Proteomics and in vivo labeling of protein thiols in Sulfolobus solfataricus during exposure to antimony

Abstract

Antimony (Sb) has a long history in both the chemical and social literature. As a metalloid it is often found in the environment with Arsenic (As). Extended exposure in humans causes heart disease, lung disease, diarrhea, severe vomiting and ulcers. In plants, it inhibits early crop growth. A large body of data on bacterial response and mechanisms for detoxification also exists. In contrast, knowledge about how archaeal species respond to Sb is much less extensive. The model crenarchaeal organism Sulfolobus solfataricus, can survive in environments with high antimony concentrations, and the genetic and biochemical mechanisms responsible for antimony tolerance have yet to be reported. As a first step in bringing to light the biological response of S. solfataricus to antimony, a set of proteomic and chemical tagging experiments were undertaken. Two-dimensional differential gel electrophoresis (2D-DIGE) showed a limited response from intracellular and membrane proteins with respect to their abundance. In contrast, chemical targeting of cysteine residues revealed that extensive oxidation had occurred to both cytosolic and membrane proteins upon exposure to antimony. To remove any possible experimental artifacts that could alter the oxidation state of protein thiols, a method for labeling cytoplasmic proteins in live S. solfataricus cells was developed. This method used the recently described Z-dye probes for quantitative comparisons. Together, our results suggest that Sb response is primarily focused on a general stress factors likely stemming from oxidative damage to proteins. No evidence for a specific transport or bioconversion was present. Cysteine residues in membrane proteins displayed the most significant oxidative changes. The demonstration that chemical biology approaches can be applied to prokaryotic cells, even those growing at extremes of temperature and Ph, should have broad appeal for microbiologists well beyond those investigating archaea.