Overexpression of diglucosyldiacylglycerol synthase leads to daptomycin resistance in Bacillus subtilis.

The lipopeptide antibiotic daptomycin exhibits bactericidal activity against Gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanisms of bacterial resistance to daptomycin has gained great importance. In this study, we aimed to identify the genetic factors contributing to daptomycin resistance in Bacillus subtilis, a model Gram-positive bacterium. Our findings demonstrated that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis. Specifically, overexpression of ugtP resulted in increased levels of diglucosyldiacylglycerol (Glc2DAG) and decreased levels of acidic phospholipids cardiolipin and PG, as well as the basic phospholipid lysylphosphatidylglycerol. However, ugtP overexpression did not alter the cell surface charge and the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide. Furthermore, by serial passaging in the presence of daptomycin, we obtained daptomycin-resistant mutants carrying ugtP mutations. These mutants showed increased levels of Glc2DAG and a >4-fold increase in the minimum inhibitory concentration of daptomycin. These results suggest that increased Glc2DAG levels, driven by ugtP overexpression, modify the phospholipid composition and confer daptomycin resistance in B. subtilis without altering the cell surface charge of the bacteria.IMPORTANCEDaptomycin is one of the last-resort drugs for the treatment of methicillin-resistant Staphylococcus aureus infections, and the emergence of daptomycin-resistant bacteria has become a major concern. Understanding the mechanism of daptomycin resistance is important for establishing clinical countermeasures against daptomycin-resistant bacteria. In the present study, we found that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis, a model Gram-positive bacteria. The overexpression of UgtP increased diglucosyldiacylglycerol levels, resulting in altered phospholipid composition and daptomycin resistance. These findings are important for establishing clinical strategies against daptomycin-resistant bacteria, including their detection and management.

Ancestry-, Sex-, and Age-Based Differences of Gene Alterations in NSCLC: From the Real-World Data of Cancer Genomic Profiling Tests.

BACKGROUND: Some genomic alterations in non-small cell lung cancer (NSCLC) are known to differ according to race, sex, or age. These studies have been limited in sample size and thus they cannot detect the differences precisely and comprehensively. METHODS: Tissue-based comprehensive genomic profiling was performed on 75,362 patients with NSCLC from the United States during routine clinical care. Additionally, we examined data of a Japanese NSCLC cohort with 1,019 patients. In the US cohort, 296 genes were examined for pathogenic alterations. Predominant genetic ancestry was inferred using a SNP-based approach, and patients were categorized into European (EUR), African (AFR), East Asian (EAS), Admixed American (AMR), and South Asian (SAS) ancestry groups. Patients were additionally stratified by histologic type, age (<40/≥40 years, <75/≥75 years), and sex. The prevalence of high tumor mutational burden (TMB-High) and microsatellite instability status was also calculated. RESULTS: Stratified by ancestry, EGFR alterations were significantly enriched in EAS versus other ancestry groups. The prevalence of ALK was significantly higher in the AMR, EAS, and SAS patients than in AFR and EUR patients. KRAS and STK11 were enriched in EUR and AFR patients versus other groups. TMB-High was significantly enriched in AFR patients versus all other groups. An analysis based on sex revealed differences in prevalence of alterations in 80 genes and TMB-High status. For example, EGFR, ALK, BRAF, and KRAS alterations were significantly enriched in females, whereas TP53, STK11, KEAP1, and TMB-High were significantly enriched in males. With respect to age, the prevalence of alterations in 41 genes, including ALK, RET, MET, EGFR, STK11, KEAP1, BRAF, and KRAS, as well as TMB-High, were significantly different between patients aged <40 years and those aged ≥40 years. CONCLUSIONS: Comprehensive analysis from a large real-world dataset revealed ancestry-associated differences in genomic alterations in NSCLC. Age- and sex-related differences in prevalence of genomic alterations and TMB-High status were also observed.

Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent manner.

The flagellar motor proteins, MotA and MotB, form a complex that rotates the flagella by utilizing the proton motive force (PMF) at the bacterial cell membrane. Although PMF affects the susceptibility to aminoglycosides, the effect of flagellar motor proteins on the susceptibility to aminoglycosides has not been investigated. Here, we found that MotB overexpression increased susceptibility to aminoglycosides, such as kanamycin and gentamicin, in Bacillus subtilis without affecting swimming motility. MotB overexpression did not affect susceptibility to ribosome-targeting antibiotics other than aminoglycosides, cell wall-targeting antibiotics, DNA synthesis-inhibiting antibiotics, or antibiotics inhibiting RNA synthesis. Meanwhile, MotB overexpression increased the susceptibility to aminoglycosides even in the motA-deletion mutant, which lacks swimming motility. Overexpression of the MotB mutant protein carrying an amino acid substitution at the proton-binding site (D24A) resulted in the loss of the enhanced aminoglycoside-sensitive phenotype. These results suggested that MotB overexpression sensitizes B. subtilis to aminoglycosides in a motility-independent manner. Notably, the aminoglycoside-sensitive phenotype induced by MotB requires the proton-binding site but not the MotA/MotB complex formation.

Knockout of adenylosuccinate synthase purA increases susceptibility to colistin in Escherichia coli.

Colistin is a cationic cyclic antimicrobial peptide used as a last resort against multidrug-resistant gram-negative bacteria. To understand the factors involved in colistin susceptibility, we screened colistin-sensitive mutants from an E. coli gene-knockout library (Keio collection). The knockout of purA, whose product catalyzes the synthesis of adenylosuccinate from IMP in the de novo purine synthesis pathway, resulted in increased sensitivity to colistin. Adenylosuccinate is subsequently converted to AMP, which is phosphorylated to produce ADP, a substrate for ATP synthesis. The amount of ATP was lower in the purA-knockout mutant than that in the wild-type strain. ATP synthesis is coupled with proton transfer, and it contributes to the membrane potential. Using the membrane potential probe, 3,3'-diethyloxacarbocyanine iodide [DiOC2(3)], we found that the membrane was hyperpolarized in the purA-knockout mutant compared to that in the wild-type strain. Treatment with the proton uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), abolished the hyperpolarization and colistin sensitivity in the mutant. The purA-knockout mutant exhibited increased sensitivity to aminoglycosides, kanamycin, and gentamicin; their uptake requires a membrane potential. Therefore, the knockout of purA, an adenylosuccinate synthase, decreases ATP synthesis concurrently with membrane hyperpolarization, resulting in increased sensitivity to colistin.

Suppression of Mast Cell Activation by GPR35: GPR35 Is a Primary Target of Disodium Cromoglycate.

Mast cell stabilizers, including disodium cromoglycate (DSCG), were found to have potential as the agonists of an orphan G protein-coupled receptor, GPR35, although it remains to be determined whether GPR35 is expressed in mast cells and involved in suppression of mast cell degranulation. Our purpose in this study is to verify the expression of GPR35 in mast cells and to clarify how GPR35 modulates the degranulation. We explored the roles of GPR35 using an expression system, a mast cell line constitutively expressing rat GPR35, peritoneal mast cells, and bone marrow-derived cultured mast cells. Immediate allergic responses were assessed using the IgE-mediated passive cutaneous anaphylaxis (PCA) model. Various known GPR35 agonists, including DSCG and newly designed compounds, suppressed IgE-mediated degranulation. GPR35 was expressed in mature mast cells but not in immature bone marrow-derived cultured mast cells and the rat mast cell line. Degranulation induced by antigens was significantly downmodulated in the mast cell line stably expressing GPR35. A GPR35 agonist, zaprinast, induced a transient activation of RhoA and a transient decrease in the amount of filamentous actin. GPR35 agonists suppressed the PCA responses in the wild-type mice but not in the GPR35-/- mice. These findings suggest that GPR35 should prevent mast cells from undergoing degranulation induced by IgE-mediated antigen stimulation and be the primary target of mast cell stabilizers. SIGNIFICANCE STATEMENT: The agonists of an orphan G protein-coupled receptor, GPR35, including disodium cromoglycate, were found to suppress degranulation of rat and mouse mature mast cells, and their antiallergic effects were abrogated in the GPR35-/- mice, indicating that the primary target of mast cell stabilizers should be GPR35.

Short-chain fatty acids stimulate dendrite elongation in dendritic cells by inhibiting histone deacetylase.

Dendritic cells activate immune responses by presenting pathogen-derived molecules. The dendrites of dendritic cells contribute to the incorporation of foreign antigens or presenting antigens to T cells. Short-chain fatty acids (SCFAs), such as acetic, propionic, butyric and valeric acids, have many effects on immune responses by activating specific receptors or inhibiting a histone deacetylase (HDAC), although their effect on dendrite formation in dendritic cells is unknown. In the present study, we aimed to investigate the effect of SCFAs on dendrite elongation using a dendritic cell line (DC2.4 cells) and mouse bone marrow-derived dendritic cells. We found that SCFAs induced dendrite elongation. The elongation was reduced by inhibitors of Src family kinase (SFK), phosphatidylinositol-3 kinase (PI3K), Rho family GTPases (Cdc42, Rac1) or actin polymerization, indicating that SCFAs promote dendrite elongation by activating actin polymerization via the SFK/PI3K/Rho family GTPase signaling pathway. We showed that agonists for SCFA receptors GPR43 and GPR109a did not promote dendrite elongation. By contrast, HDAC inhibitors, including trichostatin A, promoted dendrite elongation in DC2.4 cells, and the promoting activity of trichostatin A was decreased by inhibiting the SFK/PI3K/Rho family GTPase signaling pathway or actin polymerization. Furthermore, DC2.4 cells treated with valeric acid showed enhanced uptake of soluble proteins, insoluble beads and Staphylococcus aureus. We also found that treatment with valeric acid enhanced major histocompatibility complex class II-mediated antigen presentation in bone marrow-derived dendritic cells. These results suggest that SCFAs promote dendrite elongation by inhibiting HDAC, stimulating the SFK/PI3K/Rho family pathway and activating actin polymerization, resulting in increased antigen uptake and presentation in dendritic cells.

Budget impact analysis of comprehensive genomic profiling for untreated advanced or recurrent solid cancers in Japan.

AIMS: In Japan, the use of comprehensive genomic profiling (CGP) is only available for cancer patients who have no standard of care (SoC), or those who have completed SoC. This may lead to missed treatment opportunities for patients with druggable alterations. In this study, we evaluated the potential impact of CGP testing before SoC on medical costs and clinical outcome in untreated patients with advanced or recurrent biliary tract cancer (BTC), non-squamous non-small cell lung cancer (NSQ-NSCLC), or colorectal cancer (CRC) in Japan between 2022 and 2026. MATERIALS AND METHODS: We constructed a decision-tree model reflecting the healthcare environment of Japan, to estimate the clinical outcome and medical costs impact of CGP testing by comparing two groups (with vs without CGP testing before SoC). The epidemiological parameters, detection rates of druggable alterations, and overall survival were collected from literature and claims databases in Japan. Treatment options selected based on druggable alterations were set in the model based on clinical experts' opinions. RESULTS: In 2026, the number of untreated patients with advanced or recurrent BTC, NSQ-NSCLC, and CRC was estimated to be 8600, 32,103, and 24,896, respectively. Compared with the group without CGP testing before SoC, CGP testing before SoC increased druggable alteration detection and treatment rate with matched therapies in all three cancer types. The medical costs per patient per month were estimated to increase with CGP testing before SoC in the three cancer types by 19,600, 2900, and 2200 JPY (145, 21, and 16 USD), respectively. LIMITATIONS: Only those druggable alterations with matched therapies were considered in the analysis model, while the potential impact of other genomic alterations provided by CGP testing was not considered. CONCLUSIONS: The present study suggested that CGP testing before SoC may improve patient outcomes in various cancer types with a limited and controllable increase in medical costs.

Nicotine- and tar-removed cigarette smoke extract modulates the antigen presentation function of mouse bone marrow-derived dendritic cells.

Dendritic cells (DCs) take up antigens derived from pathogens such as bacteria and viruses, and from tumor cells and induce the activation of antigen-specific T cells through major histocompatibility complex (MHC)-mediated antigen presentation. Mainstream cigarette smoke extract (CSE) has various effects, and the effects of its major components, nicotine and tar, have been analyzed extensively. Recently, the physiological effects of nicotine- and tar-removed CSE (cCSE) have also been reported. However, the effects of cCSE on DC-mediated immune responses remain unknown. In this study, we found that cCSE enhanced lipopolysaccharide (LPS)-stimulated induction of the expression of MHC-I and MHC-II on the cell surface of mouse bone marrow-derived DCs (BMDCs). In contrast, cCSE suppressed the induction of CD86 induced by stimulation with curdlan and interferon-γ (IFN-γ). In addition, cCSE suppressed the production of IL-12, IL-23, and IL-10 by LPS and curdlan stimulation. In the presence of cCSE, LPS-stimulated BMDCs showed enhanced activation of CD4 and CD8 T cells and increased IL-2 production from T cells by antigen presentation in a mixed-leukocyte reaction assay. In contrast, cCSE did not affect the activation of T cells by curdlan- or IFN-γ-stimulated BMDCs, and curdlan-stimulated BMDCs suppressed IL-17 production from T cells and enhanced IFN-γ production. These results suggest that cCSE has different effects on the activation signals induced by LPS, curdlan, and IFN-γ in BMDCs and modulates the antigen presentation function of BMDCs.

Knockout of ribosomal protein RpmJ leads to zinc resistance in Escherichia coli.

Zinc is an essential metal for cells, but excess amounts are toxic. Other than by regulating the intracellular zinc concentration by zinc uptake or efflux, the mechanisms underlying bacterial resistance to excess zinc are unknown. In the present study, we searched for zinc-resistant mutant strains from the Keio collection, a gene knockout library of Escherichia coli, a model gram-negative bacteria. We found that knockout mutant of RpmJ (L36), a 50S ribosomal protein, exhibited zinc resistance. The rpmJ mutant was sensitive to protein synthesis inhibitors and had altered translation fidelity, indicating ribosomal dysfunction. In the rpmJ mutant, the intracellular zinc concentration was decreased under excess zinc conditions. Knockout of ZntA, a zinc efflux pump, abolished the zinc-resistant phenotype of the rpmJ mutant. RNA sequence analysis revealed that the rpmJ mutant exhibited altered gene expression of diverse functional categories, including translation, energy metabolism, and stress response. These findings suggest that knocking out RpmJ alters gene expression patterns and causes zinc resistance by lowering the intracellular zinc concentration. Knockouts of other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, also led to a zinc-resistant phenotype, suggesting that deletion of ribosomal proteins is closely related to zinc resistance.

ATP and its metabolite adenosine cooperatively upregulate the antigen-presenting molecules on dendritic cells leading to IFN-γ production by T cells.

Dendritic cells (DCs) present foreign antigens to T cells via the major histocompatibility complex (MHC), thereby inducing acquired immune responses. ATP accumulates at sites of inflammation or in tumor tissues, which triggers local inflammatory responses. However, it remains to be clarified how ATP modulates the functions of DCs. In this study, we investigated the effects of extracellular ATP on mouse bone marrow-derived dendritic cells (BMDCs) as well as the potential for subsequent T cell activation. We found that high concentrations of ATP (1 mM) upregulated the cell surface expression levels of MHC-I, MHC-II, and co-stimulatory molecules CD80 and CD86 but not those of co-inhibitory molecules PD-L1 and PD-L2 in BMDCs. Increased surface expression of MHC-I, MHC-II, CD80, and CD86 was inhibited by a pan-P2 receptor antagonist. In addition, the upregulation of MHC-I and MHC-II expression was inhibited by an adenosine P1 receptor antagonist and by inhibitors of CD39 and CD73, which metabolize ATP to adenosine. These results suggest that adenosine is required for the ATP-induced upregulation of MHC-I and MHC-II. In the mixed leukocyte reaction assay, ATP-stimulated BMDCs activated CD4 and CD8T cells and induced interferon-γ (IFN-γ) production by these T cells. Collectively, these results suggest that high concentrations of extracellular ATP upregulate the expression of antigen-presenting and co-stimulatory molecules but not that of co-inhibitory molecules in BMDCs. Cooperative stimulation of ATP and its metabolite adenosine was required for the upregulation of MHC-I and MHC-II. These ATP-stimulated BMDCs induced the activation of IFN-γ-producing T cells upon antigen presentation.