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1.
FEBS J ; 290(6): 1502-1518, 2023 03.
Article in English | MEDLINE | ID: mdl-36148483

ABSTRACT

Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing the toxicity. Recently, we identified a TA system termed TsbA/TsbT in the Staphylococcus aureus genome. The induction of the tsbT gene in Escherichia coli halted both DNA and RNA synthesis, reduced supercoiled plasmid and resulted in increasingly relaxed DNA. These results suggested that DNA gyrase was the target of TsbT. In addition, TsbT inhibited both E. coli and S. aureus DNA gyrase activity and induced linearization of plasmid DNA in vitro. Taken together, these results demonstrate that the TsbT toxin targets DNA gyrase in vivo. Site-directed mutagenesis experiments showed that the E27 and D37 residues in TsbT are critical for toxicity. Secondary structure prediction combining the analysis of vacuum-ultraviolet circular-dichroism spectroscopy and neural network method demonstrated that the 22nd-32nd residues of TsbT form an α-helix structure, and that the E27 residue is located around the centre of the α-helix segment. These findings give new insights not only into S. aureus TA systems, but also into bacterial toxins targeting DNA topoisomerases.


Subject(s)
Antitoxins , Toxin-Antitoxin Systems , Escherichia coli/genetics , Escherichia coli/metabolism , Bacterial Proteins/metabolism , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , DNA Gyrase/genetics , Toxin-Antitoxin Systems/genetics , Antitoxins/genetics , RNA
2.
Transplant Direct ; 6(12): e624, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33204822

ABSTRACT

In organ transplantation, the University of Wisconsin (UW) solution has been the gold standard for organ preservation. Quercetin (Que) has numerous antioxidant and anti-inflammatory activities, and sucrose (Suc) may be effective for cold storage (CS). This study aimed to investigate the in vitro protective effect of Que and Suc on cold injury to the kidney and to determine whether Que + Suc could improve ischemia-reperfusion injury during CS and hypothermic oxygenated perfusion (HOPE) in autologous transplantation models. METHODS: BHK-21 cells were stored at 4°C for 3 days in UW solution for CS/machine perfusion (CS/MP-UW) with Que (33.1 µM, 3.3 µM, 0.33 µM) and Suc (0.1 M). In a porcine model of renal autologous transplantation, left kidney grafts were preserved under 3 conditions: group 1, CS preservation for 24 hours; group 2, CS preservation for 22 hours and HOPE with CS/MP-UW solution for 2 hours; and group 3, identical preservation as group 2, with Que and Suc added to the solution. Animals were euthanized on day 7 after autologous transplantation. RESULTS: After 3 days of CS preservation, the CS/MP-UW solution with Que (33.1 µM, 3.3 µM) and Suc showed significant cell protection against cold injury. In the porcine model of renal autologous transplantation, the last blood Cre level and the blood lipid hydroperoxide on posttransplantation day 2 were significantly different between group 1 and group 3. Moreover, the total endothelial, glomerular, tubular, interstitial (EGTI) histology score in the kidney tissue was also significantly different. Regarding the change in renal resistance in HOPE, the decrease observed in group 3 was significantly larger than that in group 2. CONCLUSIONS: Our results suggest that the addition of Que and Suc to a UW solution can improve kidney preservation and could potentially enhance the outcome of kidney transplantation.

3.
Appl Environ Microbiol ; 85(20)2019 10 15.
Article in English | MEDLINE | ID: mdl-31375497

ABSTRACT

Toxin-antitoxin (TA) systems consist of toxin-inhibiting diverse cellular functions (e.g., DNA replication, transcription, and translation) and a noncoding RNA or protein antitoxin. TA systems are associated with various cellular events, such as stress responses, programmed cell death, and bacterial pathogenicity. Recent advances in genome sequencing and bioinformatics research have demonstrated that most bacteria harbor various kinds of TA modules on their chromosomes; however, there is little understanding of chromosomally encoded TA systems in the Gram-positive pathogen Staphylococcus aureus Here, we report on newly discovered S. aureus TA systems, each of which is composed of two proteins. Manual search and gene operon prediction analysis identified eight 2-gene operons as potential candidates for TA systems. Subsequently, using an Escherichia coli host killing and rescue assay, we demonstrated that four of the eight candidates worked as TA systems, designated tsaAT, tsbAT, tscAT, and tsdAT Moreover, the TsaT, TsbT, TscT, and TsdT toxins inhibited S. aureus growth, and the toxicity of TsbT was neutralized by coexpressing the tsbA gene in the native host, S. aureus Further, the bioinformatics analysis of the gene clusters revealed that TsaAT, TsbAT, TscAT, and TsdAT did not exhibit sequence similarity to known bacterial TA systems, and their homologues were present only within Staphylococcus species and not among any other bacteria. Our results further advance not only the understanding of S. aureus TA systems but also the study of unannotated TA systems in various bacterial species.IMPORTANCE Recent advances in genome sequencing and bioinformatics research have demonstrated that most pathogenic bacteria harbor a large number of chromosomally encoded toxin-antitoxin (TA) modules. However, little is known about the TA systems in S. aureus Here, we newly identified four S. aureus TA systems using a combination of manual base-by-base screening and functional analysis in E. coli Moreover, all toxins of the identified TA systems caused growth inhibition in the native host S. aureus Although the newly identified TA systems did not exhibit sequence similarity with known bacterial TA systems, their orthologues were conserved only among other Staphylococcus species, indicating their uniqueness to staphylococci. Our approach opens the possibility for studying unannotated TA systems in various bacterial species.


Subject(s)
Antitoxins/genetics , Bacterial Toxins/genetics , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Toxin-Antitoxin Systems/genetics , Antitoxins/pharmacology , Bacterial Proteins/genetics , Bacterial Toxins/toxicity , Escherichia coli/drug effects , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Multigene Family , Operon , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development
4.
Sci Rep ; 7(1): 2865, 2017 06 06.
Article in English | MEDLINE | ID: mdl-28588310

ABSTRACT

Recent advances in fluorescent protein technology provide a wide variety of biological imaging applications; however current tools for bio-imaging in the Gram-positive bacterium Staphylococcus aureus has necessitated further developments for fluorescence intensity and for a multicolor palette of fluorescent proteins. To enhance the expression of multicolor fluorescent proteins in clinical S. aureus strains, we developed new fluorescent protein expression vectors, containing the blaZ/sodp promoter consisting of the ß-lactamase gene (blaZ) promoter and the ribosome binding site (RBS) of superoxide dismutase gene (sod). We found S. aureus-adapted GFP (GFPsa) driven by the blaZ/sodp promoter was highly expressed in the S. aureus laboratory strain RN4220, but not in the clinical strains, MW2 and N315, harboring the endogenous blaI gene, a repressor of the blaZ gene promoter. We therefore constructed a constitutively induced blaZ/sodp promoter (blaZ/sodp(Con)) by introducing substitution mutations into the BlaI binding motif, and this modification allowed enhanced expression of the multicolor GFP variants (GFPsa, EGFP, mEmerald, Citrine, Cerulean, and BFP) as well as codon-optimized reef coral fluorescent proteins (mCherry and AmCyan) in the S. aureus clinical strains. These new fluorescent probes provide new tools to enhance expression of multicolor fluorescent proteins and facilitate clear visualization of clinical S. aureus strains.


Subject(s)
Fluorescent Antibody Technique , Genetic Vectors/genetics , Molecular Imaging , Staphylococcal Infections/diagnosis , Staphylococcal Infections/microbiology , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Codon , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mutation , Promoter Regions, Genetic
5.
Pathog Dis ; 75(5)2017 07 31.
Article in English | MEDLINE | ID: mdl-28334216

ABSTRACT

Toxin-antitoxin (TA) systems are composed of a toxin that inhibits an essential cellular process (e.g. DNA replication, transcription, membrane integrity) and its cognate antitoxin that neutralizes the effect of the toxin. Staphylococcus aureus harbors two types of chromosomally encoded TA systems, namely mazEFsa encoding a UACAU-specific mRNA interferase and two paralogous genes of yefM-yoeBsa encoding a ribosome-dependent endoribonuclease system. However, little is known about the physiological role of MazEFsa and YefM-YoeBsa in S. aureus. Upon characterizing the phenotypes of single, double and triple gene deletion mutants, we found that mazFsa deletion led to increased biofilm formation. Subsequently, transcriptional analysis revealed that expression of intercellular adhesin (ica) gene, icaADBC, increased in a mazFsa deletion mutant. mazFsa/icaADBC double gene deletion and genetic complementation approaches provided convincing evidence that increased biofilm formation was caused by an increase in polysaccharide intercellular adhesin synthesized by icaADBC-encoded proteins. Furthermore, through the use of alanine substitutions at the conserved active residues of MazFsa, our results suggested that ica-mediated biofilm formation depended on the mRNA interferase activity of MazFsa. These findings give new insights not only into the physiological role of MazEFsa in S. aureus, but also into the regulatory mechanism of ica-dependent biofilm formation.


Subject(s)
Biofilms/growth & development , Gene Deletion , Genes, Bacterial , Staphylococcus aureus/physiology , Toxin-Antitoxin Systems , Amino Acid Substitution , DNA Mutational Analysis , Gene Expression Profiling , Genetic Complementation Test , Polysaccharides, Bacterial/biosynthesis , Staphylococcus aureus/genetics
6.
mBio ; 8(1)2017 01 31.
Article in English | MEDLINE | ID: mdl-28143981

ABSTRACT

Staphylococcus aureus TF2758 is a clinical isolate from an atheroma and a super-biofilm-elaborating/polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosamine (PNAG)-overproducing strain (L. Shrestha et al., Microbiol Immunol 60:148-159, 2016, https://doi.org/10.1111/1348-0421.12359). A microarray analysis and DNA genome sequencing were performed to identify the mechanism underlying biofilm overproduction by TF2758. We found high transcriptional expression levels of a 7-gene cluster (satf2580 to satf2586) and the ica operon in TF2758. Within the 7-gene cluster, a putative transcriptional regulator gene designated rob had a nonsense mutation that caused the truncation of the protein. The complementation of TF2758 with rob from FK300, an rsbU-repaired derivative of S. aureus strain NCTC8325-4, significantly decreased biofilm elaboration, suggesting a role for rob in this process. The deletion of rob in non-biofilm-producing FK300 significantly increased biofilm elaboration and PIA/PNAG production. In the search for a gene(s) in the 7-gene cluster for biofilm elaboration controlled by rob, we identified open reading frame (ORF) SAOUHSC_2898 (satf2584). Our results suggest that ORF SAOUHSC_2898 (satf2584) and icaADBC are required for enhanced biofilm elaboration and PIA/PNAG production in the rob deletion mutant. Rob bound to a palindromic sequence within its own promoter region. Furthermore, Rob recognized the TATTT motif within the icaR-icaA intergenic region and bound to a 25-bp DNA stretch containing this motif, which is a critically important short sequence regulating biofilm elaboration in S. aureus Our results strongly suggest that Rob is a long-sought repressor that recognizes and binds to the TATTT motif and is an important regulator of biofilm elaboration through its control of SAOUHSC_2898 (SATF2584) and Ica protein expression in S. aureus IMPORTANCE: During the search for molecular mechanisms underlying biofilm overproduction of Staphylococcus aureus TF2758, we found a putative transcriptional regulator gene designated rob within a 7-gene cluster showing a high transcriptional expression level by microarray analysis. The deletion of rob in non-biofilm-producing FK300, an rsbU-repaired derivative of NCTC8325-4, significantly increased biofilm elaboration and PIA/PNAG production. The search for a gene(s) in the 7-gene cluster for biofilm elaboration controlled by rob identified ORF SAOUHSC_2898. Besides binding to its own promoter region to control ORF SAOUHSC_2898 expression, Rob recognized the TATTT motif within the icaR-icaA intergenic region and bound to a 25-bp DNA stretch containing this motif, which is a critically important short sequence regulating biofilm elaboration in S. aureus Our results strongly suggest that Rob is a long-sought repressor that recognizes and binds to the TATTT motif and is a new important regulator of biofilm elaboration through its control of SAOUHSC_2898 and Ica protein expression in S. aureus.


Subject(s)
Biofilms/growth & development , Gene Expression Regulation, Bacterial , Genetic Loci , Repressor Proteins/metabolism , Staphylococcus aureus/genetics , Staphylococcus aureus/physiology , Codon, Nonsense , Gene Deletion , Gene Expression Profiling , Genetic Complementation Test , Genome, Bacterial , Microarray Analysis , Multigene Family , Mutant Proteins/genetics , Mutant Proteins/metabolism , Operon , Polysaccharides, Bacterial/metabolism , Repressor Proteins/genetics , Sequence Analysis, DNA , Staphylococcal Infections/microbiology , Staphylococcus aureus/isolation & purification
7.
J Investig Clin Dent ; 8(1)2017 Feb.
Article in English | MEDLINE | ID: mdl-26345599

ABSTRACT

AIM: Oral microflora during pregnancy is critical to oral health care in the mother and her child. We examined the changes in the oral microbiota between pregnancy and nonpregnancy periods. METHODS: The study was performed using 132 healthy pregnant women enrolled from Hiroshima City Asa Citizens Hospital and 51 healthy nonpregnant women as control. During pregnancy, 132 subjects were assessed for seven microbial species by the cultured method and polymerase chain reaction at the early (7-16 weeks gestation), the middle (17-28 weeks), and the late (29-39 weeks) pregnancy periods. Pregnant women completed a series of questionnaires regarding oral and systemic health and lifestyle habits. RESULTS: The total cultivable microbial counts in the early pregnancy were significantly higher than that of the nonpregnant women (P < 0.05). The incidences of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in gingival sulcus during the early and middle pregnancy were significantly higher than the nonpregnant group (P < 0.05), while Prevotella intermedia and Fusobacterium nucleatum did not change. Candida species were more frequently detected during the middle and late pregnancy. CONCLUSION: The data suggest that pregnancy, especially in the early periods, promotes the proliferation of microorganisms in the oral cavity and facilitates a colonization of periodontal pathogens.


Subject(s)
Microbiota , Mouth/microbiology , Adult , Aggregatibacter actinomycetemcomitans , Bacterial Load , Candida , Case-Control Studies , Female , Fusobacterium nucleatum , Humans , Japan/epidemiology , Porphyromonas gingivalis , Pregnancy , Pregnancy Complications, Infectious/epidemiology , Pregnancy Complications, Infectious/microbiology , Pregnancy Trimesters , Prevotella intermedia
8.
Microbiol Immunol ; 60(3): 148-59, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26786482

ABSTRACT

A novel benzimidazole molecule that was identified in a small-molecule screen and is known as antibiofilm compound 1 (ABC-1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 µM ABC-1 was tested in various biofilm-forming strains of S. aureus. It was demonstrated that ABC-1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall-associated protein dependent or cell wall- associated extracellular DNA (eDNA). Of note, ABC-1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC-1 treated strains, implying that ABC-1 affects not only SpA but also other factors. Indeed, ABC-1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC-1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.


Subject(s)
Anti-Bacterial Agents/pharmacology , Benzimidazoles/pharmacology , Biofilms/drug effects , Staphylococcus aureus/drug effects , Staphylococcus aureus/physiology , Aminoacyltransferases/genetics , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Benzimidazoles/chemistry , Biofilms/growth & development , Cell Wall/metabolism , Cysteine Endopeptidases/genetics , Down-Regulation , Polysaccharides, Bacterial/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Staphylococcal Protein A/biosynthesis , Staphylococcal Protein A/drug effects , Staphylococcal Protein A/genetics , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism
9.
Antimicrob Agents Chemother ; 59(5): 2678-87, 2015 May.
Article in English | MEDLINE | ID: mdl-25712351

ABSTRACT

A 9-year surveillance for multidrug-resistant (MDR) Pseudomonas aeruginosa in the Hiroshima region showed that the number of isolates harboring the metallo-ß-lactamase gene bla(IMP-1) abruptly increased after 2004, recorded the highest peak in 2006, and showed a tendency to decline afterwards, indicating a history of an epidemic. PCR mapping of the variable regions of the integrons showed that this epidemic was caused by the clonal persistence and propagation of an MDR P. aeruginosa strain harboring the bla(IMP-1) gene and an aminoglycoside 6'-N-acetyltransferase gene, aac(6')-Iae in a class I integron (In113), whose integrase gene intl1 was disrupted by an IS26 insertion. Sequence analysis of the representative strain PA058447 resistance element containing the In113-derived gene cassette array showed that the element forms an IS26 transposon embedded in the chromosome. It has a Tn21 backbone and is composed of two segments sandwiched by three IS26s. In Japan, clonal nationwide expansion of an MDR P. aeruginosa NCGM2.S1 harboring chromosomally encoded In113 with intact intl1 is reported. Multilocus sequence typing and genomic comparison strongly suggest that PA058447 and NCGM2.S1 belong to the same clonal lineage. Moreover, the structures of the resistance element in the two strains are very similar, but the sites of insertion into the chromosome are different. Based on tagging information of the IS26 present in both resistance elements, we suggest that the MDR P. aeruginosa clone causing the epidemic in Hiroshima for the past 9 years originated from a common ancestor genome of PA058447 and NCGM2.S1 through an IS26 insertion into intl1 of In113 and through IS26-mediated genomic rearrangements.


Subject(s)
Pseudomonas aeruginosa/genetics , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Integrons/genetics , Japan , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , beta-Lactamases/genetics
10.
PLoS One ; 9(5): e98285, 2014.
Article in English | MEDLINE | ID: mdl-24865216

ABSTRACT

Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP) that was originally identified as an inositol trisphosphate-binding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs). We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Fibroblasts/metabolism , Microtubule-Associated Proteins/metabolism , Staphylococcal Infections/immunology , Staphylococcus aureus/physiology , Adaptor Proteins, Signal Transducing/genetics , Animals , Autophagy , Cells, Cultured , Fibroblasts/cytology , Immunity, Innate , Mice , Mice, Knockout , Phagosomes/metabolism , Phagosomes/microbiology , Staphylococcal Infections/microbiology
11.
Antimicrob Agents Chemother ; 57(12): 6131-40, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24080652

ABSTRACT

We report the complete nucleotide sequence and analysis of pETBTY825, a Staphylococcus aureus TY825 plasmid encoding exfoliative toxin B (ETB). S. aureus TY825 is a clinical isolate obtained from an impetigo patient in 2002. The size of pETBTY825, 60.6 kbp, was unexpectedly larger than that of the archetype pETBTY4 (∼30 kbp). Genomic comparison of the plasmids shows that pETBTY825 has the archetype pETBTY4 as the backbone and has a single large extra DNA region of 22.4 kbp. The extra DNA region contains genes for resistance to aminoglycoside [aac(6')/aph(2″)], macrolide (msrA), and penicillin (blaZ). A plasmid deletion experiment indicated that these three resistance elements were functionally active. We retrospectively examined the resistance profile of the clinical ETB-producing S. aureus strains isolated in 1977 to 2007 using a MIC determination with gentamicin (GM), arbekacin (ABK), and erythromycin (EM) and by PCR analyses for aac(6')/aph(2″) and msrA using purified plasmid preparations. The ETB-producing S. aureus strains began to display high resistance to GM, which was parallel with the detection of aac(6')/aph(2″) and mecA, after 1990. Conversely, there was no significant change in the ABK MIC during the testing period, although it had a tendency to slightly increase. After 2001, isolates resistant to EM significantly increased; however, msrA was hardly detected in ETB-producing S. aureus strains, and only five isolates were positive for both aac(6')/aph(2″) and msrA. In this study, we report the emergence of a fusion plasmid carrying the toxin gene etb and drug resistance genes. Prevalence of the pETBTY825 carrier may further increase the clinical threat, since ETB-producing S. aureus is closely related to more severe impetigo or staphylococcal scalded-skin syndrome (SSSS), which requires a general antimicrobial treatment.


Subject(s)
Drug Resistance, Multiple, Bacterial/genetics , Exfoliatins/genetics , Plasmids , Staphylococcus aureus/genetics , Anti-Bacterial Agents/pharmacology , Base Sequence , Dibekacin/analogs & derivatives , Dibekacin/pharmacology , Erythromycin/pharmacology , Exfoliatins/biosynthesis , Gentamicins/pharmacology , Humans , Impetigo/drug therapy , Impetigo/microbiology , Japan , Microbial Sensitivity Tests , Molecular Sequence Data , Retrospective Studies , Staphylococcal Scalded Skin Syndrome/drug therapy , Staphylococcal Scalded Skin Syndrome/microbiology , Staphylococcus aureus/drug effects , Staphylococcus aureus/isolation & purification
12.
PLoS One ; 8(8): e70557, 2013.
Article in English | MEDLINE | ID: mdl-23950962

ABSTRACT

In a continuing study from Dec 2006 to Apr 2008, we characterized nine multi-drug resistant Pseudomonas aeruginosa strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis of SpeI-digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-ß-lactamase. PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated as In124, that carries an array of four gene cassettes: a novel aminoglycoside (AG) resistance gene, aac(6')-Iag, blaIMP-1, a truncated form of blaIMP-1, and a truncated form of aac(6')-Iag. The aac(6')-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6')-Iz. Recombinant AAC(6')-Iag protein showed aminoglycoside 6'-N-acetyltransferase activity using thin-layer chromatography (TLC) and MS spectrometric analysis. Escherichia coli carrying aac(6')-Iag showed resistance to amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not to gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for blaIMP-1 and aac(6')-Ig strongly suggested In124 is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually all AGs, suggesting that the In124 conjugal plasmid also possesses a gene conferring resistance to gentamicin.


Subject(s)
Acetyltransferases/genetics , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Integrons , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , beta-Lactamases/genetics , Acetylation , Acetyltransferases/metabolism , Aminoglycosides/chemistry , Aminoglycosides/metabolism , Base Sequence , Cross Infection/epidemiology , Disease Outbreaks , Gene Order , Humans , Kinetics , Molecular Sequence Data , Phylogeny , Pseudomonas Infections/epidemiology , Pseudomonas aeruginosa/classification , Sequence Alignment , beta-Lactamases/metabolism
13.
PLoS One ; 8(4): e61785, 2013.
Article in English | MEDLINE | ID: mdl-23613936

ABSTRACT

IL-5 is a key cytokine that plays an important role in the development of pathological conditions in allergic inflammation. Identifying strategies to inhibit IL-5 production is important in order to establish new therapies for treating allergic inflammation. We found that SH-2251, a novel thioamide-related small compound, selectively inhibits the differentiation of IL-5-producing Th2 cells. SH-2251 inhibited the induction of active histone marks at the Il5 gene locus during Th2 cell differentiation. The recruitment of RNA polymerase II, and following expression of the Th2 cell-specific intergenic transcripts around the Il5 gene locus was also inhibited. Furthermore, Th2 cell-dependent airway inflammation in mice was suppressed by the oral administration of SH-2251. Gfi1, a transcriptional repressor, was identified as a downstream target molecule of SH-2251 using a DNA microarray analysis. The Gfi1 expression dramatically decreased in SH-2251-treated Th2 cells, and the SH-2251-mediated inhibition of IL-5-producing Th2 cell differentiation was restored by transduction of Gfi1. Therefore, our study unearthed SH-2251 as a novel therapeutic candidate for allergic inflammation that selectively inhibits active histone marks at the Il5 gene locus.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Chromatin/metabolism , Inflammation/drug therapy , Interleukin-5/genetics , Th2 Cells/metabolism , Animals , CD4-Positive T-Lymphocytes/metabolism , Chromatin/drug effects , Chromatin Immunoprecipitation , Enzyme-Linked Immunosorbent Assay , Immunoblotting , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis/methods , Respiratory Hypersensitivity/drug therapy , Reverse Transcriptase Polymerase Chain Reaction
14.
Diagn Microbiol Infect Dis ; 76(1): 119-21, 2013 May.
Article in English | MEDLINE | ID: mdl-23541689

ABSTRACT

We investigated 5 metallo-ß-lactamase (MBL)-positive Klebsiella isolates from Japan showing intermediate resistance to imipenem. Sequencing of the MBL gene identified a novel variant of IMP-1 with a single amino acid substitution, Glu87Gly. This variant is designated as IMP-34 where blaIMP-34 is located on a transmissible plasmid.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Imipenem/pharmacology , Klebsiella/enzymology , beta-Lactamases/genetics , Japan , Klebsiella/drug effects , Klebsiella/genetics , Klebsiella/isolation & purification , Microbial Sensitivity Tests , Plasmids
15.
J Microbiol Methods ; 88(1): 182-4, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22108497

ABSTRACT

Klebsiella pneumoniae resistant to almost all ß-lactams except imipenem designated as ISMRK (imipenem-susceptible meropenem-resistant Klebsiella) is emerging in Japan. All ISMRK carries bla(IMP-6) which differs from bla(IMP-1) by only a single nucleotide at position 640. We devised a rapid detection system of bla(IMP-6) by using ARMS PCR.


Subject(s)
Bacterial Proteins/genetics , Klebsiella Infections/microbiology , Klebsiella pneumoniae/isolation & purification , Mutation , Polymerase Chain Reaction/methods , beta-Lactamases/genetics , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Humans , Imipenem/pharmacology , Japan , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/genetics , Microbial Sensitivity Tests
16.
Diagn Microbiol Infect Dis ; 72(1): 109-12, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22078906

ABSTRACT

We identified 5 Klebsiella pneumoniae isolates showing high resistance to ß-lactams except imipenem and designated them ISMRK (imipenem-susceptible but meropenem-resistant Klebsiella). They carried the bla(IMP-6) and bla(CTX-M-2) on a self-transmissible plasmid. ISMRK may be falsely categorized as susceptible to carbapenems if imipenem is used to screen carbapenem resistance.


Subject(s)
Anti-Bacterial Agents/pharmacology , Imipenem/pharmacology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/enzymology , Thienamycins/pharmacology , beta-Lactam Resistance , beta-Lactamases/metabolism , Aged, 80 and over , Conjugation, Genetic , Female , Gene Transfer, Horizontal , Humans , Japan , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Male , Meropenem , Plasmids/analysis
17.
Appl Environ Microbiol ; 77(22): 8097-105, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21926198

ABSTRACT

Staphylococcus aureus produces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed by in vitro experiments using bacterial medium. However, when S. aureus infects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors in S. aureus grown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 µM FeCl3 into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant with agr inactivated grown in serum, the expression of RNA III, psm, and sec4 was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate that S. aureus expresses virulence factors in adaptation to the host environment.


Subject(s)
Gene Expression Profiling , Serum/microbiology , Staphylococcus aureus/growth & development , Staphylococcus aureus/pathogenicity , Virulence Factors/biosynthesis , Animals , Cattle , Culture Media/chemistry , Transcriptome
18.
J Microbiol Methods ; 87(1): 76-81, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21801759

ABSTRACT

Staphylococcus aureus is a Gram-positive pathogen that causes opportunistic infections and a wide variety of diseases. Methicillin-resistant S. aureus (MRSA) is frequently isolated as multidrug-resistant in nosocomial and community infections. Molecular genetic manipulation is an important tool for understanding the molecular mechanism of S. aureus infection. However the number of available antibiotic markers is limited due to multidrug resistance. In this study, we constructed two Escherichia coli-S. aureus shuttle vectors, pKFT and pKFC, that carry a temperature-sensitive origin of replication in S. aureus, lacZ(a) enabling a simple blue-white screening in E. coli, an ampicillin resistant gene, and either a tetracycline resistance gene or a chloramphenicol resistance gene. We report a simple technique using pKFT to construct a markerless gene deletion mutant in S. aureus by allelic replacement without the use of a counter-selection marker. Subculture twice at 25°C was critical to promote an allelic exchange rate in S. aureus. This technique is very simple and useful to facilitate genetic research on S. aureus.


Subject(s)
Gene Deletion , Gene Knockout Techniques/methods , Staphylococcus aureus/genetics , Bacterial Proteins/genetics , Cloning, Molecular , DNA-Binding Proteins/genetics , Genetic Engineering , Homologous Recombination , Plasmids/genetics , Staphylococcus aureus/growth & development
19.
FEMS Microbiol Lett ; 320(1): 33-9, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21517944

ABSTRACT

Bacterial two-component systems (TCSs) have been demonstrated to be associated with not only the expression of virulence factors, but also the susceptibility to antibacterial agents. In Staphylococcus aureus, 16 types of TCSs have been identified. We previously found that the inactivation of one uncharacterized TCS (designated as BceRS, MW gene ID: MW2545-2544) resulted in an increase in susceptibility to bacitracin. In this study, we focused on this TCS and tried to identify the TCS-controlled factors affecting the susceptibility to bacitracin. We found that two ABC transporters were associated with the susceptibility to bacitracin. One transporter designated as BceAB (MW2543-2542) is downstream of this TCS, while another (formerly designated as VraDE: MW2620-2621) is separate from this TCS. Both transporters showed homology with several bacitracin-resistance factors in Gram-positive bacteria. Inactivation of each of these two transporters increased the susceptibility to bacitracin. Expressions of these transporters were significantly increased by the addition of bacitracin, while this induction was not observed in the TCS-inactivated mutant. These results indicate that this TCS senses bacitracin, and also positively regulates the expression of two ABC transporters.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacitracin/pharmacology , Drug Resistance, Bacterial , Staphylococcus aureus/drug effects , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Microbial Sensitivity Tests , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism
20.
Microbiology (Reading) ; 157(Pt 6): 1786-1797, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21393369

ABSTRACT

Bacterial cell surface charge is responsible for susceptibility to cationic antimicrobial peptides. Previously, Staphylococcus aureus dlt and mprF were identified as factors conferring a positive charge upon cell surfaces. In this study, we investigated the regulation of cell surface charge during growth. Using a group of S. aureus MW2 mutants, which are gene-inactivated in 15 types of two-component systems (TCSs), we tested dltC and mprF expression and found that two TCSs, aps and agr, were associated with dltC and mprF expression in a growth phase-dependent manner. The first of these, aps, which had already been identified as a sensor of antimicrobial peptides and a positive regulator of dlt and mprF expression, was expressed strongly in the exponential phase, while its expression was significantly suppressed by agr in the stationary phase, resulting in higher expression of dltC and mprF in the exponential phase and lower expression in the stationary phase. Since both types of expression affected the cell surface charge, the susceptibility to antimicrobial peptides and cationic antibiotics was changed during growth. Furthermore, we found that the ability to sense antimicrobial peptides only functioned in the exponential phase. These results suggest that cell surface charge is tightly regulated during growth in S. aureus.


Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Quorum Sensing , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Aminoacyltransferases/genetics , Aminoacyltransferases/metabolism , Bacterial Proteins/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Membrane , Humans , Microbial Sensitivity Tests , Mutation , Staphylococcus aureus/genetics , Trans-Activators/genetics , Trans-Activators/metabolism
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