Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis.

The resistance to detergents and detergent-induced tolerance of a gastrointestinal organism, Enterococcus faecalis ATCC 19433, were examined. The most remarkable observation was the rapid response of cells in contact with bile salts and sodium dodecyl sulfate (SDS). The killing by high concentrations of detergents was nearly instantaneous. A 5-s adaptation with moderate sublethal concentrations of bile salts or SDS (0.08 or 0.01%, respectively) was sufficient to induce significant adaptation against homologous lethal conditions (0.3% bile salts or 0.017% SDS). However, resistance to a subsequent lethal challenge progressively increased further to a maximum reached after 30 min of adaptation. Furthermore, extremely strong cross-resistances were observed with bile salts- and SDS-adapted cells. However, no relationship seems to exist between levels of tolerance and de novo-synthesized proteins, since blockage of protein synthesis during adaptation had no effect on induction of resistance to bile salts and SDS. We conclude that this induced tolerance to detergent stress is independent of protein synthesis. Nevertheless, the stress-induced protein patterns of E. faecalis ATCC 19433 showed significant modifications. The rates of synthesis of 45 and 34 proteins were enhanced after treatments with bile salts and SDS, respectively. In spite of the overlap of 12 polypeptides, the protein profiles induced by the two detergents were different, suggesting that these detergents trigger different responses in E. faecalis. Therefore, bile salts cannot be substituted for SDS in biochemical detergent shock experiments with bacteria.     

Survey of genomic diversity among Enterococcus faecalis strains by microarray-based comparative genomic hybridization

We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic hybridization using microarrays (CGH). The strains used in this study (the "test" strains) originated from various environments. CGH is a powerful and promising tool for obtaining novel information on genome diversity in bacteria. By CGH, one obtains clues about which genes are present or divergent in the strains, compared to a reference strain (here, V583). The information obtained by CGH is important from both ecological and systematic points of view. CGH of E. faecalis showed considerable diversity in gene content: Compared to V583, the percentage of divergent genes in the test strains varied from 15% to 23%, and 154 genes were divergent in all strains. The main variation was found in regions corresponding to exogenously acquired or mobile DNA in V583. Antibiotic resistance genes, virulence factors, and integrated plasmid genes dominated among the divergent genes. The strains examined showed various contents of genes corresponding to the pTEF1, pTEF2, and pTEF3 genes in V583. The extensive transport and metabolic capabilities of V583 appeared similar in the test strains; CGH indicated that the ability to transport and metabolize various carbohydrates was similar in the test strains (verified by API 50 CH assays). The contents of genes related to stress tolerance appeared similar in V583 and the nine test strains, supporting the view of E. faecalis as an organism able to resist harsh conditions.     

Large-scale screening of a targeted Enterococcus faecalis mutant library identifies envelope fitness factors

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.     

Comparative genomic hybridization analysis of Enterococcus faecalis: identification of genes absent from food strains

Enterococcus faecalis, a member of the natural microbiota of animal and human intestinal tracts, is also present as a natural contaminant in a variety of fermented foods. Over the last decade, E. faecalis has emerged as a major cause of nosocomial infections. We investigated the genetic diversity in 30 clinical and food isolates, including strains V583 and MMH594, in order to determine whether clinical and food isolates could be distinguished. Data were obtained using comparative genomic hybridization and specific PCR with a total of 202 probes of E. faecalis, selected using the available V583 genome sequence and part of the MMH594 pathogenicity island. The cognate genes encoded mainly exported proteins. Hybridization data were analyzed by a two-component mixture model that estimates the probability of any given gene to be either present or absent in the strains. A total of 78 genes were found to be variable, as they were absent in at least one isolate. Most of the variable genes were clustered in regions that, in the published V583 sequence, related to prophages or mobile genetic elements. The variable genes were distributed in three main groups: (i) genes equally distributed between clinical and dairy food isolates, (ii) genes absent from dairy food-related isolates, and (iii) genes present in MMH594 and V583 strains only. Further analysis of the distribution of the last gene group in 70 other isolates confirmed that six of the probed genes were always absent in dairy food-related isolates, whereas they were detected in clinical and/or commensal isolates. Two of them corresponded to prophages that were not detected in the cognate isolates, thus possibly extending the number of genes absent from dairy food isolates. Genes specifically detected in clinical isolates may prove valuable for the development of new risk assessment markers for food safety studies and for identification of new factors that may contribute to host colonization or infection.     

LuxS-Dependent AI-2 Regulates Versatile Functions in Enterococcus faecalis V583

Bacteria utilize a quorum sensing (QS) system to coordinate gene expression by monitoring the concentration of molecules known as autoinducers (AI). In the present study, we confirmed the presence of a LuxS/AI-2 dependent QS system in vancomycin-resistant Enterococcus faecalis V583. Then, the cellular targets controlled by AI-2 were identified by comparative proteomics analysis in order to elucidate the possible role of AI-2 in E. faecalis. Results demonstrated 15 proteins that are differentially expressed upon the addition of AI-2, including proteins involved in metabolism, translation, energy production and/or conversion, and cell wall biogenesis. Glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase associated with carbohydrate metabolism and energy production were up-regulated upon inducing by AI-2. In addition, externally added AI-2 could down-regulate acetyl-coenzyme A carboxylase and ornithine carbamoyltransferase, two key enzyme involved in metabolism. All these data suggest that AI-2 signaling may play a role in the regulation of a number of important metabolic properties of E. faecali. We further investigated the role of AI-2 in biofilm formation by E. faecalis, showing the addition of AI-2 to E. faecalis V583 cultures resulted in increased biofilm formation. Our results provide important clues to the role of a LuxS/AI-2 dependent QS system in vancomycin-resistant E. faecalis.     

RNA-Seq探索低水平利奈唑胺耐药粪肠球菌机制

为了探索低水平利奈唑胺耐药粪肠球菌机制,本研究采用Illumina HiSeq 4000高通量转录组测序技术对低水平利奈唑胺耐药粪肠球菌(P10748 MIC:8 mg/L)和利奈唑胺敏感粪肠球菌(3138 MIC:2 mg/L)进行测序及生物信息学分析,以标准菌株ATCC29212作为质量评估参考。利用GO和KEGG数据库对差异表达基因进行注释与富集分析,并利用荧光定量PCR对测序中的差异表达基因进行验证。结果显示,测序共获得了3.57 Gb有效数据,通过De novo拼接获得1 920条unigenes,总长度为2 122 210 bp,平均长度为1 105 bp。差异基因模式聚类分析显示共有150个显著性差异表达基因(FDR≤0.001,|log2 Ratio|≥1),其中141个上调,9个下调。其中生物膜形成和外排泵相关基因esp、optrA、fexA在耐药菌中显著上调。GO功能注释结果显示,催化活性、代谢过程和细胞是注释最多的条目;Pathway富集分析发现,肽聚糖生物合成、缬氨酸亮氨酸和异亮氨酸的降解和硫代谢为显著性富集通路(p<0.05)。荧光定量PCR结果与测序结果基本一致。推测膜转运蛋白和生物膜形成可能在耐药机制中具有重要作用,为低水平利奈唑胺耐药肠球菌机制提供了可参考的耐药靶标。     

野生稻基因导入系W6023对白叶枯菌的抗谱及转录组差异表达基因分析

水稻白叶枯病是世界性水稻病害,严重威胁水稻的高产和稳产。为了挖掘水稻抗白叶枯病新基因,本研究对一个野生稻基因导入系W6023进行了白叶枯病多菌系接种鉴定及抗谱分析,发现W6023具有广谱抗病性。用强致病菌PXO99诱导W6023及其感病轮回亲本IR24后进行转录组测序,分别获得105644962和91022599条序列,通过GO注释及KEGG富集分析,发现差异表达基因主要富集在次生代谢产物的生物合成、植物激素信号转导及糖代谢途径等方面。在这些差异表达基因中,发现203个基因的表达有显著差异,其中在W6023中上调表达的基因有114个(56.2%),下调表达的有89个(43.8%),而且35.9%分布在第11染色体上。生物信息学分析发现在203个差异表达基因中有16个属于类抗病基因,如NBS-LRR等;14个直接或间接与水稻体内过氧化物的代谢相关,如编码过氧化物酶和金属硫蛋白等;6个编码抗病相关转录因子,如WRKY和NAC等;18个为信号传导相关基因,编码钙调素结合蛋白和萜类合成酶等。随机选择6个W6023中上调表达和3个IR24中上调表达的基因进行RT-PCR及qRT-PCR分析,结果与转录组测序结果一致,表明本研究获得的转录组测序数据结果是可靠的,为以后更深入挖掘W6023的抗病基因及分子机理研究提供了基础。     

Survey of Genomic Diversity among Enterococcus faecalis Strains by Microarray-Based Comparative Genomic Hybridization

We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic hybridization using microarrays (CGH). The strains used in this study (the “test” strains) originated from various environments. CGH is a powerful and promising tool for obtaining novel information on genome diversity in bacteria. By CGH, one obtains clues about which genes are present or divergent in the strains, compared to a reference strain (here, V583). The information obtained by CGH is important from both ecological and systematic points of view. CGH of E. faecalis showed considerable diversity in gene content: Compared to V583, the percentage of divergent genes in the test strains varied from 15% to 23%, and 154 genes were divergent in all strains. The main variation was found in regions corresponding to exogenously acquired or mobile DNA in V583. Antibiotic resistance genes, virulence factors, and integrated plasmid genes dominated among the divergent genes. The strains examined showed various contents of genes corresponding to the pTEF1, pTEF2, and pTEF3 genes in V583. The extensive transport and metabolic capabilities of V583 appeared similar in the test strains; CGH indicated that the ability to transport and metabolize various carbohydrates was similar in the test strains (verified by API 50 CH assays). The contents of genes related to stress tolerance appeared similar in V583 and the nine test strains, supporting the view of E. faecalis as an organism able to resist harsh conditions.     

Immune-related gene expression profiling of yellowtail (Seriola quinqueradiata) kidney cells stimulated with ConA and LPS using microarray analysis

To better understand the immune system of a commercially important fish (yellowtail, Seriola quinqueradiata), we constructed a cDNA microarray containing 1001 selected genes from yellowtail EST and used this to investigate gene expression of primary cultured kidney cells stimulated with ConA and LPS. The total number of up-regulated genes stimulated by LPS was apparently greater than that of ConA stimulation, whereas down-regulated genes were markedly found in ConA-stimulated group. Of the genes that were up-regulated at 3, 6, and 12h after LPS treatment, 12%, 13% and 12%, respectively, were immune-related. Immune-related genes were sorted into 4 groups based on their differential expression patterns against LPS induction. LPS induced the expression of genes related to inflammation, cytokine activity, antigen presentation and antigen binding such as, IL-1beta, CC chemokine with stalk CK2, MHC class II beta chain and immunoglobulin heavy chain. Amplified fragments of RT-PCR products of IgM, IL-1beta, nephrosin, and beta-actin had signal intensities that were comparable to those obtained with the microarray. Overall, these results show that microarrays are a promising tool for uncovering immune mechanism in teleost fish. cDNA sequences of genes were deposited in the GenBank database at DDBJ with accession numbers BB 996897-BB 997897.     

Probiotic characteristics and in vitro compatibility of a combination of Bifidobacterium breve M-16 V,Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536

The consumption of probiotic-based products has risen greatly in recent decades. Due to their probiotic characteristics, microorganisms such as lactobacilli and bifidobacteria are in daily use in the production of food supplements. In the present study, three bifidobacterial strains (Bifidobacterium breve M-16 V, Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536) were tested for growth compatibility, resistance to antimicrobial agents, antibacterial activity against pathogens, resistance to gastric acidity, bile salt hydrolysis and adhesion to the human intestinal epithelial cell line HT29. All of these strains were resistant to gentamycin, but none showed in vitro growth incompatibility or the presence of known resistance determinants. B. breve M-16 V had the best probiotic characteristics and, indeed, was the only strain possessing antibacterial activity against Escherichia coli and Klebsiella pneumoniae. All strains were resistant to simulated gastric juice, while only B. longum subsp. longum BB536 and B. breve M-16 V showed a bile salt hydrolytic activity. Interestingly, a strong adhesion to HT29 cells was observed in all Bifidobacterium strains. In conclusion, B. breve M-16 V, B. longum subsp. longum BB536 and B. longum subsp. infantis M-63 showed several promising characteristics as probiotic strains.     

Enterococcus faecalis V583 contains a cytochrome bd-type respiratory oxidase

We have cloned an Enterococcus faecalis gene cluster, cydABCD, which when expressed in Bacillus subtilis results in a functional cytochrome bd terminal oxidase. Our results indicate that E. faecalis V583 cells have the capacity of aerobic respiration when grown in the presence of heme.     

Early and specific gene expression triggered by rice resistance gene Pi33 in response to infection by ACE1 avirulent blast fungus

* Our view of genes involved in rice disease resistance is far from complete. Here we used a gene-for-gene relationship corresponding to the interaction between atypical avirulence gene ACE1 from Magnaporthe grisea and rice resistance gene Pi33 to better characterize early rice defence responses induced during such interaction. * Rice genes differentially expressed during early stages of Pi33/ACE1 interaction were identified using DNA chip-based differential hybridization and QRT-PCR survey of the expression of known and putative regulators of disease resistance. * One hundred genes were identified as induced or repressed during rice defence response, 80% of which are novel, including resistance gene analogues. Pi33/ACE1 interaction also triggered the up-regulation of classical PR defence genes and a massive down-regulation of chlorophyll a/b binding genes. Most of these differentially expressed genes were induced or repressed earlier in Pi33/ACE1 interaction than in the gene-for-gene interaction involving Nipponbare resistant cultivar. * Besides demonstrating that an ACE1/Pi33 interaction induced classical and specific expression patterns, this work provides a list of new genes likely to be involved in rice disease resistance.     

Systematic inactivation and phenotypic characterization of two-component signal transduction systems of Enterococcus faecalis V583

The ability of enterococci to adapt and respond to different environmental stimuli, including the host environment, led us to investigate the role of two-component signal transduction in the regulation of Enterococcus faecalis physiology. Using a bioinformatic approach, we previously identified 17 two-component systems (TCS), consisting of a sensory histidine kinase and the cognate response regulator, as well as an additional orphan response regulator (L. E. Hancock and M. Perego, J. Bacteriol. 184:5819-5825, 2002). In an effort to identify the potential function of each TCS in the biology of E. faecalis clinical isolate strain V583, we constructed insertion mutations in each of the response regulators. We were able to inactivate 17 of 18 response regulators, the exception being an ortholog of YycF, previously shown to be essential for viability in a variety of gram-positive microorganisms. The biological effects of the remaining mutations were assessed by using a number of assays, including antibiotic resistance, biofilm formation, and environmental stress. We identified TCS related to antibiotic resistance and environmental stress and found one system which controls the initiation of biofilm development by E. faecalis.