Organic Chemistry, Short talk
OC-013

A ratiometric pH sensor for Gram-positive and Gram-negative bacteria.

D. F. Kossmann1, A. Iizuka2,3, N. Khanna2,4, P. Rivera-Fuentes1*
1Department of Chemistry, University of Zurich, 8057, Zurich, Switzerland, 2Department of Biomedicine, University of Basel, 4031, Basel, Switzerland, 3Swiss Institute of Bioinformatics, 4051, Basel, Switzerland, 4Division of Infection Diseases, University Hospital Basel, 4031, Basel, Switzerland

Persister cells are phenotypically dormant bacteria that survive antibiotic treatment without genetic resistance. Their formation is modulated by environmental fluctuations, especially exposure to low pH. To understand how individual cells adapt to pH stress, methods are required to analyze single-cell physiology. Yet most pH probes are optimized for mammalian cells and poorly suited for bacterial applications, as bacteria preferentially take up positively charged, polar, or zwitterionic molecules.[1-3]
We report a ratiometric fluorescent probe for sensing cytoplasmic pH in bacteria.[4] Our probes are based on hemicyanine dyes that exhibit good cellular uptake in both Gram-positive and Gram-negative bacteria. Additionally, our probes exhibit improved photophysical properties for bacterial imaging, particularly increased brightness due to the apolar nature of the intracellular space. By tuning the electrophilicity of the molecular scaffold, we obtained a pH-sensing CouCy probe that preferentially reacts with OH¯ and exhibits a ratiometric emission change. The response is rapid and reversible, enabling real-time tracking of pH fluctuations around neutrality and biologically relevant acidification. We validated the live-cell imaging using Escherichia coli, Staphylococcus epidermidis, and a clinically isolated methicillin-resistant Staphylococcus aureus strain. Furthermore, the probes enabled monitoring of phagocytosis of clinical strains by immune cells and allowed distinction of virulence phenotypes based on intracellular pH. Our probes are promising tools for detecting phenotypic heterogeneity of clinically relevant bacteria.

[1] T. A. Krulwich, G. Sachs, E. Padan, Nat. Rev. Microbiol. 2011, 9, 330–343.
[2] N. Leimer, C. Rachmühl, M. Palheiros Marques, A. S. Bahlmann, A. Furrer, F. Eichenseher, K. Seidl, U. Matt, M. J. Loessner, R. A. Schuepbach, A. S. Zinkernagel, J. Infect. Dis. 2016, 213, 305–313.
[3] H.-K. Ropponen, R. Richter, A. K. Hirsch, C.-M. Lehr, Adv Drug Deliv Rev 2021, 172, 339-360.
[4] D. Kossmann, A. Iizuka, N. Khanna, P. Rivera-Fuentes, J. Am. Chem. Soc. 2026, 148, 4754–4765.