Tracking bacterial infection of macrophages using a novel red-emission pH sensor.
The relationship between bacteria and host phagocytic cells is key to the induction of immunity. To visualize and monitor bacterial infection, we developed a novel bacterial membrane permeable pH sensor for the noninvasive monitoring of bacterial entry into murine macrophages. The pH sensor was constructed using 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as an electron-withdrawing group and aniline as an electron-donating group. A piperazine moiety was used as the pH-sensitive group. Because of the strong electron-donating and -withdrawing units conjugated in the sensing moiety M, the fluorophore emitted in the red spectral window, away from the autofluorescence regions of the bacteria. Following the engulfment of sensor-labeled bacteria by macrophages and their subsequent merger with host lysosomes, the resulting low-pH environment enhances the fluorescence intensity of the pH sensors inside the bacteria. Time-lapse analysis of the fluorescent intensity suggested significant heterogeneity of bacterial uptake among macrophages. In addition, qRT-PCR analysis of the bacterial 16 S rRNA gene expression within single macrophage cells suggested that the 16 S rRNA of the bacteria was still intact 120 min after they had been engulfed by macrophages. A toxicity assay showed that the pH sensor has no cytotoxicity towards either E. coli or murine macrophages. The sensor shows good repeatability, a long lifetime, and a fast response to pH changes, and can be used for a variety of bacteria.
Jin
Yuguang
Y
Center for Ecogenomics, The Biodesign Institute, Arizona State University, PO Box 875801, Tempe, AZ 85287-5801, USA.
Tian
Yanqing
Y
Zhang
Weiwen
W
Jang
Sei-Hum
SH
Jen
Alex K-Y
AK
Meldrum
Deirdre R
DR
D000818:Animals; D001407:Bacillus / Q000502:physiology*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004926:Escherichia coli / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008264:Macrophages / Q000382:microbiology*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D018613:Microscopy, Confocal; D010587:Phagocytosis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers