scholarly journals Measurement of bacterial gene expression in vivo

2000 ◽  
Vol 355 (1397) ◽  
pp. 601-611 ◽  
Author(s):  
Isabelle Hautefort ◽  
Jay C. D. Hinton
Keyword(s):  
Gene Expression ◽  
Dynamic Environment ◽  
Subtractive Hybridization ◽  
Fluorescence Induction ◽  
Infection Process ◽  
Bacterial Gene ◽  
Technology System ◽  
Bacterial Gene Expression

The complexities of bacterial gene expression during mammalian infection cannot be addressed by in vitro experiments. We know that the infected host represents a complex and dynamic environment, which is modified during the infection process, presenting a variety of stimuli to which the pathogen must respond if it is to be successful. This response involves hundreds of ivi (in vivo– induced) genes which have recently been identified in animal and cell culture models using a variety of technologies including in vivo expression technology, differential fluorescence induction, subtractive hybridization and differential display. Proteomic analysis is beginning to be used to identify IVI proteins, and has benefited from the availability of genome sequences for increasing numbers of bacterial pathogens. The patterns of bacterial gene expression during infection remain to be investigated. Are ivi genes expressed in an organ–specific or cell–type–specific fashion ? New approaches are required to answer these questions. The uses of the immunologically based in vivo antigen technology system, in situ PCR and DNA microarray analysis are considered. This review considers existing methods for examining bacterial gene expression in vivo, and describes emerging approaches that should further our understanding in the future.

Concurrence between the gene expression pattern of Actinobacillus actinomycetemcomitans in localized aggressive periodontitis and in human epithelial cells

2005 ◽  
Vol 54 (5) ◽  
pp. 497-504 ◽  
Author(s):  
Joseph Richardson ◽  
Justin Corey Craighead ◽  
Sam Linsen Cao ◽  
Martin Handfield
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Aggressive Periodontitis ◽  
Actinobacillus Actinomycetemcomitans ◽  
Bacterial Gene ◽  
Bacterial Gene Expression

Actinobacillus actinomycetemcomitans is a facultatively intracellular pathogen and the aetiological agent of localized aggressive periodontitis. Screening of the genome of A. actinomycetemcomitans for in vivo-induced antigen determinants previously demonstrated that the proteome of this organism differs in laboratory culture compared with conditions found during active infection. The aim of the present study was to determine whether the bacterial gene expression pattern inferred with in vivo-induced antigen technology (IVIAT) in human infections was consistent with the gene expression pattern occurring upon epithelial cell association. To this end, a real-time PCR method was developed and used to quantify absolute and relative bacterial gene expression of A. actinomycetemcomitans grown extra- and intracellularly in two human epithelial cell lines (HeLa and IHGK). The amount of template used in the assay was normalized using the total count of viable bacteria (c.f.u.) as a reference point and performed in duplicate in at least two independent experiments. Controls for this experiment included 16S rRNA and gapdh. Transcription of all eight ORFs tested increased significantly (P < 0.05) in HeLa and IHGK cells compared with bacteria grown extracellularly. The concurrence of gene expression patterns found in the two models suggests that these epithelial cells are valid in vitro models of infection for the genes tested. IVIAT is an experimental platform that can be used as a validation tool to assess the reliability of animal and other models of infection and is applicable to most pathogens.

The identification of bacterial gene expression differences using mRNA-based isothermal subtractive hybridization

1995 ◽  
Vol 41 (2) ◽  
pp. 152-156 ◽  
Author(s):  
Eric A. Utt ◽  
Jeffery P. Brousal ◽  
Lynne C. Kikuta-Oshima ◽  
Frederick D. Quinn
Keyword(s):  
Gene Expression ◽  
Listeria Monocytogenes ◽  
Subtractive Hybridization ◽  
Listeriolysin O ◽  
Bacterial Strains ◽  
Bacterial Gene ◽  
Total Rna ◽  
Bacterial Gene Expression ◽  
Hybridization Probes ◽  
The Difference

We describe a method for isolating and determining differences in gene expression between related bacterial strains. The method is based upon differences in mRNA expression. To demonstrate this procedure, cDNA generated from total RNA of Listeria monocytogenes serotype 1/2a was hybridized to total RNA from a Tn916 mutant of serogroup 1/2a (M3) that was deficient in the production of listeriolysin O, the product of the hly gene. The single-stranded cDNA fragments remaining after hybridization represent the difference in expressed genes between the two strains. These subtraction products were used as hybridization probes to identify the corresponding hly gene in a Southern hybridization.Key words: subtractive hybridization, Listeria monocytogenes, hemolysin, gene expression, isogenic.

Spatiotemporal control of the bacteria to express interferon-γ by focused ultrasound for tumor immunotherapy

2021 ◽  
Author(s):  
Yuhao Chen ◽  
Meng Du ◽  
Zhen Yuan ◽  
Fei Yan ◽  
Zhiyi Chen
Keyword(s):  
Focused Ultrasound ◽  
Tumor Therapy ◽  
Tumor Immunotherapy ◽  
Interferon Γ ◽  
Genetic Switch ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
Ifn Γ

Abstract Bacteria-based tumor therapy has recently attracted wide attentions due to its unique capability in targeting tumors and preferentially colonizing the core area of the tumor. Various therapeutic genes were also harbored into these engineering bacteria to enhance their anti-tumor efficacy. However, it is difficult to spatiotemporally control the expression of these inserted genes in the tumor site. Here, we engineered an ultrasound-responsive bacterium (URB) which can induce the expression of exogenous genes in an ultrasound-controllable manner. Owing to the advantage of ultrasound in the tissue penetration, energy focusing into heating, an acoustic remote control of bacterial gene expression can be realized by designing a temperature-actuated genetic switch. Cytokine interferon-γ (IFN-γ), an important immune regulatory molecule that plays a significant role in tumor immunotherapy, was used to test the system. Our results showed a brief hyperthermia by focused ultrasound successfully induced the expression of IFN-γ gene, significantly improving anti-tumor efficacy of URB in vitro and in vivo. Our study provided a novel strategy for bacteria-mediated tumor immunotherapy.

scholarly journals Metatranscriptome profiling of the dynamic transcription of mRNA and sRNA of a probioticLactobacillusstrain in human gut

2018 ◽  
Author(s):  
Jicheng Wang ◽  
Zhihong Sun ◽  
Jianmin Qiao ◽  
Dong Chen ◽  
Chao Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lactobacillus Casei ◽  
Lactic Acid Production ◽  
Expression Patterns ◽  
Probiotic Bacteria ◽  
Gi Tract ◽  
Human Gut ◽  
Bacterial Gene

AbstractMetatranscriptomic sequencing has recently been applied to study how pathogens and probiotics affect human gastrointestinal (GI) tract microbiota, which provides new insights into their mechanisms of action. In this study, metatranscriptomic sequencing was applied to deduce thein vivoexpression patterns of an ingestedLactobacillus caseistrain, which was compared with itsin vitrogrowth transcriptomes. Extraction of the strain-specific reads revealed that transcripts from the ingestedL. caseiwere increased, while those from the residentL. paracaseistrains remained unchanged. Mapping of all metatranscriptomic reads and transcriptomic reads toL. caseigenome showed that gene expressionin vitroandin vivodiffered dramatically. About 39% (1163) mRNAs and 45% (93) sRNAs ofL. caseiwell-expressed were repressed after ingested into human gut. Expression of ABC transporter genes and amino acid metabolism genes was induced at day-14 of ingestion; and genes for sugar and SCFA metabolisms were activated at day-28 of ingestion. Moreover, expression of sRNAs specific to thein vitrolog phase was more likely to be activated in human gut. Expression of rli28c sRNA with peaked expression during thein vitrostationary phase was also activated in human gut; this sRNA repressedL. caseigrowth and lactic acid productionin vitro. These findings implicate that the ingestedL. caseimight have to successfully change its transcription patterns to survive in human gut, and the time-dependent activation patterns indicate a highly dynamic cross-talk between the probiotic and human gut including its microbe community.ImportanceProbiotic bacteria are important in food industry and as model microorganisms in understanding bacterial gene regulation. Although probiotic functions and mechanisms in human gastrointestinal tract are linked to the unique probiotic gene expression, it remains elusive how transcription of probiotic bacteria is dynamically regulated after being ingested. Previous study of probiotic gene expression in human fecal samples has been restricted due to its low abundance and the presence of of closely related species. In this study, we took the advantage of the good depth of metatranscriptomic sequencing reads and developed a strain-specific read analysis method to discriminate the transcription of the probioticLactobacillus caseiand those of its resident relatives. This approach and additional bioinformatics analysis allowed the first study of the dynamic transcriptome profiles of probioticL casei in vivo. The novel findings indicate a highly regulated repression and dynamic activation of probiotic genome in human GI tract.

USE OF DNA-DIRECTED IN VITRO SYSTEMS TO STUDY BACTERIAL GENE EXPRESSION

1981 ◽  
pp. 215-243
Author(s):  
Herbert Weissbach ◽  
Tanya Zarucki-Schulz ◽  
Hsiang-fu Kung ◽  
Carlos Spears ◽  
Betty Redfield ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
In Vitro Systems

scholarly journals Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere

2001 ◽  
Vol 91 (9) ◽  
pp. 873-881 ◽  
Author(s):  
R. Notz ◽  
M. Maurhofer ◽  
U. Schnider-Keel ◽  
B. Duffy ◽  
D. Haas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Fluorescens ◽  
Reporter Gene ◽  
Pythium Ultimum ◽  
Disease Suppression ◽  
Biotic Factors ◽  
Host Genotype ◽  
Bacterial Gene ◽  
Bacterial Gene Expression

Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA′-′lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA′-′lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.

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