72T were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c, 21.6%), iso-C15:0 (17.9%), anteiso-C15:0 (13.3%) and summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B, 11.3%). The major polar lipids were phosphatidylethanolamine, an unidentified amino lipid, and two unidentified aminophospholipids. The main respiratory quinone was menaquinone-7 (MK-7), and the main polyamine was homospermidine. The DNA G+C content was 59.8 mol%. Based on the phylogenetic, physiological, and chemotaxonomic characteristics, strain BRD72T represents a novel species, for which the name Hymenobacter baengnokdamensis sp. nov. is proposed. The type strain is BRD72T (= KCTC 72649T = JCM 33837T).

Crystal structure of the highly radiation-inducible DinB/YfiT superfamily protein DR0053 from Deinococcus radiodurans R1.

Deinococcus radiodurans is an extremophilic bacterium well-known for its extraordinary resistance to ionizing radiation and other DNA damage- and oxidative stress-generating agents. In addition to its efficient DNA damage repair and oxidative stress resistance mechanisms, protein family expansions and stress-induced genes/proteins are also regarded as important components that add to the robustness of this bacterium. D. radiodurans encodes specific expansions of 13 DinB/YfiT homologs, which is a relatively large number when compared to those found in Gram-positive bacteria. In this study, we investigated the expression profiles of 13 dinB genes after γ-irradiation, mitomycin C and H2O2 treatment. dr0053 had the highest expression levels after DNA-damage inducing γ-irradiation and MMC treatment, increasing ∼200-fold and ∼16-fold, respectively. We also determined the crystal structure of DR0053 at 2.07 Šresolution. DR0053 adopted a typical four-helix bundle structure that is characteristic of DinB/YfiT proteins. A putative metal binding site was occupied by zinc even though the highly conserved His triad of DinB/YfiT proteins was replaced by Glu-Asn-His.

ER stress stimulates production of the key antimicrobial peptide, cathelicidin, by forming a previously unidentified intracellular S1P signaling complex.

We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.

Gamma-irradiation-killed Streptococcus pneumoniae potently induces the expression of IL-6 and IL-8 in human bronchial epithelial cells.

Streptococcus pneumoniae is a major respiratory pathogen that can cause pneumonia, meningitis, and otitis media. Although capsular polysaccharide-based vaccines are commercially available, there is a need for broad-spectrum, serotype-independent, and cost-effective vaccines. Recently, an intranasal vaccine formulated with gamma-irradiated nonencapsulated S. pneumoniae whole cells has been developed and its immunogenicity is under investigation. Since innate immunity influences the subsequent adaptive immunity, in the present study, we investigated the immunostimulatory activity of gamma-irradiated S. pneumoniae (r-SP) in the human bronchial epithelial cell-line, BEAS-2B, by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP). r-SP potently induced interleukin (IL)-6 and IL-8 at both mRNA and protein levels in a dose- and time-dependent manner, whereas h-SP and f-SP poorly induced them. Of note, the mRNA levels of IL-6 and IL-8 were approximately two-fold higher when cells were stimulated with 3 × 107 CFU/ml of r-SP for 3 h, while the protein levels of IL-6 and IL-8 were approximately five-fold higher after stimulation with 3 × 107 CFU/ml of r-SP for 24 h. Furthermore, r-SP exhibited potent activation of Toll-like receptor 2 compared with h-SP or f-SP. The expression of IL-6 and IL-8 induced by r-SP was mediated through the activation of mitogen-activated protein kinases. Remarkably, when r-SP was further treated with heat or formalin, there was a decrease in the aforementioned activities. Taken together, we suggest that r-SP stimulates the human respiratory epithelial cells to produce the cytokines IL-6 and IL-8, which might influence the induction of adaptive immune responses.

Vaccination With a Latch Peptide Provides Serotype-Independent Protection Against Group B Streptococcus Infection in Mice.

Streptococcus agalactiae (group B streptococcus [GBS]) is a leading cause of invasive diseases in neonates and severe infections in elderly individuals. GBS serine-rich repeat glycoprotein 1 (Srr1) acts as a critical virulence factor by facilitating GBS invasion into the central nervous system through interaction with the fibrinogen Aα chain. This study revealed that srr1 is highly conserved, with 86.7% of GBS clinical isolates expressing the protein. Vaccination of mice with different Srr1 truncated peptides revealed that only Srr1 truncates containing the latch domain protected against GBS meningitis. Furthermore, the latch peptide alone was immunogenic and elicited protective antibodies, which efficiently enhanced antibody-mediated opsonophagocytic killing of GBS by HL60 cells and provided heterogeneous protection against 4 different GBS serogroups. Taken together, these findings indicated that the latch domain of Srr1 may constitute an effective peptide vaccine candidate for GBS.

Opsonophagocytic Antibodies to Serotype Ia, Ib, and III Group B Streptococcus among Korean Infants and in Intravenous Immunoglobulin Products.

Group B streptococcus (GBS) infection is a leading cause of sepsis and meningitis among infants, and is associated with high rates of morbidity and mortality in many countries. Protection against GBS typically involves antibody-mediated opsonization by phagocytes and complement components. The present study evaluated serotype-specific functional antibodies to GBS among Korean infants and in intravenous immunoglobulin (IVIG) products. An opsonophagocytic killing assay (OPA) was used to calculate the opsonization indices (OIs) of functional antibodies to serotypes Ia, Ib, and III in 19 IVIG products from 5 international manufacturers and among 98 Korean infants (age: 0-11 months). The GBS Ia, Ib, and III serotypes were selected because they are included in a trivalent GBS vaccine formulation that is being developed. The OI values for the IVIG products were 635-5,706 (serotype Ia), 488-1,421 (serotype Ib), and 962-3,315 (serotype III), and none of the IVIG lots exhibited undetectable OI values (< 4). The geometric mean OI values were similar for all 3 serotypes when we compared the Korean manufacturers. The seropositive rate among infants was significantly lower for serotype Ia (18.4%), compared to serotype Ib and serotype III (both, 38.8%). Infant age of ≥ 3 months was positively correlated with the seropositive rates for each serotype. Therefore, only a limited proportion of infants exhibited protective immunity against serotype Ia, Ib, and III GBS infections. IVIG products that exhibit high antibody titers may be a useful therapeutic or preventive measure for infants. Further studies are needed to evaluate additional serotypes and age groups.

The three catalases in Deinococcus radiodurans: Only two show catalase activity.

Deinococcus radiodurans, which is extremely resistant to ionizing radiation and oxidative stress, is known to have three catalases (DR1998, DRA0146, and DRA0259). In this study, to investigate the role of each catalase, we constructed catalase mutants (Δdr1998, ΔdrA0146, and ΔdrA0259) of D. radiodurans. Of the three mutants, Δdr1998 exhibited the greatest decrease in hydrogen peroxide (H2O2) resistance and the highest increase in intracellular reactive oxygen species (ROS) levels following H2O2 treatments, whereas ΔdrA0146 showed no change in its H2O2 resistance or ROS level. Catalase activity was not attenuated in ΔdrA0146, and none of the three bands detected in an in-gel catalase activity assay disappeared in ΔdrA0146. The purified His-tagged recombinant DRA0146 did not show catalase activity. In addition, the phylogenetic analysis of the deinococcal catalases revealed that the DR1998-type catalase is common in the genus Deinococcus, but the DRA0146-type catalase was found in only 4 of 23 Deinococcus species. Taken together, these results indicate that DR1998 plays a critical role in the anti-oxidative system of D. radiodurans by detoxifying H2O2, but DRA0146 does not have catalase activity and is not involved in the resistance to H2O2 stress.

Engineering Synthetic Multistress Tolerance in Escherichia coli by Using a Deinococcal Response Regulator, DR1558.

Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H2O2). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H2O2, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.

Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization.

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 "latching" domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr-fibrinogen interactions in the female reproductive tract.

Characterization of fibrinogen binding by glycoproteins Srr1 and Srr2 of Streptococcus agalactiae.

The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of KD = 2.1 × 10(-5) and 3.7 × 10(-6) M, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6-8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a "dock, lock, and latch" mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains.

Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.

The serine-rich repeat glycoprotein Srr1 of Streptococcus agalactiae (GBS) is thought to be an important adhesin for the pathogenesis of meningitis. Although expression of Srr1 is associated with increased binding to human brain microvascular endothelial cells (hBMEC), the molecular basis for this interaction is not well defined. We now demonstrate that Srr1 contributes to GBS attachment to hBMEC via the direct interaction of its binding region (BR) with human fibrinogen. When assessed by Far Western blotting, Srr1 was the only protein in GBS extracts that bound fibrinogen. Studies using recombinant Srr1-BR and purified fibrinogen in vitro confirmed a direct protein-protein interaction. Srr1-BR binding was localized to amino acids 283-410 of the fibrinogen Aα chain. Structural predictions indicated that the conformation of Srr1-BR is likely to resemble that of SdrG and other related staphylococcal proteins that bind to fibrinogen through a "dock, lock, and latch" mechanism (DLL). Deletion of the predicted latch domain of Srr1-BR abolished the interaction of the BR with fibrinogen. In addition, a mutant GBS strain lacking the latch domain exhibited reduced binding to hBMEC, and was significantly attenuated in an in vivo model of meningitis. These results indicate that Srr1 can bind fibrinogen directly likely through a DLL mechanism, which has not been described for other streptococcal adhesins. This interaction was important for the pathogenesis of GBS central nervous system invasion and subsequent disease progression.

Serotype-Dependent Inhibition of Streptococcus pneumoniae Growth by Short-Chain Fatty Acids.

Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that can cause severe infectious diseases such as pneumonia, meningitis, and otitis media. Despite the availability of antibiotics and pneumococcal vaccines against some invasive serotypes, pneumococcal infection remains a tremendous clinical challenge due to the increasing frequency of infection by antimicrobial resistant, nonencapsulated, and/or non-vaccine serotype strains. Short-chain fatty acids (SCFAs), which are produced at various mucosal sites in the body, have potent antimicrobial activity, including inhibition of pathogen growth and/or bacterial biofilm formation. In this study, we investigated the antimicrobial activity of SCFAs (acetate, propionate, and butyrate) against various serotypes pneumococci. Propionate generally inhibited the growth of S. pneumoniae serotypes included in the pneumococcal conjugate vaccine (PCV) 13, except for serotypes 3 and 7F, though butyrate and acetate showed no or low inhibition, depending on the serotypes. Of note, butyrate showed strong inhibition against serotype 3, the most prevalent invasive strain since the introduction of the PCV. No SCFAs showed inhibitory effects against serotype 7F. Remarkably, the nonencapsulated pneumococcal strain had more sensitivity to SCFAs than encapsulated parental strains. Taken together, these results suggest that propionate showing the most potent inhibition of pneumococcal growth may be used as an alternative treatment for pneumococcal infection, and that butyrate could be used against serotype 3, which is becoming a serious threat.

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.

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy.

Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4+ T, and CD8+ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.