ABSTRACT
We report the use of genetically engineered cells in a pathogen identification sensor. This sensor uses B lymphocytes that have been engineered to emit light within seconds of exposure to specific bacteria and viruses. We demonstrated rapid screening of relevant samples and identification of a variety of pathogens at very low levels. Because of its speed, sensitivity, and specificity, this pathogen identification technology could prove useful for medical diagnostics, biowarfare defense, food- and water-quality monitoring, and other applications.
Subject(s)
B-Lymphocytes , Bacteria/isolation & purification , Bacteriological Techniques , Biosensing Techniques , Viruses/isolation & purification , Aequorin/biosynthesis , Antibodies, Bacterial/immunology , Antibodies, Viral/immunology , B-Lymphocytes/immunology , Bacillus anthracis/immunology , Bacillus anthracis/isolation & purification , Bacteria/immunology , Cell Line , Colony Count, Microbial , Encephalitis Virus, Venezuelan Equine/immunology , Encephalitis Virus, Venezuelan Equine/isolation & purification , Escherichia coli O157/immunology , Escherichia coli O157/isolation & purification , Foot-and-Mouth Disease Virus/immunology , Foot-and-Mouth Disease Virus/isolation & purification , Immunoglobulin Variable Region/immunology , Light , Receptors, Antigen, B-Cell/immunology , Sensitivity and Specificity , Time Factors , Transfection , Viruses/immunology , Yersinia pestis/immunology , Yersinia pestis/isolation & purificationABSTRACT
The transcription factor C/EBP alpha regulates early steps of normal granulocyte differentiation since mice with a disruption of the C/EBP alpha gene do not express detectable levels of the granulocyte colony-stimulating factor receptor and produce no neutrophils. We have recently shown that C/EBP alpha function is also impaired in acute myeloid leukemias. However, how the transcriptional activity of C/EBP alpha is regulated both in myelopoiesis and leukemogenesis is not fully understood. The current study demonstrates that activated Ras enhances the ability of C/EBP alpha to transactivate the granulocyte colony-stimulating factor receptor promoter and a minimal promoter containing only C/EBP DNA binding sites. Ras signaling activates C/EBP alpha via the transactivation domain because it enhances the transactivation function of a fusion protein containing a Gal4 DNA binding domain and the C/EBP alpha transactivation domain and does not change C/EBP alpha DNA binding. Ras acts on serine 248 of the C/EBP alpha transactivation domain, because it does not enhance the transactivation function of a C/EBP alpha serine 248 to alanine point mutant. Interestingly, serine 248 of C/EBP alpha is a protein kinase C (PKC) consensus site, and a PKC inhibitor blocks the activation of C/EB alpha by Ras. Ras signaling leads to phosphorylation of C/EBP alpha in vivo. Finally, mutation of serine 248 to alanine obviates the ability of C/EBP alpha to induce granulocytic differentiation. These data suggest a model where Ras signaling enhances the activity of C/EBP alpha to induce granulocytic differentiation by phosphorylation of serine 248.
