WileyActa Medica Okayama0385-56002026Overexpression of Escherichia coli yaiX Confers Multidrug Resistance and Enhances Virulence in the Silkworm Infection ModelENKinukaHonguGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKazuyaIshikawaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTomokiKosakiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityShin]IchiMiyoshiResearch Center for Intestinal Health Science, Okayama UniversityKazuyukiFurutaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityChikaraKaitoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityThe emergence of bacteria with both antimicrobial resistance and high virulence has become a global health concern, underscoring the urgent need to elucidate the molecular basis underlying these traits. Here, we employed the silkworm (Bombyx mori) infection model, which is suitable for high-throughput screening, together with an Escherichia coli library containing plasmid clones of all genes from strain W3110, to identify genes whose overexpression enhances virulence. We found that overexpression of the uncharacterized protein YaiX promoted bacterial proliferation in silkworms and increased host lethality. Compared with the empty-vector control, the YaiX-overexpressing strain exhibited resistance to multiple antimicrobial agents with diverse mechanisms of action, including -lactams, tetracyclines, fluoroquinolones, aminoglycosides, cationic surfactants, and hydrogen peroxide. Sequence analysis revealed that amino acids 18–52 of YaiX contain a transferase hexapeptide domain predicted to form a left-handed parallel -helix. Overexpression of YaiX mutants lacking regions outside this domain conferred ampicillin resistance, whereas deletion of the hexapeptide domain abolished this phenotype. RNA sequencing and GO enrichment analyses further indicated that YaiX overexpression altered the expression of genes encoding RNA-binding proteins and porins. These findings suggest that YaiX overexpression, through its hexapeptide domain, modulates gene expression and contributes to both multidrug resistance and enhanced virulence in E. coli.No potential conflict of interest relevant to this article was reported.Elsevier BVActa Medica Okayama0006-291X77920251,2-naphthoquinone enhances IFN--induced MHC-I expression in dendritic cells, thereby inducing CD8 T cell activation152453ENKazuyukiFurutaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKanonMiyazatoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKaiKobataGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKazuyaIshikawaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityChikaraKaitoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityDendritic cells play a crucial role in immune responses by capturing pathogens and presenting antigens to T cells via major histocompatibility complex (MHC) molecules, thus triggering adaptive immune responses. 1,2-naphthoquinone (1,2-NQ), a quinone found in diesel exhaust and cigarette smoke, has various physiological functions. In this study, we investigated the effect of 1,2-NQ on the expression of antigen presentation-related molecules in the dendritic cell line DC2.4. The results revealed that 1,2-NQ enhanced the IFN--induced upregulation of MHC-I expression at the transcriptional level. Moreover, it upregulated the expression of NLRC5, a transcriptional activator of MHC-I. 1,2-NQ is a reactive oxygen species (ROS) producing reagent. The 1,2-NQ-induced upregulation of MHC-I expression and downregulation of MHC-II expression were abolished by the ROS scavenger N-acetylcysteine. Similar effects on MHC expression were also observed with ROS-inducing reagents, such as paraquat and diethyl maleate. In addition, dendritic cells stimulated with 1,2-NQ exhibited enhanced efficacy in CD8 T cell activation, which was accompanied by increased IFN- production by T cells. These findings demonstrate that 1,2-NQ enhances the IFN--induced activation of dendritic cells and promotes the activation of CD8 T cells.No potential conflict of interest relevant to this article was reported.WileyActa Medica Okayama1462-29122762025Sulphur]Acquisition Pathways for Cysteine Synthesis Confer a Fitness Advantage to Bacteria in Plant Extractse70126ENKazuyaIshikawaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversitySakiYamaguchiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTaketoTsukaokaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMakotoTsunodaGraduate School of Pharmaceutical Sciences, The University of TokyoKazuyukiFurutaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityChikaraKaitoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityBacteria and plants are closely associated with human society, in fields such as agriculture, public health, the food industry, and waste disposal. Bacteria have evolved nutrient-utilisation systems adapted to achieve the most efficient growth in their major habitats. However, empirical evidence to support the significance of bacterial nutrient utilisation in adaptation to plants is limited. Therefore, we investigated the genetic and nutritional factors required for bacterial growth in plant extracts by screening an Escherichia coli gene-knockout library in vegetable-based medium. Mutants lacking genes involved in sulphur assimilation, whereby sulphur is transferred from sulphate to cysteine, exhibited negligible growth in vegetable-based medium or plant extracts, owing to the low cysteine levels. The reverse transsulphuration pathway from methionine, another pathway for donating sulphur to cysteine, occurring in bacteria such as Bacillus subtilis, also played an important role in growth in plant extracts. These two sulphur-assimilation pathways were more frequently observed in plant-associated than in animal-associated bacteria. Sulphur-acquisition pathways for cysteine synthesis thus play a key role in bacterial growth in plant-derived environments such as plant residues and plant exudates.No potential conflict of interest relevant to this article was reported.WileyActa Medica Okayama0385-56002025Lytic Transglycosylase Deficiency Increases Susceptibility to -lactam Antibiotics But Reduces Susceptibility to Vancomycin in Escherichia coliENTakahikoKimuraLaboratory of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKazuyaIshikawaLaboratory of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityRyosukeNakagawaLaboratory of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKazuyukiFurutaLaboratory of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityChikaraKaitoLaboratory of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityIn Staphylococcus aureus, a gram-positive pathogen, vancomycin-resistant strains become susceptible to -lactam antibiotics, referred to as the gseesaw effect.h However, in gram-negative bacteria, the phenomenon is less clear. Here, we analyzed the gene-knockout effects of eight lytic transglycosylases (slt, mltA, mltB, mltC, mltD, mltE, mltF, mltG) on antibiotic sensitivity in Escherichia coli. Knockout of both slt and mltG increased sensitivity to -lactam antibiotics and reduced sensitivity to vancomycin. The -lactam antibiotic sensitivity and vancomycin resistance of the slt-knockout mutant were abolished by the introduction of the wild-type slt gene but remained unchanged by the introduction of the mutant slt gene encoding an amino acid substitution variant of the transglycosylase catalytic centre. The double-knockout strain for slt and mltB was more sensitive to ampicillin and more resistant to vancomycin than each single-knockout strain. The double-knockout strain for slt and mltG was more sensitive to ampicillin and more resistant to vancomycin than each single-knockout strain. These results suggest that loss of lytic transglycosylase activity causes -lactam antibiotic sensitivity and vancomycin resistance in E. coli.No potential conflict of interest relevant to this article was reported.American Society for MicrobiologyActa Medica Okayama0019-95672025Xenopus laevis as an infection model for human pathogenic bacteriaENAyanoKuriuDivision of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKazuyaIshikawaDivision of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKohsukeTsuchiyaDivision of Immunology and Molecular Biology, Cancer Research Institute, Kanazawa UniversityKazuyukiFurutaDivision of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityChikaraKaitoDivision of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityAnimal infection models are essential for understanding bacterial pathogenicity and corresponding host immune responses. In this study, we investigated whether juvenile Xenopus laevis could be used as an infection model for human pathogenic bacteria. Xenopus frogs succumbed to intraperitoneal injection containing the human pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes. In contrast, non-pathogenic bacteria Bacillus subtilis and Escherichia coli did not induce mortality in Xenopus frogs. The administration of appropriate antibiotics suppressed mortality caused by S. aureus and P. aeruginosa. Strains lacking the agr locus, cvfA (rny) gene, or hemolysin genes in S. aureus, LIPI-1-deleted mutant of L. monocytogenes, which attenuate virulence within mammals, exhibited reduced virulence in Xenopus frogs compared with their respective wild-type counterparts. Bacterial distribution analysis revealed that S. aureus persisted in the blood, liver, heart, and muscles of Xenopus frogs until death. These results suggested that intraperitoneal injection of human pathogenic bacteria induces sepsis-like symptoms in Xenopus frogs, supporting their use as a valuable animal model for evaluating antimicrobial efficacy and identifying virulence genes in various human pathogenic bacteria.No potential conflict of interest relevant to this article was reported.American Society for MicrobiologyActa Medica Okayama0099-22402025Enterobacterial common antigen repeat-unit flippase WzxE is required for Escherichia coli growth under acidic conditions, low temperature, and high osmotic stress conditionsENSakiYamaguchiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKazuyaIshikawaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKazuyukiFurutaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityChikaraKaitoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityColanic acid and enterobacterial common antigen (ECA) are cell-surface polysaccharides that are produced by many Escherichia coli isolates. Colanic acid is induced under acidic, low temperature, and high-salt conditions and is important for E. coli resistance to these stresses; however, the role of ECA in these stresses is less clear. Here, we observed that knockout of flippase wzxE, which translocates lipid-linked ECA repeat units from the cytoplasmic side of the inner membrane to the periplasmic side, resulted in the sensitivity of E. coli BW25113 to acidic conditions. The wzxE-knockout mutant showed reduced growth potential and viable counts in vegetable extracts with acidic environments, including cherry tomatoes, carrots, celery, lettuce, and spinach. A double-knockout strain of wzxE and wecF (glycosyltransferase that adds the third-and-final sugar of the lipid-linked ECA repeat unit) was not sensitive to acidic conditions, with similar results obtained for a double-knockout strain of wzxE and wcaJ (glycosyltransferase that initiates colanic acid lipid-linked repeat-unit biosynthesis). The wzxE-knockout mutant was sensitive to low temperatures or high-salt conditions, which induced colanic acid synthesis, and these sensitivities were abolished by the additional knockout of wcaJ. These results suggest that lipid-linked ECA repeat units confer E. coli susceptibility to acidic, low temperatures, and high-salt conditions in a colanic acid-dependent manner and that wzxE suppresses this negative effect.No potential conflict of interest relevant to this article was reported.Public Library of ScienceActa Medica Okayama1932-62031942024Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent mannere0300634ENMioUnemeGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKazuyaIshikawaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKazuyukiFurutaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityAtsukoYamashitaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityChikaraKaitoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityThe 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.No potential conflict of interest relevant to this article was reported.Oxford University Press (OUP)Acta Medica Okayama1574-69683712024Knockout of adenylosuccinate synthase purA increases susceptibility to colistin in Escherichia colifnae007ENTomonoriKanoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKazuyaIshikawaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKazuyukiFurutaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityChikaraKaitoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityColistin 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.No potential conflict of interest relevant to this article was reported. ElsevierActa Medica Okayama1083-351X29942023ATP and its metabolite adenosine cooperatively upregulate the antigen-presenting molecules on dendritic cells leading to IFN-gamma production by T cells104587ENKazuyukiFurutaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityHirokaOnishiGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityYukiIkadaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKentoMasakiGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversitySatoshiTanakaDepartment of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical UniversityChikaraKaitoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityDendritic 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-gamma (IFN-gamma) production by these T cells. Collectively, these results suggest that high concentrations of extracellular ATP upregulate the expression of antigenpresenting and co-stimulatory molecules but not that of coinhibitory 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-gamma-producing T cells upon antigen presentation.No potential conflict of interest relevant to this article was reported.Public Library of ScienceActa Medica Okayama1932-62031832023Knockout of ribosomal protein RpmJ leads to zinc resistance in Escherichia coliENRikoShirakawaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKazuyaIshikawaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKazuyukiFurutaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityChikaraKaitoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityZinc 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.No potential conflict of interest relevant to this article was reported.WileyActa Medica Okayama0385-56006492020Animal infection models using non]mammals585592ENChikaraKaitoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKanadeMurakamiGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityLinaImaiGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKazuyukiFurutaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityThe use of non]human animal models for infection experiments is important for investigating the infectious processes of human pathogenic bacteria at the molecular level. Mammals, such as mice and rabbits, are also utilized as animal infection models, but large numbers of animals are needed for these experiments, which is costly, and fraught with ethical issues. Various non]mammalian animal infection models have been used to investigate the molecular mechanisms of various human pathogenic bacteria, including Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. This review discusses the desirable characteristics of non]mammalian infection models and describes recent non]mammalian infection models that utilize Caenorhabditis elegans, silkworm, fruit fly, zebrafish, two]spotted cricket, hornworm, and waxworm.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama2352-3409302020Dataset for de novo transcriptome assembly of the African bullfrog Pyxicephalus adspersus105388ENNaokiYoshidaGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityChikaraKaitoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityIn this article, we report the first de novo transcriptome assembly of the African bullfrog Pyxicephalus adspersus. In this data, 75,320,390 raw reads were acquired from African bullfrog mRNA using Illumina paired-end sequencing platform. De novo assembly resulted in a total of 136,958 unigenes. In the obtained unigenes, 30,039 open reading frames (ORFs) were detected. This dataset provides basic information for molecular level analysis of this species, which undergoes a state of dormancy under dry conditions at ordinary temperatures called estivation. No potential conflict of interest relevant to this article was reported.Public Library of ScienceActa Medica Okayama1553-73661642020Non-pathogenic Escherichia coli acquires virulence by mutating a growth-essential LPS transportere1008469ENChikaraKaitoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityHironoYoshikaiGraduate School of Pharmaceutical Sciences, The University of TokyoAiWakamatsuJapan Biological Informatics Consortium (JBIC)AtsushiMiyashitaGraduate School of Pharmaceutical Sciences, The University of TokyoYasuhikoMatsumotoDepartment of Microbiology, Meiji Pharmaceutical UniversityTomokoFujiyukiThe Institute of Medical Science, The University of TokyoMasaruKatoDevision of Bioanalytical Chemistry, School of Pharmacy,Showa UniversityYoshitoshiOguraDepartment of Bacteriology, Faculty of Medical Sciences,Kyushu UniversityTetsuyaHayashiDepartment of Bacteriology, Faculty of Medical Sciences,Kyushu UniversityTakaoIsogaiTranslational Research Center, Fukushima Medical UniversityKazuhisaSekimizuInstitute of Medical Mycology, Teikyo UniversityThe molecular mechanisms that allow pathogenic bacteria to infect animals have been intensively studied. On the other hand, the molecular mechanisms by which bacteria acquire virulence functions are not fully understood. In the present study, we experimentally evaluated the evolution of a non-pathogenic strain of Escherichia coli in a silkworm infection model and obtained pathogenic mutant strains. As one cause of the high virulence properties of E. coli mutants, we identified amino acid substitutions in LptD (G580S) and LptE (T95I) constituting the lipopolysaccharide (LPS) transporter, which translocates LPS from the inner to the outer membrane and is essential for E. coli growth. The growth of the LptD and LptE mutants obtained in this study was indistinguishable from that of the parent strain. The LptD and LptE mutants exhibited increased secretion of outer membrane vesicles containing LPS and resistance against various antibiotics, antimicrobial peptides, and host complement. In vivo cross-linking studies revealed that the conformation of the LptD-LptE complex was altered in the LptD and LptE mutants. Furthermore, several clinical isolates of E. coli carried amino acid substitutions of LptD and LptE that conferred resistance against antimicrobial substances. This study demonstrated an experimental evolution of bacterial virulence properties in an animal infection model and identified functional alterations of the growth-essential LPS transporter that led to high bacterial virulence by conferring resistance against antimicrobial substances. These findings suggest that non-pathogenic bacteria can gain virulence traits by changing the functions of essential genes, and provide new insight to bacterial evolution in a host environment.
Author summary
Pathogenic bacteria developed their virulence properties by changing the functions of various genes after the emergence of the host animals on earth. The types of gene function alterations that confer bacterial virulence properties, however, have remained unclear. We utilized a silkworm infection model to perform an experimental evolution of bacterial virulence activity. From a non-pathogenic strain of Escherichia coli, we obtained a mutant strain that exhibited 500-fold higher virulence than the original strain and identified mutations of the lipopolysaccharide (LPS) transporter, which translocates LPS onto the bacterial surface, as one cause of the high virulence. The mutations changed the structure of the LPS transporter, increased the secretion of outer membrane vesicles, and enabled bacterial survival in the presence of host antimicrobial substances. This mechanism to gain high virulence occurs naturally, as several E. coli clinical isolates carried mutations of the LPS transporter that confer resistance against antimicrobial substances. Our study unveiled a novel mechanism by which bacteria increase their virulence through modifying their gene function.No potential conflict of interest relevant to this article was reported.Public Library of ScienceActa Medica Okayama1932-62031452019Staphylococcus aureus aggregation in the plasma fraction of silkworm hemolymphe0217517ENHirokiRyunoGraduate School of Pharmaceutical Sciences, The University of TokyoFukiNigoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityIsaoNaguroGraduate School of Pharmaceutical Sciences, The University of TokyoKazuhisaSekimizuInstitute of Medical Mycology, Teikyo UniversityChikaraKaitoGraduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Staphylococcus aureus formed bacterial aggregates in the plasma fraction of the hemolymph of silkworm, the larva of Bombyx mori, in a growth-dependent manner. The addition of arabinose or galactose inhibited the formation of S. aureus aggregates in the silkworm plasma. Formation of the bacterial aggregates depended on S. aureus genes required for the synthesis of bacterial surface polysaccharides-ypfP and ltaA, which are involved in lipoteichoic acid synthesis, and the tagO gene, which is involved in wall teichoic acid synthesis. These findings suggest that S. aureus forms bacterial aggregates in the silkworm plasma via bacterial surface teichoic acids.No potential conflict of interest relevant to this article was reported.